@article {pmid41454999,
year = {2025},
author = {Vadakkan, K and Raphael, R and Korattil, TT and Mapranathukaran, VO and Ramadas, V and Kizhakkepeedika, RD},
title = {A Critical Review of Quorum-Sensing Inhibition Strategies in Klebsiella Pneumoniae.},
journal = {Current microbiology},
volume = {83},
number = {2},
pages = {103},
pmid = {41454999},
issn = {1432-0991},
abstract = {Klebsiella pneumoniae is an antibiotic-resistant microbe that causes pneumonia, urinary tract infections, bloodstream infections, and liver abscesses. Its pathogenicity is fueled by the polysaccharide capsule, lipopolysaccharides, and quorum-sensing-regulated biofilm formation, which enhance immune evasion and antibiotic resistance. The rise of multidrug-resistant and hypervirulent pathogens poses a significant challenge to treatment. Targeting quorum sensing with quorum quenching is a promising method for disrupting bacterial interaction and reducing virulence without increasing resistance. This review examines the pathogenesis of K. pneumoniae, the significance of quorum sensing, and emerging therapeutics, including quorum-sensing inhibitors and advanced techniques such as CRISPR-Cas and computational drug development, while emphasizing the need for further investigation.},
}

@article {pmid41762232,
year = {2026},
author = {Deepika, and Sharma, S and Kumar, P and Rathore, V and Chauhan, A and Kumar, A and Dogra, RK and Handa, A},
title = {Insights into physiological, biochemical and molecular mechanisms of abiotic stress tolerance in Persian walnut (Juglans regia L.).},
journal = {Protoplasma},
volume = {263},
number = {4},
pages = {1099-1116},
pmid = {41762232},
issn = {1615-6102},
abstract = {Walnut (Juglans regia) is an economically and nutritionally valuable tree species that often encounters diverse abiotic stresses such as drought, salinity, cold, heat and heavy metal toxicity. These stresses adversely affect its growth, productivity and survival by altering physiological functions, disturbing cellular homeostasis and triggering oxidative damage. In response, J. regia deploy a multifaceted adaptive system comprising morphological changes, biochemical adjustments and intricate molecular signaling pathways. The review aims to analyze current knowledge on the physiological, morphological changes observed under stress with biochemical defense mechanism. These include the crucial antioxidant defense system (increase in Superoxide Dismutase (SOD), Peroxidase (POD) and Catalase (CAT) activity), the accumulation of protective soluble solutes and amino acids and the biosynthesis of secondary metabolites through the Methylerythritol Phosphate (MEP) pathway involved in mitigating oxidative stress caused by Reactive Oxygen Species (ROS). Crucially, we synthesize the understanding of molecular regulation that underpins stress adaptation. This encompasses stress-responsive gene expression including Jr (Juglans regia) VHAG1 (V-ATPase H+ transporting ATPase subunit G1), JrDREB (Dehydration-Responsive Element Binding protein), JrRD29 (Responsive to Dehydration 29), transcriptional regulation by myeloblastosis (MYB), Dehydration-responsive element-binding proteins (DREB) and WRKY-TF families and their interaction with hormonal (abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and ethylene) which play a pivotal role in signal transduction and stress tolerance. These mechanisms employed by walnut under abiotic stresses, highlighting key genetic and hormonal pathways that can be targeted for the development of stress-resilient cultivars and ensuring sustainable production under changing climatic conditions. Recent advancements in genomics, transcriptomics and proteomics got attention that provides new insights into the regulatory networks and candidate genes conferring stress resilience. Furthermore, it explores biotechnological approaches for improving stress tolerance, highlighting the prospects of latest high-throughput techniques, including molecular breeding, genetic engineering, Next-Generation Sequencing (NGS), microRNA (miRNA)-based regulation and Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas) gene editing. This integrated review connects multi-level stress response mechanisms, highlighting potential molecular markers and biotechnological interventions for accelerating walnut improvement and ensuring sustainable production under changing climatic conditions.},
}

@article {pmid41820648,
year = {2026},
author = {Hanif, N and Naveed, M and Salah Ud Din, M and Aziz, T and Shami, A and Al-Joufi, FA},
title = {Genome-wide characterization and comparative analysis of Bacillus paranthracis MBBL1 reveals genomic plasticity features.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {41820648},
issn = {1438-7948},
support = {PNURSP2026R31//Princess Nourah Bint Abdulrahman University/ ; },
abstract = {Bacillus paranthracis is a member of the Bacillus cereus group with close phylogenetic proximity to pathogenic species, yet its genomic diversity and pathogenic potential remain poorly understood. In this study, we present a comprehensive whole-genome analysis of B. paranthracis strain MBBL1, an environmental isolate, to explore its genomic architecture, evolutionary relationships, and potential virulence traits. Comparative genomic and pangenome analyses revealed that MBBL1 clusters closely with other B. paranthracis strains while maintaining measurable genomic similarity to B. cereus and B. anthracis. Notably, the genome lacks plasmids and known serotype markers, distinguishing it from many pathogenic members of the group. Despite this, MBBL1 harbors multiple chromosomally encoded virulence-associated genes, mobile genetic elements, and genomic islands, indicating considerable genomic plasticity. The presence of an active Type I CRISPR-Cas system may contribute to genome stability and the absence of plasmids. Pathogenicity prediction suggests a low but detectable potential for human pathogenicity. Collectively, these findings highlight the importance of environmental Bacillus strains as reservoirs of hidden virulence traits and provide a valuable genomic framework for future functional and risk assessment studies.},
}

@article {pmid41964738,
year = {2026},
author = {Karwa, P and Kharul, A and Parekar, V and Labhade, S and Sakle, N and Bhole, R and Kapare, H},
title = {Next-Generation Therapies for Genetic Diseases: The Synergy of CRISPR and Gene Therapy.},
journal = {Biochemical genetics},
volume = {},
number = {},
pages = {},
pmid = {41964738},
issn = {1573-4927},
abstract = {The introduction of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology has changed the game of gene therapy and promises surgical precision and efficacy in the process of addressing genetic disorders in humans. Gene therapy that commonly assumes insertion of functional DNA with the help of viral vectors has developed or rather undergone adaptation with the introduction of CRISPR-Cas mechanisms that make it possible to edit the genome, correct or regulate it and silence. It is a critical study that reviews mechanistic disparities among traditional and CRISPR bases types of gene therapy, based on benefits, shortcomings, and states of improvement till date, as far as clinical development is concerned. It talks about their historical evolution, molecular principles, delivery modes, and therapeutic promise of the CRISPR systems such as Cas9, Cas12, Cas13, base editing, and prime editing. It is of significant value to mention the application of CRISPR as a treatment in monogenic illnesses such as 2-thalassemia and Duchenne muscular dystrophy as well as the future use of CRISPR in complex and multifactorial diseases. Also, safety concerns, ethical issues, and delivery translational problems are discussed by the review and they are paramount to successful clinical translation of CRISPR-based therapeutics. The discussion highlights the revolutionary possibility of CRISPR in precision medicine and predetermines its expansion in the future healthcare genomic practice.},
}

@article {pmid42105238,
year = {2026},
author = {Wang, DM and Tiruppathi, C},
title = {Protocol to generate endothelial cell-specific knockout mouse models using Cas9/Cdh5-Cre mice coupled with sgRNA.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104551},
pmid = {42105238},
issn = {2666-1667},
mesh = {Animals ; Mice ; *Endothelial Cells/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Mice, Knockout ; Integrases/genetics/metabolism ; Cadherin 5 ; *Cadherins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Antigens, CD/genetics/metabolism ; *Gene Knockout Techniques/methods ; },
abstract = {The vascular endothelium is a critical regulator of vascular homeostasis and tissue fluid balance, and mouse models are essential for studying these processes in vivo. Here, we present a protocol to generate adult endothelial cell (EC)-specific gene knockout (KO) mouse models. We describe steps for Cas9-active and Cdh5-Cre-positive (Cas9/Cdh5-Cre) mouse line generation, single guide RNA (sgRNA) design for vector construction, plasmid DNA generation, and liposome preparation. We then detail procedures for liposome/plasmid complex injection, lung harvest, homogenization, protein quantification, and verification with western blotting. For complete details on the use and execution of this protocol, please refer to Wang et al.[1].},
}

Animals
Mice
*Endothelial Cells/metabolism
*RNA, Guide, CRISPR-Cas Systems/genetics
Mice, Knockout
Integrases/genetics/metabolism
Cadherin 5
*Cadherins/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Antigens, CD/genetics/metabolism
*Gene Knockout Techniques/methods

@article {pmid42163310,
year = {2026},
author = {Jiang, Y and Caban, KM and Peitzsch, M and Herrmann, C and Mayr, D and Stöckl, JB and Fröhlich, T and Mayerhofer, A and Müller-Taubenberger, A and Welter, H},
title = {Knockout of filamin A in KGN granulosa tumor cells impairs proliferation, cell cycle progression, migration, and cytoskeletal organization under mechanical stress.},
journal = {Biological research},
volume = {59},
number = {1},
pages = {},
pmid = {42163310},
issn = {0717-6287},
mesh = {*Filamins/genetics/metabolism ; Humans ; Female ; *Cell Movement/physiology ; *Cell Proliferation/physiology ; *Granulosa Cell Tumor/pathology/metabolism/genetics ; *Cytoskeleton/physiology ; Stress, Mechanical ; *Cell Cycle/physiology ; Cell Line, Tumor ; Immunohistochemistry ; *Ovarian Neoplasms/pathology ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Cell Adhesion ; },
abstract = {BACKGROUND: Filamin A (FLNA) is an actin-binding protein that regulates mechanosensitivity and functions as an intracellular signaling scaffold in various cell types. It has also been implicated in tumor growth. We recently reported FLNA expression in human ovarian granulosa cells and in KGN cells, a granulosa cell tumor (GCT) line.

RESULTS: Immunohistochemistry analysis of 51 GCT samples revealed heterogeneous FLNA expression, with approximately 20% showing weak, 18% strong, and the majority moderate expression. We therefore conducted functional studies in KGN cells using CRISPR/Cas9 gene editing. A proteomic approach revealed marked changes in protein abundance upon FLNA depletion: proteins with increased abundance were predominantly related to adhesion, cytoskeletal organization, regulation of cell shape, and lipid metabolic process, whereas those with decreased abundance were associated with DNA replication, cell division, and cell cycle regulation. FLNA-knockout cells showed enlarged cell sizes, reduced proliferation, and slightly affected steroidogenesis. Disruption of FLNA further reduced migration velocity, altered actin cytoskeletal alignment under flow, and modified expression of genes involved in cytoskeletal architecture, adhesion, and mechanosensing under shear stress.

