@article {pmid41543170,
year = {2026},
author = {Romanowski, JS and Myles, KM and Adelman, ZN},
title = {Microhomology-mediated end joining is the predominant form of DNA repair in the mosquito Aedes aegypti with implications for gene editing, gene drive, and transgene removal.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41543170},
issn = {1362-4962},
support = {//National Institute of Allergies and Infectious Diseases/ ; AI148787/NH/NIH HHS/United States ; //NIAID/ ; },
mesh = {Animals ; *Aedes/genetics ; *Gene Editing/methods ; *DNA End-Joining Repair ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Transgenes ; Gene Drive Technology ; DNA Repair ; },
abstract = {Programmable site-specific nucleases have revolutionized the field of genetics, and in the field of mosquito vector control, gene editing by these tools has inspired a new wave of population control approaches that aim to prevent disease transmission. Little is known of how DNA repair is prioritized in mosquitoes, which diverged from the nearest model system (Drosophila) by >200 million years, despite site-specific gene editing now being commonplace. Here, we report a scalable, high-throughput platform for studying DNA double-stranded DNA break (DSB) repair in mosquitoes by delivering CRISPR/Cas9, I-SceI, or other nucleases to Aedes aegypti embryos, capable of measuring single-strand annealing (SSA), non-homologous end joining, and microhomology-mediated end-joining (MMEJ) repair outcomes. We find CRISPR/Cas9 can induce deletions of up to 8.6 kb through SSA repair and is tolerant of resection distances of 3.5 kb. Indel events were insensitive to lig4 knockouts, and across 20 synthetic guide RNAs (sgRNAs) representing 5 locations in 2 transgenic strains were almost exclusively attributed to MMEJ repair, establishing MMEJ as the dominant form of repair in A. aegypti at CRISPR/Cas9 DSBs. This information is critical to our understanding of how DNA repair shapes processes required for genetic control strategies involving gene drive action/resistance as well as transgene stability.},
}

Animals
*Aedes/genetics
*Gene Editing/methods
*DNA End-Joining Repair
CRISPR-Cas Systems
DNA Breaks, Double-Stranded
Transgenes
Gene Drive Technology
DNA Repair

@article {pmid41542957,
year = {2026},
author = {Lee, SY and Park, HH},
title = {Structural insights into promoter recognition by Aca7.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.70405},
pmid = {41542957},
issn = {1742-4658},
support = {RS-2025-02316334//NRF/ ; RS-2025-16065724//NRF korea/ ; },
abstract = {CRISPR-Cas systems provide adaptive immunity to bacteria, although bacteriophages counter these defenses with anti-CRISPR (Acr) proteins. Acr expression is frequently regulated by anti-CRISPR associated (Aca) proteins, which repress transcription by binding inverted repeat (IR) sequences in operon promoters. Here, we report the first identification of an IR motif within the AcrIF11-Aca7 operon promoter from Halomonas caseinilytica and present the crystal structure of Aca7 bound to this IR DNA. Biochemical assays demonstrated that Aca7 specifically recognizes the IR element, and structural analysis revealed a symmetric Aca7 dimer engaging both major grooves via helix-turn-helix motifs while stabilizing DNA bending through minor groove contacts. Residue-level interactions, including those mediated by R38, Q42, K46, and K49, establish a detailed basis for sequence-specific recognition. Comparison with Aca2 highlights distinct dimer architectures and DNA deformation strategies among Aca proteins. Our findings uncover the molecular mechanism by which Aca7 represses AcrIF11 expression and broaden the understanding of Aca-mediated transcriptional regulation.},
}

@article {pmid41541975,
year = {2025},
author = {Hummel, L and Carr, C and Biow, S and Asher, K and Sauer, T and Maylin, M},
title = {Re-examining the Diagnostic Criteria for Wilson's Disease: A Case Report and Literature Review.},
journal = {Cureus},
volume = {17},
number = {12},
pages = {e99271},
pmid = {41541975},
issn = {2168-8184},
abstract = {Wilson's disease (WD) is a heterogeneous genetic disorder for which diagnosis is challenging. We present the case of a 54-year-old woman with a complicated medical history, including hepatitis C, cirrhosis, hepatic encephalopathy, and extensive psychiatric disease, who was transferred to our hospital for management of a spinal epidural abscess. Further findings suggested undiagnosed WD, including bilateral rings around Descemet's membrane and modestly low ceruloplasmin but normal urinary copper excretion. Many algorithms have been proposed for Wilson disease diagnosis, including clinical, laboratory, imaging, and genetic findings; however, no single test is diagnostic. The European Association for the Study of the Liver (EASL) guidelines are the most commonly employed algorithm. Updated 2022 guidelines by both the American Association for the Study of Liver Diseases (AASLD) and the British Association for the Study of the Liver (BASL) build upon this algorithm. Our patient scored 5 points on the EASL scale, which is sufficient for diagnosis. Nevertheless, we believe that she met the diagnostic criteria without having the actual disease. Newer guidelines by AASLD and BASL do not provide additional conclusivity. Our case demonstrates the need for re-evaluation of the diagnostic criteria of WD, where uncertainty can mean permanent hepatic and brain damage. Diagnostic guidelines should incorporate new biomarkers, ophthalmological techniques, and advanced technologies such as next-generation sequencing or CRISPR-Cas-based tools.},
}

@article {pmid41499124,
year = {2026},
author = {Wang, K and Wang, W and Hu, Y and Li, X and Yu, H and Xia, X and Kai, T and Wen, M},
title = {A Relay CRISPR/Cas12a System for Extraction-Free and Ultrasensitive Detection of Staphylococcus aureus in Complex Food Samples.},
journal = {Analytical chemistry},
volume = {98},
number = {2},
pages = {1657-1666},
doi = {10.1021/acs.analchem.5c06345},
pmid = {41499124},
issn = {1520-6882},
mesh = {*Staphylococcus aureus/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Food Microbiology ; Electrochemical Techniques ; DNA, Bacterial/genetics/analysis ; Limit of Detection ; RNA, Ribosomal, 16S/genetics ; },
abstract = {Staphylococcus aureus (SA) is a predominant foodborne pathogen, frequently associated with nutrient-rich matrices. However, rapid and ultrasensitive detection of SA in complex food samples remains a formidable challenge. In this work, we developed a relay biosensing system for detecting SA-16S rDNA that combines a magnetic bead-anchored APE1 DNA walker with an asymmetric CRISPR/Cas12a system. The magnetic bead-anchored DNA walker enables efficient separation of SA-targets from complex food matrices, while generating abundant activators through APE1-mediated catalytic cleavage. Employing spatially decoupled reactions with differential crRNAs, the asymmetric Cas12a system achieves programmable cascade amplification. Dual-mode fluorescence/electrochemical readout supports both laboratory analysis and on-site detection. The system demonstrates exceptional sensitivity, capable of detecting trace amounts of SA-16S rDNA (7.5 aM by fluorescence, 1.0 fM by electrochemistry) and SA (4 CFU/mL by fluorescence, 34 CFU/mL by electrochemistry). Overall, this work provides a modular and programmable biosensing strategy for bacterial identification, thereby facilitating advanced nucleic acid analysis within complex biological matrices.},
}

*Staphylococcus aureus/isolation & purification/genetics
*CRISPR-Cas Systems
*Biosensing Techniques/methods
*Food Microbiology
Electrochemical Techniques
DNA, Bacterial/genetics/analysis
Limit of Detection
RNA, Ribosomal, 16S/genetics

@article {pmid41439322,
year = {2026},
author = {Wang, Z and Yang, F and Zeng, S and Sun, R and Hu, Q and Du, Y},
title = {An integrated valved microfluidic platform for rapid and simultaneous nucleic acid detection.},
journal = {Lab on a chip},
volume = {26},
number = {2},
pages = {507-514},
doi = {10.1039/d5lc01096a},
pmid = {41439322},
issn = {1473-0189},
mesh = {Humans ; *Lab-On-A-Chip Devices ; *Nucleic Acid Amplification Techniques/instrumentation ; Human papillomavirus 16/genetics/isolation & purification ; Human papillomavirus 18/genetics/isolation & purification ; *DNA, Viral/analysis/genetics ; *Nucleic Acids/analysis ; *Microfluidic Analytical Techniques/instrumentation ; CRISPR-Cas Systems ; },
abstract = {Applying CRISPR-based diagnostics to point-of-care pathogen detection remains challenging because of the multi-step and time-consuming sample preparation process. This study presents a low-cost, integrated valved microfluidic device that combines recombinase polymerase amplification (RPA), CRISPR signal amplification, and lateral flow readout for simultaneous nucleic acid detection. The core advantage of the platform lies in its ability to sequentially control the entire multi-step assay through simple valve operation, significantly minimizing user intervention. All key reagents, including the RPA mix, Cas12a/crRNA complex, and proteinase K lysis buffer, are pre-lyophilized, ensuring stability and ready-to-use functionality. The platform demonstrates a sensitivity of 20 copies/reaction for HPV16/18 plasmids and accurately genotypes HPV in lysates of cervical cancer cells within one hour, showing complete concordance with quantitative PCR results. This integrated device, achieving a user-friendly protocol and visual readout, provides a powerful tool for nucleic acid-based point-of-care testing and self-testing in resource-limited settings.},
}

Humans
*Lab-On-A-Chip Devices
*Nucleic Acid Amplification Techniques/instrumentation
Human papillomavirus 16/genetics/isolation & purification
Human papillomavirus 18/genetics/isolation & purification
*DNA, Viral/analysis/genetics
*Nucleic Acids/analysis
*Microfluidic Analytical Techniques/instrumentation
CRISPR-Cas Systems

@article {pmid41422144,
year = {2025},
author = {Felício, D and Osório, H and Pereira, C and Brandão, AF and Freixo, JP and Carvalho, I and Sousa, AP and Castro-Caldas, M and Sequeiros, J and Lemos, C and Santos, M},
title = {Missense variant in TTBK2 kinase domain causes loss of function and impaired protein phosphorylation.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {2501},
pmid = {41422144},
issn = {2045-2322},
support = {UI/BD/154402/2023//Fundação para a Ciência e a Tecnologia/ ; UIDB/00215/2020, UIDP/00215/2020, LA/P/0064/2020 , UID/215/2025//Fundação para a Ciência e a Tecnologia/ ; DL 57/2016 - Norma Transitória//Fundação para a Ciência e a Tecnologia/ ; ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125//Rede Nacional de Espectrometria de Massa/ ; ZGRACA//Ataxia UK/ ; },
mesh = {Humans ; Phosphorylation ; *Mutation, Missense ; *Protein Serine-Threonine Kinases/genetics/metabolism/chemistry ; Protein Domains ; CRISPR-Cas Systems ; *Loss of Function Mutation ; HEK293 Cells ; },
abstract = {Tau tubulin kinase 2 (TTBK2) is a ubiquitous serine-threonine protein kinase implicated in diverse cellular processes, including microtubule regulation, ciliogenesis, synaptic signaling, and the phosphorylation of key proteins like TDP-43. Despite its relevance, many aspects of TTBK2 function in both physiological and pathological conditions remain poorly understood. Truncating variants in TTBK2 gene cause spinocerebellar ataxia type 11 (SCA11), a rare form of autosomal dominant cerebellar ataxia. However, the functional consequences and pathogenic potential of missense variants have yet to be elucidated. In this study, we developed a CRISPR/Cas9 knock-in cell model harboring a missense variant in TTBK2 kinase domain (NM_173500.4:c.625 C > T; p.Leu209Phe) to evaluate its impact on TTBK2 expression, associated protein levels, and phosphoproteomic profiles. TTBK2 missense variant (TTBK2-L209F) was associated with reduced TTBK2 protein levels, altered levels of cytoskeleton-related proteins, and impaired kinase activity, namely toward TDP-43. Phosphoproteomic analyses identified dysregulation in pathways linked to gene regulation, protein degradation, cytoskeletal organization, and TGF-β signaling. These findings provide valuable insights into the biological roles of TTBK2 in cellular signaling. Moreover, this study underscores the importance of functional studies to better understand the consequences of TTBK2 missense variants, particularly those affecting the kinase domain, and their potential contribution to disease.},
}

Humans
Phosphorylation
*Mutation, Missense
*Protein Serine-Threonine Kinases/genetics/metabolism/chemistry
Protein Domains
CRISPR-Cas Systems
*Loss of Function Mutation
HEK293 Cells

@article {pmid41388295,
year = {2025},
author = {Ji, T and Zhang, Y and Wang, Y and Yuan, K and Wang, M and Ye, J and Zhang, H and Zhang, N and Zhang, H},
title = {AND logic-gated CRISPR/Cas9 and hybridization chain reaction system for precise ctDNA detection.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {43},
pmid = {41388295},
issn = {1477-3155},
support = {2022YFB3808200//National Key Research and Development Program of China/ ; },
mesh = {*Circulating Tumor DNA/genetics/blood/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; Polymorphism, Single Nucleotide ; Nucleic Acid Amplification Techniques/methods ; Biomarkers, Tumor/genetics/blood ; Nucleic Acid Hybridization ; Mutation ; Proto-Oncogene Proteins p21(ras)/genetics ; Limit of Detection ; },
abstract = {Circulating tumor DNA (ctDNA) is a critical biomarker for liquid biopsies, enabling the non-invasive acquisition of cancer-related information from blood samples. Precise detection of ctDNA, particularly the identification of single-nucleotide variations (SNVs), is crucial for early cancer diagnosis, therapeutic monitoring, and prognostic evaluation. However, current ctDNA detection methods often encounter challenges such as complex procedures, difficult data analysis, and false-positive signals during pre-amplification. In this study, we introduce a novel detection method based on AND logic-gated integration of interspaced short palindromic repeats and associated proteins (CRISPR/Cas9) system with hybridization chain reaction (HCR) isothermal amplification. This strategy enhances the specific and sensitive detection of ctDNA. The incorporation of the AND logic gate effectively minimizes the off-target effects of Cas9 and enables the differentiation of single-nucleotide mutations, such as KRAS G12D, even in complex serum environments. Our system exhibits high sensitivity and specificity, achieving a limit of detection as low as 1 fM and capable of identifying SNVs mutations with allele fractions as low as 0.1% among wild-type sequences. Furthermore, we validated the specificity of our approach by successfully detecting various mutations, including KRAS G12C, KRAS G12D, EGFR T790M and TP53 R273H, in simulated clinical samples. These findings highlight a reliable method for precise ctDNA detection, offering high specificity, selectivity, and accuracy, thus paving the way for potential cancer diagnostic application.},
}

*Circulating Tumor DNA/genetics/blood/analysis
Humans
*CRISPR-Cas Systems/genetics
Polymorphism, Single Nucleotide
Nucleic Acid Amplification Techniques/methods
Biomarkers, Tumor/genetics/blood
Nucleic Acid Hybridization
Mutation
Proto-Oncogene Proteins p21(ras)/genetics
Limit of Detection

@article {pmid41387738,
year = {2025},
author = {Zhang, X and Zhu, T and Zhang, W and Zhang, Y and Zhang, J and Yang, J and Xia, C and Zhao, H and Yu, Y and Wen, C},
title = {Ethylene promotes branch formation but inhibits tendril development in cucumber.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {745},
pmid = {41387738},
issn = {2041-1723},
support = {JKZX202207//Beijing Academy of Agricultural and Forestry Sciences (BAAFS)/ ; },
mesh = {*Ethylenes/pharmacology/metabolism ; *Cucumis sativus/growth & development/genetics/metabolism/drug effects ; Gene Expression Regulation, Plant/drug effects ; Plant Proteins/genetics/metabolism ; Signal Transduction/drug effects ; CRISPR-Cas Systems ; *Plant Growth Regulators/pharmacology/metabolism ; Mutation ; Promoter Regions, Genetic ; },
abstract = {Ethylene coordinates numerous plant growth processes, particularly in cucurbit crops, yet its role in vegetative growth regulation remains largely unexplored. Here, we report the function of ethylene in controlling branch and tendril development in cucumber. We find that ethylene promotes branches formation but inhibits tendrils development in a dose-dependent manner. CRISPR-Cas9-generated gene-edited Csein2 and Csein3/Cseil1 mutants exhibit few branches and more tendrils. Exogenous ethylene can recover the branch/tendril defective phenotypes of the Csein3 and Cseil1 mutants but not those of the Csein2 mutant or the Csein3/Cseil1 double mutant. Transcriptomic and metabolic analyses reveal that CsCYP707A4 and CsTL are the key downstream targets of ethylene signaling. We show that CsEIN3 can bind to its promoters to activate the expression of CsCYP707A4 but inhibit the expression of CsTL, which leads to the opposite effect on branch and tendril development. The study sets the foundation for designing ideal plant architecture to increase production efficiency.},
}

*Ethylenes/pharmacology/metabolism
*Cucumis sativus/growth & development/genetics/metabolism/drug effects
Gene Expression Regulation, Plant/drug effects
Plant Proteins/genetics/metabolism
Signal Transduction/drug effects
CRISPR-Cas Systems
*Plant Growth Regulators/pharmacology/metabolism
Mutation
Promoter Regions, Genetic

@article {pmid41382257,
year = {2025},
author = {Chen, W and Wu, P and Champer, J},
title = {Strategies to improve the efficiency of homing gene drives with multiplexed gRNAs.},
journal = {BMC biology},
volume = {24},
number = {1},
pages = {12},
pmid = {41382257},
issn = {1741-7007},
support = {32270672//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics ; *Gene Drive Technology/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; },
abstract = {BACKGROUND: CRISPR homing gene drive holds great potential for pest control, but its success is challenged by the generation of resistance alleles through end-joining repair. Using multiple gRNAs to target adjacent sites within a conserved gene can prevent functional resistance by allowing repeated cleavage events, but poor homology during DNA repair may compromise efficiency.

RESULTS: We first assessed the efficiency of single gRNA drives with truncated homology arms in Drosophila melanogaster mimicking a multiplexed system in which only one site is cleaved. Integrating results into a detailed gRNA multiplexing model, we found that efficiency loss was greater than expected. To mitigate this, we evaluated two new strategies: (1) extended homology arms to span all target sites (with mutations in the PAMs to prevent self-cleavage) and (2) a population-level gRNA multiplexing system involving two or more drives, each carrying two gRNAs. Extended homology arms did not result in notable improvement in conversion efficiency, and the extended region could be lost during drive conversion. The population-level multiplexing gRNAs strategy was more promising, though the intentionally mutated PAM also could not be consistently inherited. Simulations of homing suppression drives applying population-level multiplexed gRNAs increased the success rate of population elimination and reduced the time required for suppression.

CONCLUSIONS: Future drive designs requiring a larger number of gRNAs could potentially be improved. The design relying on extended homology arms may not represent an optimal strategy. However, population-level multiplexing gRNAs could serve as a promising alternative, enhancing efficiency while maintaining tolerance to functional resistance.},
}

Animals
*Drosophila melanogaster/genetics
*Gene Drive Technology/methods
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems

@article {pmid41370122,
year = {2026},
author = {Pfisterer, L and Boyle, C and Cole, A and Mitchell, I and Flanagan, M and Gromley, Z and Gromley, A},
title = {Disruption of the centriolin/Cep110 gene (CNTRL) with CRISPR/Cas9 leads to cell cycle arrest and cell death of rhabdomyosarcoma cells in vitro.},
journal = {Molecular biology of the cell},
volume = {37},
number = {2},
pages = {br4},
doi = {10.1091/mbc.E25-08-0365},
pmid = {41370122},
issn = {1939-4586},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; *Rhabdomyosarcoma/genetics/metabolism/pathology ; Cell Line, Tumor ; *Cell Cycle Checkpoints/genetics ; *Cell Cycle Proteins/genetics/metabolism ; Gene Editing/methods ; Centrosome/metabolism ; Cell Death/genetics ; *Microtubule-Associated Proteins/genetics/metabolism ; Cilia/metabolism ; Cell Survival/genetics ; Cell Proliferation/genetics ; Apoptosis/genetics ; },
abstract = {Rhabdomyosarcoma is the most common pediatric soft tissue cancer, thought to arise from primitive mesenchymal cells that differentiate into skeletal muscle. Previous studies suggest that primary cilia may play a role in the development of rhabdomyosarcoma. Primary cilia are cellular structures that arise from the centrosome and serve important functions in sensory signaling, cell migration, and developmental processes. However, most rhabdomyosarcoma cell lines do not have primary cilia. Because primary cilia are derived from centrosomes, the development of rhabdomyosarcoma may, in fact, be due to the function of centrosome proteins rather than the primary cilia itself. Therefore, this study sought to determine if the centrosomal protein centriolin/Cep110, which is localized to both centrosomes and primary cilia, plays a role in rhabdomyosarcoma biology. The gene editing tool CRISPR/Cas9 was used to disrupt the centriolin/Cep110 gene in the rhabdomyosarcoma cell line CCL-136, and the effects on cell viability and cell cycle progression were assayed. Our results show that loss of centriolin/Cep110 leads to cell cycle arrest and apoptotic cell death in rhabdomyosarcoma cells. These findings suggest that centriolin/Cep110 plays a key role in rhabdomyosarcoma cell proliferation and viability and that this centrosome protein may represent a potential target for future rhabdomyosarcoma therapies.},
}

CRISPR-Cas Systems/genetics
Humans
*Rhabdomyosarcoma/genetics/metabolism/pathology
Cell Line, Tumor
*Cell Cycle Checkpoints/genetics
*Cell Cycle Proteins/genetics/metabolism
Gene Editing/methods
Centrosome/metabolism
Cell Death/genetics
*Microtubule-Associated Proteins/genetics/metabolism
Cilia/metabolism
Cell Survival/genetics
Cell Proliferation/genetics
Apoptosis/genetics

@article {pmid41540035,
year = {2026},
author = {Zhang, H and Shang, R and Zhang, Z and Zhou, M and Bigot, A and Cai, Y and Zhao, Y and Wang, Y and Deshmukh, A and Kudryashova, E and Kudryashov, DS and He, C and Mouly, V and Bi, P},
title = {Development of a split-toxin CRISPR screening platform to systematically identify regulators of human myoblast fusion.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {547},
pmid = {41540035},
issn = {2041-1723},
support = {GM147209//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; AR080330//U.S. Department of Health & Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; },
mesh = {Humans ; *Myoblasts/metabolism/cytology ; *Muscle Development/genetics ; Cell Differentiation/genetics ; Cell Fusion ; *CRISPR-Cas Systems/genetics ; Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Muscle, Skeletal/metabolism ; High-Throughput Screening Assays/methods ; Single-Cell Analysis ; Cell Line ; },
abstract = {Muscle defects are common in human developmental disorders and often cause severe functional impairment. These defects arise from intricate tissue crosstalk and rare genetic mutations, underscoring the need to systematically identify cell-autonomous mechanisms regulating human myogenesis. Here we show a rationally designed, high-throughput genetic screening platform that integrates human myoblast models, customized CRISPR libraries, and a split-toxin strategy that enables quantitative selection of fusion-defective myocytes. Leveraging this platform, our initial screen uncovers a large group of hits essential for human myoblast fusion. The majority of these hits converge into 23 protein complexes. Notably, mutations in 41 screen hits are associated with human diseases marked by abnormal skeletal-muscle morphology. Applying a new single-cell CRISPR & RNA-seq approach, we show that majority of these hits control human myoblast fusion as well as influence early-stage myogenic differentiation. This work establishes a scalable approach to identify cell-autonomous regulators of human muscle differentiation and fusion.},
}

Humans
*Myoblasts/metabolism/cytology
*Muscle Development/genetics
Cell Differentiation/genetics
Cell Fusion
*CRISPR-Cas Systems/genetics
Mutation
*Clustered Regularly Interspaced Short Palindromic Repeats
Muscle, Skeletal/metabolism
High-Throughput Screening Assays/methods
Single-Cell Analysis
Cell Line

@article {pmid41538317,
year = {2026},
author = {Ham, DT and Browne, TS and Zhang, CQ and Foo, GW and Uruthirapathy, AS and Gloor, GB and Edgell, DR},
title = {Machine learning reveals sequence and methylation determinants of SaCas9-PAM interactions in bacteria.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41538317},
issn = {1362-4962},
support = {PJT 159708/CAPMC/CIHR/Canada ; PJT 191939/CAPMC/CIHR/Canada ; },
mesh = {*Machine Learning ; Staphylococcus aureus/genetics/enzymology ; *CRISPR-Associated Protein 9/metabolism/genetics ; Escherichia coli/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Cas Systems ; Nucleotide Motifs ; DNA Methylation ; Methylation ; Gene Editing/methods ; },
abstract = {Cas9 nucleases defend bacteria against invading DNA and can be used with single guide RNAs (sgRNAs) as antimicrobials and genome-editing tools. However, bacterial applications are limited by incomplete knowledge of Cas9-target interactions. Here, we generated large-scale Staphylococcus aureus Cas9 (SaCas9)/sgRNA activity datasets in bacteria and trained a machine learning model (crispr macHine trAnsfer Learning) to predict SaCas9 activity. Incorporating downstream sequences flanking the canonical NNGRRN protospacer adjacent motif (PAM) at positions [+1] and [+2] improved predictive performance, with T-rich dinucleotides at these positions correlating with higher in vivo activity. Crucially, SaCas9 showed $\sim$10-fold reduced activity at sites containing a 5$^{\prime}$
-NNGGAT[C]-3$^\prime$ PAM [+1] sequence in pooled sgRNA experiments in Escherichia coli and Citrobacter rodentium. Plasmid cleavage assays in DNA adenine methyltransferase (DAM)-deficient E. coli confirmed that adenine methylation at GATC motifs inhibited SaCas9 activity. Removal of a DAM site within a PAM sequence enhanced cleavage, while introduction of a site reduced activity, directly linking adenine methylation to SaCas9 activity. These findings demonstrate that machine learning can uncover biologically relevant determinants of Cas9 activity. Avoidance of methylated PAMs may reflect an evolutionary adaptation by SaCas9 to discriminate self from nonself or to counter methylation as a phage and plasmid antirestriction strategy.},
}

*Machine Learning
Staphylococcus aureus/genetics/enzymology
*CRISPR-Associated Protein 9/metabolism/genetics
Escherichia coli/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
CRISPR-Cas Systems
Nucleotide Motifs
DNA Methylation
Methylation
Gene Editing/methods

@article {pmid41538311,
year = {2026},
author = {Xiao, Z and Sun, Y},
title = {Illuminating the genome: emerging approaches in CRISPR-Cas live-cell imaging.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41538311},
issn = {1362-4962},
support = {2022YFA1303103//National Key Research and Development Program of China/ ; 2022YFA3401100//National Key Research and Development Program of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Genome ; Gene Editing/methods ; Animals ; },
abstract = {CRISPR-Cas-based live-cell imaging has rapidly become a central technology for studying genome dynamics with high specificity and flexibility. By coupling nuclease-deactivated Cas (dCas) with programmable guide RNAs, genomic loci can be tracked in living cells, providing direct insights into nuclear organization and chromatin behavior. While repetitive regions such as telomeres and centromeres are readily visualized, labeling non-repetitive loci remains more challenging due to weak signals and high background. Recent advances, including multicolor labeling strategies, innovative amplification systems based on dCas9 and single-guide RNA (sgRNA) engineering, and integration with novel fluorescent reporters, have markedly expanded the applicability of CRISPR imaging across the genome. These developments have expanded the multiplexing capacity of CRISPR imaging, improved signal-to-background ratios, and even enabled the visualization of non-repetitive genomic loci. Nonetheless, key challenges remain, including cellular toxicity, replication stress, and genomic instability associated with prolonged CRISPR expression. In this review, we summarize recent advances in CRISPR live-cell imaging and highlight key design trade-offs and biological constraints.},
}