CONCLUSIONS: Our results identify crucial roles of FLNA in shaping the cellular architecture, motility, and proliferation of KGN cells. Consequently, alterations in FLNA expression may influence intracellular signaling, and responsiveness to mechanical cues in both physiological and pathological contexts.},
}

*Filamins/genetics/metabolism
Humans
Female
*Cell Movement/physiology
*Cell Proliferation/physiology
*Granulosa Cell Tumor/pathology/metabolism/genetics
*Cytoskeleton/physiology
Stress, Mechanical
*Cell Cycle/physiology
Cell Line, Tumor
Immunohistochemistry
*Ovarian Neoplasms/pathology
CRISPR-Cas Systems
Gene Knockout Techniques
Cell Adhesion

@article {pmid42172127,
year = {2026},
author = {Hong, JP and Nussenzweig, MC},
title = {Protocol for an in vivo CRISPR screen for germinal center B cells in mice using ecotropic retrovirus.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104586},
pmid = {42172127},
issn = {2666-1667},
mesh = {Animals ; Mice ; *Germinal Center/cytology/immunology ; *B-Lymphocytes/metabolism/cytology ; *Retroviridae/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The lack of an in vitro model that recapitulates germinal center (GC) B cell biology necessitates the use of animal models for genetic studies. Here, we present a protocol for an in vivo CRISPR screen for GC B cells in mice using an ecotropic retrovirus. We describe steps for constructing a single guide (sgRNA) library and performing a genetic screen in a mouse model of protein immunization, including procedures for sequencing and data analysis.},
}

Animals
Mice
*Germinal Center/cytology/immunology
*B-Lymphocytes/metabolism/cytology
*Retroviridae/genetics
*CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42213768,
year = {2026},
author = {Hassan, AZ and Zhang, X and Ward, HN and Billmann, M and Bajjali, S and Brown, KR and Bhojoo, U and Chan, K and Lin, K and Costanzo, M and Andrews, B and Boone, C and Moffat, J and Myers, CL},
title = {Orobas: A computational approach for scoring and analysis of quantitative chemical-genetic interactions from CRISPR-Cas9 screens.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104594},
pmid = {42213768},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Computational Biology/methods ; *Software ; },
abstract = {We present Orobas, a computational approach for transforming raw read count data from CRISPR-Cas9 chemical-genetic screens into quantitative interaction scores. We describe steps for computing differential interaction scores with statistical tests that account for multiple CRISPR guides per gene. We then outline approaches for post-processing differential log2-fold-change scores across multiple screens, incorporating normalization to reduce technical artifacts and correct batch effects.},
}

*CRISPR-Cas Systems/genetics
*Computational Biology/methods
*Software

@article {pmid42213769,
year = {2026},
author = {Li, Z and Roberts, A and Nimse, U and Vu, L and Fei, J},
title = {Protocol for generating endogenous degron tags in essential transcription factors in human iPSCs via CRISPR-Cas9.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104602},
pmid = {42213769},
issn = {2666-1667},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Transcription Factors/genetics/metabolism ; Degrons ; *Gene Editing/methods ; Gene Knock-In Techniques/methods ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Precise genome engineering in human pluripotent stem cells remains inefficient, limiting endogenous fluorescent tagging needed to study phase-separated membraneless nuclear compartments. Here, we present a protocol for generating precise knockin alleles of essential genes in human induced pluripotent stem cells (iPSCs) via CRISPR-Cas9 editing. We describe steps for combining transient p53 inhibition, optimized transfection conditions, and fluorescence-activated cell sorting. This protocol enables recovery of viable edited clones that would otherwise be lost due to editing-induced stress or essential gene perturbation. For complete details on the use and execution of this protocol, please refer to Li et al.[1].},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Transcription Factors/genetics/metabolism
Degrons
*Gene Editing/methods
Gene Knock-In Techniques/methods
Tumor Suppressor Protein p53/genetics

@article {pmid42226724,
year = {2026},
author = {Jong, U and Lim, D and Lee, SH},
title = {Prime editing updates: technological evolution, methodological expansion, and delivery strategies for in vivo applications.},
journal = {BMB reports},
volume = {59},
number = {6},
pages = {313-320},
pmid = {42226724},
issn = {1976-670X},
mesh = {*Gene Editing/methods/trends ; CRISPR-Cas Systems/genetics ; Humans ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Prime editing is a next-generation genome editing technology that enables precise base substitutions, insertions, and deletions at target genomic loci without inducing double-strand breaks or requiring exogenous donor DNA. This system operates through a fusion protein composed of Cas9 nickase and reverse transcriptase together with prime editing guide RNA; it has emerged as a precise genome editing platform that overcomes the limitations of conventional double-strand break-inducing CRISPR-Cas9 systems. Since first reported in 2019, diverse methodological improvements from PE1 to PE7 have been achieved, leading to rapid advances in editing efficiency, expansion of the editable target range, correction of large genomic regions, and development of in vivo delivery technologies. In this review, we comprehensively discuss the fundamental working mechanism of prime editing, its methodological evolution, recent expansion strategies, and delivery platforms for therapeutic applications, and provide perspectives for future development. [BMB Reports 2026; 59(6): 313-320].},
}

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

@article {pmid42264452,
year = {2026},
author = {Li, Y and Du, H and Chen, J and Xiao, J and Ma, Y and Zhang, Y and Wang, Q},
title = {The development of RPA-CRISPR/Cas12a assay for the detection of Pseudomonas plecoglossicida.},
journal = {FEMS microbiology letters},
volume = {373},
number = {},
pages = {},
doi = {10.1093/femsle/fnag067},
pmid = {42264452},
issn = {1574-6968},
support = {T2023328//Shanghai Agricultural Science and Technology Innovation Project/ ; //MOF/ ; },
mesh = {*Pseudomonas/genetics/isolation & purification ; Animals ; *Fish Diseases/microbiology/diagnosis ; *CRISPR-Cas Systems ; *Pseudomonas Infections/veterinary/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Perciformes/microbiology ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; Bacterial Proteins/genetics ; DNA, Bacterial/genetics ; Recombinases/metabolism ; },
abstract = {Pseudomonas plecoglossicida is the etiological agent of visceral white spot disease, which induces significant mortality in economically important fish such as the large yellow croaker. In this study, we integrated recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated detection to establish a fluorescence-based assay for rapid identification of P. plecoglossicida. The complete single-tube, two-stage RPA-CRISPR/Cas12a workflow can be performed within ~45 min. Using purified genomic DNA, the assay achieved an analytical detection limit of 1.65 copies μl-1 and showed no cross-reactivity with several other common fish pathogens. Its applicability was further evaluated using crude DNA extracts from spleen, liver, and kidney tissues of experimentally infected large yellow croakers. Overall, with its rapid turnaround, minimal equipment requirement, and high sensitivity, the RPA-CRISPR/Cas12a assay represents a promising diagnostic tool for rapid detection of P. plecoglossicida, thereby helping to control the spread of infection.},
}

*Pseudomonas/genetics/isolation & purification
Animals
*Fish Diseases/microbiology/diagnosis
*CRISPR-Cas Systems
*Pseudomonas Infections/veterinary/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
Perciformes/microbiology
Rapid Diagnostic Tests
Sensitivity and Specificity
Bacterial Proteins/genetics
DNA, Bacterial/genetics
Recombinases/metabolism

@article {pmid42265371,
year = {2026},
author = {Su, F and Dong, Y and Guo, R and Xie, H and Zhang, Y and Liu, J and Cao, X and Xie, H and Zhou, M and Sun, X and Wang, M and Zhang, J and Zhu, JK},
title = {Efficient prime editors for heritable multiplex precision genome editing in soybean.},
journal = {Nature plants},
volume = {12},
number = {6},
pages = {1252-1263},
pmid = {42265371},
issn = {2055-0278},
support = {32188102//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Glycine max/genetics ; Plants, Genetically Modified/genetics ; *Genome, Plant ; CRISPR-Cas Systems ; Genetic Engineering/methods ; RNA Editing ; },
abstract = {Prime editing (PE) is limited by low efficiency in dicot plants. Here we develop an optimized PE system for soybean, GmPEplus, by deleting the RNase H domain, introducing a V223A substitution within the reverse transcriptase domain, inserting a viral nucleocapsid protein between Cas9 and reverse transcriptase, and co-expressing a dominant-negative engineered allele of the endogenous GmMLH1. GmPEplus achieves editing efficiencies of up to 81.3% in stable transgenic lines. Subsequently, we show that nicking the non-edited strand using an additional sgRNA via the tRNA processing system enhances editing efficiency, and that optimizing its expression with an independent AtU6 cassette boosts efficiency by 13.1-fold. Importantly, a Csy4-mediated multiplex PE system (CMMPE) is established for simultaneous editing of 2-12 genes in soybean hairy roots and up to 3 genes in stable transgenic lines. GmPEplus and CMMPE offer powerful, versatile tools for precise, multiplex and heritable genome editing in soybean breeding.},
}

*Glycine max/genetics
Plants, Genetically Modified/genetics
*Genome, Plant
CRISPR-Cas Systems
Genetic Engineering/methods
RNA Editing

@article {pmid42267397,
year = {2026},
author = {Xie, X and Meng, X and Han, B and Zhang, X and Qiu, M and Zhang, J and Zhao, F and Jiang, Y and Zhang, X},
title = {A Near-Infrared Light-Driven Photoelectrochemical Biosensor Based on CRISPR/Cas12a for Highly Sensitive Detection of Pseudomonas fluorescens in Dairy Products.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {24},
pages = {19094-19104},
doi = {10.1021/acs.jafc.6c01081},
pmid = {42267397},
issn = {1520-5118},
mesh = {*Pseudomonas fluorescens/genetics/isolation & purification/radiation effects ; *Biosensing Techniques/methods/instrumentation ; *Electrochemical Techniques/methods/instrumentation ; *Dairy Products/microbiology/analysis ; CRISPR-Cas Systems ; Infrared Rays ; Food Contamination/analysis ; Gold/chemistry ; Limit of Detection ; },
abstract = {Pseudomonas fluorescens (P. fluorescens) is a psychrotrophic bacterium causing dairy spoilage. Plate count and PCR methods, as conventional detection standards, are either time-consuming or dependent on expensive instruments, restricting widespread application. Herein, a near-infrared (NIR)-driven photoelectrochemical (PEC) biosensor based on CRISPR/Cas12a was constructed by using SH-ssDNA/AuNPs/AgBiS2 as the working electrode. AgBiS2 possesses broad-spectrum absorption and an appropriate bandgap for efficient NIR utilization. In the presence of P. fluorescens, the CRISPR/Cas12a system was activated by the LAMP amplification products of the target DNA to perform the trans-cleavage activity toward the SH-ssDNA modified on the AuNPs/AgBiS2 electrode surface. This cleavage released SH-ssDNA from the electrode, reducing steric hindrance and accelerating interfacial electron transfer, thereby enhancing the PEC signal. The constructed biosensor achieved sensitive detection of P. fluorescens with a limit of detection (LOD) of 1 CFU/mL in the range of 10[1]-10[8] CFU/mL, offering a reliable strategy for detecting the psychrotrophic bacteria in dairy products.},
}

*Pseudomonas fluorescens/genetics/isolation & purification/radiation effects
*Biosensing Techniques/methods/instrumentation
*Electrochemical Techniques/methods/instrumentation
*Dairy Products/microbiology/analysis
CRISPR-Cas Systems
Infrared Rays
Food Contamination/analysis
Gold/chemistry
Limit of Detection

@article {pmid42274413,
year = {2026},
author = {Liu, L and Wu, M and Sun, X and Lu, X and Yuan, C and Li, CY},
title = {Hierarchical Amplification-Interlinked CRISPR-Cas14a Luminescent Biosensor Coupled with Portable Photonic Crystal Biochip-Boosted Time-Delayed Signaling for the Diagnosis of Pediatric Mycoplasma pneumoniae Pneumonia.},
journal = {Analytical chemistry},
volume = {98},
number = {24},
pages = {18288-18298},
doi = {10.1021/acs.analchem.6c03088},
pmid = {42274413},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Pneumonia, Mycoplasma/diagnosis ; *Mycoplasma pneumoniae/isolation & purification/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; Luminescent Measurements ; Nucleic Acid Amplification Techniques ; Limit of Detection ; Luminescence ; Rapid Diagnostic Tests ; },
abstract = {Mycoplasma pneumoniae pneumonia (MPP) is a respiratory infection that readily propagates within pediatric populations, and thus, the development of a robust assay approach for its timely diagnosis is of clinical importance. In this contribution, we build a hierarchical amplification-interlinked CRISPR-Cas14a luminescent biosensor coupled with portable photonic crystal biochip-boosted time-delayed signaling to fulfill this need. To first perform highly sensitive detection, an initial DNA walker module and a later rolling circle amplification module are integrated to construct a hierarchical amplification, which is then utilized to interlink CRISPR-Cas14a systems. To further improve applicability in complicated biosamples, a time-delayed signaling actualized by afterglow luminescence is introduced to circumvent background autoluminescence from biological media. After employing a photonic crystal self-assembled from polystyrene nanospheres to manufacture a portable biochip, the naturally attenuated afterglow luminescence is dramatically boosted. Leveraging these strategies, our biosensing platform achieves a limit of detection as low as 2.27 fM for the M. pneumoniae sequence while maintaining good specificity. Moreover, the luminescent biosensor permits precise analysis of targets in throat swab samples from a pediatric cohort (n = 250) comprising severe and mild MPP patients as well as healthy controls, based on which a convolutional neural network-implemented deep learning model is finally established for accurate automated disease stratification, thereby holding great potential as an efficient and promising diagnostic tool.},
}

*Biosensing Techniques/methods
*Pneumonia, Mycoplasma/diagnosis
*Mycoplasma pneumoniae/isolation & purification/genetics
Humans
*CRISPR-Cas Systems/genetics
Luminescent Measurements
Nucleic Acid Amplification Techniques
Limit of Detection
Luminescence
Rapid Diagnostic Tests

@article {pmid42299927,
year = {2026},
author = {Xia, Q and Guo, R and Xiao, J and Zeng, L and Huang, Y and Li, J and Chen, X and Huang, T and Xiao, B and Miao, C and Liu, W and Liang, QL and Lau, CH and Zhu, H},
title = {A rapid, multiplex, one-pot CRISPR/Dx system for visual detection of influenza A, influenza B, and respiratory syncytial viruses.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {24},
pages = {5052-5060},
doi = {10.1039/d6ay00425c},
pmid = {42299927},
issn = {1759-9679},
mesh = {*Influenza B virus/isolation & purification/genetics ; *Influenza A virus/isolation & purification/genetics ; Humans ; *CRISPR-Cas Systems ; *Respiratory Syncytial Viruses/isolation & purification/genetics ; Rapid Diagnostic Tests ; Respiratory Syncytial Virus Infections/diagnosis/virology ; },
abstract = {The detection of Influenza A Virus (IAV), Influenza B Virus (IBV), and Respiratory Syncytial Virus (RSV) presents significant diagnostic challenges due to the high similarity of clinical symptoms with other respiratory infections, leading to the need for multiplexed, rapid testing. Herein, we have established a one-pot, multiplex CRISPR/Dx detection system based on reverse transcription-recombinase polymerase amplification (RT-RPA) and CRISPR/Cas12a. It completes the detection within 30 minutes at a constant temperature of 40 °C, with the ability to detect 10 copies per µL of IAV, 10 copies per µL of IBV, and 8 copies per µL of RSV. No cross-reactivity between these respiratory viruses was observed. When equipped with our customized miniature device, it allows visual fluorescence readout without specialized equipment. Compared with conventional RT-qPCR and two-tube RT-RPA-CRISPR/Cas12a approaches, this one-pot detection system offers a simplified workflow and shorter detection time and enables visual detection, making it especially suitable for point-of-care testing and field deployment. In essence, our CRISPR/Dx system provides a novel and practical molecular diagnostic strategy for rapid and multiplex detection of respiratory pathogens to improve patient management, rational antiviral use, and epidemic control.},
}

*Influenza B virus/isolation & purification/genetics
*Influenza A virus/isolation & purification/genetics
Humans
*CRISPR-Cas Systems
*Respiratory Syncytial Viruses/isolation & purification/genetics
Rapid Diagnostic Tests
Respiratory Syncytial Virus Infections/diagnosis/virology

@article {pmid42325637,
year = {2026},
author = {Adegoke, SC and Karim, MA and Jr, MC and Yao Yawlui, IS and LaJeunesse, D},
title = {Advancements in Technologies Targeting Horizontal Gene Transfer(?)Routes to Control Drug Resistance Evolution.},
journal = {ACS bio & med chem Au},
volume = {6},
number = {3},
pages = {210-236},
pmid = {42325637},
issn = {2694-2437},
abstract = {The global rise of multidrug-resistant (MDR) bacteria poses a major public health crisis, threatening the effectiveness of modern medicine. Traditional antibiotic development struggles to keep pace with bacterial evolution, largely due to the rapid dissemination of antibiotic resistance genes via horizontal gene transfer (HGT). HGT mechanisms both canonical and noncanonical enable bacteria to acquire resistance traits defining species and even special challenges. In this review, we cover the current understanding of HGT in spreading antibiotic resistance and explore possible strategies to control HGT and slow the spread of antimicrobial resistance. Recent advances highlight the potential of synthetic competence inhibitors, advanced oxidation processes (AOPs), CRISPR-Cas technologies, gene drives, and antiplasmids to disrupt horizontal gene flow and mitigate resistance evolution. Despite promising laboratory results, challenges remain in translating these approaches into clinical and environmental applications. Blocking HGT could complement antimicrobial stewardship programs and traditional antibiotic therapies by curbing the emergence of new resistant strains at their genetic roots. By targeting the foundational mechanisms of resistance acquisition, these strategies offer a proactive pathway to extend the efficacy of existing antibiotics and prevent a "postantibiotic" era. Ongoing research into bacterial pathogenesis, genome defense systems, and innovative gene-editing technologies will be critical to developing effective, scalable solutions for managing MDR infections worldwide.},
}

@article {pmid42326420,
year = {2026},
author = {Alzahrani, AJ},
title = {Bacteriophage therapy against multidrug resistant bacterial infections demonstrates clinical advances and engineering innovations between 2020-2026.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1865548},
pmid = {42326420},
issn = {1664-302X},
abstract = {PURPOSE: The global crisis of antimicrobial resistance has reached critical levels, with multi-drug resistant (MDR) bacterial pathogens threatening to render conventional antibiotics ineffective. This mini review synthesizes recent evidence from 2020 to 2026 on bacteriophage therapy against MDR bacteria, examining clinical applications, engineering advances, mechanistic insights, and emerging technologies.

METHODS: A comprehensive literature search was conducted across Embase, Scopus, and Cochrane Library databases, supplemented by PubMed, Google Scholar, and ArXiv searches, focusing on phage therapy for MDR bacterial infections published between 2020 and 2026.

RESULTS: Recent developments include expanded clinical experience through specialized phage centers, sophisticated genetic engineering techniques including CRISPR-based systems, successful compassionate-use programs, and innovative combination therapies with antibiotics. Clinical reports demonstrate safety and preliminary efficacy signals in selected refractory infections, though randomized controlled data remain limited. Engineering advances have produced phage-delivered CRISPR antimicrobials, hybrid delivery platforms, and synthetic phage particles that expand therapeutic capabilities.