*CRISPR-Cas Systems/genetics
Humans
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Genome
Gene Editing/methods
Animals

@article {pmid41536960,
year = {2026},
author = {van der Wilt, CN and Veltrop, RJA and Janssens, MH and Bakker, I and Stillitano, F and Sluijter, JPG and van Laake, LW and van der Velden, J and Villard, E and Montag, J and Denning, C and van Tintelen, JP and Te Riele, ASJM and van der Harst, P and Schurgers, LJ and van Steenbeek, FG and Harakalova, M},
title = {Setting the stage for cardiomyopathy gene editing trials: a systematic review of isogenic pair use in human induced pluripotent stem cell-derived cardiomyocyte research.},
journal = {European heart journal open},
volume = {6},
number = {1},
pages = {oeaf161},
pmid = {41536960},
issn = {2752-4191},
abstract = {In vitro gene editing using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has demonstrated the feasibility of introducing or correcting specific pathogenic variants. These successes represent a key first step towards therapeutic genome editing for cardiomyopathies, showing that precise, variant-specific interventions are achievable. To translate in vitro findings to the clinic, it is essential to develop robust disease models that yield meaningful, translatable data. The next challenge is systematically identifying disease-causing variants amenable to gene editing with strong pre-clinical support. Therefore, we conducted a systematic search of published studies on isogenic hiPSC-CM pairs in cardiomyopathy research with specific criteria, including (likely) pathogenic variants causing cardiomyopathy, correction and/or introduction of variants, differentiation into CMs, and functional follow-up. We systematically assessed 785 papers and highlighted 101 studies meeting our inclusion criteria reporting 69 patients carrying 56 unique variants across 31 genes, most commonly MYH7, MYBPC3, and DMD. This expanded to 91 variants across 38 genes upon inclusion of the introduced variants in a donor line. However, reported clinical data were often incomplete, underscoring the need for standardized phenotypic documentation. We reveal a lack of patient details, which creates an incomplete picture of underlying disease variables that hinder the design of targeted personalized treatments. Omitted key clinical data can lead to misinterpretations or overlooked variables that impact treatment outcomes. This systematic review integrates current evidence from successful in vitro studies using isogenic hiPSC-CM models and proposes a reporting framework for variant prioritization and the rigorous application of isogenic controls in cardiomyopathy research.},
}

@article {pmid41536809,
year = {2026},
author = {Liou, RH and Urrutia-Cabrera, D and Liu, CF and Wu, S and Westin, IM and Golovleva, I and Liu, GS and Kumar, S and McLenachan, S and Chen, FK and Hsu, FT and Huang, CL and Edwards, T and Martin, KR and Cheng, AW and Wong, RCB},
title = {Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to target ABCA4 splice variants in Stargardt disease.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {1},
pages = {102789},
pmid = {41536809},
issn = {2162-2531},
abstract = {Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in the ABCA4 gene. The splicing variants c.5461-10T>C and c.4773+3A>G in ABCA4 cause the skipping of exon 39-40 and exon 33-34, respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate these ABCA4 splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C, and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containing ABCA4 variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-spliced ABCA4 c.5461-10T>C variants and up to 78% reduction of the ABCA4 c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced ABCA4 c.4773+3A>G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts for ABCA4, providing an important step to advance the development of gene therapy to treat STGD1.},
}

@article {pmid41535388,
year = {2026},
author = {Rolando, JC and Thieme, A and Weckman, NE and Kim, N and de Puig, H and Tan, X and Cotnoir, E and Chaturvedi, V and Collins, JJ and Walt, DR},
title = {Digital CRISPR-based diagnostics for quantification of Candida auris and resistance mutations.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {41535388},
issn = {2157-846X},
support = {WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; },
abstract = {Candida auris, an increasingly prevalent fungal pathogen, requires both rapid identification and antifungal susceptibility testing to enable proper treatment. This study introduces digital SHERLOCK (dSHERLOCK), a platform that combines CRISPR/Cas nucleic acid detection, single-template quantification and real-time kinetics monitoring. Assays implemented on this platform display excellent sensitivity to C. auris from major clades 1-4, while maintaining specificity when challenged with common environmental and pathogenic fungi. dSHERLOCK detects C. auris within 20 min in minimally processed swab samples and achieves sensitive quantification (1 c.f.u. µl[-1]) within 40 min. To address antifungal susceptibility testing, we develop assays that detect mutations that are commonly associated with azole and echinocandin multidrug resistance. We use machine learning and real-time monitoring of reaction kinetics to achieve highly accurate simultaneous quantification of mutant and wild-type FKS1 SNP alleles in fungal populations with mixed antifungal susceptibility, which would be misdiagnosed as completely susceptible or resistant under standard reaction conditions. Our platform's use of commercially available materials and common laboratory equipment makes C. auris diagnostics widely deployable in global healthcare settings.},
}

@article {pmid41526513,
year = {2026},
author = {Ngo, W and Wu, JLY and Wasko, KM and Doudna, JA},
title = {Targeted delivery of genome editors in vivo.},
journal = {Nature biotechnology},
volume = {44},
number = {1},
pages = {49-59},
pmid = {41526513},
issn = {1546-1696},
support = {DE-AC52-07NA27344//DOE | LDRD | Lawrence Livermore National Laboratory (LLNL)/ ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; Animals ; *Genetic Therapy/methods ; *Gene Transfer Techniques ; *Drug Delivery Systems ; },
abstract = {Genome editing has revolutionized the treatment of genetic diseases, yet the difficulty of tissue-specific delivery currently limits applications of editing technology. In this Review, we discuss preclinical and clinical advances in delivering genome editors with both established and emerging delivery mechanisms. Targeted delivery promises to considerably expand the therapeutic applicability of genome editing, moving closer to the ideal of a precise 'magic bullet' that safely and effectively treats diverse genetic disorders.},
}

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

@article {pmid41468873,
year = {2026},
author = {Zhang, J and Zhang, X and Xie, X and Han, B and Zhao, F and Yang, X and Zhang, W and Jiang, Y and Zhang, X},
title = {CRISPR/Cas12a-mediated marker-free fluorescent biosensing platform based on a lightful copper nanocluster for highly sensitive detection of mycotoxin.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129326},
doi = {10.1016/j.talanta.2025.129326},
pmid = {41468873},
issn = {1873-3573},
mesh = {*Copper/chemistry ; *Biosensing Techniques/methods ; *Aflatoxin B1/analysis ; *CRISPR-Cas Systems ; *Metal Nanoparticles/chemistry ; Limit of Detection ; Food Contamination/analysis ; Fluorescence ; *Mycotoxins/analysis ; },
abstract = {The problem of mycotoxin contamination in foodstuffs has attracted widespread attention and posed a great threat to human health. Therefore, the sensitive and effective detection of mycotoxins is of great importance for preserving public health worldwide. In this study, a CRISPR/Cas12a-mediated marker-free fluorescent biosensing platform was constructed for highly sensitive and fast detection of aflatoxin B1. The copper nanoclusters were synthesized with marker-free DNA single strands within 5 min, showing the outstanding fluorescence properties. With the existence of aflatoxin B1, the released complementary DNA (cDNA) triggered multiple isothermal amplification reaction. Subsequently, the obtained amplification products triggered the trans-cleavage activity of CRISPR/Cas12a system, which degraded the DNA single strands for synthesis of copper nanoclusters, leading to a decreased fluorescent signal. Benefiting from good fluorescence properties of copper nanoclusters, the established biosensing platform for aflatoxin B1 detection had a high specificity and a limit of detection of 47.51 pg/mL in the linear range of 0.05-10 ng/mL. The proposed platform provided a new insight for the detection of non-nucleic acid targets.},
}

*Copper/chemistry
*Biosensing Techniques/methods
*Aflatoxin B1/analysis
*CRISPR-Cas Systems
*Metal Nanoparticles/chemistry
Limit of Detection
Food Contamination/analysis
Fluorescence
*Mycotoxins/analysis

@article {pmid41454886,
year = {2026},
author = {Yang, H and Gao, X and Jin, ZC and Zhang, R and Ning, B and Yan, X},
title = {Simple and Versatile Toolkit for Genetic Manipulation of Bacillus licheniformis.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {262-270},
doi = {10.1021/acssynbio.5c00699},
pmid = {41454886},
issn = {2161-5063},
mesh = {*Bacillus licheniformis/genetics ; Plasmids/genetics ; *Genetic Engineering/methods ; DNA Transposable Elements/genetics ; Mutagenesis ; CRISPR-Cas Systems/genetics ; },
abstract = {Bacillus licheniformis is a spore-forming bacterium with probiotic, environmental, and industrial applications. Many wild strains with diverse functions have been described in recent years. Nevertheless, the lack of efficient and universal genetic manipulation tools hinders the study and engineering of these strains. Here, a versatile and simple genetic manipulation toolkit is established for B. licheniformis. The cornerstone of this toolkit is a conjugative DNA transfer system. This system could effectively transfer temperature-sensitive plasmid pTSMK into all ten tested B. licheniformis strains, with efficiencies ranging from 10[-5] to 10[-3]. Based on this DNA transfer system, the tools for maker-free knockout and knock-in, CRISPRi, as well as transposon mutagenesis, were built. A transposition frequency of 7.68 × 10[-3] was observed. The toolkit developed in this study fulfills most tasks in the engineering of this species and will promote the basic and applied research of B. licheniformis.},
}

*Bacillus licheniformis/genetics
Plasmids/genetics
*Genetic Engineering/methods
DNA Transposable Elements/genetics
Mutagenesis
CRISPR-Cas Systems/genetics

@article {pmid41418786,
year = {2026},
author = {Sherman, A and Benvenisty, N},
title = {Genetic screening of long non-coding RNAs in human embryonic stem cells reveals novel regulators of pluripotency.},
journal = {Stem cell reports},
volume = {21},
number = {1},
pages = {102743},
doi = {10.1016/j.stemcr.2025.102743},
pmid = {41418786},
issn = {2213-6711},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Differentiation/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; Octamer Transcription Factor-3/genetics/metabolism ; *Genetic Testing ; Cell Line ; CRISPR-Cas Systems ; Apoptosis/genetics ; },
abstract = {The human genome encodes thousands of long non-coding RNAs (lncRNAs), transcripts of over 200 nucleotides that lack protein-coding potential. lncRNAs are emerging as key players in diverse cellular processes, particularly in tissue-specific contexts, yet their functionality remained poorly understood. Here, we performed a CRISPR interference (CRISPRi) screen in human embryonic stem cells (hESCs), identifying over 100 essential and about 150 growth-restricting lncRNAs. We show that growth-modifying lncRNAs display distinctive properties, including unique expression signatures, genomic structure, evolutionary conservation, chromosomal distribution, and potential involvement in teratoma formation. Notably, we uncovered two primate-conserved, uncharacterized, essential lncRNAs that regulate neighboring pluripotency transcription factors: lncOCT4, which positively regulates OCT4 and induces p53-mediated apoptosis upon knockdown, and lncVRTN, which acts as a putative negative regulator of VRTN, affecting cell fate determination. These findings shed light on the contribution of lncRNAs to the human-specific pluripotency network and provide insights into lncRNA-mediated regulation of hESC growth and differentiation.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*Human Embryonic Stem Cells/metabolism/cytology
Cell Differentiation/genetics
*Pluripotent Stem Cells/metabolism/cytology
Octamer Transcription Factor-3/genetics/metabolism
*Genetic Testing
Cell Line
CRISPR-Cas Systems
Apoptosis/genetics

@article {pmid41391726,
year = {2026},
author = {Liao, XR and Han, D and Qi, LJ and Huang, QY and Gao, QY and He, XY and Guo, T and Lei, JJ and Cheng, SX},
title = {Aptamer-functionalized nanoparticles for CRISPR-Cas9 delivery to circulating malignant cells for therapeutic efficacy evaluation.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {390},
number = {},
pages = {114542},
doi = {10.1016/j.jconrel.2025.114542},
pmid = {41391726},
issn = {1873-4995},
mesh = {Humans ; *CRISPR-Cas Systems ; *Nanoparticles/chemistry/administration & dosage ; *Aptamers, Nucleotide/administration & dosage/chemistry ; Cell Line, Tumor ; Hyaluronic Acid/chemistry ; *Neoplastic Cells, Circulating/metabolism ; Gene Editing/methods ; *Neoplasms/therapy/genetics ; Hyaluronan Receptors/metabolism ; Epithelial Cell Adhesion Molecule/genetics ; Proto-Oncogene Proteins c-met/genetics ; *Gene Transfer Techniques ; ErbB Receptors/genetics ; Plasmids ; },
abstract = {Genome editing therapies targeting oncogenic pathways represent a promising alternative to small-molecule inhibitors, enabling durable therapeutic responses without inducing drug resistance. However, their success hinges on overcoming tumor heterogeneity, as malignant cells of cancer patients exhibit significant phenotypic variability. To advance personalized research on genome editing efficacy, tailored delivery systems capable of precisely targeting heterogeneous cancer cell populations are essential. Herein, we developed a facile modification strategy to construct a multiplexed surface-functionalized gene delivery system targeting heterogeneous cancer cells for personalized therapeutic studies. The system integrates the EGFR-targeting TuTu22 aptamer with SYL3C-conjugated hyaluronic acid (SYL3C-HA) for EpCAM and CD44 recognition. This triple-targeting platform enables efficient delivery of genome editing plasmid for c-Met knockout in both cancer cell lines and circulating malignant cells (CMCs) from cancer patients. The c-Met knockout not only reduces tumor malignancy but also reverses immune suppression, evidenced by PD-L1 downregulation and restored immune surveillance. By combining gene delivery with an ex vivo patient-derived evaluation platform, this system provides a robust tool for personalized research on the therapeutic strategies for tumor progression inhibition and immunity restoration.},
}

Humans
*CRISPR-Cas Systems
*Nanoparticles/chemistry/administration & dosage
*Aptamers, Nucleotide/administration & dosage/chemistry
Cell Line, Tumor
Hyaluronic Acid/chemistry
*Neoplastic Cells, Circulating/metabolism
Gene Editing/methods
*Neoplasms/therapy/genetics
Hyaluronan Receptors/metabolism
Epithelial Cell Adhesion Molecule/genetics
Proto-Oncogene Proteins c-met/genetics
*Gene Transfer Techniques
ErbB Receptors/genetics
Plasmids

@article {pmid41387457,
year = {2025},
author = {Cheng, F and Soleimani Samarkhazan, H and Khazaei, Y},
title = {CRISPR-engineered microbiome: living therapeutics revolutionize blood cancer immunotherapy.},
journal = {NPJ biofilms and microbiomes},
volume = {12},
number = {1},
pages = {17},
pmid = {41387457},
issn = {2055-5008},
mesh = {Humans ; *Immunotherapy/methods ; Animals ; *Hematologic Neoplasms/therapy/immunology/microbiology ; *CRISPR-Cas Systems ; *Gastrointestinal Microbiome/genetics ; *Microbiota ; },
abstract = {Blood cancers such as leukemia, lymphoma, and myeloma remain refractory in many patients due to immune escape, antigen heterogeneity, and therapy‑related toxicities. To address these challenges, we review recent strategies that harness CRISPR‑engineered gut commensals as precision "living therapeutics" to modulate host immunity and directly target malignant clones. We frame this review around three principal themes: (1) mechanistic strategies whereby CRISPR-engineered commensals modulate host immunity and directly antagonize malignant clones; (2) the enabling technologies and delivery/containment platforms, CRISPR variants, phage/LNP delivery, genetic circuits and biocontainment, that make living therapeutics feasible; and (3) translational progress, outstanding technical and safety barriers, and ethical/regulatory challenges that must be addressed for clinical deployment. To illustrate these themes, we discuss three concrete therapeutic modalities: engineered microbial secretion of immunomodulators, targeted delivery of tumor-lytic payloads, and engineered production of anticancer metabolites, and how these are enabled by contemporary CRISPR and synthetic-biology toolkits. Selected preclinical models report substantial antitumor effects, often >60% tumor reduction in rodent studies, and restoration of CAR-T cell function in controlled settings; however, effect sizes vary across models, and human translation remains unproven. We also analyze key technical barriers, strain stability, biocontainment, off‑target effects, and propose solutions, including auxotrophic kill-switches and AI‑guided strain optimization. Finally, we outline future directions, from in situ phage delivery to multi‑omics-driven patient stratification. CRISPR‑microbiome editing represents a paradigm shift in hematologic oncology, offering localized, sustained therapy with reduced systemic toxicity.},
}

Humans
*Immunotherapy/methods
Animals
*Hematologic Neoplasms/therapy/immunology/microbiology
*CRISPR-Cas Systems
*Gastrointestinal Microbiome/genetics
*Microbiota

@article {pmid41319963,
year = {2026},
author = {Kong, H and Wang, S and Zhuo, C and Zhong, Q and Xu, Y and Lao, YH and Lv, S and Xie, X and Yuan, Q and Li, K and Tao, Y and Li, M},
title = {Nanovesicles integrating PD-1-mediated targeting and CRISPR/Cas9-based CD47 editing for dual immune checkpoint blockade.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {390},
number = {},
pages = {114480},
doi = {10.1016/j.jconrel.2025.114480},
pmid = {41319963},
issn = {1873-4995},
mesh = {*CD47 Antigen/genetics ; CRISPR-Cas Systems ; Animals ; Gene Editing ; *Immune Checkpoint Inhibitors/administration & dosage ; *Programmed Cell Death 1 Receptor/genetics/immunology ; Humans ; B7-H1 Antigen/immunology ; Mice ; Cell Line, Tumor ; *Neoplasms/therapy/immunology/genetics ; Mice, Inbred C57BL ; Female ; Immunotherapy/methods ; },
abstract = {Immunotherapy with immune checkpoint inhibitors has revolutionized cancer treatment, yet many tumors evade immune surveillance through multiple suppressive mechanisms. In particular, the adaptive immune checkpoint programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) and the innate "don't eat me" signal CD47/signal-regulatory protein alpha (SIRPα) represent two distinct pathways that cancers exploit to avoid T-cell attack and macrophage phagocytosis, respectively. Herein, we present BITE (Biomimetic Immune Targeting and Editing), a genetically engineered biomimetic nanoplatform designed to concurrently blockade both pathways by combining PD-1-mediated tumor targeting with CRISPR/Cas9 gene editing of CD47. BITE nanovesicles display PD-1 on their surface, enabling selective binding to PD-L1-expressing tumor cells and local disruption of PD-1/PD-L1 signaling. Simultaneously, they deliver a CRISPR/Cas9 payload that knocks out the CD47 gene in tumor cells, abolishing the anti-phagocytic signal and thus activating innate immune clearance. We demonstrate that BITE efficiently homes to PD-L1-positive tumors in vitro and in vivo, achieves significant CD47 gene disruption in tumor cells, and triggers robust phagocytosis by macrophages. In a mouse tumor model, dual checkpoint blockade by BITE reshapes the tumor microenvironment, yielding increased infiltration of CD4[+] T cells, CD8[+] T cells, and M1 macrophages; treatment with BITE induces pronounced tumor regression and extended survival, outperforming single-target controls. Our results establish a proof-of-concept for this dual-function nanovesicle approach, highlighting its potential to engage both adaptive and innate immunity synergistically. The BITE platform offers a versatile and targeted strategy to overcome immune resistance in cancer, representing a promising therapeutic avenue in biomedical engineering and nanomedicine.},
}

*CD47 Antigen/genetics
CRISPR-Cas Systems
Animals
Gene Editing
*Immune Checkpoint Inhibitors/administration & dosage
*Programmed Cell Death 1 Receptor/genetics/immunology
Humans
B7-H1 Antigen/immunology
Mice
Cell Line, Tumor
*Neoplasms/therapy/immunology/genetics
Mice, Inbred C57BL
Female
Immunotherapy/methods

@article {pmid41147788,
year = {2026},
author = {Saika, H and Hara, N and Yasumoto, S and Muranaka, T and Yoshimi, K and Mashimo, T and Toki, S},
title = {Versatile genome editing using Type I-E CRISPR-Cas3 in rice.},
journal = {Plant & cell physiology},
volume = {67},
number = {1},
pages = {82-92},
doi = {10.1093/pcp/pcaf138},
pmid = {41147788},
issn = {1471-9053},
support = {JPJ008000//Ministry of Agriculture, Fisheries and Food/ ; //Cross-ministerial Strategic Innovation Promotion Program/ ; JPJ012287//Bio-oriented Technology Research Advancement Institution/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; Plants, Genetically Modified ; },
abstract = {The Type I-E CRISPR-Cas3 derived from Escherichia coli (Eco CRISPR-Cas3) can introduce large deletions in target sites and is available for mammalian genome editing. The use of Eco CRISPR-Cas3 in plants is challenging because seven CRISPR-Cas3 components (six Cas proteins and CRISPR RNA) must be expressed simultaneously in plant cells. To date, application has been limited to maize protoplasts, and no mutant plants have been produced. In this study, we developed a genome editing system in rice using Eco CRISPR-Cas3 via Agrobacterium-mediated transformation. Deletions in the target gene were detected in 39%-71% of transformed calli by polymerase chain reaction (PCR) analysis, and the frequency of alleles lacking a region 7.0 kb upstream of the protospacer adjacent motif sequence was estimated as 21%-61% by quantifying copy number by droplet digital PCR, suggesting that mutant plants could be obtained with reasonably high frequency. Deletions were determined in plants regenerated from transformed calli, and stably inherited to the progenies. Sequencing analysis showed that deletions of 0.1-7.2 kb were obtained, as reported previously in mammals. Interestingly, deletions separated by intervening fragments or with short insertion and inversion were also determined, suggesting the creation of novel alleles. Moreover, we demonstrated C to T base editing based on Type I-E CRISPR-Cas3 in rice, whereas base editing based on Type I-C and Type I-F2 CRISPR-Cas3 has been reported previously only in human cells. Overall, Eco CRISPR-Cas3 could be a promising genome editing tool for gene knockout, gene deletion, base editing, and genome rearrangement in plants.},
}

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

@article {pmid40971496,
year = {2026},
author = {V Pillai, V and Koganti, PP and Gurung, S and Cheong, SH and Selvaraj, V},
title = {Transformed bovine trophoblast stem cell lines, characterization, gene editing and secretion†.},
journal = {Biology of reproduction},
volume = {114},
number = {1},
pages = {273-286},
doi = {10.1093/biolre/ioaf212},
pmid = {40971496},
issn = {1529-7268},
support = {USDA-NIFA 2023-08329//United States Department of Agriculture/ ; NE-2227//USDA-Multistate Program/ ; },
mesh = {Animals ; *Trophoblasts/cytology/physiology/metabolism ; Cattle ; *Gene Editing/veterinary ; Female ; *Stem Cells/cytology/physiology/metabolism ; Pregnancy ; Cell Differentiation ; Cell Line, Transformed ; CRISPR-Cas Systems ; },
abstract = {Trophoblast stem cells (TSCs) serve as a critical model for understanding placental development, early embryo-maternal interactions, and pregnancy establishment in mammals. In cattle, the developing trophectoderm plays an essential role in conceptus elongation and secretion of factors necessary for maternal recognition of pregnancy. Building on previous work identifying signaling pathways regulating bovine TSC self-renewal and differentiation, we report the generation and characterization of transformed bovine TSC (bTSC) lines derived from blastocysts via lentiviral transduction of simian vacuolating virus 40 large T antigen. These rapidly proliferating TSC cell lines, maintained in the presence of Rho-associated protein kinase (ROCK) inhibition, retain key morphological and transcriptional characteristics of bovine TSCs. Upon transforming growth factor β-induced differentiation, they exhibit morphological and molecular changes consistent with trophoblast maturation. To evaluate their utility for functional studies, we demonstrated stable gene introduction of tdTomato and EGFP using lentiviral vectors and employed CRISPR/Cas9-mediated gene editing to target lentiviral EGFP integration sites, confirming efficient gene deletion. Additionally, proteomic analysis of conditioned medium identified secreted proteins with potential roles in embryo-uterine interactions, aligning with factors previously reported in bovine conceptus secretomes. These findings establish transformed bTSC lines as a valuable model for investigating bovine trophoblast biology, functional gene studies, and trophoblast-endometrial signaling. By providing a renewable in vitro system with stable proliferative capacity, these cell lines enable further exploration of the molecular mechanisms governing early pregnancy in cattle.},
}

Animals
*Trophoblasts/cytology/physiology/metabolism
Cattle
*Gene Editing/veterinary
Female
*Stem Cells/cytology/physiology/metabolism
Pregnancy
Cell Differentiation
Cell Line, Transformed
CRISPR-Cas Systems

@article {pmid41535270,
year = {2026},
author = {Taguchi, J and Kikuchi, M and Jeon, H and Shimizu, R and Mori, H and Ikawa, M and Yamada, Y and Sato, K and Ikeda, T and Yamazaki, S and Ozawa, M},
title = {A scalable two-step genome editing strategy for generating full-length gene-humanized mice at diverse genomic loci.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {356},
pmid = {41535270},
issn = {2041-1723},
support = {23K27084//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 25K18393//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21H05033//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21H05033//Japan Society for the Promotion of Science London (JSPS London)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Humans ; Mice ; CRISPR-Cas Systems/genetics ; Gene Knock-In Techniques/methods ; Male ; Mice, Transgenic ; Alleles ; Homologous Recombination ; Embryonic Stem Cells/metabolism ; Genetic Loci ; Hematopoiesis/genetics ; Female ; },
abstract = {Full-length gene-humanized mice engineered by completely replacing mouse loci with human counterparts, including untranslated and regulatory regions, provide a robust in vivo platform for human gene function studies. However, reliably humanizing large genomic regions remains challenging due to limited DNA insert sizes, complex protocols, and specialized material requirements. This study introduces a streamlined approach that enables full-length gene humanization through two sequential CRISPR-assisted homologous recombination steps in embryonic stem cells. This method supports targeted knock-in of genomic fragments (> 200 kbp) and is applicable across multiple mouse strains. Humanized alleles generated using the developed method recapitulate human-like splicing isoforms and organ-specific gene expression while restoring essential functions in hematopoiesis, spermatogenesis, and survival. Furthermore, disease-associated mutations can be engineered into humanized alleles to model human genetic disorders in vivo. This versatile platform enables the creation of physiologically relevant, fully gene-humanized mouse models for broad applications in biomedical research.},
}

Animals
*Gene Editing/methods
Humans
Mice
CRISPR-Cas Systems/genetics
Gene Knock-In Techniques/methods
Male
Mice, Transgenic
Alleles
Homologous Recombination
Embryonic Stem Cells/metabolism
Genetic Loci
Hematopoiesis/genetics
Female