CONCLUSION: While challenges remain in regulatory standardization, scalable manufacturing, and resistance management, the field has demonstrated significant progress toward clinical translation. The convergence of synthetic biology, personalized medicine approaches, and growing clinical evidence positions phage therapy as a viable complementary strategy in the fight against MDR bacterial infections.},
}

@article {pmid42328080,
year = {2026},
author = {Singh, AP and Haider, S and Sawarkar, A and Karki, S and Atta, K and Al-Zharani, M and Barasarathi, J and Rebouh, NY and Ahmed, ZFR},
title = {Programmable nanocarriers for precision plant engineering: converging nanotechnology, CRISPR, and next-generation breeding.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1845328},
pmid = {42328080},
issn = {1664-462X},
abstract = {The convergence of nanotechnology and genome editing in plant sciences is redefining modern precision breeding through efficient, transgene free, tissue culture independent pathways for genetic improvement in crops. Conventional breeding and transgenic tools are limited to genotype dependency, inefficient gene delivery, unpredictable transgene insertions, thereby restricting their application in elite germplasm. Nanoparticles-mediated gene delivery systems have revolutionized the genetic transformation in plants through targeted and transgene free delivery of CRISPR/Cas ribonucleoproteins (RNPs), DNA, and RNA into plant cells, while minimizing genome interference. Nanocarriers are the engineered delivery systems wherein the material component is a nanoparticle. DNA-free delivery refers to the absence of exogenous DNA during editing, whereas transgene free plants are those that do not retain integrated foreign DNA after regeneration. Firstly, this review summarizes current progress in designing nanocarriers, including lipid, polymeric, mesoporous silica nanoparticles, carbon-based nanoparticles, layered double hydroxides, and DNA-based nanoparticles; harnessing the function of their physicochemical traits in modulating plant cellular uptake, cargo stability, controlled delivery, and tissue specific targeting in plants. Secondly, the broad-spectrum roles of nano particles in genome editing, crop protection via RNA interference, organelle-targeted modifications are discussed, stressing transgene free approaches to mitigate somaclonal variation and regulatory concerns to foster public acceptance. The integration of nano-mediated delivery with speed breeding, meristem transformation, multiplexed editing in elite germplasm is proposed as an approach for prompt trait stacking and validation. Thirdly, the collaborative roles of experts in the field of nanotechnology, plant breeding, plant physiology, and agronomy are mentioned for mitigating multifaceted climatic effects and glitches. Moreover, current challenges including nanotoxicity, scalability and field translation, regulatory concerns, and public perception are also discussed. While nanocarrier mediated delivery shows strong potential for improving plant genome engineering, current evidence is largely confined to controlled experimental systems, and significant challenges remain before routine integration into breeding pipelines becomes feasible.},
}

@article {pmid42328450,
year = {2026},
author = {Corno, C and Costantino, M and Pettinari, P and Mirra, L and Stucchi, S and Arrighetti, N and Perta, N and Di Muccio, G and Robin, M and Beretta, GL and Corna, E and Carenini, N and Cleris, L and Colombo, D and Luison, E and Ciniselli, CM and Lecchi, M and Verderio, P and Figini, M and Linder, S and Tosi, D and La Teana, A and D'Arcy, P and Perego, P},
title = {USP18 Impacts Cisplatin Resistance in Ovarian Cancer Cells by Modulating DNA Repair.},
journal = {International journal of biological sciences},
volume = {22},
number = {11},
pages = {5780-5798},
pmid = {42328450},
issn = {1449-2288},
mesh = {Female ; *Cisplatin/therapeutic use/pharmacology ; Humans ; *Ovarian Neoplasms/metabolism/drug therapy/genetics ; *DNA Repair/genetics/drug effects ; *Drug Resistance, Neoplasm/genetics ; Cell Line, Tumor ; Animals ; *Ubiquitin Thiolesterase/metabolism/genetics ; Mice ; CRISPR-Cas Systems ; Antineoplastic Agents/therapeutic use/pharmacology ; DNA Damage ; },
abstract = {Deubiquitinases (DUBs) are proteases with emerging roles in cancer, yet their contribution to drug resistance in ovarian cancer remains underexplored. Ovarian cancer patients often fail to benefit from platinum-based therapy, highlighting the need to identify novel factors driving drug resistance. Thus, we performed a CRISPR/Cas9 screen targeting the DUB family to identify genes essential for cisplatin-resistant ovarian carcinoma cell survival. CRISPR/Cas9 DUB knockout screens, preclinical pharmacology approaches, RNA sequencing, proteomic analyses, computational tools, surface plasma resonance were applied. We identified USP18 as a survival factor in cisplatin-resistant ovarian cancer cells. USP18 expression was elevated at the mRNA and protein levels across five cisplatin-resistant variants. Knockdown and CRISPR/Cas9 editing of USP18 sensitized cells to cisplatin, coinciding with impaired repair of cisplatin-induced DNA damage. Enhanced sensitivity to cisplatin was evident from studies in mice. RNA-seq of USP18 RNA interfered and edited cells revealed the modulation of pathways including DNA repair. A peptide-based USP18 inhibitor suppressed growth of cisplatin-resistant cells, supporting USP18 role in sustaining their growth. We identified USP18 as a novel mediator of cisplatin resistance in ovarian cancer, acting through DNA repair modulation. Targeting USP18 may offer a therapeutic strategy to improve outcomes in platinum-resistant ovarian cancer.},
}

Female
*Cisplatin/therapeutic use/pharmacology
Humans
*Ovarian Neoplasms/metabolism/drug therapy/genetics
*DNA Repair/genetics/drug effects
*Drug Resistance, Neoplasm/genetics
Cell Line, Tumor
Animals
*Ubiquitin Thiolesterase/metabolism/genetics
Mice
CRISPR-Cas Systems
Antineoplastic Agents/therapeutic use/pharmacology
DNA Damage

@article {pmid42328522,
year = {2026},
author = {Wang, W and Zheng, S and Xiang, G and Feng, T and Peng, Y and Wu, Y and Du, X and Zhu, P and Ru, Y and Zhang, J and Wu, S and Han, H and Huang, T and Cao, G and Zheng, H and Zhao, Y and Wang, H and Zhang, R},
title = {Robust production of heavy-chain-only antibodies in mice by CRISPR/Cas mediated in situ modification of IgH locus.},
journal = {National science review},
volume = {13},
number = {11},
pages = {nwag270},
pmid = {42328522},
issn = {2053-714X},
}

@article {pmid42328613,
year = {2025},
author = {Selim, HMRM and Gomaa, FAM and Alshahrani, MY and Aboshanab, KM},
title = {Role of CRISPR-Cas system as a new approach in fighting the antimicrobial resistance of bacterial and viral pathogens.},
journal = {Infectious diseases & immunity},
volume = {5},
number = {2},
pages = {127-137},
pmid = {42328613},
issn = {2693-8839},
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)-Caspase (Cas) system acts as a natural defense of bacteria against invasion by mobile genetic elements, such as plasmids, transposons, and bacteriophages. The review discusses the different classes and types of CRISPR-Cas systems in terms of principles of their action, limitations, and future perspectives. Also, the role of the CRISPR-Cas system as a new arsenal in fighting multidrug-resistant pathogens and clinically relevant pathogenic viruses and up-to-date clinical trials have been discussed and highlighted. Moreover, the utilization mode, regulation, and the link of CRISPR-Cas to quorum sensing for targeting cell chromosome or antimicrobial-resistant gene(s) of some clinically relevant pathogens, including Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa have been discussed. The information provided in this review can act as a platform for researchers for the future use of the CRISPR-Cas system as a smart antimicrobial agent for combating infections caused by life-threatening pathogens, particularly those with limited therapeutic options.},
}

@article {pmid41061927,
year = {2026},
author = {Sun, H and Zou, J and Tu, S and Luo, D and Xiao, R and Du, Y and Xiong, C and Xie, S and Liu, H and Jin, M and Chen, H and Zhou, H},
title = {Genome-wide CRISPR screen identifies STK11 as a critical regulator of sialic acid clusters important for influenza A virus attachment.},
journal = {Journal of advanced research},
volume = {85},
number = {},
pages = {1207-1222},
doi = {10.1016/j.jare.2025.09.059},
pmid = {41061927},
issn = {2090-1224},
mesh = {Animals ; *Protein Serine-Threonine Kinases/genetics/metabolism ; Virus Replication ; Swine ; *Influenza A virus/physiology ; CRISPR-Cas Systems ; *N-Acetylneuraminic Acid/metabolism ; *Virus Attachment ; AMP-Activated Protein Kinase Kinases ; Dogs ; Mice ; *Orthomyxoviridae Infections/virology/genetics ; Host-Pathogen Interactions ; Humans ; },
abstract = {INTRODUCTION: Swine influenza virus (SIV) is a highly contagious respiratory pathogen in pigs that causes substantial economic losses in the pig industry. Importantly, pigs act as "mixing vessels" for diverse influenza A viruses (IAVs), facilitating the emergence of novel pandemic strains through reassortment, which represents a continuous global public health threat. IAV replication relies heavily on host cellular machinery, underscoring the importance of elucidating virus-host protein interactions for the development of targeted antiviral therapeutics.

OBJECTIVES: This study aims to identify host genes required for SIV replication via a genome-wide CRISPR screen and elucidate the mechanism by which STK11 modulates viral replication.

METHODS: A pig genome-scale CRISPR knockout (PigGeCKO) screen was performed in newborn pig trachea (NPTr) cells to identify host genes required for SIV replication. Candidate genes were further validated by generating knockout cell lines using CRISPR/Cas9-mediated gene editing, followed by assessing their impact on IAV replication. The specific lifecycle stage regulated by STK11 and its mechanistic role in viral attachment were determined via Western blotting, confocal microscopy, transmission electron microscopy, and stimulated emission depletion (STED) imaging. In vivo validation of STK11 knockdown effects on IAV replication was conducted in BALB/c mice treated with STK11-targeting siRNA, with outcomes evaluated by survival analysis, body weight monitoring, lung viral titers quantification, immunofluorescence, and histopathology.

RESULTS: STK11 promotes replication of different IAV subtypes in vitro, and STK11 knockdown significantly suppresses SIV replication in vivo. Mechanistically, STK11 depletion impairs viral attachment by altering the organization of sialic acid clusters, mediated through reduced intracellular actin stress fibers via inhibition of RhoA signaling pathway.

CONCLUSION: We identify STK11 as a novel regulator of IAV attachment and elucidate its mechanistic role in facilitating viral entry. These findings highlight the potential of STK11 to serve as an ideal antiviral target against IAV infection.},
}

Animals
*Protein Serine-Threonine Kinases/genetics/metabolism
Virus Replication
Swine
*Influenza A virus/physiology
CRISPR-Cas Systems
*N-Acetylneuraminic Acid/metabolism
*Virus Attachment
AMP-Activated Protein Kinase Kinases
Dogs
Mice
*Orthomyxoviridae Infections/virology/genetics
Host-Pathogen Interactions
Humans

@article {pmid41692276,
year = {2026},
author = {Piseddu, I and Endres, R and Lanzl, F and Hammann, L and Bérouti, M and Thaler, M and Fahr, L and Fischer, H and Varlamova, V and Gärtig, J and Nixdorf, D and Layritz, P and Marx, C and Hörth, C and Witte, C and Bulut, A and Illig, D and Senz, AM and Holdt, L and Regel, I and Gottschlich, A and Subklewe, M and Mayerle, J and Anz, D and Kobold, S and Linder, A and Hornung, V},
title = {STING Ablation in T Cells Is Required for the Efficacy of STING Agonists in CAR-T Cell Immunotherapy of Pancreatic Cancer.},
journal = {Gastroenterology},
volume = {171},
number = {1},
pages = {50-65},
doi = {10.1053/j.gastro.2026.01.031},
pmid = {41692276},
issn = {1528-0012},
mesh = {Animals ; *Pancreatic Neoplasms/therapy/immunology/pathology/genetics/metabolism ; STING Protein ; *Membrane Proteins/agonists/genetics/metabolism ; *Immunotherapy, Adoptive/methods ; Humans ; *Receptors, Chimeric Antigen/immunology/metabolism/genetics ; Cell Line, Tumor ; cGAS-STING Signaling Pathway ; Mice ; Xenograft Model Antitumor Assays ; T-Cell Exhaustion ; Tumor Microenvironment/immunology ; Interferon-gamma/metabolism ; *T-Lymphocytes/immunology/metabolism/transplantation/drug effects ; Tumor Necrosis Factor-alpha/metabolism ; Cell Proliferation ; CRISPR-Cas Systems ; Signal Transduction ; Female ; Immunologic Memory ; },
abstract = {BACKGROUND & AIMS: Chimeric antigen receptor (CAR) T cells have shown great potential in hematological cancers, but lack efficacy in solid tumors, highlighting the need for novel strategies. Stimulator of interferon genes (STING) activation was shown to inflame the tumor microenvironment, but combination of STING agonists and CAR-T cells might be limited by detrimental outcomes of T cell-intrinsic STING activation. In this study, we evaluated the potential of combining STING agonists and CAR-T cells in the context of pancreatic cancer.

METHODS: We assessed the synergy of CRISPR-Cas9-edited CAR-T cells and the STING agonist diABZI within a T cell exhaustion model in vitro and both xenograft and syngeneic mouse models in vivo.

RESULTS: Combination of STING-ablated CAR-T cells and diABZI resulted in enhanced cancer cell killing, increased CAR-T cell proliferation, reduced exhaustion, and expansion of an effector-memory phenotype in vitro. Mechanistically, superior CAR-T cell functionality required genetic ablation of STING in CAR-T cells and was dependent on cancer cell-intrinsic STING signaling on STING-agonistic treatment. Moreover, we identified a synergistic feedback loop comprising the T cell-secreted cytokines interferon-γ and tumor necrosis factor, which prime STING signaling within cancer cells, thereby potentiating the outcomes of cancer cell-intrinsic STING activation in inducing ameliorated CAR-T cell states. Ultimately, we could demonstrate that combination of STING deficient CAR-T cells and diABZI was able to provide enhanced tumor control in both xenograft and syngeneic mouse models. This was accompanied by increased intratumoral CAR-T cell numbers and reprogramming of the tumor microenvironment in vivo.

CONCLUSIONS: Our findings suggest that STING deficient CAR-T cells stand to benefit from STING agonists to improve CAR-T cell therapy for immune-deprived cancers such as pancreatic cancer.},
}

Animals
*Pancreatic Neoplasms/therapy/immunology/pathology/genetics/metabolism
STING Protein
*Membrane Proteins/agonists/genetics/metabolism
*Immunotherapy, Adoptive/methods
Humans
*Receptors, Chimeric Antigen/immunology/metabolism/genetics
Cell Line, Tumor
cGAS-STING Signaling Pathway
Mice
Xenograft Model Antitumor Assays
T-Cell Exhaustion
Tumor Microenvironment/immunology
Interferon-gamma/metabolism
*T-Lymphocytes/immunology/metabolism/transplantation/drug effects
Tumor Necrosis Factor-alpha/metabolism
Cell Proliferation
CRISPR-Cas Systems
Signal Transduction
Female
Immunologic Memory

@article {pmid41886459,
year = {2026},
author = {Yang, M and Chen, G and Xiao, J and Zhang, X and Hu, Z and Zhou, B},
title = {Protocol for enhancing Cas9 efficiency and fidelity through structure-guided phosphate-locking loop engineering.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104452},
pmid = {41886459},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Phosphates/chemistry/metabolism ; DNA/genetics/chemistry/metabolism ; *Protein Engineering/methods ; },
abstract = {The phosphate-locking loop (PLL), stabilizing Cas9-DNA interactions, is a key target for optimizing efficiency and specificity. Here, we present a protocol for enhancing Cas9 efficiency and fidelity through structure-guided PLL engineering. We describe steps for identifying PLL engineering targets through sequence alignment and structural analysis, constructing variants via inverse PCR, evaluating efficiency using amplicon sequencing, and assessing specificity through Genome-wide Unbiased Identification of DSBs Evaluated by sequencing (GUIDE-seq (GUIDE-seq). This protocol provides a generalizable framework for Cas9 engineering across orthologs. For complete details on the use and execution of this protocol, please refer to Yang et al.[1].},
}

*CRISPR-Cas Systems/genetics
*Phosphates/chemistry/metabolism
DNA/genetics/chemistry/metabolism
*Protein Engineering/methods

@article {pmid41903144,
year = {2026},
author = {Zhou, Z and Zhu, S and Pan, D and Lee, HY and Liu, N},
title = {Protocol to identify SINE-VNTR-Alu regulators using genome-wide screening in human K562 cells.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104468},
pmid = {41903144},
issn = {2666-1667},
mesh = {Humans ; K562 Cells ; CRISPR-Cas Systems/genetics ; *DNA Transposable Elements/genetics ; Genome, Human/genetics ; },
abstract = {The composite transposon SINE-VNTR-Alu (SVA) is hominid-specific and composed of five parts, but the regulatory mechanism of SVA transcription is still unclear. Here, we present a protocol to identify SVA regulators using genome-wide screening in human K562 cells. We describe steps for constructing an SVA-GFP reporter and performing genome-wide CRISPR-Cas9 screening in human K562 cells to identify genes that control SVA transcription. This protocol provides a representative procedure of genome-wide screening for transposon regulators. For complete details on the use and execution of this protocol, please refer to Zhou et al.[1].},
}

Humans
K562 Cells
CRISPR-Cas Systems/genetics
*DNA Transposable Elements/genetics
Genome, Human/genetics

@article {pmid41957359,
year = {2026},
author = {Wang, Y and Zhao, Y and Hu, J and Wang, Z and Pattarayan, D and Li, S and Zhang, Y and Wang, X and Wang, Y and Xie, W and Zhang, M and Yang, D},
title = {CRISPR activation screens identify oncogenic lncRNAs that are susceptible to CDK4/6 inhibitor treatment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41957359},
issn = {2041-1723},
support = {R01CA255196//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01CA222274//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01 CA255196/CA/NCI NIH HHS/United States ; R01CA272866//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01 CA282704/CA/NCI NIH HHS/United States ; R01CA282704//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {Humans ; *Cyclin-Dependent Kinase 4/antagonists & inhibitors/metabolism/genetics ; *Cyclin-Dependent Kinase 6/antagonists & inhibitors/metabolism ; *RNA, Long Noncoding/genetics/metabolism ; *Breast Neoplasms/genetics/drug therapy/pathology/metabolism ; Piperazines/pharmacology/therapeutic use ; Cell Line, Tumor ; Pyridines/pharmacology/therapeutic use ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; Cell Proliferation/drug effects/genetics ; Animals ; Estrogen Receptor alpha/metabolism/genetics ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The roles of long non-coding RNAs (lncRNAs) in tumorigenesis and therapeutic response remain largely unknown. Here we perform genome-wide and focused CRISPR activation screens to identify lncRNAs regulating palbociclib response in breast cancer cells. A synchronized two-stage proliferation screen not only characterizes tumor growth-regulating lncRNAs, but also reveals a strong negative correlation between lncRNA-mediated regulation of tumor proliferation and CDK4/6 inhibitor sensitivity. By integrating CRISPRa screen results with drug response data from 815 cancer cell lines, we identify and functionally validate that TENM3-AS1, LINC01117, and ENSG00000226706 can increase breast cancer sensitivity to CDK4/6i while promoting tumor proliferation. In breast cancer patients, all three lncRNA signatures are associated with CDK4/6 inhibitor response. Mechanistically, we have shown that lncRNA TENM3-AS1 is a potential ERα-interacting lncRNA, and its regulation of CDK4/6 inhibitor sensitivity is dependent on ERα expression. Our integrated strategy characterizes oncogenic lncRNAs as potential therapeutic biomarkers for CDK4/6 inhibitor treatment in cancer.},
}