@article {pmid41533983,
year = {2026},
author = {Zhang, RR and Wang, YH and Peng, XF and Sun, YJ and Xu, ZS and Liu, H and Xiong, AS},
title = {Expansion protein DcEXP22 regulates taproot enlargement via mediating root cell extension in carrot.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {1},
pages = {e70677},
doi = {10.1111/tpj.70677},
pmid = {41533983},
issn = {1365-313X},
support = {KYLH2025002//Fundamental Research Funds for the Central Universities/ ; 2023-SYS-02//Open Research Project of Key Laboratory of Biotechnology of Qinghai-Tibet Plateau Biotechnology of the Ministry of Education/ ; //Priority Academic Program Development of Jiangsu Higher Education Institutions Project PAPD/ ; //Bioinformatics Center of Nanjing Agricultural University/ ; },
mesh = {*Daucus carota/genetics/growth & development/metabolism ; *Plant Roots/growth & development/genetics/cytology/metabolism ; *Plant Proteins/genetics/metabolism/physiology ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; CRISPR-Cas Systems ; },
abstract = {Carrot (Daucus carota L.) is an important root vegetable crop of the Daucus genus in the Apiaceae. As the main product organ of carrot, the taproot has high nutritional and economic value. Expansins, a class of proteins involved in plant cell wall relaxation and cell extension, are mainly found in growing tissues and organs. Expansins play an important role in plant root development. Here, the DcEXP22 gene with a length of 789 bp was cloned from the carrot cultivar 'Kurodagosun'. Based on the stable genetic transformation system and CRISPR/Cas9 gene-editing technology, the DcEXP22 gene was overexpressed and knocked out in carrots. The results indicated that overexpression of the DcEXP22 gene increased carrot root fresh weight, root diameter, and root-shoot ratio, and enlarged the perimeter and area of taproot phloem cells. In contrast, knockout of the DcEXP22 gene inhibited the development of carrot taproots and the extension of phloem cells, suggesting that the DcEXP22 gene might promote the enlargement of carrot taproots by regulating the size of phloem cells. RNA-seq analysis identified several genes that were co-expressed with DcEXP22, including DcCYP734A1, DcERF1, DcMAP2K1, and DcSAD9. It was hypothesized that the DcEXP22 gene might influence the enlargement of carrot taproot by participating in the signal transduction of phytohormones such as brassinosteroids, cell wall synthesis and modification, and fatty acid metabolisms. These findings will advance our knowledge of the molecular mechanisms of carrot taproot enlargement.},
}

*Daucus carota/genetics/growth & development/metabolism
*Plant Roots/growth & development/genetics/cytology/metabolism
*Plant Proteins/genetics/metabolism/physiology
Gene Expression Regulation, Plant
Plants, Genetically Modified
CRISPR-Cas Systems

@article {pmid41500123,
year = {2026},
author = {Zang, Z and Chen, J and Dong, Y and Chen, L and Yang, M and Mu, M and Zhou, L and Zhang, W and Zou, G and Liu, C},
title = {CRISPR-Cas12a biosensing technology advances and applications in precision diagnostics and cancer research.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129298},
doi = {10.1016/j.talanta.2025.129298},
pmid = {41500123},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/diagnosis/genetics ; *Precision Medicine/methods ; Biomarkers, Tumor/genetics ; Gene Editing ; },
abstract = {CRISPR-Cas12a has become a versatile biotechnology platform with important applications in biosensing, diagnostics, and precision genome editing. This system is activated by a single crRNA, exhibits target-responsive trans-cleavage activity, and recognizes programmable PAM sequences. These features provide a robust basis for accurate detection of diverse biomarkers. Its detection capabilities include nucleic acid targets such as viral RNA and cancer mutations, as well as non-nucleic acid molecules like exosomes and proteins. Recent advancements have shown significant advantages, including multi-temperature adaptability, rapid kinetics, and compatibility with both DNA and RNA targets. Technical improvements include machine learning-assisted crRNA design for enhanced prediction accuracy and engineered EnAsCas12a variants that overcome conventional PAM restrictions. Notable achievements involve entropy-driven circuits that achieve attomolar-level sensitivity, smartphone-compatible four-channel quantitative detection systems, and streamlined integrated workflows completed within 30 min. Advances in sensor design, such as metal-organic framework encapsulation and high-performance aptamer-based sensors, have further expanded detection capabilities. In oncology research, CRISPR-Cas12a technology provides powerful tools to comprehensively analyze complex molecular networks within the tumor microenvironment (TME) and facilitate ultrasensitive detection of early-stage cancer biomarkers. Additionally, in genome editing, CRISPR-Cas12a enables precise genomic modifications due to distinct repair pathways, versatile delivery methods, and efficient creation of transgenic models. Thus, it expands its functional scope beyond diagnostics. With ongoing development, this technology is expected to evolve into an integrated platform combining TME research, point-of-care cancer diagnostics, and programmable genome engineering, offering innovative solutions for both biomedical research and clinical translation.},
}

*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*Neoplasms/diagnosis/genetics
*Precision Medicine/methods
Biomarkers, Tumor/genetics
Gene Editing

@article {pmid41478231,
year = {2026},
author = {Liu, Y and Mei, H and Gao, C and Yang, Y},
title = {Rapid detection of respiratory syncytial virus using RT-LAMP-CRISPR/Cas12b on a gravity-driven microfluidic chip.},
journal = {Diagnostic microbiology and infectious disease},
volume = {114},
number = {3},
pages = {117244},
doi = {10.1016/j.diagmicrobio.2025.117244},
pmid = {41478231},
issn = {1879-0070},
mesh = {Humans ; *Respiratory Syncytial Virus Infections/diagnosis/virology ; *Molecular Diagnostic Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; *Respiratory Syncytial Virus, Human/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Lab-On-A-Chip Devices ; RNA, Viral/genetics ; Limit of Detection ; Point-of-Care Systems ; },
abstract = {AIMS: Respiratory syncytial virus (RSV) is the most significant pathogen causing acute lower respiratory tract infections in children. Early detection of RSV can help control disease progression and reduce complications. However, RT-qPCR-based detection methods cannot provide accurate results within one hour and are not suitable for use in resource-limited settings. Therefore, there is a pressing need to develop a rapid and precise bedside RSV detection assay to meet clinical demands.

METHODS AND RESULTS: Firstly, we developed a RT-LAMP-assisted CRISPR/Cas12b method for detecting RSV M gene, capable of identifying target RNA with a limit of detection as low as 100 copies/μL within 40 min. Secondly, we created and tested a sampling lysis reagent, demonstrating its effectiveness in enabling direct detection without the need for nucleic acid extraction, thus improving bedside detection efficiency. Lastly, to facilitate use in resource-limited areas, we designed and developed a gravity-driven microfluidic chip that simplifies the stepwise process of RT-LAMP amplification and CRISPR/Cas12b detection. This chip allows for visual recognition of results without the need for an external power source when used in point-of-care (POC) settings. This assay showed 99% agreement with RT-qPCR, highlighting its potential for practical application. Additionally, no cross-reactivity was observed with other respiratory pathogens infection, demonstrating good clinical specificity.

CONCLUSIONS: In summary, the platform we developed is faster and more user-friendly than RT-qPCR, while achieving comparable sensitivity.

IMPACT STATEMENT: Our finding fills the gap in the inability to detect RSV in POC setting, safeguarding the health of children, and offers new insights for the innovation of CRISPR diagnostics.},
}

Humans
*Respiratory Syncytial Virus Infections/diagnosis/virology
*Molecular Diagnostic Techniques/methods/instrumentation
*CRISPR-Cas Systems
*Respiratory Syncytial Virus, Human/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*Lab-On-A-Chip Devices
RNA, Viral/genetics
Limit of Detection
Point-of-Care Systems

@article {pmid41476394,
year = {2026},
author = {Zhuang, M and Song, J and Hu, X and Wang, X},
title = {Metabolic Engineering of Escherichia coli Nissle 1917 for Efficient Production of p-Coumaric Acid.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {210-222},
doi = {10.1021/acssynbio.5c00639},
pmid = {41476394},
issn = {2161-5063},
mesh = {*Escherichia coli/metabolism/genetics ; *Metabolic Engineering/methods ; *Coumaric Acids/metabolism ; *Propionates/metabolism ; Ammonia-Lyases/genetics/metabolism ; Tyrosine/biosynthesis/metabolism ; CRISPR-Cas Systems/genetics ; Plasmids/genetics/metabolism ; Escherichia coli Proteins/genetics/metabolism ; Quorum Sensing/genetics ; Phenylalanine/biosynthesis ; },
abstract = {p-Coumaric acid is a valuable phytochemical with significant roles in anticancer cell proliferation, antianxiety, and neuroprotection and as a key precursor for various flavonoids. However, the production of p-coumaric acid in microorganisms is often limited by enzyme compatibility and its antimicrobial effects. In this study, a p-coumaric acid producing Escherichia coli strain was constructed. First, the cryptic plasmids pMUT1 and pMUT2 were eliminated from E. coli Nissle 1917 by using the CRISPR/Cas9 method to mitigate their interference with heterologous gene expression, and the resulting strain WEN01 was used to screen for the genes encoding for tyrosine ammonia-lyase with superior host compatibility. Next, the gene tyrR encoding a global regulator was knocked out to alleviate the repression of l-tyrosine production. The key genes pheL and pheA involved in phenylalanine biosynthesis were knocked out to reduce byproduct formation, resulting in the strain WEN06. Finally, the quorum sensing system was used to overexpress the key genes aroG[fbr] and tyrA[fbr] in the l-tyrosine biosynthetic pathway, and the resulting strain WEN06/pWT101-AT, pWT104F could produce 462.6 mg/L p-coumaric acid in shake flask fermentation. In fed-batch fermentation, the engineered strain WEN06/pWT101-AT, pWT104F could produce 10.3 g/L p-coumaric acid with a glucose conversion yield of 0.13 g/g and a productivity of 0.14 g/L/h. This work provides a novel strategy for the efficient production of p-coumaric acid and lays a foundation for the efficient production of antimicrobial natural products in bacteria.},
}

*Escherichia coli/metabolism/genetics
*Metabolic Engineering/methods
*Coumaric Acids/metabolism
*Propionates/metabolism
Ammonia-Lyases/genetics/metabolism
Tyrosine/biosynthesis/metabolism
CRISPR-Cas Systems/genetics
Plasmids/genetics/metabolism
Escherichia coli Proteins/genetics/metabolism
Quorum Sensing/genetics
Phenylalanine/biosynthesis

@article {pmid41455263,
year = {2026},
author = {Liu, F and Cheng, AX and Zhang, C and Li, J and Huang, YF and Zhang, YP and Li, CP and Zhao, H},
title = {Amplification-free, CRISPR-Cas12a-mediated fluorescence biosensor using mesoporous silica nanomaterials for ultrasensitive detection of nucleic acid biomarkers.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129289},
doi = {10.1016/j.talanta.2025.129289},
pmid = {41455263},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *Silicon Dioxide/chemistry ; *CRISPR-Cas Systems ; Humans ; *Nanostructures/chemistry ; Porosity ; Limit of Detection ; Fluorescent Dyes/chemistry ; Rhodamines/chemistry ; Fluorescence ; ErbB Receptors/genetics ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Sensitive detection of nucleic acid biomarkers is crucial in many fields, including biomedical diagnosis, veterinary medicine, and food safety. Thus, developing an accurate and cost-effective detection method for nucleic acid biomarkers is essential. Here, we developed a sensitive CRISPR-Cas12a-based fluorescence biosensor using mesoporous silica nanomaterials (MSNs). A large quantity of rhodamine B (RB) was enriched on the MSNs to synthesize RB@MSN nanocomposites, which served as fluorescent probe materials, and Au NPs acted as fluorescence quenching materials. Combined with the high specific recognition capability of the CRISPR-Cas12a system, we detected three important nucleic acids without requiring amplification: the EGFR exon 19 deletion mutation (EGFR 19Del, found in circulating tumor DNA), African swine fever virus (ASFV), and human papilloma virus (HPV). Under optimal conditions and using quantitative analysis, there were strong linear correlations between the concentrations of the targets and their respective fluorescence intensities. The lowest detection limits were 55 aM for EGFR 19Del, 51 aM for ASFV, and 24 aM for HPV. By enriching and encapsulating MSNs with RB, our method avoided the problems of fluorescence modifications in typical CRISPR-Cas12a systems, such as professional outsourcing requirements and easily quenched fluorescence. Moreover, the results exhibited good repeatability and stability. This method provides a novel approach to nucleic acid fluorescence detection using the CRISPR-Cas12a system.},
}

*Biosensing Techniques/methods
*Silicon Dioxide/chemistry
*CRISPR-Cas Systems
Humans
*Nanostructures/chemistry
Porosity
Limit of Detection
Fluorescent Dyes/chemistry
Rhodamines/chemistry
Fluorescence
ErbB Receptors/genetics
Gold/chemistry
Metal Nanoparticles/chemistry
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41448060,
year = {2026},
author = {Long, Y and Sun, S and Mei, H and Zhou, D and Zhou, H and Fang, Z and Li, X and Li, N and Zhuang, T and Guo, C},
title = {RT-LAMP-CRISPR/Cas12b-based hand-pressure-actuated microfluidic chip for rapid and portable detection of severe fever with thrombocytopenia syndrome virus.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129277},
doi = {10.1016/j.talanta.2025.129277},
pmid = {41448060},
issn = {1873-3573},
mesh = {*Phlebovirus/genetics/isolation & purification ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology ; *Nucleic Acid Amplification Techniques/instrumentation/methods ; Humans ; *Lab-On-A-Chip Devices ; *Molecular Diagnostic Techniques/instrumentation/methods ; *CRISPR-Cas Systems ; Pressure ; RNA, Viral/genetics/blood ; },
abstract = {Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, highly pathogenic tick-borne virus causing severe viral hemorrhagic fever, posing a significant public health threat. Rapid and accurate detection of SFTSV in resource-limited settings is critical for early diagnosis and effective control of severe fever with thrombocytopenia syndrome (SFTS). Here, we developed the RT-LAMP-CRISPR/Cas12b-based Hand-Pressure-Actuated Microfluidic Chip for Rapid and Portable Detection of Severe Fever with Thrombocytopenia Syndrome Virus (HARD). The hand-pressure-actuated microfluidic chip integrates reverse transcription loop-mediated isothermal amplification (RT-LAMP), clustered regularly interspaced short palindromic repeats (CRISPR) and its associated proteins (CRISPR associated proteins, Cas) 12b in order to achieve rapid, low-cost, and contamination-free point-of-care testing. The HARD system achieves a detection limit of 5 copies per reaction, utilizing direct RNA lysis from blood samples and a hand warmer as a heat source, enabling electricity-free operation. Clinical validation with blood samples from vector-borne infectious diseases demonstrated high concordance with laboratory RT-qPCR, with 88.9 % sensitivity, 100 % specificity, and 95 % accuracy. Thus, the HARD platform offers a rapid, portable, and efficient solution for the early diagnosis of SFTSV in resource-limited settings, with potential for broader application in POCT for infectious diseases.},
}

*Phlebovirus/genetics/isolation & purification
*Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology
*Nucleic Acid Amplification Techniques/instrumentation/methods
Humans
*Lab-On-A-Chip Devices
*Molecular Diagnostic Techniques/instrumentation/methods
*CRISPR-Cas Systems
Pressure
RNA, Viral/genetics/blood

@article {pmid41439415,
year = {2026},
author = {Zhao, R and Wan, P and Huang, H and Li, Q and Zeng, Z and Xiong, W},
title = {Harnessing CRISPR-Cas9 and CRISPRi systems to reverse antibiotic resistance in a clinical multidrug-resistant Escherichia coli isolate.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {81},
number = {1},
pages = {},
doi = {10.1093/jac/dkaf442},
pmid = {41439415},
issn = {1460-2091},
support = {2022YFD1800400//National Key R&D Program/ ; },
mesh = {*Escherichia coli/drug effects/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Drug Resistance, Multiple, Bacterial/genetics ; Microbial Sensitivity Tests ; *Anti-Bacterial Agents/pharmacology ; Escherichia coli Proteins/genetics ; Escherichia coli Infections/microbiology ; Humans ; Multidrug Resistance-Associated Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Deletion ; },
abstract = {OBJECTIVES: To evaluate the CRISPR-Cas9 and CRISPR interference (CRISPRi) systems as an antibiotic re-sensitization strategy for reversing multidrug resistance in a clinical Escherichia coli isolate.

MATERIALS AND METHODS: The CRISPR-Cas9 system was applied for precise deletion of the acrB gene from clinical E. coli isolate GP53 and homologous recombination (HR) was provided for accurate repairs of double-strand breaks. An arabinose-inducible CRISPRi system was developed and optimized using fluorescent reporter strain GH01. Multiple guide RNAs (gRNAs) targeting acrB were designed, and the most effective gRNA was selected based on its transcriptional suppression of gene acrB. The minimum inhibitory concentrations (MICs) of selected antibiotics in GP53ΔacrB, CRISPRi strains, WT and WT combined with efflux pump inhibitor PAβN were evaluated.

RESULTS: The CRISPR-Cas9 system precisely deleted the acrB gene in clinical E. coli isolate GP53 with 11.46% knockout efficiency. The constructed arabinose-inducible CRISPRi system effectively repressed fluorescent protein expression in strain GH01. Although dCas9 expression increased with L-arabinose concentration, the transcriptional repression efficiency of the target gene under 1 mM induction reached a significant inhibitory level. The CRISPRi system targeting gene acrB exhibited 44.9%, 5.4% and 23.5% inhibition rates on the transcriptional levels with 1 mM L-arabinose for three distinct gRNAs. Both the knockout and CRISPRi strains successfully restored susceptibility of the multidrug-resistant E. coli GP53 to quinolones and tetracyclines, outperforming the effect of PAβN combination therapy.

CONCLUSIONS: In this study, CRISPR-based systems effectively reversed multidrug resistance in a clinical E. coli isolate, advancing the applications of CRISPR systems in controlling bacterial multidrug resistance.},
}

*Escherichia coli/drug effects/genetics/isolation & purification
*CRISPR-Cas Systems
*Drug Resistance, Multiple, Bacterial/genetics
Microbial Sensitivity Tests
*Anti-Bacterial Agents/pharmacology
Escherichia coli Proteins/genetics
Escherichia coli Infections/microbiology
Humans
Multidrug Resistance-Associated Proteins/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Gene Deletion

@article {pmid41433963,
year = {2026},
author = {Schneider, O and Zehl, M and Miele, M and Pace, V and Brungs, C and Cheng, JF and Hummelbrunner, S and Dirsch, VM and Zotchev, SB},
title = {Heterologous Expression and CRISPR/Cas9-Assisted Manipulation of the Hybrid Gene Cluster Specifying the Biosynthesis of Meroterpenoids and Phenazines.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {137-148},
doi = {10.1021/acssynbio.5c00531},
pmid = {41433963},
issn = {2161-5063},
mesh = {*Multigene Family/genetics ; *CRISPR-Cas Systems/genetics ; *Phenazines/metabolism/chemistry ; *Terpenes/metabolism/chemistry ; Streptomyces/genetics/metabolism ; Streptomyces coelicolor/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Anti-Bacterial Agents/biosynthesis ; },
abstract = {A hybrid gene cluster, mfq, predicted to govern the biosynthesis of both meroterpenoids and phenaziterpenes, was cloned from the genome of Streptomyces sp. S4.7 and introduced into the heterologous host Streptomyces coelicolor M1154. The biosynthesis of the meroterpenoids marfuraquinocins C and D, previously isolated from Streptomyces niveus SCSIO 3406, as well as a new congener, marfuraquinocin E, which exhibited antibacterial activity, was activated upon overexpression of the regulatory protein MfqF. However, production of neither phenaziterpenes nor phenazines was detected. The structure of marfuraquinocin E was elucidated, revealing the attachment of a terpene moiety at C-2, in contrast to C-6 as seen in the known congeners A-D. Using the CRISPR/Cas9 system, several genes in the mfq cluster were inactivated, confirming the role of MfqW as a prenyltransferase specific to the meroterpenoid pathway. Both gene overexpression and further knockouts provided the first insights into the complex regulation of this hybrid gene cluster. To restore the presumably deficient phenazine biosynthetic pathway, a gene encoding a PhzF homologue from another gene cluster in S4.7 was heterologously expressed alongside the mfq cluster, leading to the production of 1,6-phenazine dicarboxylic acid upon MfqF overexpression. This work lays the foundation for elucidating the complete biosynthetic pathway of marfuraquinocins and its potential coregulation with that of phenazines.},
}

*Multigene Family/genetics
*CRISPR-Cas Systems/genetics
*Phenazines/metabolism/chemistry
*Terpenes/metabolism/chemistry
Streptomyces/genetics/metabolism
Streptomyces coelicolor/genetics/metabolism
Bacterial Proteins/genetics/metabolism
Anti-Bacterial Agents/biosynthesis

@article {pmid41396964,
year = {2026},
author = {Jiang, C and Liu, Y and Han, W and Zou, D and Chen, K and Jiang, X and Ma, A and Wei, X},
title = {Regulation of Single and Multiple Genes in Bacillus amyloliquefaciens by an Evolution System In Vivo.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {88-98},
doi = {10.1021/acssynbio.5c00480},
pmid = {41396964},
issn = {2161-5063},
mesh = {*Bacillus amyloliquefaciens/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Directed Molecular Evolution/methods ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Cytidine Deaminase/genetics/metabolism ; Mutation ; },
abstract = {With the development of synthetic biology, an evolution system in vivo has been applied to accelerate the construction of cell factories. In this study, an efficient in vivo evolution system was developed for regulation of single and multiple genes in Bacillus amyloliquefaciens. First, the CRISPR/Cas9n-AID base editor was constructed through integration expression of the fused Cas9n protein and activation-induced cytidine deaminase (AID), and the base conversion efficiency from C to T was as high as 90% in single-gene editing. Subsequently, the evolution template (XP43) with an editable RBS sequence (GGGGGGGG) was designed for in vivo evolution through two strategies. By next-generation sequencing of RBS mutation libraries, the extended sgRNA strategy was confirmed to be the optimal evolution scheme. Using the alkaline protease gene (aprE) as the single gene target, the evolution program was initiated to successfully obtain a series of mutant strains with gradient AprE activities. Furthermore, multiple key genes (dhemA, SAM2, and hemEHY) were evolved simultaneously to balance the heme metabolic network, and the optimal mutant strain (HZHA-C2) produced 14.02 mg/L heme, 93% higher than the control strain. Finally, the overexpression of the hemH gene further increased the heme titer by 49%. By a fed-batch fermentation strategy, the heme titer of the optimal engineered strain (HZHA2/pHY-hemH) was improved by 64%, achieving 32.61 mg/L.},
}

*Bacillus amyloliquefaciens/genetics/metabolism
CRISPR-Cas Systems/genetics
Gene Editing/methods
*Directed Molecular Evolution/methods
Bacterial Proteins/genetics/metabolism
Gene Expression Regulation, Bacterial
Cytidine Deaminase/genetics/metabolism
Mutation

@article {pmid41352420,
year = {2026},
author = {Tang, M and Liang, R and Wu, Z and Chen, C and He, B and Zhou, N and Wang, S and Xiao, X and Li, G and Jiang, Y and Gong, G and Zhou, Y},
title = {Deciphering OCT4A-dose-dependent transcriptional profiles associated with tumorigenic potential in somatic cancer cells.},
journal = {SLAS technology},
volume = {36},
number = {},
pages = {100381},
doi = {10.1016/j.slast.2025.100381},
pmid = {41352420},
issn = {2472-6311},
mesh = {*Octamer Transcription Factor-3/genetics/metabolism ; Humans ; *Neoplasms/genetics/pathology ; Cell Line, Tumor ; *Carcinogenesis/genetics ; Gene Expression Regulation, Neoplastic ; Gene Expression Profiling ; *Transcriptome ; CRISPR-Cas Systems ; Gene Regulatory Networks ; Prognosis ; },
abstract = {AIMS: The transcription factor OCT4A, a well-established master pluripotency factor, exerts regulatory effects on cell fate determination that are closely associated with its protein levels. This study aims to uncover the downstream gene profile features relevant to tumorigenic potential mediated by OCT4A under varying protein abundance in somatic cancer cells (SCCs).

MATERIALS AND METHODS: CRISPR-Cas9-mediated knockout and doxycycline-inducible OCT4A expression systems were established in cervical (HeLa) and hepatocellular (HepG2, Huh7) cancer cells. Single-cell sequencing, spatial transcriptomic and survival analysis data were used to elucidate the expression pattern of OCT4 in somatic cancer tissues and its prognostic relevance. The plate colony formation assay was performed to assess the tumorigenic capacity of SCCs, and Bulk RNA sequencing coupled with weighted gene co-expression network analysis (WGCNA) identified dose-relevant downstream pathways. Functional enrichment, survival modeling, and RT-qPCR validation were used to construct OCT4A-dose-dependent transcriptional regulatory networks.

KEY FINDINGS: OCT4 transcript, is heterogeneously present and confined to a small subset of tumor cells within somatic cancer tissues, with a significantly higher proportion of OCT4-positive cells in tumor tissues compared to paired paraneoplastic tissues and is significantly correlated with poor prognosis in SCCs. Endogenous low-level OCT4A positively regulates tumorigenic capacity predominantly through targeting non-coding genes, whereas high-level OCT4A suppresses tumorigenic capacity primarily via protein-coding genes in SCCs. A prognostic model based on high-level OCT4A-regulated protein-coding genes was associated with favorable clinical outcomes, aligning with in vitro phenotypic results.