Humans
*Cyclin-Dependent Kinase 4/antagonists & inhibitors/metabolism/genetics
*Cyclin-Dependent Kinase 6/antagonists & inhibitors/metabolism
*RNA, Long Noncoding/genetics/metabolism
*Breast Neoplasms/genetics/drug therapy/pathology/metabolism
Piperazines/pharmacology/therapeutic use
Cell Line, Tumor
Pyridines/pharmacology/therapeutic use
Female
Gene Expression Regulation, Neoplastic/drug effects
*Protein Kinase Inhibitors/pharmacology/therapeutic use
Cell Proliferation/drug effects/genetics
Animals
Estrogen Receptor alpha/metabolism/genetics
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41986330,
year = {2026},
author = {Nguyen, QV and Lan, YJ and Chang, JC and Shih, HA and Faustine, J and Chen, CC and Ho, SY and Cheng, CW and Chao, TL and Lin, S},
title = {Genome-wide CRISPR screens in primary human natural killer cells identify countermeasures against immunosuppressive environment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41986330},
issn = {2041-1723},
mesh = {Humans ; *Killer Cells, Natural/immunology/metabolism ; *CRISPR-Cas Systems ; Dinoprostone/metabolism ; Tumor Microenvironment/immunology/genetics ; Interleukin-2/metabolism ; Cell Proliferation ; Clustered Regularly Interspaced Short Palindromic Repeats ; Protein Serine-Threonine Kinases/genetics ; Signal Transduction ; },
abstract = {Natural killer (NK) cells are promising effectors for cancer immunotherapy, but their efficacy is limited by immunosuppressive tumor microenvironments. To uncover strategies for enhancing NK cell function, we establish a CRISPR loss-of-function screening platform for primary human NK cells by combining BaEVRless-pseudotyped lentiviral transduction of sgRNA libraries with Cas9 protein electroporation. This platform enables genome-scale interrogation of gene function in non-transformed NK cells. Kinome-focused and genome-wide screens identify key regulators of NK cell proliferation, cytotoxicity, and resistance to prostaglandin E2 (PGE2)-mediated suppression. STK17B deletion enhances NK cell expansion, while loss of CCDC53 boosts degranulation and cytotoxicity. We also uncover the CRL5 complex-including RNF7, UBE2F, and CISH-as critical inhibitors of IL-2 signaling and effector function under PGE2 stress. These findings establish a scalable platform for CRISPR-based functional genomics in primary NK cells and reveal engineering targets to enhance NK cell persistence and efficacy in tumor microenvironments.},
}

Humans
*Killer Cells, Natural/immunology/metabolism
*CRISPR-Cas Systems
Dinoprostone/metabolism
Tumor Microenvironment/immunology/genetics
Interleukin-2/metabolism
Cell Proliferation
Clustered Regularly Interspaced Short Palindromic Repeats
Protein Serine-Threonine Kinases/genetics
Signal Transduction

@article {pmid42012982,
year = {2026},
author = {Djamshidi, M and Tanida, R and Heshmatzad, K and Krowicki, H and Hill, A and Yang, Y and Riabowol, K},
title = {Protocol for enhancing CRISPR-Cas9 genome editing using histone deacetylase inhibition and engineered virus-like particle delivery.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104493},
pmid = {42012982},
issn = {2666-1667},
mesh = {*CRISPR-Cas Systems/genetics ; *Histone Deacetylase Inhibitors/pharmacology ; *Gene Editing/methods ; Humans ; *Virion/genetics ; },
abstract = {We present a 10-fold faster, accurate, and more efficient (FAME)-CRISPR-Cas9 gene editing workflow utilizing histone deacetylase inhibitor (HDACi)-mediated chromatin relaxation and engineered virus-like particle (eVLP) delivery of Cas9. We describe steps for optimizing HDACi concentration, euchromatinization timing, and Cas9 delivery/expression to improve CRISPR-Cas9 editing efficiency and efficacy. This protocol can eliminate the need for single-cell cloning and reduce experimental timelines up to 10-fold while minimizing HDACi-mediated toxicity. For complete details on the use and execution of this protocol, please refer to Djamshidi et al.[1].},
}

*CRISPR-Cas Systems/genetics
*Histone Deacetylase Inhibitors/pharmacology
*Gene Editing/methods
Humans
*Virion/genetics

@article {pmid42025580,
year = {2026},
author = {Shukla, SK and Singh, A and Yadav, R and Kumar, A},
title = {Advances in molecular diagnostic strategies during the SARS-CoV-2 pandemic.},
journal = {Expert review of molecular diagnostics},
volume = {26},
number = {4},
pages = {293-308},
doi = {10.1080/14737159.2026.2665263},
pmid = {42025580},
issn = {1744-8352},
mesh = {Humans ; *COVID-19/diagnosis/epidemiology/virology ; *SARS-CoV-2/genetics/isolation & purification ; *Molecular Diagnostic Techniques/methods ; Pandemics ; COVID-19 Testing/methods ; COVID-19 Nucleic Acid Testing/methods ; Rapid Diagnostic Tests ; Pandemic Preparedness ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: The SARS-CoV-2 pandemic provided critical insights into pandemic preparedness. The community spread can be slowed down or contained through effective, rapid, and robust diagnosis of infected individuals.

AREA COVERED: During the pandemic, substantial advances were made in developing rapid and cost-effective diagnostic approaches. Self-collected gargle samples offer clear advantages over conventional NSP/OPS methods by reducing reliance on trained personnel and personal protective equipment. Colorimetric assays further improve accessibility, enabling rapid, instrument-free, and visually interpretable detection at low cost. CRISPR-based diagnostics present a promising alternative to RT-PCR, facilitating scalable mass screening with reduced technical dependence. Concurrently, optimization of RT-PCR workflows-particularly through minimization of pre-PCR steps-can enhance speed and affordability. The integration of digital technologies and artificial intelligence further leverages diagnostic capabilities. Despite this, improved regulatory frameworks and resilient supply chains are critical for ensuring scalable, equitable access, and effective pandemic preparedness.

EXPERT OPINION: Global efforts were made to develop sensitive, rapid, cost-effective, and noninvasive technologies to identify the pandemic virus; however, variations in sensitivity/specificity and limited sample size validation hampered their utility in routine diagnostics. The COVID-19 pandemic has ended, but global efforts are still needed to combat the early infection of subsequent waves or similar disease waves.},
}

Humans
*COVID-19/diagnosis/epidemiology/virology
*SARS-CoV-2/genetics/isolation & purification
*Molecular Diagnostic Techniques/methods
Pandemics
COVID-19 Testing/methods
COVID-19 Nucleic Acid Testing/methods
Rapid Diagnostic Tests
Pandemic Preparedness
CRISPR-Cas Systems

@article {pmid42033729,
year = {2026},
author = {He, Z and Chen, S and Huang, A and Jiang, H},
title = {Protocol for the generation of DDT signaling reporter cell line for CRISPR screening.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104523},
pmid = {42033729},
issn = {2666-1667},
mesh = {*Signal Transduction/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Genes, Reporter/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Line ; High-Throughput Nucleotide Sequencing/methods ; },
abstract = {Cells respond to perturbations through signaling pathways that often induce characteristic transcriptional changes. Here, we present a protocol for generating a double death trap (DDT) reporter that converts pathway activity into a binary survival-death outcome. The DDT reporter employs puromycin resistance and FKBP12(F36V)-ΔCaspase9 constructs driven by pathway-specific response elements. We describe the steps for DDT reporter plasmid construction, cell line generation, and genome-wide CRISPR screening in DDT cells. We further detail procedures for next-generation sequencing (NGS) sample preparation, sequencing, and downstream analysis. For complete details on the use and execution of this protocol, please refer to He et al.[1].},
}

*Signal Transduction/genetics
Humans
*CRISPR-Cas Systems/genetics
*Genes, Reporter/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Cell Line
High-Throughput Nucleotide Sequencing/methods

@article {pmid42033731,
year = {2026},
author = {Sarmah, H and Iannello, G and Wantroba, R and Wu, C and Idiarte, J and Munoz, A and Déry, O and Rubio de la Torre, E and Lebayle, E and Seminara, S and Sirabella, D and Huynh, S and Issa, R and Kissner, M and Corneo, B},
title = {Protocol for clonal isolation of gene-edited hiPSCs using droplet and microfluidic sorting.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104518},
pmid = {42033731},
issn = {2666-1667},
mesh = {Humans ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Microfluidics/methods ; Electroporation ; *Cell Separation/methods ; },
abstract = {Genetically engineered human induced pluripotent stem cells (hiPSCs) are vital for disease modeling and drug discovery, yet generating clonal lines efficiently post-editing remains challenging. Here, we present a protocol to generate clonal hiPSC lines after gene editing using either electrostatic droplet- or microfluidics-based sorting platforms. We describe steps for culturing hiPSCs, CRISPR-RNP electroporation, single-cell sorting, and expansion of gene-edited clones. Using this protocol, we generated over 100 clonal lines across seven knock-in/knock-out experiments, demonstrating broad utility and reproducibility. For additional details on the use and execution of this protocol, please refer to Patel et al.[1].},
}

Humans
*Induced Pluripotent Stem Cells/cytology/metabolism
*Gene Editing/methods
CRISPR-Cas Systems/genetics
*Microfluidics/methods
Electroporation
*Cell Separation/methods

@article {pmid42044055,
year = {2026},
author = {Li, X and Gao, X and Dong, J and Gu, T and Li, Q and Wang, L and Deng, F and Hou, J and Hou, C and Huo, D},
title = {Aptamer Nanoswitch-Mediated Lock-Expose Mechanism Enables Highly Sensitive In Vitro Detection and Precise In Situ Membrane Imaging of HER2 Protein.},
journal = {ACS sensors},
volume = {11},
number = {5},
pages = {4014-4023},
doi = {10.1021/acssensors.6c00371},
pmid = {42044055},
issn = {2379-3694},
mesh = {Humans ; *Erb-b2 Receptor Tyrosine Kinases/metabolism/analysis/genetics ; *Aptamers, Nucleotide/chemistry/metabolism/genetics ; Cell Line, Tumor ; Limit of Detection ; *Biosensing Techniques/methods ; Neoplastic Cells, Circulating ; Fluorescent Dyes/chemistry ; CRISPR-Cas Systems ; Breast Neoplasms ; },
abstract = {Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer correlates with high aggressiveness, an increased recurrence rate, and poor survival, holding significant diagnostic value. In this work, a HER2-specific aptamer (Apt) was engineered into a Apt nanoswitch (hApt) and revealed the interaction of HER2-aptamers through molecular docking and quantified the binding energy and dynamic behavior through molecular dynamics simulations. A lock-expose mechanism was designed by combining the target-induced conformational switch of hApt with template-prefabricated rolling circle amplification (rRCA), forming a T-hApt-rRCA sandwich structure. Coupled with CRISPR/Cas12a and fluorescent probes, this sensor enabled highly sensitive detection, with a linear range of 10 fg/mL to 10 ng/mL and a limit of detection of 1.42 fg/mL. Using HUVEC, A549, MCF-7, and SK-BR-3 cell lines to model HER2 heterogeneity in circulating tumor cells (CTCs) enabled in situ imaging, differentiation, and quantitative detection of membrane HER2 expression, thereby providing direct visualization of expression levels and highlighting the translational promise of this approach. The accurate detection and clear differentiation between the eight healthy samples and the twenty-eight breast cancer patient samples further underscore the practical applicability of this sensing strategy.},
}