SIGNIFICANCE: Our findings further confirm in SCCs that the functional pleiotropy of OCT4A is closely linked to its protein abundance, and further systematically elucidate the molecular signatures of OCT4A-regulated downstream gene networks associated with tumorigenic phenotypes at differential protein levels, providing novel insights for its translational exploitation in both oncological intervention and regenerative medicine strategies.},
}

*Octamer Transcription Factor-3/genetics/metabolism
Humans
*Neoplasms/genetics/pathology
Cell Line, Tumor
*Carcinogenesis/genetics
Gene Expression Regulation, Neoplastic
Gene Expression Profiling
*Transcriptome
CRISPR-Cas Systems
Gene Regulatory Networks
Prognosis

@article {pmid41313840,
year = {2026},
author = {Mohammad, SI and Kareem, AK and Vasudevan, A and Rekha, MM and Jabir, MS and Nayak, P and AlKhafaje, Z and Arora, V and Kadhum, W and Chennakesavulu, K},
title = {Genome editing of immune checkpoints: CRISPR-mediated PD-1 inhibition in cancer.},
journal = {Seminars in oncology},
volume = {53},
number = {1},
pages = {152438},
doi = {10.1016/j.seminoncol.2025.152438},
pmid = {41313840},
issn = {1532-8708},
mesh = {Humans ; *Gene Editing/methods ; *Neoplasms/genetics/therapy/immunology ; *Programmed Cell Death 1 Receptor/antagonists & inhibitors/genetics ; Animals ; *CRISPR-Cas Systems ; *Immune Checkpoint Inhibitors/therapeutic use/pharmacology ; B7-H1 Antigen/antagonists & inhibitors/genetics ; Immunotherapy, Adoptive/methods ; },
abstract = {The programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint is a primary mechanism by which tumors evade immune surveillance, limiting the efficacy of cytotoxic T lymphocytes (CTLs) and tumor-infiltrating lymphocytes (TILs). Although immune checkpoint blockade therapies have revolutionized cancer treatment, their efficacy is restricted by acquired resistance, T-cell exhaustion, and tumor heterogeneity. The advent of CRISPR-Cas9 genome editing provides a precise and versatile approach to disrupt PD-1 or PD-L1, directly enhancing anti-tumor immune responses. Preclinical studies demonstrate that ex vivo PD-1 knockout in primary human T cells or TILs enhances proliferation, cytokine production, and cytotoxicity, resulting in improved tumor clearance in xenograft and humanized mouse models. In chimeric antigen receptor (CAR) T cell therapy, CRISPR-mediated disruption of PD-1 improves effector function, persistence, and resistance to exhaustion, with universal and allogeneic CAR-T platforms benefiting from multiplex genome editing. Direct PD-L1 knockout in tumor cells, often facilitated via nanoparticle- or biomaterial-assisted delivery, reshapes the immunosuppressive tumor microenvironment, promotes T cell infiltration, and enhances the efficacy of adoptive cellular therapy. Combination approaches integrating PD-1 editing with viral antigen targeting, long noncoding RNA (lncRNA) modulation, or conventional checkpoint blockade demonstrate synergistic anti-tumor effects. Clinically, early-phase trials in non-small cell lung cancer, mesothelin-positive solid tumors, and hematological malignancies establish the feasibility, safety, and preliminary efficacy of PD-1-deficient T cells. Despite these promising outcomes, challenges such as off-target effects, delivery efficiency, immunogenicity, long-term persistence, and regulatory considerations remain. This review aims to comprehensively evaluate preclinical and clinical studies investigating CRISPR-mediated PD-1/PD-L1 inhibition across various cancers, summarize mechanistic insights, and highlight translational opportunities and challenges for clinical implementation.},
}

Humans
*Gene Editing/methods
*Neoplasms/genetics/therapy/immunology
*Programmed Cell Death 1 Receptor/antagonists & inhibitors/genetics
Animals
*CRISPR-Cas Systems
*Immune Checkpoint Inhibitors/therapeutic use/pharmacology
B7-H1 Antigen/antagonists & inhibitors/genetics
Immunotherapy, Adoptive/methods

@article {pmid41286106,
year = {2026},
author = {Angelini Stewart, A and Ahrens-Nicklas, RC and Tsai, SQ and Musunuru, K and Giannikopoulos, P and Clelland, CD},
title = {Measurement and clinical interpretation of CRISPR off-targets.},
journal = {Nature genetics},
volume = {58},
number = {1},
pages = {20-27},
pmid = {41286106},
issn = {1546-1718},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods/adverse effects ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; },
abstract = {CRISPR genetic therapies are revolutionizing the landscape of preclinical research and clinical studies, providing new potential routes for curative intervention for a range of previously untreatable diseases. As with any therapy, the therapeutic benefits and risks must be weighed against consideration of the disease threat. Genome-related adverse events are an inherent risk of CRISPR genetic therapies, including off-target edits. The perception that CRISPR therapies ought to have near-zero off-targets belies clinical medicine, therapy development and biology, which demonstrate that 'perfect' therapeutics do not exist. Given that not all genomic off-target events are equal, we provide a practical framework to evaluate and assess off-target safety based on the tools available today and ones that will be developed in the future. With the comprehensive information and assessment gathered using these guidelines, we aim to streamline the transition of CRISPR therapeutics from bench to bedside.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Genetic Therapy/methods/adverse effects
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Animals

@article {pmid41270550,
year = {2026},
author = {Hu, Q and Liu, J and Zhang, W and Liao, X and Guo, Y and Lu, Q and Zhang, R and Yang, B and Zhang, T and Zhai, X and Luo, Q},
title = {Rapid nucleic acid detection of Mycoplasma synoviae using dual-mode RAA-CRISPR/Cas12a system.},
journal = {Poultry science},
volume = {105},
number = {1},
pages = {106126},
pmid = {41270550},
issn = {1525-3171},
mesh = {Animals ; *Mycoplasma synoviae/isolation & purification ; *Mycoplasma Infections/veterinary/diagnosis/microbiology ; *Poultry Diseases/diagnosis/microbiology ; *Chickens ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; Ducks ; Columbidae ; },
abstract = {Mycoplasma synoviae is a significant avian pathogen implicated in diverse clinical manifestations, including air sacculitis, infectious synovitis with arthritis, and eggshell apex abnormalities, which significantly threaten the poultry industry. To enable the rapid and accurate detection of M. synoviae, we developed a dual-mode diagnostic platform that combines recombinant enzyme-assisted amplification (RAA) with CRISPR/Cas12a-based fluorescence and lateral flow dipsticks (LFD). The Qsep100 automatic nucleic acid analysis system was used to screen for RAA primers by evaluating the amplicon size and concentration. Fluorescence monitoring revealed that the optimal reaction time for the RAA-CRISPR/Cas12a system was 28 min. After optimizing the concentrations of LbaCas12a, crRNA, and the ssDNA reporter, the assay achieved a sensitivity of 5.2 copies/µL. Cross-reactivity testing with seven common avian pathogens confirmed the high specificity of the established method for detecting M. synoviae. In the clinical validation, the method perfectly matched the results of quantitative real time polymerase chain reaction (qPCR). Furthermore, an epidemiological investigation revealed that chickens had the highest positivity rate for M. synoviae among the chickens, ducks, and pigeons. In summary, we developed a rapid, accurate, and portable diagnostic platform for M. synoviae detection, which provides a valuable tool for disease prevention and control in resource-limited settings.},
}

Animals
*Mycoplasma synoviae/isolation & purification
*Mycoplasma Infections/veterinary/diagnosis/microbiology
*Poultry Diseases/diagnosis/microbiology
*Chickens
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/veterinary/methods
Sensitivity and Specificity
Ducks
Columbidae

@article {pmid41265135,
year = {2026},
author = {Wright, M and Choi, W and Deng, Z and Kim, D and Lee, K},
title = {CRISPR/Cas9 Gene Editing of Turkey Cells Using Adenoviral Delivery Running Head: RESEARCH NOTE.},
journal = {Poultry science},
volume = {105},
number = {1},
pages = {106096},
pmid = {41265135},
issn = {1525-3171},
mesh = {Animals ; *Gene Editing/veterinary/methods ; *CRISPR-Cas Systems ; *Turkeys/genetics ; *Adenoviridae/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Line ; Receptors, Leptin/genetics ; Genetic Vectors ; },
abstract = {Poultry, including birds such as chicken, duck, turkey, geese, and other game birds is the most widely consumed animal protein globally, with per capita poultry consumption steadily rising in the United States over the past several decades. To meet the demand for poultry, many avenues of improving poultry production have been explored including genetic modification solutions. While genetic modification in many poultry species has been well explored, gene editing in turkey has no research into its application. This study investigated the feasibility of using CRISPR/Cas9 mediated gene editing using adenoviral delivery in vitro, a method that has been proven successful for other species of poultry but never turkey. Three guide RNAs (gRNAs) were designed, targeting the leptin receptor (LEPR) gene matching the gene sequences of both chicken and turkey. Following the assessment of each gRNA's editing efficiency in turkey embryonic fibroblast (TEF) cells and commercially available chicken DF-1 cells via liposome-mediated transfection of the CRISPR vector, the most effective gRNA was chosen for recombinant adenovirus type 5 production. From there, both DF-1 and TEF cells were transduced using the adenovirus and the genome editing efficiency was assessed. The results show that turkey cells can be genetically modified in vitro with indel frequencies comparable to those observed in chicken cells, using both CRISPR plasmids and adenoviral vectors. These findings can lay the groundwork for future experiments and adaptation to in vivo modification of turkey in the future.},
}

Animals
*Gene Editing/veterinary/methods
*CRISPR-Cas Systems
*Turkeys/genetics
*Adenoviridae/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Cell Line
Receptors, Leptin/genetics
Genetic Vectors

@article {pmid41236017,
year = {2026},
author = {Zhang, C and Goldsmith, SR and Htut, M and Rhee, JW and Vyas, V and Clark, MC and Armenian, SH and Forman, SJ and Wang, X},
title = {CRISPR Knockdown of CHIP Genes in Macrophages Drives Increased Production of Inflammatory Cytokines in CAR-T Cell Therapy.},
journal = {Transplantation and cellular therapy},
volume = {32},
number = {1},
pages = {10.e1-10.e5},
doi = {10.1016/j.jtct.2025.08.022},
pmid = {41236017},
issn = {2666-6367},
mesh = {Humans ; *Macrophages/metabolism/immunology ; *Cytokines/metabolism ; DNA Methyltransferase 3A ; *Immunotherapy, Adoptive/methods/adverse effects ; *CRISPR-Cas Systems ; Dioxygenases/genetics ; Receptors, Chimeric Antigen ; Gene Knockdown Techniques ; Proto-Oncogene Proteins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Binding Proteins/genetics ; Repressor Proteins/genetics ; DNA (Cytosine-5-)-Methyltransferases/genetics ; Gene Editing ; },
abstract = {Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by somatic mutations in leukemogenic genes (e.g., DNMT3A, TET2, and ASXL1) in hematopoietic stem cells and is linked to age-related clonal expansion and inflammation. We previously showed that patients with CHIP receiving chimeric antigen receptor (CAR)-T cell therapy have a higher risk of developing clinically significant cytokine release syndrome (CRS) compared to those without CHIP. Here, we investigated the mechanisms through which CHIP mutations can contribute to CAR-T-related toxicities. To model the interaction of CAR-T cell-mediated inflammatory response and macrophages harboring CHIP gene mutations, we employed CRISPR-based gene editing to knock down three key CHIP-associated genes (DNMT3A, TET2, and ASXL1) in human macrophages, using multiple guide RNAs for each. We co-cultured gene-edited macrophages with autologous CS1 and BCMA CAR-T cells and multiple myeloma (MM) tumor cells. Compared to nonedited macrophages, DNMT3A-, TET2-, and ASXL1-edited macrophages secreted significantly higher levels of pro-inflammatory cytokines characteristic of CRS, including IL-6, MCP-1, and IL-1β (P < .05 to .01). These results suggest that the presence of CHIP mutations in human macrophages may exacerbate the inflammatory response during CAR-T therapy. This study highlights CHIP mutations as potential biomarkers for identifying patients at high risk of developing CRS and for guiding personalized prophylactic interventions in MM CAR-T therapy.},
}

Humans
*Macrophages/metabolism/immunology
*Cytokines/metabolism
DNA Methyltransferase 3A
*Immunotherapy, Adoptive/methods/adverse effects
*CRISPR-Cas Systems
Dioxygenases/genetics
Receptors, Chimeric Antigen
Gene Knockdown Techniques
Proto-Oncogene Proteins/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA-Binding Proteins/genetics
Repressor Proteins/genetics
DNA (Cytosine-5-)-Methyltransferases/genetics
Gene Editing

@article {pmid41150843,
year = {2026},
author = {Butt, H and Sathish, S and London, E and Le, A and Li, Q and Gudmundsdottir, B and Lee, DY and Burke, EV and Yates, BP and Liu, DR and Hsieh, M and Leonard, A and Eaton, WA and Uchida, N and Pierciey, FJ and Newby, GA and Tisdale, JF and Demirci, S},
title = {Comparative analysis of CRISPR-Cas9, lentiviral transduction, and base editing for sickle cell disease in a murine model.},
journal = {Blood advances},
volume = {10},
number = {2},
pages = {289-300},
doi = {10.1182/bloodadvances.2025017321},
pmid = {41150843},
issn = {2473-9537},
mesh = {Animals ; *Anemia, Sickle Cell/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; *Lentivirus/genetics ; Disease Models, Animal ; Humans ; *Transduction, Genetic ; Hematopoietic Stem Cells/metabolism ; Hematopoietic Stem Cell Transplantation ; },
abstract = {Sickle cell disease (SCD) is a red blood cell disorder caused by a mutation in the β-globin gene, leading to sickle hemoglobin polymerization under low oxygen conditions. Both CRISPR-Cas9 editing and lentiviral transduction have shown promising clinical outcomes, but it remains unclear which approach is superior. Alternatively, new editing tools such as base editing may also be promising and reduce risks of genotoxicity. To compare these approaches, we studied them in an immunocompromised mouse model. We optimized ex vivo conditions in CD34+ hematopoietic stem and progenitor cells (HSPC) and infused edited SCD HSPC into busulfan-conditioned nonirradiated NOD,B6.SCID Il2rγ-/-KitW41/W41 (NBSGW) mice. Ex vivo analysis confirmed successful editing and transduction. At 16 weeks, bone marrow analysis showed similar human CD45+ cell engraftment across all groups (75%-90%). In the competitive transplantation group, there was a lower amount of B-cell lymphoma/leukemia 11A enhancer editing than base editing and lentiviral transduction. A secondary transplantation model yielded similar results. An antisickling assay showed significantly higher red blood cell sickling reduction in the base editing, transduction, and competitive transplantation groups compared to CRISPR-Cas9. In conclusion, although all methods showed therapeutic potential, base editing and lentiviral transduction provided superior outcomes over CRISPR-Cas9-mediated editing in a competitive murine transplantation model.},
}

Animals
*Anemia, Sickle Cell/therapy/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Mice
*Lentivirus/genetics
Disease Models, Animal
Humans
*Transduction, Genetic
Hematopoietic Stem Cells/metabolism
Hematopoietic Stem Cell Transplantation

@article {pmid40971839,
year = {2026},
author = {Lv, X and Wei, Q and Zhi, Q and Liu, X and Li, F and Niu, Y and Sun, H and Jin, K and Chen, GH and Li, B and Zuo, Q},
title = {CRISPR/Cas9-mediated TOP1 knockout in chicken DF-1 cells reveals its critical role in apoptosis regulation and genomic stability.},
journal = {Journal of animal science},
volume = {104},
number = {},
pages = {},
doi = {10.1093/jas/skaf315},
pmid = {40971839},
issn = {1525-3163},
mesh = {Animals ; *Apoptosis/genetics/physiology ; *Chickens/genetics ; *CRISPR-Cas Systems ; *Genomic Instability/genetics ; *DNA Topoisomerases, Type I/genetics/metabolism ; Cell Line ; Gene Knockout Techniques/veterinary ; DNA Damage ; },
abstract = {The role of topoisomerase I (encoded by TOP1) in avian cell survival and apoptosis regulation remains unclear, limiting its potential application in poultry biotechnology. This study aimed to establish a CRISPR/Cas9-mediated TOP1 knockout platform in chicken DF-1 cells and evaluate its functional impact on apoptosis. Three sgRNAs targeting TOP1 were designed and delivered via liposome vectors, achieving knockout efficiencies up to 50% as confirmed by T7 Endonuclease I (T7E1) assay and Sanger sequencing, with no detectable off-target effects. Functional analysis revealed that TOP1 knockout significantly increased apoptosis rates and upregulated DNA damage markers (γH2AX) and apoptotic genes (Caspase 8 and BRCA1). These results demonstrate that TOP1 is essential for maintaining genomic stability in avian somatic cells, and its depletion triggers apoptosis through DNA damage accumulation. Although synthetic lethality-based sex control was not directly tested here, our findings provide critical evidence that TOP1 dysfunction could theoretically enable selective elimination of specific cell populations (e.g., primary germ cells) via CRISPR editing. Notably, developing targeted delivery systems for PGCs-a focus of future research not addressed in this study-will be essential to achieve such selectivity in vivo, highlighting a significant technological hurdle to overcome.},
}

Animals
*Apoptosis/genetics/physiology
*Chickens/genetics
*CRISPR-Cas Systems
*Genomic Instability/genetics
*DNA Topoisomerases, Type I/genetics/metabolism
Cell Line
Gene Knockout Techniques/veterinary
DNA Damage

@article {pmid39962280,
year = {2026},
author = {Zheng, C and Zhang, G and Dean, LJ and Sontheimer, EJ and Xue, W},
title = {The reverse transcriptase domain of prime editors contributes to DNA repair in mammalian cells.},
journal = {Nature biotechnology},
volume = {44},
number = {1},
pages = {146-153},
pmid = {39962280},
issn = {1546-1696},
support = {R01 CA275945/CA/NCI NIH HHS/United States ; UH3HL147367//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; P01HL158506//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 GM150273/GM/NIGMS NIH HHS/United States ; UH3 HL147367/HL/NHLBI NIH HHS/United States ; R01GM150279//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {Humans ; *DNA Repair/genetics ; *RNA-Directed DNA Polymerase/metabolism/genetics ; *Gene Editing/methods ; HEK293 Cells ; Animals ; DNA Damage ; Protein Domains ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {Reverse transcriptase (RT) has been shown to play a role in double-strand break repair in bacteria, yet the impact of the RT component of prime editors (PEs) on normal mammalian cellular functions is unclear. Here we show that overexpressed RT or PE increases short insertions and diminishes homology-directed repair following Cas9 cleavage at multiple loci in multiple cell lines. Live-cell imaging shows that RT and PEs are rapidly recruited to laser-induced DNA damage sites and promote endogenous repair, independent of known DNA damage sensors. Interestingly, RT-mCherry partially impairs green fluorescent protein-PARP1 recruitment. A compact PE without an RNase H domain shows reduced DNA repair activity and may therefore be more suitable for clinical application. These data reveal a role for untethered RT or the RT domain of PEs in the repair of chromosomal breaks, calling for evaluation of the long-term effect of PEs and retroviral RT in mammalian cells.},
}

Humans
*DNA Repair/genetics
*RNA-Directed DNA Polymerase/metabolism/genetics
*Gene Editing/methods
HEK293 Cells
Animals
DNA Damage
Protein Domains
DNA Breaks, Double-Stranded
CRISPR-Cas Systems
Cell Line

@article {pmid39934271,
year = {2026},
author = {Hart, SK and Müller, S and Wessels, HH and Méndez-Mancilla, A and Drabavicius, G and Choi, O and Sanjana, NE},
title = {Precise RNA targeting with CRISPR-Cas13d.},
journal = {Nature biotechnology},
volume = {44},
number = {1},
pages = {64-69},
pmid = {39934271},
issn = {1546-1696},
support = {DP2HG010099//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA/genetics ; *Gene Editing/methods ; HEK293 Cells ; Transcriptome/genetics ; },
abstract = {The possibility of collateral RNA degradation poses a concern for transcriptome perturbations and therapeutic applications using CRISPR-Cas13. We show that collateral activity only occurs with high RfxCas13d expression. Using low-copy RfxCas13d in transcriptome-scale and combinatorial pooled screens, we achieve high on-target knockdown without extensive collateral activity. Furthermore, analysis of a high-fidelity Cas13 variant suggests that its reduced collateral activity may be due to overall diminished nuclease capability.},
}

*CRISPR-Cas Systems/genetics
Humans
*RNA/genetics
*Gene Editing/methods
HEK293 Cells
Transcriptome/genetics

@article {pmid41533919,
year = {2026},
author = {Sun, X and Liang, Y and Liang, Y and Li, W and Chen, H and Ding, C and Duan, C and Zhou, Y and Zhang, R and Xu, H and Chen, J},
title = {PRPF8 Mutation-Induced Defects in Human iPSC-Derived RPE Are Rescued by Adenine Base Editing.},
journal = {Investigative ophthalmology & visual science},
volume = {67},
number = {1},
pages = {21},
pmid = {41533919},
issn = {1552-5783},
mesh = {Humans ; *Retinal Pigment Epithelium/metabolism/ultrastructure/pathology ; *Induced Pluripotent Stem Cells/metabolism ; *Mutation ; *Gene Editing/methods ; *Retinitis Pigmentosa/genetics/pathology/metabolism ; *Adenine ; Cell Differentiation ; Cells, Cultured ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {PURPOSE: The pathological effects of pre-mRNA processing factor 8 (PRPF8) mutations on the retinal pigment epithelium (RPE) are not fully understood. We aimed to identify disease-specific cellular and molecular phenotypes in PRPF8 retinitis pigmentosa (RP) patient-derived induced pluripotent stem cell (iPSC)-RPE and to test whether adenine base editing (ABE), which corrects the PRPF8 mutation in iPSCs, can reverse abnormal RPE phenotypes.

METHODS: We obtained patient-derived iPSCs with the heterozygous PRPF8 (c.5792C>T) mutation and created an induced mutation iPSC line by introducing the same mutation into wild-type iPSCs using CRISPR/Cas9. These cells were differentiated into RPE cells. We measured PRPF8 expression, barrier integrity, and apicobasal polarity. Electron microscopy examined apical microvilli and pigment granules. RNA sequencing quantified splicing events and affected pathways. ABE corrected the PRPF8 mutation in patient iPSCs, and the corrected clones were re-differentiated into RPE cells for evaluation.

RESULTS: PRPF8-mutant RPE cells exhibited decreased PRPF8 mRNA and protein levels, weakened barrier function, and disrupted cell polarity. Ultrastructural analysis showed loss of apical microvilli and pigment granules. Transcriptomic analysis identified abnormal splicing events, with enrichment in cilium assembly and melanosome pathways. ABE correction restored PRPF8 expression, normalized barrier integrity, apicobasal polarity, and rescued the defects in apical microvilli and pigment granules.

CONCLUSIONS: PRPF8 mutations in patient-derived iPSC RPE cause functional and ultrastructural defects driven by splicing abnormalities. ABE correction of the PRPF8 mutation in iPSCs can restore PRPF8 expression and alleviate cellular and molecular defects in RPE and highlights the therapeutic potential of precise gene editing correction strategies for RP.},
}

Humans
*Retinal Pigment Epithelium/metabolism/ultrastructure/pathology
*Induced Pluripotent Stem Cells/metabolism
*Mutation
*Gene Editing/methods
*Retinitis Pigmentosa/genetics/pathology/metabolism
*Adenine
Cell Differentiation
Cells, Cultured
Phenotype
CRISPR-Cas Systems

@article {pmid41287270,
year = {2026},
author = {Yao, W and Xu, X and Zhai, X and Ji, T and Zhang, R and Xu, S and Luo, X},
title = {Autocatalytic Circular DNA Powered Plasmonic CRISPR/Cas12a Platform for Ultrasensitive Non-Nucleic Acid Target Sensing.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {65},
number = {3},
pages = {e16838},
doi = {10.1002/anie.202516838},
pmid = {41287270},
issn = {1521-3773},
support = {21505081//National Natural Science Foundation of China/ ; 22374085//National Natural Science Foundation of China/ ; ZR2023MB110//Natural Science Foundation of Shandong Province/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA, Circular/chemistry/metabolism/genetics ; *Biosensing Techniques/methods ; Nucleic Acid Amplification Techniques ; Catalysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a-based detection of non-nucleic acid targets faces two major challenges: 1) limited sensitivity due to the inherent inability to pre-amplify non-nucleic acid targets, and 2) suboptimal performance of traditional reporters caused by photobleaching of fluorescent dyes, rapid degradation, and slow reaction kinetics resulting from random molecular collisions. To overcome these limitations, we developed an innovative plasmonic CRISPR/Cas12a platform featuring positive-feedback autocatalytic circular DNA (cir-DNA) amplification. This system synergistically combines spatial confinement effects with plasmon-enhanced fluorescence (PEF) to achieve ultrasensitive detection of non-nucleic acid targets. The engineered cir-DNA enables continuous Cas12a regeneration for autocatalytic signal amplification, while the designed plasmonic spherical nucleic acids significantly accelerate reaction kinetics while enhancing fluorescence signals. This integrated approach reduced the required reaction time to 15 min while improving the detection limit by approximately 52-fold compared to conventional methods. Furthermore, by leveraging a convolutional neural network (CNN) machine learning model, not only the assessment of the risk level of perfluorooctanoic acid (PFOA) based on threshold-positive and threshold-negative serum concentrations but also highly accurate blind testing were both achieved, highlighting its potential for clinical applications such as pregnancy risk assessment.},
}

*CRISPR-Cas Systems
*DNA, Circular/chemistry/metabolism/genetics
*Biosensing Techniques/methods
Nucleic Acid Amplification Techniques
Catalysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40759200,
year = {2026},
author = {He, J and Zhang, M and Shi, Y and Ji, Z and Lu, Y and Yu, W and Wang, F and Guan, Q and Yan, H and Chen, G and Zhang, E and Lian, S and Pu, X and Zhang, J and Ge, S and Zhou, R and Li, J and Wang, P and Sun, B and Zhang, Z},
title = {Targeting xanthine oxidoreductase reverses resistance to EGFR tyrosine kinase inhibitors in intrahepatic cholangiocarcinoma.},
journal = {Journal of hepatology},
volume = {84},
number = {2},
pages = {355-369},
doi = {10.1016/j.jhep.2025.07.016},
pmid = {40759200},
issn = {1600-0641},
mesh = {*Cholangiocarcinoma/drug therapy/genetics/pathology ; Animals ; Mice ; *Drug Resistance, Neoplasm/drug effects/genetics ; Humans ; ErbB Receptors/antagonists & inhibitors/metabolism ; *Bile Duct Neoplasms/drug therapy/pathology/genetics ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; *Xanthine Dehydrogenase/antagonists & inhibitors/genetics/metabolism ; Cell Line, Tumor ; CRISPR-Cas Systems ; Male ; Tyrosine Kinase Inhibitors ; },
abstract = {BACKGROUND & AIMS: Despite the overexpression and aberrant activation of epidermal growth factor receptor (EGFR) in intrahepatic cholangiocarcinoma (iCCA), the disease remains refractory to EGFR tyrosine kinase inhibitors (TKIs). Multiple clinical trials involving EGFR-targeting agents have been conducted; however, none have demonstrated clinically significant efficacy. The aim of this study was to elucidate the mechanisms underlying EGFR TKI resistance in iCCA.

METHODS: Genome-scale CRISPR-Cas9 chemical-genetic screens were conducted, identifying xanthine oxidoreductase (XOR) as a critical modulator. XOR expression was evaluated in surgical samples from patients with iCCA and matched peritumor tissue via immunohistochemistry, western blotting, and quantitative reverse-transcription PCR. iCCA models were established in Xdh[f/f] and Xdh[hep-/-] mice, as well as in adeno-associated virus-treated mice via hydrodynamic tail vein injection of the oncogenic driver myr-AKT combined with either Yap[S127A] or NICD1 using the Sleeping Beauty system.

RESULTS: CRISPR-Cas9 screening revealed that XOR, which is upregulated in iCCA compared to peritumor tissue and bile ducts, is required for EGFR TKI resistance. XOR knockdown inhibited iCCA cell proliferation, increased DNA damage, reduced tumor progression in iCCA models, and significantly enhanced the anti-tumor efficacy of gefitinib both in vitro and in vivo. CEBPβ was found to regulate XOR transcription. Mechanistically, XOR promoted EGFR stability via USP8-mediated deubiquitination and MUC1 upregulation, thereby enhancing signaling cascades and DNA damage repair - collectively driving resistance to TKIs.

CONCLUSIONS: Targeting XOR-mediated EGFR stabilization and MUC1 upregulation represents a promising therapeutic strategy to inhibit iCCA progression and enhance sensitivity to EGFR TKIs in patients with iCCA.

IMPACT AND IMPLICATIONS: This study identifies XOR as a key driver of resistance to EGFR tyrosine kinase inhibitors (TKIs) in intrahepatic cholangiocarcinoma (iCCA), addressing a major limitation of current targeted therapies. By promoting EGFR stability through USP8-mediated deubiquitination and MUC1 upregulation, XOR enhances DNA repair and sustains oncogenic signaling. Inhibition of XOR sensitizes iCCA cells to EGFR TKIs, suppresses tumor progression, and exacerbates DNA damage. These findings suggest that targeting XOR could overcome TKI resistance and offer a novel therapeutic strategy for iCCA, potentially improving outcomes in a patient population with limited effective treatment options.},
}

*Cholangiocarcinoma/drug therapy/genetics/pathology
Animals
Mice
*Drug Resistance, Neoplasm/drug effects/genetics
Humans
ErbB Receptors/antagonists & inhibitors/metabolism
*Bile Duct Neoplasms/drug therapy/pathology/genetics
*Protein Kinase Inhibitors/pharmacology/therapeutic use
*Xanthine Dehydrogenase/antagonists & inhibitors/genetics/metabolism
Cell Line, Tumor
CRISPR-Cas Systems
Male
Tyrosine Kinase Inhibitors

@article {pmid41533584,
year = {2026},
author = {Zhang, J and He, X and Huang, J and Cheng, C and He, G and Xia, R and Yang, J and Chen, J and Guo, L and Xiang, D and Li, F and Shi, J and Li, P},
title = {Steric regulation of CRISPR/Cas12a trans-cleavage kinetics via split-activator extensions.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41533584},
issn = {1362-4962},
support = {2025ZDXM001//Chongqing Municipal Health Commission/ ; KJZD-K202400103//Chongqing Municipal Education Commission/ ; CSTB2024TIAD-CYKJCXX0031//Scientific and Technological Innovation Cooperation Program/ ; YXGD2025029//Chongqing Young and Middle-aged Medical High-level Talent Project/ ; YXQN2025049//Chongqing Youth Outstanding Medical Talent Project/ ; 2026MSXM018//Chongqing Science and Technology Bureau/ ; 2024YCXM010//2024 Hospital-level Cultivation Project of Chongqing University Jiangjin Hospital/ ; 2025qdjfxm001//Chongqing University Jiangjin Hospital/ ; 2025qdjfxm002//Chongqing University Jiangjin Hospital/ ; PS202511//2025 Key Research Project for Enhancing Medical Service Capabilities of County-level Medical Institutions/ ; CSTB2024NSCQ-KJFZMSX0018//Chongqing Science and Technology Development Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Kinetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism/chemistry ; MicroRNAs/genetics/analysis ; DNA/genetics/chemistry/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a holds substantial promise for molecular diagnostics, yet its rapid and uncontrolled activation often results in background leakage and disrupts the coordination of upstream reaction modules. Here, we established a steric-regulation framework that enables predictable tuning of Cas12a trans-cleavage kinetics through rationally engineered extensions on split activators. Systematic analysis of extension orientation, length, and hybridization state revealed quantitative and direction-dependent rules governing steric control of activator assembly and Cas12a activation. Guided by these insights, we integrated the sterically regulated split activator into an entropy-driven DNA circuit to construct a fully one-pot cascaded detection system. The engineered steric barriers effectively suppressed premature activation and established precise kinetic matching between the DNA circuit and Cas12a. The resulting platform achieved a detection limit of 1.24 pM for microRNA-21 and demonstrated high fidelity. This work defines a predictable steric-gating mechanism for Cas12a activation and delivers a nucleic-acid-only regulatory module that can be incorporated into diverse CRISPR architectures, supporting the development of robust, leakage-resistant one-pot diagnostic systems.},
}

*CRISPR-Cas Systems/genetics
Kinetics
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
*Endodeoxyribonucleases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism/chemistry
MicroRNAs/genetics/analysis
DNA/genetics/chemistry/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41533581,
year = {2026},
author = {Sugimoto, Y and Kachi, T and Watanabe, Y and Kubokawa, M and Ogami, K and Kawamata, M and Yoshino, S and Suzuki, HI},
title = {Optimized CRISPR-Cas9 system for efficient engineering of ecDNA in cancer cells.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41533581},
issn = {1362-4962},
support = {JP24H00614//Japan Society for the Promotion of Science/ ; JP22K06925//Japan Society for the Promotion of Science/ ; JP25K10464//Japan Society for the Promotion of Science/ ; 19K24694//Japan Society for the Promotion of Science/ ; JP22ama221111//Japan Agency for Medical Research and Development/ ; JP23ck0106791//Japan Agency for Medical Research and Development/ ; JP23tk0124003//Japan Agency for Medical Research and Development/ ; JP24ck0106875//Japan Agency for Medical Research and Development/ ; JP25ck0106019//Japan Agency for Medical Research and Development/ ; JP25ak0101291//Japan Agency for Medical Research and Development/ ; JP23kk0305026//Japan Agency for Medical Research and Development/ ; JP25kk0305028//Japan Agency for Medical Research and Development/ ; //Takeda Science Foundation/ ; 22-6304//oray Science Foundation/ ; //Inamori Research Institute for Science/ ; 24KJ1238//JSPS/ ; //Nagoya University/ ; //JST-SPRING/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Neoplasms/genetics ; Cell Line, Tumor ; Gene Knock-In Techniques ; *DNA/genetics ; },
abstract = {Extrachromosomal DNA (ecDNA) amplification represents an emerging mechanism underlying oncogene amplification, tumor heterogeneity, and drug resistance in cancer. However, the biology of ecDNA remains poorly understood because tools to engineer ecDNAs and precisely monitor their dynamics are limited. In particular, genome engineering strategies have not been established for ecDNA, which exists in tens to hundreds of copies within a single cell. Here, we report a systematic validation of ecDNA editing using standard CRISPR-Cas9 system and optimized CRISPR-Cas9 system with safeguard single-guide RNAs (sgRNAs), in which the addition of cytosine extensions finely reduces excessive Cas9 activity. The conventional CRISPR-Cas9 system induced severe cytotoxicity and markedly reduced ecDNA copy number, together with frequent micronucleus formation. Knock-in efficiency was remarkably low, highlighting an intrinsic difficulty in editing ecDNA. In contrast, the safeguard sgRNA strategy not only alleviated cytotoxicity and ecDNA loss in a cytosine-length-dependent manner but also enabled efficient knock-in into multiple ecDNA per cell. Computational simulations suggested that the degree and temporal patterns of multiple DNA cleavage events shape cell death, micronucleus formation, and rapid expansion of knock-in ecDNA. Collectively, optimization of Cas9 activity using safeguard sgRNAs enables efficient and nondisruptive ecDNA engineering, providing a powerful tool to study ecDNA biology.},
}

*CRISPR-Cas Systems
Humans
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*Neoplasms/genetics
Cell Line, Tumor
Gene Knock-In Techniques
*DNA/genetics

@article {pmid41533573,
year = {2026},
author = {Kammerdiener, EK and Garcia, SK and Bales, MK and Klingeman, DM and Guss, AM and Giannone, RJ and Hettich, RL and Eckert, CA and Alexander, WG},
title = {Multilayered regulation by RNA thermometers enables precise control of Cas9 expression in E. coli.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41533573},
issn = {1362-4962},
support = {ERKP886//U.S. Department of Energy/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Promoter Regions, Genetic ; Temperature ; Escherichia coli Proteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics ; Gene Editing/methods ; Heat-Shock Proteins/genetics/metabolism ; },
abstract = {Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We also show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs in Escherichia coli. This strategy should be applicable to all bacterial Cas9-based methods and technologies.},
}

*Escherichia coli/genetics/metabolism
*Gene Expression Regulation, Bacterial
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Promoter Regions, Genetic
Temperature
Escherichia coli Proteins/genetics/metabolism
*CRISPR-Associated Protein 9/genetics
Gene Editing/methods
Heat-Shock Proteins/genetics/metabolism

@article {pmid41533202,
year = {2026},
author = {Kumar, P and Yadav, H and Mahakalkar, B and Mandlik, R and Vats, S and Thakral, V and Kumar, V and Nishad, SK and Nichal, S and Deshmukh, R and Sharma, TR and Sonah, H},
title = {Challenges and Opportunities with CRISPR-Based Genome Editing in Legume Crops.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {24},
pmid = {41533202},
issn = {1438-7948},
support = {HSCSIT/R&D/2024/511//Haryana State Council for Science and Technology/ ; HSCSIT/R&D/2024/511//Haryana State Council for Science and Technology/ ; Agril. Edn / 27/05/NP(VP)-2023-HRD//Indian Council of Agricultural Research/ ; BT/PR38279/GET/119/351/2020//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; *Fabaceae/genetics ; *Genome, Plant ; Plants, Genetically Modified/genetics ; Plant Breeding ; },
abstract = {Over the last couple of decades, tremendous progress has been made in legume genomics. Genomics information generated for legume crops is being explored through molecular breeding and transgenic approaches. However, the gap between knowledge generation and its utilization is increasing. In this regard, recent developments in genome editing techniques provide an excellent opportunity to utilize the available knowledge for the improvement of legume crops. This review highlights recent developments with Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based genome-editing approaches, including Cas variants/orthologs and Protospacer adjacent motif-less (PAMless) Genome Editing, multiplex genome editing, base editing, prime editing, transcriptional regulation, methylome editing, and DNA-free editing methods. Furthermore, the applications of non-homologous end joining (NHEJ) and homology-directed repair (HDR)- based editing, are addressed which enable targeted and precise genomic modifications. Moreover, virus-mediated genome editing, in planta transformation, and mobile guide RNAs are increasingly being leveraged to enhance the efficiency and heritability of genome editing. Additionally, the role of artificial intelligence in guide RNA design, off-target prediction, and the development of novel Cas variants is also discussed, which can speed up the legume improvement. This article highlights the successful examples of efforts utilizing CRISPR/Cas9 for the development of legume crops with biotic and abiotic stress tolerance, desirable plant architecture, improved nutrient uptake, and enhanced yield and quality. The biggest limitation in the genome editing of legume crops is their recalcitrance to both transformation and tissue culture. This article discusses how this particular limitation can be addressed in the context of genome editing of legume crops. Finally, the possibilities of integrating these recently developed tools with translational breeding have also been discussed, which will facilitate the legume production for sustainable agriculture and food security.},
}

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

@article {pmid41533153,
year = {2026},
author = {Ricci, ML and Fillo, S and Giordani, F and Ciammaruconi, A and Girolamo, A and Anselmo, A and Monte, A and Mentasti, M and Cusimano, V and Caporali, MG and Rota, MC and Petzold, M and Afshar, B and Lista, F and Luck, C and Scaturro, M},
title = {Genomic characterization of Legionella pneumophila serogroup 1 ST901 isolates responsible for recurrent travel-associated Legionnaires' disease cases and clusters.},
journal = {Pathogens and global health},
volume = {},
number = {},
pages = {1-12},
doi = {10.1080/20477724.2025.2610657},
pmid = {41533153},
issn = {2047-7732},
abstract = {Cases of travel-associated Legionnaires' disease (TALD) are frequently reported in Italy. From 1987 to 2021, 61 cases of TALD were linked to 22 hotels in a municipality in northern Italy. Legionella pneumophila serogroup 1 (Lp1) strains isolated from both patients and hotel water systems were identified as sequence type (ST) 901, a genotype rarely associated with travel-related infections in Italy or elsewhere. Whole-genome sequencing was used to analyze 41 isolates, and phylogenetic relationships were inferred by core genome multilocus sequence typing (cgMLST), single nucleotide polymorphisms (SNP) and pangenome analyses. The Lp ST901 isolates were found to form a clade characterized by some accessory genomic islands (AGI) already described in other epidemic strains, such as Alcoy, Corby, Paris and Philadelphia; other islands, containing either transposase/recombinase or transcriptional regulator factors or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-cas systems, were specific to Lp ST901. Lp ST901 also appears to have undergone possible recombination with other strains, such as Lp ST47 (Lorraine strain). Additionally, CRISPR-Cas systems may have contributed to the protection of Lp ST901 from external dangers, while the colonized hotel water systems may have provided an ideal environmental protective niche. Our findings highlight that Lp ST901 has public health significance and deserves attention in Legionnaires' disease surveillance and risk assessment.},
}

@article {pmid41532605,
year = {2026},
author = {Chen, Q and Gou, H and Xu, C and Wang, S and Zhang, H and Song, M and Wang, M and Ji, X and Wei, X and Tan, Y and Quan, H and Luo, P and Shou, H and Liu, P and Liang, Y and Zhu, JK},
title = {Structure-Guided Engineering of a Cas12i Nuclease Unlocks Near-PAMless Genome Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e16670},
doi = {10.1002/advs.202516670},
pmid = {41532605},
issn = {2198-3844},
support = {KJZD20240903102703005//Shenzhen Science and Technology Program/ ; JCYJ20241202125332043//Shenzhen Science and Technology Program/ ; 2024B1111130001//Guangdong S&T Program/ ; },
abstract = {The therapeutic and research applications of CRISPR-Cas nucleases are constrained by their reliance on specific Protospacer Adjacent Motifs (PAMs), which limit the accessible sites in the genome. To overcome this critical barrier, we performed structure-guided engineering of SF01, a compact Cas12i nuclease. Using AlphaFold-predicted structural models, we identified and systematically mutagenized 38 residues at the PAM-interacting interface. This iterative engineering process yielded three superior variants-KR, IKRR, and STKRR-that exhibit dramatically relaxed PAM specificity, enabling efficient editing at a broad spectrum of 5'-NNTN-3' sites. Importantly, while the most broad-spectrum variant (STKRR) shows a trade-off at canonical sites, the IKRR variant retains high activity at canonical 5'-NTTN-3' PAMs while simultaneously enabling efficient editing at 5'-NNTN-3' sites. This near-PAMless activity expands the targetable portion of the genome to over 25%, a four-fold increase over the parental nuclease. Furthermore, adenine base editors (ABEs) constructed with these variants achieve high-efficiency editing (∼80%) at endogenous loci with expanded targeting scope. Comprehensive off-target analysis using GUIDE-tag and Digenome-seq revealed that the enhanced on-target activity of the SF01 variants is not accompanied by a loss of specificity. These engineered nucleases represent a powerful and versatile expansion of the genome editing toolkit, enabling applications previously inaccessible due to PAM constraints.},
}

@article {pmid41528700,
year = {2026},
author = {Wei, W and Yang, Y and Shih, J},
title = {Rapid and Sensitive Detection of Phytoplasma Diseases Using a CRISPR/Cas12a DETECTR Assay Combined with Isothermal Recombinase Polymerase Amplification.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3008},
number = {},
pages = {63-74},
pmid = {41528700},
issn = {1940-6029},
mesh = {*Phytoplasma/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism ; DNA, Bacterial/genetics ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases/genetics ; Bacterial Proteins/genetics ; },
abstract = {Our protocol outlines a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay, which combines CRISPR/Cas12a technology with isothermal Recombinase Polymerase Amplification (RPA) for the rapid and specific detection of phytoplasma diseases in plants. This isothermal method utilizes RPA to amplify the target DNA fragment from the genomic DNA of phytoplasmas, followed by incubation with Cas12a nuclease and CRISPR RNAs (crRNAs) specifically designed to target unique phytoplasma DNA sequences. Upon initial cleavage of the amplified target DNA, Cas12a gains enzymatic activity to indiscriminately cleave single-stranded fluorescent oligonucleotide reporters, generating a fluorescent signal for highly sensitive detection of the pathogen. The protocol provides detailed instructions on: (i) sample collection and preparation; (ii) assay reaction setup, including RPA and Cas12a detection steps; (iii) reaction and detection conditions; and (iv) guidelines for accurately interpreting fluorescence data to detect phytoplasma DNA. This protocol is designed for researchers and agricultural professionals to effectively adopt and implement this advanced diagnostic technique.},
}

*Phytoplasma/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
*Plant Diseases/microbiology
*Nucleic Acid Amplification Techniques/methods
Recombinases/metabolism
DNA, Bacterial/genetics
CRISPR-Associated Proteins/genetics
Endodeoxyribonucleases/genetics
Bacterial Proteins/genetics

@article {pmid41505468,
year = {2026},
author = {Ma, J and Zhang, Y and Li, Z and Liu, L and A, J and Liang, R and Cao, C and Zhou, J and Cheng, P and Li, Y and Li, Z and Ma, L and Jiang, L and A, X},
title = {CRISPR/Cas12a-RCA enables ultrasensitive detection of circulating free DNA for noninvasive diagnosis of echinococcosis.},
journal = {PLoS neglected tropical diseases},
volume = {20},
number = {1},
pages = {e0013069},
doi = {10.1371/journal.pntd.0013069},
pmid = {41505468},
issn = {1935-2735},
mesh = {Humans ; *CRISPR-Cas Systems ; *Echinococcosis/diagnosis/blood/parasitology ; *Echinococcus/genetics/isolation & purification ; *Cell-Free Nucleic Acids/blood/genetics ; Sensitivity and Specificity ; *DNA, Helminth/blood/genetics ; *Molecular Diagnostic Techniques/methods ; Animals ; Male ; Female ; Limit of Detection ; Middle Aged ; High-Throughput Nucleotide Sequencing ; Adult ; },
abstract = {OBJECTIVE: To develop a novel non-invasive CRISPR/Cas12a-RCA assay for the primary screening of human echinococcosis via detection of circulating Echinococcus cell-free DNA (cfDNA) in peripheral blood.

METHODS: Plasma cfDNA from 20 AE patients was analyzed via high-throughput sequencing to identify conserved repetitive Echinococcus fragments.A one-pot RCA system coupled with CRISPR/Cas12a was optimized for Echinococcus-cfDNA detection. The limit of detection (LOD) was determined using serially diluted synthetic standards, while specificity was validated through mismatch probes and cross-reactivity testing. Clinical validation included 50 AE cases, 22 cystic echinococcosis (CE) cases, 43 non-Echinococcus hepatic disease (HD) cases, and 53 healthy controls (CON).

RESULTS: A conserved repetitive 28S rDNA fragment (pan-Echinococcus-28S) was identified as a biomarker. The CRISPR/Cas12a-RCA assay achieved amplification within 30 minutes at 37 °C, with a linear range of 1 aM to 100 pM and an LOD of 1.41 aM. Visual detection limits were 10 aM (UV light) and 1 aM (blue light). The assay demonstrated high sensitivity (87.5%) and specificity (96.9%, AUC = 0.933) in distinguishing Echinococcus infection (AE/CE) from HD and CON.

CONCLUSION: The optimized one-pot CRISPR/Cas12a-RCA system enables rapid and ultrasensitive detection of pan-Echinococcus cfDNA, providing a cost-effective and highly accurate solution for the primary screening of echinococcosis.},
}

Humans
*CRISPR-Cas Systems
*Echinococcosis/diagnosis/blood/parasitology
*Echinococcus/genetics/isolation & purification
*Cell-Free Nucleic Acids/blood/genetics
Sensitivity and Specificity
*DNA, Helminth/blood/genetics
*Molecular Diagnostic Techniques/methods
Animals
Male
Female
Limit of Detection
Middle Aged
High-Throughput Nucleotide Sequencing
Adult

@article {pmid41494531,
year = {2026},
author = {Wan, W and Ji, X and Song, H and Zhang, Z and Kwok, CK and Fang, X and Li, X},
title = {Fluorogenic CRISPR for DNA imaging in live mammalian cells.},
journal = {Cell chemical biology},
volume = {33},
number = {1},
pages = {33-44},
doi = {10.1016/j.chembiol.2025.10.013},
pmid = {41494531},
issn = {2451-9448},
mesh = {Humans ; *DNA/genetics/analysis/metabolism ; *Fluorescent Dyes/chemistry ; Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Optical Imaging ; },
abstract = {Spatiotemporal imaging of genomic DNA dynamics in live mammalian cells is essential for elucidating eukaryotic organization and processes relevant to health and disease. CRISPR systems greatly facilitate the development of live cell DNA imaging tools. However, conventional CRISPR imaging tools typically utilize constitutively fluorescent proteins, resulting in high background noise, nonspecific nucleolar signals, and low signal-to-noise ratios. To address this, fluorogenic CRISPR-based imaging tools have been developed. These tools remain non-fluorescent until they bind to the target DNA, thus significantly reducing the background and enhancing the sensitivity. This review summarizes four fluorogenic CRISPR strategies, each utilizing different fluorogenic reporters, including fluorogenic proteins, fluorogenic RNA aptamers, split fluorescent proteins, and molecular beacons. These fluorogenic CRISPR approaches successfully monitored the subnucleus gene loci localization, dynamics, and DNA breaks and repairs. We anticipate that this review can inspire researchers to expand the fluorogenic CRISPR for cellular DNA imaging and diverse bioapplications.},
}

Humans
*DNA/genetics/analysis/metabolism
*Fluorescent Dyes/chemistry
Animals
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
*Optical Imaging

@article {pmid41444344,
year = {2025},
author = {Li, X and Guo, J and Yang, H and Wu, Y and Xie, Z and Li, D},
title = {A CRISPR-assisted passive microfluidic chip for rapid, visual detection of multiple respiratory viruses.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {2033},
pmid = {41444344},
issn = {2045-2322},
support = {GJHZ20220913143207014//Science and Technology Innovation Commission of Shenzhen/ ; 12274197//China National Natural Science Fund/ ; 2022B1515020093//Guangdong Scientific and Technological Project/ ; },
mesh = {Humans ; SARS-CoV-2/genetics/isolation & purification ; *Lab-On-A-Chip Devices ; *CRISPR-Cas Systems ; COVID-19/diagnosis/virology ; *Respiratory Tract Infections/virology/diagnosis ; Coinfection/diagnosis/virology ; Nucleic Acid Amplification Techniques ; Influenza B virus/genetics/isolation & purification ; Influenza A virus/genetics/isolation & purification ; },
abstract = {In recent years, viral co-infections, particularly with respiratory viruses, have resulted in more complex symptoms, a greater disease burden, and increased challenges in clinical decision-making. These complexities underscore the urgent need for improved diagnostic tools in the managing acute respiratory infections. To address the limitations of conventional qPCR and current POCT methodologies, we developed a passively driven microfluidic chip capable of rapidly screening multiple respiratory viruses. This platform is particularly suited for the point-of-care diagnosis of viral co-infections. Our device integrates nucleic acid amplification and CRISPR-based detection within a single, passively operated system. By utilizing a rapid, 10-minute sample preparation protocol and a 35-minute on-chip assay, this platform enables the multiplex detection of influenza A/B, human parainfluenza virus, and SARS-CoV-2. The total assay time from sample to answer is approximately 45 min, with equipment requirements minimized to a heating block. The assay demonstrated a detection sensitivity of about 10 copies/µL for viral RNA in dilution series experiments. The sensitivity of the assay was 98.44% (95% CI: 91.6%-99.96%), and the specificity was 100% (95% CI: 79.4%-100%). The system combines CRISPR-Cas12a-mediated sensing with reverse transcription recombinase polymerase amplification (RPA) for highly specific nucleic acid detection. The chip design utilizes capillary action and gravity-driven flow for autonomous fluid control, while lyophilized reagent preloading ensures storage stability and minimizes user intervention.},
}

Humans
SARS-CoV-2/genetics/isolation & purification
*Lab-On-A-Chip Devices
*CRISPR-Cas Systems
COVID-19/diagnosis/virology
*Respiratory Tract Infections/virology/diagnosis
Coinfection/diagnosis/virology
Nucleic Acid Amplification Techniques
Influenza B virus/genetics/isolation & purification
Influenza A virus/genetics/isolation & purification

@article {pmid41435708,
year = {2026},
author = {Fang, J and Chen, Q and Ran, M and Chen, R and Chen, W and Cui, J and Wang, J and Zhong, K and Shi, L and Lu, C and Jiang, H},
title = {RPA-CRISPR/Cas12a-coupled microfluidic biosensor enabling on-site, sensitive quantification of Vibrio parahaemolyticus.},
journal = {Biosensors & bioelectronics},
volume = {296},
number = {},
pages = {118327},
doi = {10.1016/j.bios.2025.118327},
pmid = {41435708},
issn = {1873-4235},
mesh = {*Vibrio parahaemolyticus/isolation & purification/genetics/pathogenicity ; *Biosensing Techniques/instrumentation ; *Nucleic Acid Amplification Techniques/instrumentation ; Limit of Detection ; *CRISPR-Cas Systems/genetics ; Animals ; Seafood/microbiology ; Lab-On-A-Chip Devices ; *Vibrio Infections/microbiology/diagnosis ; Equipment Design ; },
abstract = {Vibrio parahaemolyticus is a major cause of seafood-associated gastroenteritis and aquatic animal diseases, posing persistent threats to public health and aquaculture. Rapid and accurate on-site quantitative detection is essential for risk assessment and early intervention. Although qPCR and digital PCR provide reliable quantification, their reliance on complex instrumentation limits field deployment. RPA-CRISPR-based isothermal assays offer a low-equipment alternative; however, existing approaches lack robust methodological strategies to achieve standard-curve-based quantitative reliability under field-deployable conditions. Here, we propose a methodological framework that enables in-run calibration and variance control for quantitative isothermal amplification by integrating reaction-volume locking and simultaneous standard-curve generation, implemented here within a closed centrifugal microfluidic system. A multi-unit microfluidic platform preloaded with gradient concentration standard plasmids allows concurrent construction of standard curves and sample analysis in a single run, reducing the impact of environmental and batch-to-batch variability. Signal generation was achieved using a one-pot RPA-CRISPR/Cas12a assay, in which balanced amplification and cleavage kinetics were obtained by screening crRNAs targeting suboptimal PAM sites and optimizing reaction conditions. The platform achieved a detection limit of 6.08 copies/μL and a linear quantitative range of 10[0]-10[4] copies/μL (R[2] > 0.96), with performance comparable to qPCR (AUC = 0.984), and acceptable intra- and inter-assay variability under the tested conditions, with relative standard deviations of 2.63-6.07 %, at a cost of approximately $3.30 per test. Validation using spiked and real seafood samples demonstrated reliable on-site quantification. This work establishes a transferable quantitative methodology for RPA-CRISPR-based isothermal assays, advancing field-deployable pathogen detection in aquaculture and food safety.},
}

*Vibrio parahaemolyticus/isolation & purification/genetics/pathogenicity
*Biosensing Techniques/instrumentation
*Nucleic Acid Amplification Techniques/instrumentation
Limit of Detection
*CRISPR-Cas Systems/genetics
Animals
Seafood/microbiology
Lab-On-A-Chip Devices
*Vibrio Infections/microbiology/diagnosis
Equipment Design

@article {pmid41414712,
year = {2026},
author = {Congdon, ST and Bennett, J and Opinya, R and Agosto, AR and Dossias, O and Kokko, C and Levesque, AA and Koob, AO and Silver, AC and Thomas-Charles, CA},
title = {Investigating and correcting a rare pathogenic mutation in GDF11.},
journal = {HGG advances},
volume = {7},
number = {1},
pages = {100559},
doi = {10.1016/j.xhgg.2025.100559},
pmid = {41414712},
issn = {2666-2477},
mesh = {Humans ; HEK293 Cells ; *Growth Differentiation Factors/genetics/metabolism ; *Gene Editing/methods ; *Bone Morphogenetic Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Mutation ; Golgi Apparatus/metabolism ; Codon, Nonsense ; },
abstract = {Single-nucleotide variants (SNVs) and small insertions or deletions (indels) underlie most rare monogenic disorders, yet therapeutic strategies to precisely correct these mutations remain limited. Prime editing enables the repair of such pathogenic variants without introducing double-stranded breaks. Here, we applied CRISPR prime editing to model and correct a de novo GDF11 nonsense mutation (Tyr336∗) identified in a participant from the Undiagnosed Diseases Network with growth delay and multisystem abnormalities. Using HEK293T cells, we generated heterozygous (HET) GDF11 Tyr336∗ clones, which exhibited reduced GDF11 protein levels due to post-translational degradation likely mediated by endoplasmic reticulum- and Golgi-associated quality control pathways. These cells displayed marked Golgi abnormalities, including an increased number of compact, irregularly shaped Golgi structures, findings consistent with Golgi fragmentation and stress. Transcriptomic profiling of HET cells revealed a broad dysregulation of gene networks, including downregulation of metabolic and Golgi-linked biosynthetic genes, and upregulation of cell-adhesion and extracellular matrix genes. These transcriptional shifts paralleled the participant's developmental, neural, and cardiovascular phenotypes. To correct the mutation, we tested multiple bespoke prime editing strategies and identified PE7, in combination with a prime editing guide RNA designed by Pridict, as the most effective ribonucleoprotein complex for rescue. Editing efficiency was further enhanced by introducing an additional silent protospacer-adjacent motif-disrupting mutation, likely preventing both Cas9 re-binding and mismatch repair. Together, these findings support a haploinsufficiency mechanism for the GDF11 Tyr336∗ allele and establish a generalizable framework for disease modeling and allele-specific correction of pathogenic variants in human cells.},
}