Humans
*Erb-b2 Receptor Tyrosine Kinases/metabolism/analysis/genetics
*Aptamers, Nucleotide/chemistry/metabolism/genetics
Cell Line, Tumor
Limit of Detection
*Biosensing Techniques/methods
Neoplastic Cells, Circulating
Fluorescent Dyes/chemistry
CRISPR-Cas Systems
Breast Neoplasms

@article {pmid42066814,
year = {2026},
author = {Nie, H and Zheng, Z and Fan, W and Yan, M and Shao, W and Meng, Y and An, D and Zhao, S and Yuan, L and Yang, J and Wang, H},
title = {Homozygous IbGBSS1 knockouts in hexaploid sweet potato enable amylose-free starch without a yield trade-off.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {369},
number = {},
pages = {113172},
doi = {10.1016/j.plantsci.2026.113172},
pmid = {42066814},
issn = {1873-2259},
mesh = {*Ipomoea batatas/genetics/metabolism/growth & development ; *Starch Synthase/genetics/metabolism ; *Amylose/metabolism ; *Starch/metabolism ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; Homozygote ; Polyploidy ; CRISPR-Cas Systems ; },
abstract = {Sweet potato (Ipomoea batatas) is a major crop and an important industrial starch source; however, its hexaploid genome has hindered the generation of complete gene knockouts. Because the amylose-to-amylopectin ratio determines starch functionality, the production of amylose-free (waxy) starch is of considerable interest for food, pharmaceutical, and industrial applications. It was hypothesized that the complete knockout of all six alleles of IbGBSS1, which encodes granule-bound starch synthase I, would abolish amylose biosynthesis without compromising plant growth or yield. CRISPR/Cas9 mutagenesis combined with the Hi-TOM high-throughput mutation detection platform was used to generate homozygous Ibgbss1 mutants with confirmed edits across all alleles. These mutants contained < 1% amylose and exhibited normal growth and unchanged yield under both greenhouse and field conditions. Physicochemical analyses showed that amylose-free starch displayed larger granules, an altered amylopectin chain-length distribution (reduced DP 6-12 and enriched > DP 36), and numerous surface pores. Differential scanning calorimetry indicated increased gelatinization onset and peak temperatures, along with higher gelatinization enthalpy. Transcriptome analysis revealed broad reprogramming of starch and sucrose metabolism, accompanied by increased accumulation of glucose, fructose, and sucrose in storage roots. These results demonstrate that IbGBSS1 is essential for amylose biosynthesis and establish a strategy for generating complete multi-allelic knockouts in hexaploid sweet potato. Amylose-free germplasm was obtained without a yield penalty, providing potential for food and industrial applications.},
}

*Ipomoea batatas/genetics/metabolism/growth & development
*Starch Synthase/genetics/metabolism
*Amylose/metabolism
*Starch/metabolism
Gene Knockout Techniques
*Plant Proteins/genetics/metabolism
Homozygote
Polyploidy
CRISPR-Cas Systems

@article {pmid42080968,
year = {2026},
author = {Yang, Y and Arro, J and Zou, C and Oravec, M and Reisch, B and Zhong, GY},
title = {First brassinosteroid-based dwarf mutant discovered and characterized in grapevine.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {5},
pages = {},
pmid = {42080968},
issn = {1432-2242},
mesh = {*Vitis/genetics/growth & development ; *Brassinosteroids/biosynthesis ; Phenotype ; Chromosome Mapping ; *Cytochrome P-450 Enzyme System/genetics ; Mutation ; *Plant Proteins/genetics/metabolism ; Genes, Plant ; CRISPR-Cas Systems ; Genetic Association Studies ; },
abstract = {In this study, we investigated the genetic control of dwarfism in naturally occurring dwarf mutant lines of grapevines. Through trait-segregation and marker-trait association analyses, we identified a major locus on Chromosome 14 tightly associated with the dwarf trait. Subsequently, we conducted a bulked RNA-seq analysis, fine mapped the dwarf trait and identified VviBR6OX1, a cytochrome P450 enzyme involved in brassinosteroid synthesis, as a candidate gene for the observed dwarfism. RNA-seq sequence analyses revealed two in-frame deletions in the gene: a 12-bp deletion in exon 1 and a 9-bp deletion in exon 4. A survey of the two indels in Vitis germplasm suggested that the 9-bp deletion is most likely the cause of dwarfism in the mutant. We recreated similar dwarf grapevines by knocking out VviBR6OX1 using CRISPR/Cas9 gene editing and confirmed VviBR6OX1's role in controlling vine architecture. Additionally, we observed several vines with an extreme compact dwarf phenotype and determined that the compact dwarfing phenotype was a result of simultaneous editing of a second BR6OX gene, VviBR6OX2. The discovery of BR-related dwarfism in grapevine provides an important genetic avenue for developing desirable vine architecture for various breeding purposes.},
}

*Vitis/genetics/growth & development
*Brassinosteroids/biosynthesis
Phenotype
Chromosome Mapping
*Cytochrome P-450 Enzyme System/genetics
Mutation
*Plant Proteins/genetics/metabolism
Genes, Plant
CRISPR-Cas Systems
Genetic Association Studies

@article {pmid42084357,
year = {2026},
author = {Stone, S and Elsharkawy, A and Patterson, LD and Natekar, JP and Jiang, H and Viktoria Hyddmark, E and Camarillo, J and Zhao, G and Kumar, M},
title = {A Novel Humanized Lethal Mouse Model of SARS-CoV-2-Associated Disease.},
journal = {Journal of medical virology},
volume = {98},
number = {5},
pages = {e70959},
doi = {10.1002/jmv.70959},
pmid = {42084357},
issn = {1096-9071},
mesh = {Animals ; *Disease Models, Animal ; Humans ; *COVID-19/pathology/virology ; Mice ; *SARS-CoV-2/pathogenicity ; *Angiotensin-Converting Enzyme 2/genetics/metabolism ; Mice, Inbred C57BL ; Lung/pathology/virology ; Mice, Transgenic ; Female ; Gene Knock-In Techniques ; Brain/pathology/virology ; Viral Load ; CRISPR-Cas Systems ; Cytokines ; },
abstract = {Mice are valuable small animal models for studying SARS-CoV-2 pathogenesis. Ancestral SARS-CoV-2 strains do not efficiently utilize murine Ace2, rendering wild-type mice resistant to infection. Although human ACE2 transgenic models such as K18-hACE2 have provided critical insights, they express multiple copies of both murine and human ACE2, and random transgene insertion can result in non-physiological receptor expression. To overcome these limitations, we employed a human ACE2 knock-in (hACE2-KI) model in which the murine Ace2 coding sequence is replaced with human ACE2 using CRISPR/Cas9 technology, generating an mAce2-null background. This design allows human ACE2 expression under endogenous regulatory control while eliminating murine Ace2 expression, thereby providing a more physiologically relevant platform to investigate SARS-CoV-2 pathogenesis and evaluate therapeutic and preventive strategies. In this study, SARS-CoV-2-associated disease was evaluated and compared among hACE2-KI, K18-hACE2 and C57BL/6J mice. Mice were intranasally inoculated with 10[5] plaque-forming units of SARS-CoV-2 lineages B.1 or B.1.351. Both hACE2-KI and K18-hACE2 mice developed severe disease after SARS-CoV-2 infection. Following infection with B.1, both K18-hACE2 mice and hACE2-KI mice exhibited significant weight loss and mortality, with high viral loads detected in the lungs and brain. hACE2-KI mice infected with SARS-CoV-2 B.1.351 also showed significant weight loss and viral loads, resulting in high mortality. The pathology and inflammatory response within the lungs and brain of infected hACE2-KI mice revealed robust expression of viral nucleocapsid protein, histopathological changes, and upregulated cytokine and chemokine responses. Together, these findings demonstrate that the hACE2-KI knock-in mouse model supports robust SARS-CoV-2 replication and mimics severe COVID-19 disease.},
}

Animals
*Disease Models, Animal
Humans
*COVID-19/pathology/virology
Mice
*SARS-CoV-2/pathogenicity
*Angiotensin-Converting Enzyme 2/genetics/metabolism
Mice, Inbred C57BL
Lung/pathology/virology
Mice, Transgenic
Female
Gene Knock-In Techniques
Brain/pathology/virology
Viral Load
CRISPR-Cas Systems
Cytokines

@article {pmid42096138,
year = {2026},
author = {Zhang, Q and Li, Y and Li, Y and Han, L and Yan, J and Zhan, M and Liu, T and Ke, P and Wang, Q and Huang, X},
title = {Anti-CRISPR Protein Regulates CRISPR/Cas12a Fusogenic-Nanovesicle-Based Platform for Extracellular Vesicle-Encapsulated Non-Nucleic Acid Target In-Vesicle Detection.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {65},
number = {26},
pages = {e6795217},
doi = {10.1002/anie.6795217},
pmid = {42096138},
issn = {1521-3773},
support = {2024ZD0533400//National Key Research and Development Program of China/ ; 2024ZD0533401//National Key Research and Development Program of China/ ; 0720240230//Guangdong Special Support Program Health and health talents List Project of Provincial Health Commission/ ; 2024A1515030043//Natural Science Foundation of Guangdong Province/ ; 2026A1515010500//Natural Science Foundation of Guangdong Province/ ; SZ2022QN09//"Young Talents Program" of Guangdong Academy of Traditional Chinese Medicine/ ; SKLKY2024B0011//State Key Laboratory of Traditional Chinese Medicine Syndrome Projects/ ; 2023A03J0755//Guangzhou Science and Technology Plan Projects/ ; 2023B110008//Guangdong Provincial Clinical Research Guangdong Provincial Clinical Research Center for Laboratory Medicine/ ; 2025JY-A1004//Guangdong Provincial Clinical Research Guangdong Provincial Clinical Research Center for Laboratory Medicine/ ; //Science and Technology Innovation Center of Guangzhou University of Chinese Medicine/ ; },
mesh = {*Extracellular Vesicles/chemistry/metabolism ; *CRISPR-Cas Systems ; Humans ; *CRISPR-Associated Proteins/metabolism ; *Bacterial Proteins/metabolism ; Aptamers, Nucleotide/chemistry/metabolism ; *Endodeoxyribonucleases/metabolism ; },
abstract = {The detection of non-nucleic acid targets encapsulated in extracellular vesicles (EVs) faces two major challenges: (1) difficulties in efficient isolation and the risk of content degradation, and (2) the low abundance of target molecules encapsulated in EVs always leads to failed signal transduction and inadequate output signal intensity. To overcome these limitations, we propose a high-efficiency in-vesicle analysis strategy that integrates targeting probe delivery and regulation by protein signal amplification. By applying aptamer-mediated membrane fusion and "locked-activated" CRISPR-Cas12a-AcrVA1 (LACA) for protein signal regulation, we fabricated a yly12-aptamer-functionalized self-assembled nanovesicle which encapsulate LACA-system (yly12-lipo@Cas12a nanovesicle) as an in-vesicle bioanalytical platform. Leveraging the high specificity of the aptamer and the regulatory function of AcrVA1 in selectively modulating Cas12a activity, the platform enables highly specifiec and sensitive detection, offering advantages of simple operation and versatility across platforms within only 2.5 h. Clinical analysis demonstrated effective differentiation between patients and healthy controls, yielding high diagnostic performance with an AUC of 0.965. The proposed platform shows great potential for EV-carrying protein biomarker analysis and has broad prospects for the disease's diagnosis in clinical settings.},
}