Humans
HEK293 Cells
*Growth Differentiation Factors/genetics/metabolism
*Gene Editing/methods
*Bone Morphogenetic Proteins/genetics/metabolism
CRISPR-Cas Systems
*Mutation
Golgi Apparatus/metabolism
Codon, Nonsense

@article {pmid41401738,
year = {2026},
author = {Feng, J and Lin, X and Kang, L and Duan, M and Duan, N and Wang, Z and Wu, S},
title = {Integrating a microfluidic chip@LFA biosensor enabled by Pt3Sn@MGO nanocomposites for RAA/CRISPR-Cas12b mediated food adulteration monitoring.},
journal = {Biosensors & bioelectronics},
volume = {296},
number = {},
pages = {118315},
doi = {10.1016/j.bios.2025.118315},
pmid = {41401738},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; Animals ; *Food Contamination/analysis ; *Nanocomposites/chemistry ; Salmon/genetics ; CRISPR-Cas Systems/genetics ; Limit of Detection ; Lab-On-A-Chip Devices ; Nucleic Acid Amplification Techniques ; Food Analysis ; Recombinases/chemistry ; Myoglobin/genetics ; },
abstract = {Traditional methods for food species genetic authentication typically involve time-consuming laboratory procedures and inconsistent operations, easily resulting in gene damage and inaccurate diagnostics. Here, by fully integrating reagent flow and reactions through micromachining technology, a standardized and lab-free operational PMMA-based microfluidic chip@ lateral flow assay (LFA) biosensor was developed for salmon adulteration detection. The primers recognizing the myoglobin nuclear gene of salmon were designed and optimized, enabling efficient recombinase-aided amplification (RAA) of target gene and subsequent activation of the CRISPR-Cas12b system. A multifunctional Pt3Sn@MGO nanocomposite was synthesized with enhanced FRET efficiency and photothermal properties, then employed as a signal probe in LFA test strip, achieving fluorescent, photothermal, and colorimetric quantitative detection of salmon contents in mixed samples, with detection limits of 0.007 %, 0.092 %, and 0.153 %, respectively, salmon contents in commercially products were evaluated to verify the practicality. This work presents an integrated, portable, and automation-enabled platform for standardized genetic authentication of food adulteration, which would be utilized as a universal lab-free method for on-site adulteration monitoring and species gene diagnostics by matching conserved genes and primer designs.},
}

*Biosensing Techniques/instrumentation
Animals
*Food Contamination/analysis
*Nanocomposites/chemistry
Salmon/genetics
CRISPR-Cas Systems/genetics
Limit of Detection
Lab-On-A-Chip Devices
Nucleic Acid Amplification Techniques
Food Analysis
Recombinases/chemistry
Myoglobin/genetics

@article {pmid41372199,
year = {2025},
author = {Nan, AX and Chickering, M and Bartolome, CL and Shadija, N and Li, D and Estes, BJG and Stetina, JV and Li, W and Andresen, J and Molugu, K and Amunugama, R and Fang, M and Bai, C and Wang, J and Norouzi, D and Cochrane, JC and Gatlin, JT and Dunyak, MT and Kumar, S and Chavez, L and Seth, A and Halperin, S and Finn, JD and Xie, J},
title = {Ligase-mediated programmable genomic integration (L-PGI).},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {563},
pmid = {41372199},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mice ; Humans ; Genomics/methods ; *Ligases/metabolism/genetics ; HEK293 Cells ; },
abstract = {Since their discovery, CRISPR systems have been repurposed for programmable targeted genomic editing, leading to applications for gene disruption, single base editing, insertion, deletion, and manipulation of short genomic sequences. Pairing Cas9 nickase with reverse transcriptase allows applications for insertion, substitution, and deletion of short genomic sequences from an RNA template without generating double stranded breaks however this technology typically shows reduced efficacy in post mitotic cells, limiting its translatability in vivo. Here we present a novel, ligase-based method that addresses these limiations. We introduce edits through delivery and ligation of a synthetic DNA donor to genomic nicks created with Cas9 nickase and report editing activity in cell lines, primary cell cultures, and adult mice via nonviral delivery. With favorable on target outcomes compared to transcription-based editing in key cell types, good tolerability, and deliverability, ligation-mediated gene editing has the potential to further advance genomic medicine.},
}

Animals
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Mice
Humans
Genomics/methods
*Ligases/metabolism/genetics
HEK293 Cells

@article {pmid41353404,
year = {2025},
author = {Burgold, T and Karakoc, E and Gonçalves, E and Barrio-Hernandez, I and Dwane, L and Silva, R and Souster, E and Sharma, M and Beck, A and Koh, GCC and Zalmas, LP and Garnett, MJ and Bassett, AR},
title = {A next-generation dual guide CRISPR system for genetic interaction library screening.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {561},
pmid = {41353404},
issn = {2041-1723},
support = {220540/Z/20/A//Wellcome Trust (Wellcome)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Library ; Cell Line, Tumor ; Streptococcus pyogenes/genetics ; Colorectal Neoplasms/genetics ; CRISPR-Associated Protein 9/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Transfer/genetics ; },
abstract = {Pairwise perturbation of gene function using the CRISPR/Cas9 system has potential in screening for genetic interactions and synthetic lethal gene pairs to identify combination therapies for cancer. However, existing dual guide expression systems are cumbersome to clone, often result in a large proportion of undesired guide pairs and have an imbalance of guide expression from the two positions. Here, we demonstrate a next-generation system for dual guide delivery based around a tRNA spacer that allows a single-step cloning strategy, as little as 2% of undesired guide pairs, and highly balanced expression of the two guides. This system allows efficient library-scale screening for hundreds of thousands of genetic interactions using the well-understood Streptococcus pyogenes Cas9 (SpCas9) system. We use this to screen a 100,136 guide pair library in colorectal cancer cells and successfully identify synthetic lethal genetic interactions between paralogs or other known interacting genes, establishing our method for performing efficient large-scale genetic interaction screens. This system is versatile and could be used with most guide RNA vector systems, and for other uses of paired guide delivery, such as improving single gene knockout efficiency or improving guide detection in single cell or optical CRISPR screens.},
}

*CRISPR-Cas Systems/genetics
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Library
Cell Line, Tumor
Streptococcus pyogenes/genetics
Colorectal Neoplasms/genetics
CRISPR-Associated Protein 9/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
RNA, Transfer/genetics

@article {pmid41338219,
year = {2026},
author = {Yang, Z and Zhang, L and Jiang, X and Yang, X and Ma, K and Yoo, D and Lu, Y and Zhang, S and Chen, J and Nie, Y and Bian, X and Han, J and Fu, L and Zhang, J and Ventura, M and Zhang, G and Sun, Q and Eichler, EE and Mao, Y},
title = {Incomplete lineage sorting of segmental duplications defines the human chromosome 2 fusion site early during African great ape speciation.},
journal = {Cell genomics},
volume = {6},
number = {1},
pages = {101079},
doi = {10.1016/j.xgen.2025.101079},
pmid = {41338219},
issn = {2666-979X},
mesh = {Animals ; Humans ; *Hominidae/genetics ; *Segmental Duplications, Genomic/genetics ; *Chromosomes, Human, Pair 2/genetics ; *Genetic Speciation ; Chromosome Inversion/genetics ; Evolution, Molecular ; CRISPR-Cas Systems ; },
abstract = {All great apes differ karyotypically from humans due to the fusion of chromosomes 2a and 2b, resulting in human chromosome 2. Here, we show that the fusion was associated with multiple pericentric inversions, segmental duplications (SDs), and the turnover of subterminal repetitive DNA. We characterized the fusion site at the single-base-pair resolution and identified three distinct SDs that originated more than 5 million years ago. These three distinct SDs were differentially distributed among African great apes as a result of incomplete lineage sorting (ILS) and lineage-specific duplication. One of these SDs shares homology to a hypomethylated SD spacer sequence present in the subterminal heterochromatin of Pan but is completely absent subtelomerically in both humans and orangutans. CRISPR-Cas9-mediated depletion of the fusion site in human neural progenitor cells alters the expression of genes, indicating a potential regulatory consequence to this human-specific karyotypic change. Overall, this study offers insights into how complex regions subject to ILS may contribute to speciation.},
}

Animals
Humans
*Hominidae/genetics
*Segmental Duplications, Genomic/genetics
*Chromosomes, Human, Pair 2/genetics
*Genetic Speciation
Chromosome Inversion/genetics
Evolution, Molecular
CRISPR-Cas Systems

@article {pmid40795361,
year = {2026},
author = {Prillo, S and Ravoor, A and Yosef, N and Song, YS},
title = {ConvexML: Fast and Accurate Branch Length Estimation under Irreversible Mutation Models, Illustrated through Applications to CRISPR/Cas9-Based Lineage Tracing.},
journal = {Systematic biology},
volume = {75},
number = {1},
pages = {115-134},
pmid = {40795361},
issn = {1076-836X},
support = {R56-HG013117/NH/NIH HHS/United States ; R01-HG013117/NH/NIH HHS/United States ; 101089213//ERC/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Mutation ; *Classification/methods ; *Phylogeny ; *Models, Genetic ; Software ; Cell Lineage/genetics ; },
abstract = {Branch length estimation is a fundamental problem in statistical phylogenetics and a core component of tree reconstruction algorithms. Traditionally, general time-reversible mutation models are employed, and many software tools exist for this scenario. With the advent of CRISPR/Cas9 lineage tracing technologies, there has been significant interest in the study of branch length estimation under irreversible mutation models. Under the CRISPR/Cas9 mutation model, irreversible mutations-in the form of DNA insertions or deletions-are accrued during the experiment, which are then read out at the single-cell level to reconstruct the cell lineage tree. However, most of the analyses of CRISPR/Cas9 lineage tracing data have so far been limited to the reconstruction of single-cell tree topologies, which depict lineage relationships between cells, but not the amount of time that has passed between ancestral cell states and the present. Time-resolved trees, known as chronograms, would allow one to study the evolutionary dynamics of cell populations at an unprecedented level of resolution. Indeed, time-resolved trees would reveal the timing of events on the tree, the relative fitness of subclones, and the dynamics underlying phenotypic changes in the cell population-among other important applications. In this work, we introduce the first scalable and accurate method to refine any given single-cell tree topology into a single-cell chronogram by estimating its branch lengths. To do this, we perform regularized maximum likelihood estimation (MLE) under a general irreversible mutation model, paired with a conservative version of maximum parsimony that reconstructs only the ancestral states that we are confident about. To deal with the particularities of CRISPR/Cas9 lineage tracing data-such as double-resection events, which affect runs of consecutive sites-we avoid making our model more complex and instead opt for using a simple but effective data encoding scheme. Similarly, we avoid explicitly modeling the missing data mechanisms-such as heritable missing data-by instead assuming that they are missing completely at random. We stabilize estimates in low-information regimes by using a simple penalized version of MLE using a minimum branch length constraint and pseudocounts. All this leads to a convex MLE problem that can be readily solved in seconds with off-the-shelf convex optimization solvers. We benchmark our method using both simulations and real lineage tracing data, and show that it performs well on several tasks, matching or outperforming competing methods such as TiDeTree and LAML (Lineage Analysis via Maximum Likelihood) in terms of accuracy, while being $10\sim 100\times$ faster. Notably, our statistical model is simpler and more general, as we do not explicitly model the intricacies of CRISPR/Cas9 lineage tracing data. In this sense, our contribution is 2-fold: (1) a fast and robust method for branch length estimation under a general irreversible mutation model and (2) a data encoding scheme specific to CRISPR/Cas9 lineage tracing data, which makes it amenable to the general model. Our branch length estimation method, which we call "ConvexML," should be broadly applicable to any evolutionary model with irreversible mutations (ideally, with high diversity) and an approximately ignorable missing data mechanism. "ConvexML" is available through the convexml open-source Python package.},
}

*CRISPR-Cas Systems/genetics
*Mutation
*Classification/methods
*Phylogeny
*Models, Genetic
Software
Cell Lineage/genetics

@article {pmid41528123,
year = {2025},
author = {Dong, Q and Luo, C},
title = {A recombinase polymerase amplification-coupled Cas12a for detection of Salmonella Typhi - a preliminary report.},
journal = {Folia histochemica et cytobiologica},
volume = {63},
number = {4},
pages = {185-192},
doi = {10.5603/fhc.108406},
pmid = {41528123},
issn = {1897-5631},
mesh = {*Salmonella typhi/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems/genetics ; *Typhoid Fever/diagnosis/microbiology ; *Recombinases/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *Bacterial Proteins/genetics/metabolism ; DNA, Bacterial/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; CRISPR-Associated Proteins ; },
abstract = {INTRODUCTION: . Typhoid fever, a disease resulting from an infection with Salmonella Typhi (S. Typhi) remains widespread in economically disadvantaged regions, where it continues to be a critical public health concern. As the symptoms and signs are non-specific, they are difficult to diagnose directly based on the clinical picture. Therefore, laboratory examinations are essential for diagnosis.

MATERIAL AND METHODS: . This research introduces a fast and equipment-independent approach for detecting S. Typhi by employing CRISPR/Cas12a-based technology. The optimized CRISPR/Cas12a system achieved a detection limit of 103 copies/μL of S. Typhi DNA per reaction, with the entire assay completed within 60 min.

RESULTS: . Four clinical isolates cultured from patients with typhoid fever were collected and evaluated using our CRISPR/Cas12a-based detection system. The assay results demonstrated that all four samples were accurately identified as positive.

CONCLUSIONS: . We showed that the developed CRISPR/Cas12a-based detection method provides a promising alternative for the on-site and simple detection of S. Typhi.},
}

*Salmonella typhi/genetics/isolation & purification
Humans
*CRISPR-Cas Systems/genetics
*Typhoid Fever/diagnosis/microbiology
*Recombinases/metabolism
*Nucleic Acid Amplification Techniques/methods
*Bacterial Proteins/genetics/metabolism
DNA, Bacterial/genetics
*Endodeoxyribonucleases/metabolism/genetics
CRISPR-Associated Proteins

@article {pmid41527500,
year = {2026},
author = {Atceken, N and Kahya, A and Yigci, D and Tasoglu, S},
title = {CRISPR-on-Chip for Point-of-Care Diagnostics.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.5c19771},
pmid = {41527500},
issn = {1936-086X},
abstract = {CRISPR-based diagnostic platforms have gained significant momentum in recent years, enabling highly sensitive and specific detection of pathogens and diseases. Due to their practical benefits, these platforms have become widely adopted in point-of-care (PoC) applications. CRISPR-on-chip technology integrates CRISPR-Cas platforms with diverse microfluidic systems, allowing scalability and portable, real-time, and precise biomolecule detection. This approach enhances diagnostic accuracy, reduces processing times, and minimizes the need for complex laboratory infrastructures, unlike in conventional diagnostics. Using CRISPR-Cas enzymes in microfluidic systems, CRISPR-on-chip platforms offer key advantages such as single-molecule sensitivity, multiplex detection, and applicability. However, integration with microfluidics for PoC applications is still poorly understood, despite CRISPR-Cas being widely used. This study reviews recent developments in CRISPR-on-chip-based diagnostics and highlights its potential applications in infectious diseases, biosensors, and personalized medicine. Furthermore, challenges and future perspectives in achieving an ideal diagnostic solution are discussed.},
}

@article {pmid41527434,
year = {2026},
author = {Stewart, C and Liddle, TA and Tolla, E and Lewis, JE and Marshall, C and Evans, NP and Morgan, PJ and Ebling, FJP and Stevenson, TJ},
title = {Hypothalamic deiodinase type-3 establishes the period of circannual interval timing in mammals.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {41527434},
issn = {2050-084X},
support = {LT-RL-2019-06//Leverhulme Trust/ ; Institutional Strategic Support Fund/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; *Iodide Peroxidase/metabolism/genetics ; *Hypothalamus/enzymology/physiology ; Photoperiod ; Seasons ; Phodopus/physiology ; Male ; Cricetinae ; },
abstract = {Animals respond to environmental cues to time phenological events, but the intrinsic mechanism of circannual timing remains elusive. We used transcriptomic sequencing and frequent sampling of multiple hypothalamic nuclei in Djungarian hamsters to examine the neural and molecular architecture of circannual interval timing. Our study identified three distinct phases of transcript changes, with deiodinase type-3 (Dio3) expression activated during the early induction phase. Subsequent work demonstrated that targeted mutation of Dio3 using CRISPR-Cas resulted in a shorter period for circannual interval timing. Hamsters that are non-responsive to short photoperiods and fail to show any winter adaptations do not display changes in Dio3 expression and do not show any change in body mass or pelage. Our work demonstrates that changes in Dio3 induction are essential for setting the period of circannual interval timing.},
}

Animals
*Iodide Peroxidase/metabolism/genetics
*Hypothalamus/enzymology/physiology
Photoperiod
Seasons
Phodopus/physiology
Male
Cricetinae

@article {pmid41526382,
year = {2026},
author = {Van Vu, T and Thi Nguyen, N and Kim, J and Hoai Nguyen, T and Kim, JY},
title = {Development of an ultra-efficient prime editing system in tomato.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {95},
pmid = {41526382},
issn = {2041-1723},
support = {RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; },
mesh = {*Solanum lycopersicum/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Genome, Plant ; },
abstract = {Prime editing (PE) enables precise genome modifications without donor DNA or double-strand breaks, but its application in dicot plants has faced challenges due to low efficiency, locus dependence, and poor heritability. Here, we develop an ultra-efficient prime editing (UtPE) system for dicots by integrating evolved PE6 variants (PE6c and PE6ec), an altered pegRNA (aepegRNA), an RNA chaperone, and a geminiviral replicon. UtPE significantly improves editing performance in tomatoes, with UtPEv1 excelling in simple edits (unstructured RTTs) and UtPEv3 effective for complex targets (structured RTTs or multiple nucleotide changes). Compared to a PE2max-based tool, UtPE increases desired editing efficiency by 3.39 to 8.89-fold, enables editing at previous inaccessible sites, achieves an average of 16.0% desired editing efficiency in calli, and produces high-frequency desired edits in up to 87.5% of T0 plants. Multiplexed editing at up to three loci and stable T1 inheritance are also achieved, resulting in traits such as jointless pedicels and glyphosate resistance, while minimizing off-target effects.},
}

*Solanum lycopersicum/genetics
*Gene Editing/methods
CRISPR-Cas Systems/genetics
Plants, Genetically Modified/genetics
Genome, Plant

@article {pmid41525963,
year = {2026},
author = {Habib, AH and Sain, ZM and Rafeeq, M and Karami, MM and Alsufyani, HA and Iqbal, J and Chaieb, K and Altayb, HN and Nadeem, MS and Al-Abbasi, FA and Kazmi, I},
title = {MicroRNA-CRISPR biosensors for cancer diagnostics.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {583},
number = {},
pages = {120837},
doi = {10.1016/j.cca.2026.120837},
pmid = {41525963},
issn = {1873-3492},
abstract = {Circulating microRNAs (miRNAs) are promising minimally invasive biomarkers for cancer and cardiovascular disorders. However, their low sequence length, low abundance, high sequence homology (including iso-miRs), and strong matrix and preanalytical effects in biofluids require highly sensitive and robust analytical technologies. CRISPR-Cas systems, particularly Cas12a, Cas12b, Cas13a, and Cas9, offer programmable nucleic acid recognition with high mismatch discrimination combined with collateral nuclease activity, enabling versatile signal amplification through fluorescence, electrochemical, electrochemiluminescent (ECL), photoelectrochemical (PEC), colorimetric, and lateral-flow readouts. This review critically evaluates the latest advances in CRISPR-based miRNA biosensors, emphasizing their analytical performance and translational potential in clinical diagnostics across plasma/serum, saliva, whole blood, and extracellular vesicle samples. The detection limits are typically within the femtomolar to attomolar range. The requirements for clinical translation are equally influenced by factors such as sample preparation, inhibitor tolerance, miRNA panel multiplexing, quantitative readout, and reagent stability. We compared CRISPR-based workflows with RT-qPCR and digital PCR and provided a roadmap for standardization and quality control, as well as the minimal analytical and clinical validation standards required for adopting CRISPR technology in clinical chemistry laboratories.},
}

@article {pmid41524770,
year = {2026},
author = {Basit, A and Zhu, J and Zheng, W},
title = {Assessing off-target effects in CRISPR/Cas9: challenges and strategies for precision DNA editing.},
journal = {Archives of microbiology},
volume = {208},
number = {2},
pages = {114},
pmid = {41524770},
issn = {1432-072X},
support = {YZ2024220//Work was partly supported by the 2024 Annual Special Funds for Municipal-School Cooperation Projects of Yangzhou City (YZ2024220) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)./ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The emergence of CRISPR/Cas9 technology has transformed the landscape of gene editing, allowing for precise alterations in DNA that hold great promise for research and potential therapies. However, a significant concern is the occurrence of off-target effects, which can lead to unintended genetic modifications with potentially harmful consequences. This paper explores the nature of off-target effects in CRISPR/Cas9, discussing how they arise and their implications for the reliability of gene editing. We identify the challenges faced in detecting and predicting these off-target interactions, including limitations in current detection techniques and the complexities of cellular biology. We present strategies aimed at minimizing off-target effects, such as careful design of guide RNAs, the use of computational tools for prediction, and improved delivery methods. Through a review of case studies, we highlight successful cases where off-target activity has been significantly reduced, offering insights into best practices for enhancing the accuracy of CRISPR/Cas9 applications. Moreover, we provide a comparative overview of Cas9, Cas12, and Cas13 systems, emphasizing their distinct target specificities, mechanisms of action, and off-target profiles. This comparison offers a broader understanding of how alternative CRISPR effectors may be leveraged to improve genome and transcriptome editing precision. This study underscores the importance of continued research to address the challenges of off-target effects, ultimately supporting the development of safer and more effective gene editing methods for clinical use.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
Humans
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41524535,
year = {2026},
author = {Batra, SS and Cabrera, A and Spence, JP and Goell, J and Anand, SS and Hilton, IB and Song, YS},
title = {Predicting the effect of CRISPR-Cas9-based epigenome editing.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {41524535},
issn = {2050-084X},
support = {R35 GM134922/GM/NIGMS NIH HHS/United States ; R35 GM143532/GM/NIGMS NIH HHS/United States ; R35-GM143532/GM/NIGMS NIH HHS/United States ; R35-GM134922/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; HEK293 Cells ; *Epigenome ; Protein Processing, Post-Translational ; Machine Learning ; Histones/metabolism ; K562 Cells ; *Epigenesis, Genetic ; *Epigenomics/methods ; Epigenome Editing ; },
abstract = {Epigenetic regulation orchestrates mammalian transcription, but functional links between them remain elusive. To tackle this problem, we use epigenomic and transcriptomic data from 13 ENCODE cell types to train machine learning models to predict gene expression from histone post-translational modifications (PTMs), achieving transcriptome-wide correlations of ∼0.70-0.79 for most cell types. Our models recapitulate known associations between histone PTMs and expression patterns, including predicting that acetylation of histone subunit H3 lysine residue 27 (H3K27ac) near the transcription start site (TSS) significantly increases expression levels. To validate this prediction experimentally and investigate how natural vs. engineered deposition of H3K27ac might differentially affect expression, we apply the synthetic dCas9-p300 histone acetyltransferase system to 8 genes in the HEK293T cell line and to 5 genes in the K562 cell line. Further, to facilitate model building, we perform MNase-seq to map genome-wide nucleosome occupancy levels in HEK293T. We observe that our models perform well in accurately ranking relative fold-changes among genes in response to the dCas9-p300 system; however, their ability to rank fold-changes within individual genes is noticeably diminished compared to predicting expression across cell types from their native epigenetic signatures. Our findings highlight the need for more comprehensive genome-scale epigenome editing datasets, better understanding of the actual modifications made by epigenome editing tools, and improved causal models that transfer better from endogenous cellular measurements to perturbation experiments. Together, these improvements would facilitate the ability to understand and predictably control the dynamic human epigenome with consequences for human health.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
HEK293 Cells
*Epigenome
Protein Processing, Post-Translational
Machine Learning
Histones/metabolism
K562 Cells
*Epigenesis, Genetic
*Epigenomics/methods
Epigenome Editing

@article {pmid41524418,
year = {2026},
author = {Raftopoulou, O and Malmstrom, K and Pan, M and Barrangou, R},
title = {Enhanced editing of Bifidobacterium lactis using the endogenous Type I-G CRISPR-Cas system.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0183925},
doi = {10.1128/aem.01839-25},
pmid = {41524418},
issn = {1098-5336},
abstract = {Diverse Bifidobacterium animalis subsp. lactis strains are widely used as commercial probiotics. While proof-of-concept studies have shown that some strains can be edited using several CRISPR-Cas approaches, this species remains difficult to engineer, hindering functional genomic studies to establish their molecular mode of action and enhance their probiotic functionalities. Here, we show that >95% of available B. lactis genomes harbor a conserved Type I-G CRISPR-Cas system, which we leverage to develop and validate a broadly applicable genome editing framework. We redesigned backbone plasmids with different replicons and antibiotic resistance markers and evaluated performance across six commercial strains for transformation efficiency. A vector carrying the pBC1 origin coupled with a chloramphenicol resistance marker improved transformation in most strains. Using synthetic CRISPR arrays with self-targeting spacers in combination with homologous editing templates, we tested multiple spacers and evaluated short (600 bp) versus long (1,000 bp) homology arms. To demonstrate applicability, we generated knockouts in three glycoside hydrolases within the Balac 1593-1601 cluster, readily cured editing plasmids in non-selective medium, and performed iterative genome editing. Growth phenotyping across carbohydrates confirmed that the GH36 α-galactosidase Balac 1601 knockout abolished melibiose and raffinose utilization, and that deletions within Balac 1596 and Balac 1593 carbohydrate hydrolases produced non-canonical phenotypes, suggestive of a modulatory role associated with shift in carbon use and compensation by other pathways. These results establish a practical toolkit for editing diverse B. lactis strains, unravel the genomics underlying probiotic attributes, and provide a blueprint for genome engineering in other non-model probiotic bacteria.IMPORTANCEBifidobacterium animalis subsp. lactis strains are prominent probiotics widely formulated in foods and dietary supplements, yet remain difficult to engineer, limiting efforts to connect genes to probiotic traits and to build strains with enhanced functions. Here, we harness the native Type I-G CRISPR Cas system to enable genome editing across commercial B. lactis strains by optimizing a compact plasmid backbone, testing multiple spacers to achieve efficient editing, and selecting homology arms of the appropriate length for recombination. With this framework, we generate knockouts at multiple, functionally distinct loci, demonstrating target-agnostic applicability, and we cure the CRISPR-editing vectors efficiently, enabling sequential edits. This toolkit enables systematic genotype-to-phenotype mapping in B. lactis and provides a practical framework for strain improvement in organisms of industrial relevance.},
}

@article {pmid41523156,
year = {2025},
author = {Altpeter, F},
title = {Gene editing to enhance biotic stress tolerance in sugarcane.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1750169},
pmid = {41523156},
issn = {1664-462X},
}

@article {pmid41522810,
year = {2026},
author = {Heer, CD and Elia, JL and Menon, V and Johnson, SS and Arbelaez, SR and Friedman, S and Lopez-Giraldez, F and Sundaram, RK and Herzon, SB and Bindra, RS and Gueble, SE},
title = {Targeted CRISPR knockout screening identifies known and novel chemogenomic interactions between DNA damaging agents and DNA repair genes.},
journal = {NAR cancer},
volume = {8},
number = {1},
pages = {zcaf052},
pmid = {41522810},
issn = {2632-8674},
mesh = {Humans ; *DNA Repair/genetics/drug effects ; *DNA Damage/drug effects/genetics ; Cell Line, Tumor ; Gene Knockout Techniques ; CRISPR-Cas Systems ; *Glioma/genetics/drug therapy ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Genetic instability is a hallmark of cancer, often arising from mutations to DNA damage repair and response (DDR) genes. Classical genetic, biochemical, and structural approaches elucidated the foundational mechanisms of DDR pathways and provided a scientific understanding of their involvement in repair of lesions induced by broad classes of DNA-damaging agents (DDAs). However, given the chemical diversity of DDAs and resultant DNA lesions, along with the multitude of interconnected DDR factors, the chemogenomic landscape of DDA-DDR interactions remains incompletely mapped. To this end, we developed a DDR-targeted, CRISPR knockout screening approach and assessed relationships amongst 353 DNA repair genes and 15 DDAs in LN229 glioma cells. Within this dataset of 5295 DDR-related chemogenomic interactions, we identified many established interactions and discovered novel ones. For example, we observed a specific role of transcription-coupled nucleotide excision repair in the repair of adducts generated by monofunctional alkylating agents, a role for the Fanconi anemia pathway in addressing methyl lesions, overt differences in DSB repair following treatment with topoisomerase I versus II poisons, and repair dependencies associated with the imidazotetrazines temozolomide, mitozolomide, and KL-50. Future directions will continue to investigate the mechanisms of novel chemogenomic interactions that we have uncovered as well as work to identify chemogenomic interactions amenable to clinical translation.},
}

Humans
*DNA Repair/genetics/drug effects
*DNA Damage/drug effects/genetics
Cell Line, Tumor
Gene Knockout Techniques
CRISPR-Cas Systems
*Glioma/genetics/drug therapy
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41522435,
year = {2025},
author = {Zalila-Kolsi, I},
title = {Engineered bacteria as living therapeutics: Next-generation precision tools for health, industry, environment, and agriculture.},
journal = {AIMS microbiology},
volume = {11},
number = {4},
pages = {946-962},
pmid = {41522435},
issn = {2471-1888},
abstract = {Synthetic biology has revolutionized precision medicine by enabling the development of engineered bacteria as living therapeutics, dynamic biological systems capable of sensing, responding to, and functioning within complex physiological environments. These microbial platforms offer unprecedented adaptability, allowing for real-time detection of disease signals and targeted therapeutic delivery. This review explores recent innovations in microbial engineering across medical, industrial, environmental, and agricultural domains. Key advances include CRISPR-Cas systems, synthetic gene circuits, and modular plasmid architectures that provide fine-tuned control over microbial behavior and therapeutic output. The integration of computational modeling and machine learning has further accelerated design, optimization, and scalability. Despite these breakthroughs, challenges persist in maintaining genetic stability, ensuring biosafety, and achieving reproducibility in clinical and industrial settings. Ethical and regulatory frameworks are evolving to address dual-use concerns, public perception, and global policy disparities. Looking forward, the convergence of synthetic biology with nanotechnology, materials science, and personalized medicine is paving the way for intelligent, responsive, and sustainable solutions to global health and environmental challenges. Engineered bacteria are poised to become transformative tools not only in disease treatment but also in diagnostics, biomanufacturing, pollution mitigation, and sustainable agriculture.},
}

@article {pmid41479230,
year = {2026},
author = {Zhang, J and Liu, Y and Cao, W and Ruan, R and Wang, M},
title = {Establishment of a Novel CRISPR/Cas9-Based Multiplex Editing System in the Citrus Postharvest Pathogen Penicillium digitatum.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {1},
pages = {1167-1174},
doi = {10.1021/acs.jafc.5c11144},
pmid = {41479230},
issn = {1520-5118},
mesh = {*Penicillium/genetics/pathogenicity/metabolism ; *Citrus/microbiology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Plant Diseases/microbiology ; Fungal Proteins/genetics/metabolism ; },
abstract = {Penicillium digitatum, the causal fungus of the citrus green mold, leads to substantial postharvest losses in the citrus industry. In this study, we engineered a versatile CRISPR/Cas9-mediated gene editing platform capable of generating multiple sgRNAs from a single polycistronic transcript. By incorporating endogenous tRNAs and the strong promoter PdMLE1 into the CRISPR/Cas9 system, the efficiency of single gene editing can reach up to 94.2%. By taking advantage of the efficient shearing and processing capabilities of tRNA, the platform enabled multiplex editing with efficiencies of 44.4% for two-gene and 33.3% for three-gene modifications, respectively. After two rounds of three-gene editing, we were able to successfully obtain hextuple-gene mutants. Finally, functional characterization revealed that the target polysaccharide-lyase-encoding genes play limited roles in pathogenicity in P. digitatum. Taken together, our results represent a powerful tool for genome engineering in P. digitatum, facilitating research into its pathogenesis.},
}

*Penicillium/genetics/pathogenicity/metabolism
*Citrus/microbiology
*CRISPR-Cas Systems
*Gene Editing/methods
*Plant Diseases/microbiology
Fungal Proteins/genetics/metabolism

@article {pmid41447470,
year = {2026},
author = {Liu, T and Ye, B and Zhang, Y and Yan, X},
title = {Finely Tuned CRISPRi Module for Upgrading the Performance of Constitutive Promoters in the Bacillus subtilis Protein Expression System.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {1},
pages = {1046-1052},
doi = {10.1021/acs.jafc.5c03424},
pmid = {41447470},
issn = {1520-5118},
mesh = {*Bacillus subtilis/genetics/metabolism/growth & development ; *Promoter Regions, Genetic ; *Bacterial Proteins/genetics/metabolism ; Xylose/metabolism ; Gene Expression Regulation, Bacterial ; CRISPR-Cas Systems ; Fermentation ; },
abstract = {Bacillus subtilis is a critical host for protein production, with many industrial strains relying on strong constitutive promoters. However, this kind of promoter typically imposes a heavy burden on the host from the early stage of fermentation, leading to reduced growth rate and biomass. To overcome the drawbacks of these promoters, we developed a xylose-inducible CRISPRi module to dynamically control the activity of these promoters. The strength of this module was finely tuned via promoter engineering and the xylose concentration. The addition of xylose inhibited the target promoter and favored cell growth at an early stage, while the consumption of xylose recovered the strength of the promoter and facilitated protein expression, resulting in better balance between cell growth and protein production. The yield of a target protein was increased by 38% using this module. Our work provides a simple and effective method to upgrade industrial strains driven by strong constitutive promoters.},
}

*Bacillus subtilis/genetics/metabolism/growth & development
*Promoter Regions, Genetic
*Bacterial Proteins/genetics/metabolism
Xylose/metabolism
Gene Expression Regulation, Bacterial
CRISPR-Cas Systems
Fermentation

@article {pmid41397982,
year = {2025},
author = {Okuwa, T and Himeda, T and Kobayashi, K and Nomura, N and Utani, K and Koike, S and Nakamura, A and Higuchi, M},
title = {Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {534},
pmid = {41397982},
issn = {2041-1723},
support = {25K10386//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21K07045//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 24K10234//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP25fk0108716//Japan Agency for Medical Research and Development (AMED)/ ; S2023-4//Kanazawa Medical University/ ; K2024-3//Kanazawa Medical University/ ; },
mesh = {Humans ; *Integrins/metabolism/genetics ; *Glycosaminoglycans/metabolism ; HeLa Cells ; Virus Internalization ; CRISPR-Cas Systems ; *Receptors, Virus/metabolism ; Virus Attachment ; Host-Pathogen Interactions ; Gene Knockout Techniques ; },
abstract = {Saffold virus (SAFV), a member of the species Cardiovirus saffoldi within the Picornaviridae family, causes acute respiratory and gastrointestinal illnesses as well as hand, foot, and mouth disease. It is also suspected to be associated with neuronal disorders, such as encephalitis and meningitis, in severe cases. Despite its clinical significance, the virus-host interactions underlying SAFV pathogenicity remain largely unknown. Using a genome-wide CRISPR-Cas9 knockout screen, we identify the following receptors for SAFV infection: sulfated glycosaminoglycans (GAGs) and integrin αVβ8. Single knockouts of SLC35B2, an essential gene for sulfated GAG synthesis, or the integrin genes ITGAV or ITGB8 partially reduce SAFV-3 and SAFV-2 susceptibility in HeLa cells, and a double knockout confers complete resistance. Furthermore, we demonstrate that SAFV-3 virions bind directly to sulfated GAGs and integrin αVβ8. Based on these findings, we propose a model of SAFV infection in which sulfated GAGs and integrin αVβ8 act through dual and cooperative pathways to facilitate viral entry.},
}

Humans
*Integrins/metabolism/genetics
*Glycosaminoglycans/metabolism
HeLa Cells
Virus Internalization
CRISPR-Cas Systems
*Receptors, Virus/metabolism
Virus Attachment
Host-Pathogen Interactions
Gene Knockout Techniques

@article {pmid41390353,
year = {2025},
author = {Howe, LJ and Aulchenko, YS and Davey Smith, G and Davies, NM and Esparza-Gordillo, J and Johnson, T and Liu, JZ and Richardson, TG and Sanseau, P and Scott, RA and Seaton, DD and Sharma, A and Cortes, A},
title = {Evaluating transportability of in vitro cellular models to in vivo human phenotypes using gene perturbation data.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {513},
pmid = {41390353},
issn = {2041-1723},
mesh = {Humans ; Phenotype ; Lysosomes/metabolism ; *Models, Biological ; Cholesterol, LDL/blood/metabolism ; CRISPR-Cas Systems ; Cholesterol/metabolism ; },
abstract = {Gene perturbation screens (e.g. CRISPR-Cas9) assess the impact of gene disruption on in-vitro cellular phenotypes (e.g., proliferation, anti-viral response). In-vitro experiments can be useful models for in-vivo (organismal) phenotypes (e.g., immune cell anti-viral response and infectious diseases). However, assessing whether an in-vitro cellular model effectively captures in-vivo biology is challenging. An in-vitro model is 'transportable' to an in-vivo phenotype if perturbations impacting the in-vitro phenotype also impact the in-vivo phenotype with mechanism-consistent directionality and effect sizes. We propose a framework; Gene Perturbation Analysis for Transportability (GPAT), to assess model transportability using gene perturbation effect estimates from perturbation screens (in-vitro) and loss-of-function burden tests (in-vivo). In hypothesis-driven analyses, GPAT provides evidence for model transportability of higher lysosomal cholesterol accumulation in-vitro to lower human plasma LDL-cholesterol (P = 0.0006), consistent with the known role of lysosomes in lipid biosynthesis. In contrast, there was limited evidence for other putative in-vitro models. In hypothesis-free analyses, we find evidence for transportability of cancer cell line proliferation to in-vivo human plasma cellular phenotypes (e.g. erythroleukemia proliferation and plasma lymphocyte percentage). Here we show that perturbation data can be used to evaluate transportability of in-vitro cellular models, informing assay prioritisation and supporting novel hypothesis generation.},
}

Humans
Phenotype
Lysosomes/metabolism
*Models, Biological
Cholesterol, LDL/blood/metabolism
CRISPR-Cas Systems
Cholesterol/metabolism

@article {pmid41387726,
year = {2025},
author = {Srinivasan, R and Sun, T and Sandles, A and Wu, D and Wang, L and Patel, H and Pabalate, R and Bader, M and Heidersbach, A and Ho, C and Xie, S and Ng, A and Haley, B},
title = {Chemically-inducible CRISPR/Cas9 circuits for ultra-high dynamic range gene perturbation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {504},
pmid = {41387726},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {CRISPR/Cas9 technologies provide unique capabilities for modeling disease and understanding gene-to-phenotype connections. In cultured cells, chemical-mediated control of Cas9 activity can limit off-target effects and enable mechanistic study of essential genes. However, widely-used Tet-On systems often show leaky Cas9 expression, leading to unintended edits, as well as weak activity upon induction. Leakiness can be problematic in the context of Cas9 nuclease activity, which may result in cumulative DNA damage and degradation of the target cell genome over time. To overcome these deficiencies, we have established transgenic platforms that minimize Cas9 functionality in the OFF-state along with maximized and uncompromised ON-state gene editing efficiency. By combining conditional destabilization and inhibition of Cas9, we have developed an all-in-one (one or multiple guide RNAs and Cas9) ultra-tight, Tet-inducible system with exceptional dynamic range (ON vs. OFF-state) across various cell lines and targets. As an alternative to Tet-mediated induction, we have created a Branaplam-regulated splice switch module for low-baseline and robust Cas9 activity control. Lastly, for circumstances where DNA damage needs to be avoided, we have constructed a dual-control, Tet-inducible CRISPRi module for tight and potent transcriptional silencing. This upgraded suite of inducible CRISPR systems has broad applications for numerous cell types and experimental conditions.},
}

*CRISPR-Cas Systems/genetics
Humans
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
HEK293 Cells
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid41372159,
year = {2025},
author = {Pan, R and Ren, J and Chen, X and Flores, LF and Gonzalez, RVL and Adonnino, AA and Lofts, B and Waldo, J and Halmai, J and Devinsky, O and Fink, K and Liu, XS},
title = {Editing DNA methylation in vivo.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {527},
pmid = {41372159},
issn = {2041-1723},
support = {R01MH134519//U.S. Department of Health & Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R01NS126185//U.S. Department of Health & Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; },
mesh = {Animals ; *DNA Methylation/genetics ; *Gene Editing/methods ; Mice ; DNA Methyltransferase 3A ; Mice, Transgenic ; Promoter Regions, Genetic ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Proprotein Convertase 9/genetics/metabolism ; Liver/metabolism ; DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism ; CRISPR-Cas Systems ; Proto-Oncogene Proteins/genetics/metabolism ; Male ; DNA-Binding Proteins/genetics/metabolism ; Epigenesis, Genetic ; Neurons/metabolism ; },
abstract = {DNA methylation is a crucial epigenetic mechanism that regulates gene expression. Precise editing of DNA methylation has emerged as a promising tool for dissecting its biological function. However, challenges in delivery have limited most applications of DNA methylation editing to in vitro systems. Here, we develop two transgenic mouse lines harboring an inducible dCas9-DNMT3A or dCas9-TET1 editor to enable tissue-specific DNA methylation editing in vivo. We demonstrate that targeted methylation of the Psck9 promoter in the liver of dCas9-DNMT3A mice results in decreased Pcsk9 expression and a subsequent reduction in serum low-density lipoprotein cholesterol level. Targeted demethylation of the Mecp2 promoter in dCas9-TET1 mice reactivates Mecp2 expression from the inactive X chromosome and rescues neuronal nuclear size in Mecp2[+/-] mice. Genome-wide sequencing analyses reveal minimal transcriptional off-targets, demonstrating the specificity of the system. These results demonstrate the feasibility and versatility of methylation editing, to functionally interrogate DNA methylation in vivo.},
}

Animals
*DNA Methylation/genetics
*Gene Editing/methods
Mice
DNA Methyltransferase 3A
Mice, Transgenic
Promoter Regions, Genetic
Methyl-CpG-Binding Protein 2/genetics/metabolism
Proprotein Convertase 9/genetics/metabolism
Liver/metabolism
DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism
CRISPR-Cas Systems
Proto-Oncogene Proteins/genetics/metabolism
Male
DNA-Binding Proteins/genetics/metabolism
Epigenesis, Genetic
Neurons/metabolism

@article {pmid41365890,
year = {2025},
author = {Li, W and Liu, S and Fang, X and Zou, J and Jiang, Q and Min, X and Zhu, X and Cao, Y and Gao, X and Han, W and Azhar, M and Xing, X and Li, F and Zhang, Y and Liu, H and Cheng, L and Wang, C and Bao, J},
title = {Efficient high-precision transgene knock-in by Recombinases (Redα/β)-enhanced DNA integration-CRISPR-Cas9 (RED-CRISPR).},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {538},
pmid = {41365890},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; *Gene Knock-In Techniques/methods ; *Transgenes/genetics ; Mice ; Humans ; Recombinational DNA Repair/genetics ; *Gene Editing/methods ; *Recombinases/metabolism/genetics ; DNA/genetics/metabolism ; HEK293 Cells ; },
abstract = {CRISPR-Cas9 tools have revolutionized genetic engineering, yet the efficient precise integration of DNA cargos, particularly for large DNA payloads (>1 kilobase, kb), remains a technical bottleneck. Herein, we develop a Recombinases (Redα/β)-enhanced DNA integration-CRISPR-Cas9 approach, referred to as RED-CRISPR, which offers a versatile yet robust homology-directed repair (HDR) strategy enabling efficient and precise kb-scale DNA insertion across various cell types, including immortalized and primary cells of variable origins. RED-CRISPR significantly enhances HDR efficiencies by 2- to 5-fold change across diverse loci and further elevates HDR rates by 1.5- to 2.5-fold when synergizing with other HDR-enhancing strategies. We achieved up to 45% knock-in efficiency for CAR-T cell manufacturing, and attained 43% knock-in rate for generation of genetically modified mice using an 8-kb DNA cargo. Through a head-to-head comparison, RED-CRISPR profoundly mitigates off-target mutational burden and chromosomal translocations. We envision RED-CRISPR as a powerful genome-editing tool with broad biomedical and therapeutic applications.},
}

*CRISPR-Cas Systems/genetics
Animals
*Gene Knock-In Techniques/methods
*Transgenes/genetics
Mice
Humans
Recombinational DNA Repair/genetics
*Gene Editing/methods
*Recombinases/metabolism/genetics
DNA/genetics/metabolism
HEK293 Cells

@article {pmid41521661,
year = {2026},
author = {Newman, A and Starrs, L and Burgio, G},
title = {Nuc domain electrostatics drive the trans cleavage activity of CRISPR-Cas12a.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41521661},
issn = {1362-4962},
support = {2018833//National Health and Medical Research Council/ ; 2027987//National Health and Medical Research Council/ ; //The Gordon and Gretel Bootes foundation/ ; //National Computing Infrastructure/ ; //Australian Government Research Training Program/ ; },
mesh = {Static Electricity ; *Endodeoxyribonucleases/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *Bacterial Proteins/chemistry/genetics/metabolism ; Catalytic Domain ; Gene Editing ; DNA Cleavage ; DNA/metabolism ; Protein Domains ; Kinetics ; Amino Acid Substitution ; Models, Molecular ; Mutation ; },
abstract = {The trans cleavage activity of Cas12a has been extensively used for the detection of biomolecules. Different Cas12a orthologues exhibit faster or slower trans cleavage kinetics, making some orthologues more suited for sensitive molecular detection. Ionic strength of reaction buffers and mutations that change the electrostatic environment near the RuvC active site have also been reported to strongly influence trans cleavage kinetics. Studying three commonly used Cas12a orthologues (FnCas12a, AsCas12a, and LbCas12a), we report that electrostatic interactions near the RuvC active site are critical for their trans cleavage activity. Alanine substitution of arginine and lysine residues in the Nuc domain can abolish trans cleavage while modestly reducing cis cleavage. Substitutions in the RuvC lid and substitutions to introduce positively charged residues in the Nuc could enhance both cis and trans cleavage. These Cas12a variants improved DNA detection and genome editing efficacy. Overall, this study provides a blueprint for rationally engineering the DNase activities of Cas12a.},
}

Static Electricity
*Endodeoxyribonucleases/chemistry/genetics/metabolism
*CRISPR-Cas Systems
*CRISPR-Associated Proteins/chemistry/genetics/metabolism
*Bacterial Proteins/chemistry/genetics/metabolism
Catalytic Domain
Gene Editing
DNA Cleavage
DNA/metabolism
Protein Domains
Kinetics
Amino Acid Substitution
Models, Molecular
Mutation

@article {pmid41521468,
year = {2026},
author = {Hsieh, JA and Wu, FH and Yang, DX and Wu, AE and Liu, CA and Chen, CH and Lin, SZ and Lin, YJ and Lin, CS},
title = {Protoplast-Based Functional Genomics and Genome Editing: Progress, Challenges and Applications.},
journal = {Plant, cell & environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.70375},
pmid = {41521468},
issn = {1365-3040},
support = {//Academia Sinica/ ; //Buddhist Tzu Chi Medical Foundation/ ; NSTC 113-2313-B-001-006-//National Science and Technology Council, Taiwan/ ; NSTC 114-2628-B-002-008-//National Science and Technology Council, Taiwan/ ; NSTC 114-2314-B-303-006-//National Science and Technology Council, Taiwan/ ; },
abstract = {Protoplast-based systems provide a powerful and versatile platform for exploring how plants sense and respond to their environment. By enabling the direct delivery of proteins, DNA, and RNA into plant cells after cell wall removal, this approach facilitates precise molecular dissection of signaling, stress adaptation, and gene regulation across both model species and economically important crops. In this review, we analyzed 1050 published articles and categorizing them by delivery methods, research focus, plant species, and tissue types. We further highlight recent advances, including the application of single-cell transcriptomics, which provides unprecedented resolution for dissecting cellular responses and offers deeper insights into the mechanisms underlying stress resilience. Importantly, protoplast regeneration is gaining renewed attention not only as a model system for studying cellular reprogramming but also as a practical platform for crop improvement. Applications of protoplast regeneration include protoplast fusion, which integrates nuclear and organellar DNA/genomes from divergent parents to accelerate breeding and enhance tolerance to both biotic and abiotic stresses. Another important application is CRISPR/Cas ribonucleoprotein (RNP)-based editing targeting stress-resilience-related genes. In asexually propagated or highly heterozygous perennial crops with limited sexual reproduction, protoplast-based RNP delivery offers a viable and regulation-compliant strategy. This approach may help address public concerns over transgenic technologies while enabling the rapid development of stress-tolerant cultivars.},
}

@article {pmid41521358,
year = {2026},
author = {S Marques, B and Vitorino, C and V Ventura, F},
title = {CRISPR Applications in HIV Management - Prevention, Diagnosis, Monitoring and Treatment.},
journal = {Current HIV/AIDS reports},
volume = {23},
number = {1},
pages = {1},
pmid = {41521358},
issn = {1548-3576},
mesh = {Humans ; *HIV Infections/diagnosis/prevention & control/therapy ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Despite global efforts to combat the human immunodeficiency virus (HIV) epidemic, acquired immunodeficiency syndrome (AIDS) still claims one life every minute, underscoring the persistent need for improved control strategies. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) technologies have emerged as promising tools that may transform HIV management. The objective of this review is to summarise recent advancements in CRISPR/Cas-based approaches for HIV prevention, diagnosis, monitoring, and treatment, and to evaluate their potential and current challenges. A systematic literature search was conducted to identify relevant CRISPR/Cas applications in HIV infection. In prevention, CRISPR/Cas strategies aim to hinder viral integration and enhance host immune response, although substantial development is required before clinical translation. In diagnosis, CRISPR/Cas methods show high specificity and sensitivity, yet their reliance on specialised equipment and expertise limits their accessibility. In HIV monitoring, CRISPR/Cas-based methods have not yet demonstrated superiority over the quantitative PCR. In treatment, two ongoing clinical trials - one targeting a viral co-receptor on hematopoietic stem cells (HSCs) and the other excising proviral DNA - illustrate the potential of CRISPR/Cas-mediated cures, despite challenges such as low editing efficiency and off-target effects. Overall, CRISPR/Cas technologies hold considerable promise for advancing HIV management, but issues of accessibility, affordability, and scalability must be addressed to ensure global impact.},
}

Humans
*HIV Infections/diagnosis/prevention & control/therapy
*CRISPR-Cas Systems
Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41431922,
year = {2026},
author = {Chen, K and Zhu, J and Fan, C and Zhou, A and Li, B and Ge, H and Ning, X},
title = {A Nanoimprinted Photothermal Chip for On-Demand Spatiotemporal Activation of CRISPR/Cas9 Gene Editing.},
journal = {Nano letters},
volume = {26},
number = {1},
pages = {532-542},
doi = {10.1021/acs.nanolett.5c05571},
pmid = {41431922},
issn = {1530-6992},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Mice ; Gold/chemistry ; Sheep ; Infrared Rays ; Humans ; },
abstract = {Precise control of gene editing in target cells is essential for CRISPR/Cas9 applications. Here, we present a nanoimprinted photothermal chip (NPC) engineered for on-demand delivery and activation of CRISPR/Cas9 complexes with high spatial and temporal precision. Fabricated by nanoimprint lithography and subsequent surface modification, NPC features a customized PEGylated plasmonic gold nanopillar array, which provides both optimal cellular adhesion and efficient photothermal conversion. Upon NIR irradiation, NPC generates spatially confined thermal microdomains that transiently permeabilizes cell membranes, thereby facilitating cytosolic delivery of CRISPR/Cas9 complexes and synchronously modulating genome-editing kinetics. In vitro studies demonstrate robust gene knockout in both mouse and sheep cell lines while preserving high cell viability and editing fidelity. Remarkably, NPC-mediated PD-1 gene disruption in cytotoxic T cells markedly enhance their antitumor activity. Overall, this work establishes NPC as a transformative platform for precise and controllable CRISPR/Cas9 gene editing with broad therapeutic potential.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Animals
Mice
Gold/chemistry
Sheep
Infrared Rays
Humans

@article {pmid41416985,
year = {2026},
author = {Han, DH and Lee, SY and Kim, Y and Oh, J and Park, J and Park, YM and Kim, SG and Kim, TS and Park, JK},
title = {Ultrasensitive Detection of Multiple Foodborne Pathogens Using CRISPR-Cas12a on a Finger-Actuated Microfluidic Device Integrated with a Modular Pressurizing Pump.},
journal = {Analytical chemistry},
volume = {98},
number = {1},
pages = {531-542},
doi = {10.1021/acs.analchem.5c05295},
pmid = {41416985},
issn = {1520-6882},
mesh = {*Lab-On-A-Chip Devices ; *CRISPR-Cas Systems ; *Listeria monocytogenes/isolation & purification/genetics ; Nucleic Acid Amplification Techniques ; Limit of Detection ; *Food Microbiology ; *Escherichia coli O157/isolation & purification/genetics ; Salmonella/isolation & purification/genetics ; *Microfluidic Analytical Techniques/instrumentation ; Milk/microbiology ; Foodborne Diseases/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Foodborne pathogens pose a serious threat to global health and the economy, causing gastrointestinal illnesses and potentially leading to fatalities. Here, we present a recombinase polymerase amplification (RPA)-CRISPR-Cas12a-based method for the detection of foodborne pathogens using target-specific CRISPR RNAs (crRNAs) on a reusable, reconfigurable finger-actuated microfluidic device. Unlike previous finger-actuated pushbutton-based microfluidic devices, the device incorporates a modular pressurizing pump (MoPP), a standalone, reconfigurable actuation module that not only enhances reusability and reduces cross-contamination risks but also provides a flexible interface that allows user-defined fluidic routing and multiplexed assay workflows. Using a MoPP-integrated finger-actuated microfluidic device, the RPA-CRISPR-Cas12a-based detection of three foodborne pathogens was validated with an optimized crRNA and RPA primer sequence. Genomic DNA (gDNA) extracted from pathogen-spiked milk samples further demonstrated real-world applicability, achieving a limit of detection (LOD) of 1.62, 1.84, and 1.01 CFU/mL for Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes, respectively. The developed microfluidic RPA-CRISPR-Cas12a-based detection platform is expected to be a reconfigurable, user-friendly, and highly sensitive point-of-care testing system for monitoring foodborne pathogens throughout the food supply chain.},
}

*Lab-On-A-Chip Devices
*CRISPR-Cas Systems
*Listeria monocytogenes/isolation & purification/genetics
Nucleic Acid Amplification Techniques
Limit of Detection
*Food Microbiology
*Escherichia coli O157/isolation & purification/genetics
Salmonella/isolation & purification/genetics
*Microfluidic Analytical Techniques/instrumentation
Milk/microbiology
Foodborne Diseases/microbiology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41362221,
year = {2026},
author = {Yao, X and Wang, L and Su, L and Rao, W and Luo, Z and Li, Y},
title = {Localized CRISPR/Cas13a powered DNA walker for sensitive and high-throughput detection of norovirus.},
journal = {Journal of materials chemistry. B},
volume = {14},
number = {2},
pages = {572-579},
doi = {10.1039/d5tb02131f},
pmid = {41362221},
issn = {2050-7518},
mesh = {*Norovirus/isolation & purification/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *DNA/chemistry/genetics ; Metal Nanoparticles/chemistry ; Gold/chemistry ; High-Throughput Screening Assays ; Limit of Detection ; RNA, Viral/analysis ; },
abstract = {The development of sensitive and high-throughput methods for detecting foodborne viruses is crucial for disease prevention and public health protection. In this study, we present a novel localized Cas13a-based DNA walker (LCas13a-DNA walker) for the ultrasensitive, stable, and rapid detection of norovirus (NoV). When the DNA walker was confined in AuNPs, the spatial confinement effect improved the local concentration of reaction substrates, accelerated the reaction speed, and enhanced the sensitivity of the DNA walker. Besides, an original design of uracil-rich hairpin (UH)-modified AuNPs as the walking track significantly improves the stability of the detection system. Meanwhile, employing CRISPR/Cas13a as the driving force streamlines viral RNA recognition and substantially reduces the reaction time down to 30 minutes by eliminating the reverse transcription step. Additionally, a biomimetic array, formed by photonic crystals (PCs), enabled high-throughput signal acquisition with a microplate reader, and concurrently amplified the fluorescence signal. The proposed assay realized ultra-sensitivity of NoV with a detection limit as low as 4.1 pM and a wide linear range from 10 pM to 5 nM. Due to the advantages of high sensitivity, high-throughput, stability, and rapid analysis, this proposed method provides a potential strategy for point-of-care detection of pathogenic viruses in food safety monitoring and disease diagnosis.},
}

*Norovirus/isolation & purification/genetics
*CRISPR-Cas Systems
*Biosensing Techniques/methods
*DNA/chemistry/genetics
Metal Nanoparticles/chemistry
Gold/chemistry
High-Throughput Screening Assays
Limit of Detection
RNA, Viral/analysis

@article {pmid41294335,
year = {2026},
author = {Paulin, OKA and Tsavou, A and Priest, EL and Griffiths, JS and Lortal, L and Kempf, A and Chow, EWL and Pang, LM and Wickramasinghe, DN and Lyon, CM and Hernday, AD and Wang, Y and Richardson, JP and Naglik, JR},
title = {The combinatorial action of hyphal growth and candidalysin is critical for promoting Candida albicans oropharyngeal infection.},
journal = {mBio},
volume = {17},
number = {1},
pages = {e0330425},
doi = {10.1128/mbio.03304-25},
pmid = {41294335},
issn = {2150-7511},
support = {214229_Z_18_Z/WT_/Wellcome Trust/United Kingdom ; R15AI185747//National Institute of Allergy and Infectious Diseases/ ; BBSRC: UKRI717/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Candida albicans/genetics/growth & development/pathogenicity ; *Hyphae/growth & development/genetics ; Animals ; *Fungal Proteins/genetics/metabolism ; Mice ; *Virulence Factors/genetics/metabolism ; Humans ; Gene Deletion ; Disease Models, Animal ; Aspartic Acid Endopeptidases/genetics/metabolism ; Epithelial Cells/microbiology ; Female ; *Candidiasis, Oral/microbiology ; Virulence ; Oropharynx/microbiology ; Candidiasis/microbiology ; CRISPR-Cas Systems ; },
abstract = {Candida albicans is one of the most common fungal pathogens, yet much remains unknown about how its virulence factors cooperate to promote pathogenicity. To investigate this, CRISPR-Cas9 technology was used to create a panel of 19 single, double, triple, and quadruple deletion mutant strains targeting four established virulence factors: ALS3 (adhesin/invasin), ECE1 (candidalysin toxin), HGC1 (hypha formation regulator), and SAP2 (protease). In vitro, the deletion of each gene had differing impacts across multiple characterization assays. The hgc1∆/∆ mutant was unable to form hyphae under inducing conditions, leading to downstream impairment of epithelial invasion. The als3∆/∆ mutant exhibited significantly reduced adhesion and invasion into epithelial cells, resulting in attenuated cellular damage. The ece1∆/∆ mutant displayed significantly reduced epithelial damage, cell signaling, and immune activation. The phenotype of the sap2∆/∆ mutant resembled that of wild type but was unable to degrade protein. In an immunocompromised murine model of oropharyngeal infection, hyphal growth and candidalysin production were the dominant drivers of elevated fungal burden, innate immune responses, and mortality. Following a 5-day infection with hgc1∆/∆ and ece1∆/∆ single gene deletion strains, mice had survival rates of 100% and 80%, respectively, compared to 15% in wild-type infected mice. Notably, 100% survival was also observed following challenge with all hgc1∆/∆ and ece1∆/∆ combination mutants. This study demonstrates that specific C. albicans virulence attributes act in combination to promote mucosal infection, with hyphal growth and candidalysin production being a critical driver of oropharyngeal infection.IMPORTANCECandida albicans has been classified by the WHO as a "critical priority" pathogen, highlighting the urgent need for a greater understanding of the mechanisms that enable it to cause disease. C. albicans possesses numerous virulence attributes, but how they synergize during infection is not well understood. Here, using reverse genetics, we dissect the individual and combinatorial roles of four C. albicans virulence factors (Als3p, candidalysin, hyphal growth, and Sap2p) in vitro and in an in vivo murine model of oropharyngeal candidiasis. Increasing the number of C. albicans gene deletions correlated with reduced oral fungal burden, with hyphal growth and candidalysin together being critical for infection, inflammation, and mortality during oropharyngeal infection. These findings demonstrate that virulence attributes act cooperatively as a collective network to promote pathogenicity, a finding also observed in plant fungal pathogens. Our approach has identified specific fungal virulence factors that can be targeted for new treatment strategies against C. albicans infections.},
}

*Candida albicans/genetics/growth & development/pathogenicity
*Hyphae/growth & development/genetics
Animals
*Fungal Proteins/genetics/metabolism
Mice
*Virulence Factors/genetics/metabolism
Humans
Gene Deletion
Disease Models, Animal
Aspartic Acid Endopeptidases/genetics/metabolism
Epithelial Cells/microbiology
Female
*Candidiasis, Oral/microbiology
Virulence
Oropharynx/microbiology
Candidiasis/microbiology
CRISPR-Cas Systems

@article {pmid41520043,
year = {2026},
author = {Senger, J and Keutgen, M and Roth, N and Seitl, I and Fischer, L},
title = {Toward food-grade production of the Bacteroides helcogenes protein-glutamine glutaminase with an optimized Bacillus subtilis strain.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {8},
pmid = {41520043},
issn = {1432-0614},
mesh = {*Bacillus subtilis/genetics/metabolism ; *Glutaminase/genetics/biosynthesis/metabolism ; *Bacteroides/enzymology/genetics ; *Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Bioreactors/microbiology ; },
abstract = {Protein-glutamine glutaminases (PGs; EC 3.5.1.44) have gained attention in the food industry due to their application in plant protein products. The recently discovered PG from Bacteroides helcogenes (PGB) has especially been shown to provide promising characteristics for improving the techno-functional properties of plant proteins. A prerequisite for food enzymes, such as the PG, is their production with an expression host that meets food safety and yield requirements. The antibiotic-free and secretory production of the PGB was targeted in this study using the undomesticated Bacillus subtilis 007. The CRISPR/Cas9-mediated approach enabled specific genomic PGB integrations, while simultaneously deleting unwanted B. subtilis traits. Firstly, the PGB expression cassette was integrated into the sigF gene, leading to an asporogenic strain and extracellular activity of 4.1 µkat/Lculture in bioreactor cultivations. However, excessive foaming hampered the production process tremendously. Consequently, a second PGB copy was integrated into the sfp locus, which is involved in the production of lipopeptides, such as surfactin. As a result, the PGB activity was increased to 5.4 µkat/Lculture, and foaming during cultivation was reduced significantly. The introduction of a third PGB copy for preventing cell motility did not increase production; however, the integration into the well-established amyE locus improved the PGB yield during reactor cultivations. A final extracellular activity of 9.5 µkat/Lculture was reached. The multiple genomic integrations of the PGB gene enabled the efficient PGB secretion in an optimized B. subtilis host without the need for antibiotics. KEY POINTS: • Site-specific PGB integration enabled by genome sequencing of B. subtilis 007. • Antibiotic-free and secretory PGB production with an optimized B. subtilis host. • Increased PGB production reaching 9.5 µkat/Lculture.},
}

*Bacillus subtilis/genetics/metabolism
*Glutaminase/genetics/biosynthesis/metabolism
*Bacteroides/enzymology/genetics
*Bacterial Proteins/genetics/metabolism
CRISPR-Cas Systems
Bioreactors/microbiology

@article {pmid41518500,
year = {2026},
author = {Álvarez, E and Franco-Zorrilla, JM},
title = {Targeted DNA Affinity Purification for Quantifying DNA-Binding Specificities of Transcription Factors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2985},
number = {},
pages = {89-104},
pmid = {41518500},
issn = {1940-6029},
mesh = {*Transcription Factors/metabolism/genetics ; Binding Sites ; *DNA, Plant/metabolism/genetics ; High-Throughput Nucleotide Sequencing/methods ; Protein Binding ; *Chromatography, Affinity/methods ; Gene Expression Regulation, Plant ; *DNA/metabolism ; Arabidopsis/genetics/metabolism ; },
abstract = {Plant growth relies on flexible gene regulation to adapt to environmental changes. This process is ultimately controlled by transcription factors (TFs), which bind to specific DNA motifs, known as TF-binding sites (TFBS), located in the gene regulatory regions to regulate their expression. These interactions play crucial roles in plant development and responses to environmental cues, as well as in plant evolution and domestication, making both cis- (i.e., TFBS) and trans-regulatory factors as potential molecular targets in plant breeding for traits such as yield, quality, and stress resilience. These biotechnological approaches require precise knowledge of the target gene sets and TFBS specifically recognized by TFs. Recent advances in high-throughput sequencing techniques have enabled precise identification of TF target genes, especially thanks to methodologies that combine the main features of both in vitro and in vivo approaches. However, small scale and targeted approaches are still needed to evaluate the relative contribution of specific nucleotide positions in TF recognition. In this chapter, we describe a modified version of DNA Affinity Purification sequencing (DAP-seq) that replaces genomic DNA with a PCR-generated library of TFBS variants. This approach, termed targeted-DAP, allows a flexible and quantitative analysis of TF-binding using next-generation sequencing. Additionally, expressing TFs in Escherichia coli provides an economical source of proteins, enabling scalable and cost-effective analysis of DNA-binding specificities. We showed the benefits of this technique to demonstrate the contribution of the genomic context around the TFBS for specific recognition of a bHLH TF. Development of targeted DAP-seq would be of interest for the evaluation of nucleotide variation-either allelic or generated by CRISPR/Cas-within TFBS in TF recognition with predictable consequences on plant phenotypes.},
}

*Transcription Factors/metabolism/genetics
Binding Sites
*DNA, Plant/metabolism/genetics
High-Throughput Nucleotide Sequencing/methods
Protein Binding
*Chromatography, Affinity/methods
Gene Expression Regulation, Plant
*DNA/metabolism
Arabidopsis/genetics/metabolism

@article {pmid41518477,
year = {2026},
author = {Ghazaei, C},
title = {The role of bacteriophages and CRISPR-Cas in combating multidrug-resistant bacteria.},
journal = {Natural products and bioprospecting},
volume = {16},
number = {1},
pages = {14},
pmid = {41518477},
issn = {2192-2195},
abstract = {The alarming increase of multidrug-resistant (MDR) bacteria presents a serious global health crisis, reducing the effectivenessof traditional antibiotics and requiring alternative therapeutic strategies. Among the most promising innovations are bacteriophages-viruses that specifically infect bacteria-and CRISPR-Cas systems, molecular tools enabling precise genome editing. These technologies individually offer targeted antibacterial activity with minimal disturbance to the host microbiota. When combined, they forma synergistic platform capable of overcoming many limitations of conventional antibiotics, including broad-spectrum activity, resistance development, and limited adaptability. This review examinesmechanisms of bacterial resistance, the biological foundation of bacteriophages and CRISPR-Cas systems, and their application in fighting MDR pathogens. However, significant challenges remain, including delivery barriers, off-target effects, regulatory uncertainty, and public acceptance of gene-editing tools. Antimicrobial resistance now tanks among the top threats to global health, with an estimated burden exceeding one million deaths annually, surpassing many other infectious diseases. The article concludes with a discussion of the clinical prospects of phage-CRISPR therapies and highlights key areas for future research. By merging the specificity of phages with the programmable strength of CRISPR, these biotechnological advances provide a powerful and approach to address the growing threat of antibiotic resistance.},
}

@article {pmid41518077,
year = {2026},
author = {Liu, Y and Wang, H and Chen, L and Wu, X and Xu, Z and Huang, Q and Zhang, H and Cao, X and Liang, X and Zhong, X and Luo, C},
title = {CRISPR-Cas9-Generated TXNDC15 c.560delA Homozygous Mouse Model Exhibits Meckel-Gruber Syndrome Phenotype.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {64},
number = {1},
pages = {e70040},
pmid = {41518077},
issn = {1526-968X},
support = {SZSM202311005//Sanming Project of Medicine in Shenzen Municipality/ ; 2021A1515012495//Natural Science Foundation of Guangdong Province/ ; JCYJ20210324135009024//Shenzhen Science and Technology Innovation Commission/ ; },
mesh = {Animals ; Mice ; *Polycystic Kidney Diseases/genetics/pathology ; CRISPR-Cas Systems ; *Ciliary Motility Disorders/genetics/pathology ; *Encephalocele/genetics/pathology ; Disease Models, Animal ; Phenotype ; Homozygote ; *Retinitis Pigmentosa/genetics/pathology ; Frameshift Mutation ; },
abstract = {To determine whether TXNDC15 variation causes Meckel-Gruber syndrome (MKS), we assessed the pathogenicity of the frameshift variant c.560delA. A CRISPR-Cas9 generated mouse model carrying the equivalent Txndc15 c.512delA mutation was analyzed at embryonic day 15.5. Homozygous Txndc15[mt/mt] embryos displayed the complete MKS phenotype-fetal lethality, exencephaly, omphalocele, post-axial polydactyly, and polycystic kidneys-together with markedly reduced TXNDC15 protein in brain, liver, and kidney. These findings confirm TXNDC15 as a bona fide MKS disease gene.},
}

Animals
Mice
*Polycystic Kidney Diseases/genetics/pathology
CRISPR-Cas Systems
*Ciliary Motility Disorders/genetics/pathology
*Encephalocele/genetics/pathology
Disease Models, Animal
Phenotype
Homozygote
*Retinitis Pigmentosa/genetics/pathology
Frameshift Mutation

@article {pmid41329281,
year = {2025},
author = {Kolesov, DE and Orlova, NA and Vorobiev, II},
title = {Generation of Long-Lived CHO Cells Suitable for Production of Afucosylated Antibodies and Fc-Fusion Proteins.},
journal = {Doklady biological sciences : proceedings of the Academy of Sciences of the USSR, Biological sciences sections},
volume = {525},
number = {1},
pages = {359-367},
pmid = {41329281},
issn = {1608-3105},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Immunoglobulin Fc Fragments/genetics/metabolism ; *Fucosyltransferases/genetics/metabolism ; CRISPR-Cas Systems ; *Recombinant Fusion Proteins/genetics/biosynthesis ; *Antibodies/metabolism ; Gene Knockout Techniques ; },
abstract = {Using genome editing, we created a homozygous α-(1,6)-fucosyltransferase (FUT8[-]/[-]) knockout in apoptosis-resistant CHO 4BGD cells, yielding the new 4BGD-F cell line. Combining CRISPR/Cas9 with paired gRNAs and non-specific puromycin selection yielded a cell population with an exceptionally high FUT8 knockout frequency, obviating the need for metabolic enrichment with lentil lectin (Lens culinaris agglutinin, LCA). Despite impaired clonogenicity of the knockout cells, we successfully isolated multiple clonal cell lines harboring extensive biallelic FUT8 deletions. Isolated clones with biallelic deletions retained key parental line characteristics: viability >90% in 17-day fed-batch cultures at high densities (>15 × 10[6] cells/mL), and rapid selectability using both dihydrofolate reductase and glutamine synthetase systems. Mass spectrometric analysis of the test protein GLP1-Fc secreted by 4BGD-F cells confirmed the absence of N-glycan fucosylation. The CHO 4BGD-F cell line provides a valuable platform for producing afucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity.},
}

Animals
CHO Cells
Cricetulus
*Immunoglobulin Fc Fragments/genetics/metabolism
*Fucosyltransferases/genetics/metabolism
CRISPR-Cas Systems
*Recombinant Fusion Proteins/genetics/biosynthesis
*Antibodies/metabolism
Gene Knockout Techniques

@article {pmid41517680,
year = {2026},
author = {Rehman, SU and Abbas, GH},
title = {CRISPR/CAS9-based gene editing in cancer therapy: A systematic review and meta-analysis on current status and future directions.},
journal = {Medicine},
volume = {105},
number = {2},
pages = {e47114},
doi = {10.1097/MD.0000000000047114},
pmid = {41517680},
issn = {1536-5964},
mesh = {*Gene Editing/methods/trends ; *CRISPR-Cas Systems ; *Neoplasms/therapy/genetics ; Humans ; *Genetic Therapy/methods/trends ; },
abstract = {BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has recently been discovered for gene editing and cancer therapy and its applications are expanding. This review and meta-analysis aim to assess the present and future of CRISPR/Cas9 based gene editing in cancer treatment and the way forward.

METHODS: The search was conducted in PubMed from 2015 to 2025 and 89 relevant studies were identified. The study design, CRISPR/Cas9 targets, delivery methods, therapeutic efficacy and limitations were extracted from the studies.

RESULTS: We reviewed the efficacy, challenges, and potential for translation of CRISPR/Cas9 in oncogene and tumor suppressor gene targeting and immune modulation. Several preclinical researches showed that CRISPR/Cas9 mediated disruption of oncogenes or restoration of tumor suppressor genes led to significant tumor regression. The evaluation was also extended to off target effects and integration with immunotherapy.

CONCLUSION: From the findings of this work, it can be concluded that CRISPR/Cas9 is a promising tool, but there are several limitations including off target effects, delivery systems and ethical issues that need to be solved in order to improve the clinical significance.},
}

*Gene Editing/methods/trends
*CRISPR-Cas Systems
*Neoplasms/therapy/genetics
Humans
*Genetic Therapy/methods/trends

@article {pmid41516423,
year = {2026},
author = {Kongsomboonchoke, P and Ariyachet, C and Kaewsapsak, P and Sirichindakul, P and Tangkijvanich, P},
title = {Development of a Sensitive and Specific RPA-CRISPR/Cas12a Assay for Intrahepatic Quantification of HBV cccDNA.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516423},
issn = {1422-0067},
support = {//Second Century Fund (C2F)/ ; //Center of Excellence in Hepatitis and Liver Cancer/ ; },
mesh = {Humans ; *Hepatitis B virus/genetics ; *DNA, Circular/genetics/analysis ; *CRISPR-Cas Systems/genetics ; *DNA, Viral/genetics/analysis ; *Hepatitis B/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; *Liver/virology ; Sensitivity and Specificity ; Recombinases/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Hepatitis B virus (HBV) persists in infected hepatocytes through covalently closed circular DNA (cccDNA), a stable episomal form that serves as the transcriptional template for viral replication. Accurate and sensitive quantification of intrahepatic cccDNA is crucial for evaluating antiviral therapies, particularly those targeting a functional cure. Here, we report the development of a novel, cccDNA-specific detection system combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a-based fluorescence detection. We designed and validated CRISPR RNAs (crRNAs) targeting HBV cccDNA-specific regions conserved across genotypes A-D. Reaction conditions for both RPA and Cas12a detection were optimized to enhance sensitivity, specificity, and accuracy. The system reliably detected as few as 10 copies of cccDNA-containing plasmid per reaction and showed no cross-reactivity with non-cccDNA forms in serum or plasma, indicating assay specificity. When applied to liver tissue samples from 10 HBV-infected and 6 non-HBV patients, the RPA-CRISPR/Cas12a assay exhibited a high sensitivity (90%) and a strong correlation with qPCR results (R[2] = 0.9155), confirming its accuracy. In the conclusion, the RPA-CRISPR/Cas12a system provides a robust, cost-effective, and scalable platform for sensitive and specific quantification of intrahepatic HBV cccDNA. This method holds promises for research and high-throughput therapeutic screening applications targeting cccDNA clearance.},
}

Humans
*Hepatitis B virus/genetics
*DNA, Circular/genetics/analysis
*CRISPR-Cas Systems/genetics
*DNA, Viral/genetics/analysis
*Hepatitis B/virology/diagnosis
*Nucleic Acid Amplification Techniques/methods
*Liver/virology
Sensitivity and Specificity
Recombinases/metabolism
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41516375,
year = {2026},
author = {Firdaus, F and Yadav, V and Ramakrishnan, M and Wasi, A and Ganie, IB and Upadhyay, A and Shahzad, A and Ahmad, Z},
title = {2-Hydroxy-4-Methoxybenzaldehyde (2H4MB): Integrating Cell Culture, Metabolic Engineering, and Intelligent Genome Editing.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516375},
issn = {1422-0067},
support = {Y20240114//Young Foreign Talent Program/ ; },
mesh = {*Metabolic Engineering/methods ; *Gene Editing/methods ; *Benzaldehydes/metabolism/chemistry ; Cell Culture Techniques/methods ; CRISPR-Cas Systems ; Biosynthetic Pathways ; },
abstract = {2-Hydroxy-4-Methoxybenzaldehyde (2H4MB) is a valuable aromatic compound with applications in flavour, fragrance, and pharmaceuticals. Because of its endangered status and root-specific accumulation, its production in native plants is restricted. In order to increase 2H4MB yield, this study emphasises recent developments in metabolic engineering, synthetic biology, in vitro culture methods, and AI-assisted route prediction. This review discussed about how CRISPR-based genome editing can be used to modify important biosynthetic genes and regulatory components, as well as how predictive machine learning techniques can be used to improve production conditions. Inadequate genetic resources, poorly understood biosynthetic pathways, and a dearth of reliable transformation systems are among the present constraints. The work highlights the importance of using integrative plant biotechnology techniques to fully realise the industrial and medicinal potential of this underutilised chemical.},
}

*Metabolic Engineering/methods
*Gene Editing/methods
*Benzaldehydes/metabolism/chemistry
Cell Culture Techniques/methods
CRISPR-Cas Systems
Biosynthetic Pathways

@article {pmid41516361,
year = {2026},
author = {Deriglazova, IO and Shepelev, MV and Kruglova, NA and Georgiev, PG and Maksimenko, OG},
title = {The Chimeric Nuclease SpRYc Exhibits Highly Variable Performance Across Biological Systems.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516361},
issn = {1422-0067},
support = {075-15-2024-539//Ministry of Science and Education of the Russian Federation/ ; },
mesh = {Animals ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; HEK293 Cells ; Drosophila melanogaster/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {The CRISPR-Cas9 system has significantly advanced genome editing but remains constrained by its requirement for specific protospacer adjacent motifs (PAMs). To overcome this limitation, PAM-relaxed nucleases, including the novel near-PAMless chimeric SpRYc, have been developed. Here, we evaluated SpRYc editing activity across multiple experimental systems, including human HEK293 and CEM-R5 cells, as well as Drosophila melanogaster S2 cells and embryos. In HEK293 cells, SpRYc exhibited broad PAM compatibility, enabling editing at non-canonical PAMs, albeit with reduced and variable efficiency at canonical NGG sites compared to SpCas9. This context dependency was more pronounced in CEM-R5 T cells, where SpRYc activity at endogenous CXCR4 and B2M loci was largely restricted to NGG PAMs. In contrast, unlike SpCas9, SpRYc displayed negligible genome-editing activity in Drosophila embryos in vivo. Notably, the transcriptional activator dSpRYc-VPR showed robust activity in Drosophila S2 cells at both canonical and non-canonical PAMs. Reduced chromatin occupancy of dSpRYc-VPR suggests a balance between expanded PAM recognition and DNA-binding stability, providing a mechanistic explanation for context-dependent performance of SpRYc. Overall, our results highlight that expanded targeting flexibility comes at the cost of variable efficiency, underscoring the need for extensive locus- and context-specific validation of PAM-relaxed genome-editing tools.},
}

Animals
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
HEK293 Cells
Drosophila melanogaster/genetics
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid41516323,
year = {2025},
author = {Evseev, PV and Podoprigora, IV and Chaplin, AV and Khabadze, ZS and Malkov, AA and Kafarskaia, LI and Shagin, DA and Urban, YN and Borisova, OY and Efimov, BA},
title = {Bulleidia extructa PP_925: Genome Reduction, Minimalist Metabolism, and Evolutionary Insights into Firmicutes Diversification.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516323},
issn = {1422-0067},
mesh = {*Genome, Bacterial ; Phylogeny ; *Evolution, Molecular ; Humans ; *Firmicutes/genetics/metabolism/classification ; },
abstract = {Bulleidia extructa strain PP_925, isolated from the periodontal pocket of a patient with periodontitis, is a Gram-positive Bacillota with an unusually compact genome of 1.38 Mb. Phylogenomic analyses place PP_925 within Erysipelotrichales and show close relatedness of Bulleidia to Solobacterium and Lactimicrobium, as well as the existence of previously undescribed related clades. The metabolic repertoire of PP_925 is strongly reduced: it retains glycolysis, the phosphotransacetylase-acetate kinase pathway, and arginine catabolism but lacks the tricarboxylic acid cycle and most de novo biosynthetic pathways for amino acids, nucleotides, fatty acids, cofactors, and vitamins, implying reliance on salvage and cross-feeding. Phylogenetic inference indicates independent peptidoglycan losses in multiple mycoplasma Erysipelotrichia-related lineages, while PP_925 has retained an ancestral Gram-positive cell wall despite extensive genomic reduction. The genome preserves systems crucial for host interaction and adaptability, including a horizontally acquired tad locus encoding type IV pili, a comG competence system, and several adherence-associated virulence factors. Defense mechanisms are diverse and include a CRISPR-Cas II-A system, a type II restriction-modification module adjacent to Gao_Qat-like genes, and the Wadjet system in a genome without prophages; CRISPR spacers indicate repeated encounters with Bacillota phages. Comparative genomics of PP_925 and related strains reveals a small core genome with lineage-specific adhesion and defense modules, indicating recent shared ancestry combined with adaptive flexibility under substantial genome reduction.},
}

*Genome, Bacterial
Phylogeny
*Evolution, Molecular
Humans
*Firmicutes/genetics/metabolism/classification