*Extracellular Vesicles/chemistry/metabolism
*CRISPR-Cas Systems
Humans
*CRISPR-Associated Proteins/metabolism
*Bacterial Proteins/metabolism
Aptamers, Nucleotide/chemistry/metabolism
*Endodeoxyribonucleases/metabolism

@article {pmid42017197,
year = {2026},
author = {Wafer, R and Tandon, P and Minchin, J},
title = {A quantitative in vivo CRISPR-imaging platform identifies regulators of hyperplastic and hypertrophic adipose morphology in zebrafish.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {42017197},
issn = {2050-084X},
support = {BB/X009467/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; REA PhD Studentship/BHF_/British Heart Foundation/United Kingdom ; },
mesh = {Animals ; *Zebrafish/genetics ; *Adipose Tissue/metabolism/pathology ; Hyperplasia/genetics ; Humans ; Hypertrophy/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Zebrafish Proteins/genetics/metabolism ; Adipocytes ; CRISPR-Cas Systems ; },
abstract = {Adipose tissues exhibit a remarkable capacity to expand, regress, and remodel in response to energy status. The cellular mechanisms underlying adipose remodelling are central to metabolic health. Hypertrophic remodelling - characterised by the enlargement of existing adipocytes - is associated with insulin resistance, type 2 diabetes, and cardiovascular disease. In contrast, hyperplastic remodelling - in which new adipocytes are generated - is linked to improved metabolic outcomes. Despite its clinical importance, the regulation of hypertrophic and hyperplastic adipose morphology remains poorly understood. Here, we integrate human transcriptomic data with a quantitative CRISPR-imaging platform in zebrafish to identify regulators of adipose morphology. We developed an image-based phenotyping pipeline that captures lipid droplet size, number, and spatial patterning, and applied generalised additive modelling to quantify hyperplastic versus hypertrophic morphology signatures. Using this platform, we conducted an F0 CRISPR screen targeting 25 candidate genes and identified three that induced hypertrophic morphology (txnipa, mmp14b, and foxp1b) and an additional candidate that altered total adiposity (kazna). For functional validation, we generated stable loss-of-function alleles for both zebrafish foxp1 paralogues. Spatial analysis along the anterior-posterior axis revealed that foxp1b mutants display developmental hypertrophy but profoundly blunted adaptive responses to high-fat diet (~68% reduction across all spatial zones), while foxp1a mutants show normal baseline morphology but disrupted spatial patterning of diet-induced hypertrophy. Together, these findings establish a scalable CRISPR-imaging platform for in vivo genetic screening of adipose morphology and reveal distinct roles for Foxp1 paralogues in developmental patterning and adaptive responses to dietary challenge in adipose tissue.},
}

Animals
*Zebrafish/genetics
*Adipose Tissue/metabolism/pathology
Hyperplasia/genetics
Humans
Hypertrophy/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Zebrafish Proteins/genetics/metabolism
Adipocytes
CRISPR-Cas Systems

@article {pmid41742419,
year = {2026},
author = {Zhu, M and Yuan, J and Meng, Q and Yu, J and Xu, X and Xu, M and Ren, X and Hu, Y and Wei, G and Jia, Z and Yuan, G and Zang, L and Liu, S and Yang, Y and Zheng, Y and Wang, J and Cong, T and Xie, W and Lan, X and Cong, L and Ma, T and Ding, S and Guo, W and Zhang, X and Li, Y},
title = {Minimizing far-extending chromatin perturbation in genome editing preserves stem cell identity.},
journal = {Cell stem cell},
volume = {33},
number = {3},
pages = {470-486.e14},
doi = {10.1016/j.stem.2026.01.015},
pmid = {41742419},
issn = {1875-9777},
mesh = {Animals ; *Chromatin/metabolism/genetics ; *Gene Editing/methods ; Mice ; Cell Differentiation ; *Neural Stem Cells/metabolism/cytology ; CRISPR-Cas Systems/genetics ; Mouse Embryonic Stem Cells/metabolism/cytology ; },
abstract = {Although CRISPR-Cas9 holds therapeutic promise, broader application demands an understanding of complications in vast non-coding regions. We found that CRISPR-Cas9 can cause premature differentiation of neural stem cells in vivo and mouse embryonic stem cells in vitro, even when cleavage occurred at distant sites tens of kilobases away from the nearest regulatory elements. To investigate this, we employed an integrated assay for transposase-accessible chromatin (ATAC)/RNA sequencing (AR-seq) approach and identified editing-induced chromatin accessibility changes, with their scale varying by cell type. Cells with stemness are most affected, experiencing perturbations that extend over a hundred kilobases. Furthermore, even local DNA perturbations can disrupt CTCF- and condensate-associated chromatin architecture, causing distal transcriptional rewiring and, ultimately, loss of stemness identity. To minimize chromatin perturbations and preserve cell identity, we refined gene-editing strategies, including distance-aware sgRNA design, pharmacological attenuation of DNA resection, and alternative editing systems. This work paves the way for the safer and broader application of genome-editing technologies.},
}

Animals
*Chromatin/metabolism/genetics
*Gene Editing/methods
Mice
Cell Differentiation
*Neural Stem Cells/metabolism/cytology
CRISPR-Cas Systems/genetics
Mouse Embryonic Stem Cells/metabolism/cytology

@article {pmid41744735,
year = {2026},
author = {Hu, X and Su, J and Song, S},
title = {CRISPR/Cas System-Based Biosensors.},
journal = {Biosensors},
volume = {16},
number = {2},
pages = {},
pmid = {41744735},
issn = {2079-6374},
abstract = {Over the past decade, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, originally identified as adaptive immune systems in bacteria and archaea that defend against invading nucleic acids, have revolutionized biological research [...].},
}

@article {pmid41747377,
year = {2026},
author = {Pan, MX and Lv, MM and Nie, YG and Su, M and Zha, CJ and Mei, RY and Ying, ZM},
title = {Ultrasensitive miRNA detection via magnetic bead-confined catalytic hairpin assembly enabling transcription-driven crRNA assembly and CRISPR/Cas12a activation.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118559},
doi = {10.1016/j.bios.2026.118559},
pmid = {41747377},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/analysis/isolation & purification/blood ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/chemistry/genetics ; DNA/chemistry/genetics ; Nucleic Acid Hybridization ; *Endodeoxyribonucleases/chemistry/genetics ; Limit of Detection ; Inverted Repeat Sequences ; Transcription, Genetic ; Catalysis ; Bacterial Proteins ; },
abstract = {The integration of CRISPR/Cas12a with catalytic hairpin assembly (CHA), a strategy that predominantly relies on CHA to generate dsDNA activators for direct Cas12a activation, has emerged as a powerful tool in molecular diagnostics. However, two major challenges remain: the strict protospacer adjacent motif (PAM) dependence of the dsDNA and background leakage from hairpin hybridization. Herein, we report a bead-confined platform that transcription mediates crRNA reassembly and template activation of Cas12a for ultrasensitive miRNA detection. The target-triggered CHA assembly dynamically constructed a T7 transcription template from three initially locked hairpins (H1, H2, and H3), which not only transcribed scaffold RNA but also hybridized with its own product to form a DNA/RNA complex that activates Cas12a. The integration of the split T7 promoter with CHA effectively suppressed background suppression and enhanced detection sensitivity. Additionally, the magnetic beads increase local concentration and reaction kinetics, collectively contributing to a substantially enhanced detection sensitivity. Moreover, a crRNA assembly strategy designed for transcription-powered Cas12a not only circumvents the conventional PAM-dependent dsDNA activation pathway of Cas12a but also enables self-supplied crRNA without requiring additional activators. We demonstrated that the biosensor exhibits exceptional sensitivity for miRNA-21 detection, achieving a limit of 65.3 aM. Furthermore, the practicality of this method was preliminarily confirmed through accurately quantifying target levels in cell lines and human serum. Our method presents a viable solution with transformative potential, designed to address complex challenges in contemporary diagnostic applications.},
}

*MicroRNAs/genetics/analysis/isolation & purification/blood
Humans
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/chemistry/genetics
DNA/chemistry/genetics
Nucleic Acid Hybridization
*Endodeoxyribonucleases/chemistry/genetics
Limit of Detection
Inverted Repeat Sequences
Transcription, Genetic
Catalysis
Bacterial Proteins

@article {pmid41747626,
year = {2026},
author = {Yoo, DH and Bayarsaikhan, D and Lee, J and Im, YS and Bayarsaikhan, G and Kang, HA and Lee, B and Kim, YO},
title = {Generation and characterization of SOX17-specific EGFP expressing human induced pluripotent stem cell line, KSCBi017-A-4, using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103943},
doi = {10.1016/j.scr.2026.103943},
pmid = {41747626},
issn = {1876-7753},
mesh = {Humans ; *SOXF Transcription Factors/metabolism/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; Cell Differentiation ; Endoderm/cytology/metabolism ; },
abstract = {We generated a human induced pluripotent stem cell (hiPSC) reporter line in which EGFP was inserted in-frame at the C-terminus of the endogenous SOX17 locus using CRISPR/Cas9-mediated homologous recombination. The targeted clone, KSCBi017-A-4, was isolated by puromycin selection and validated by PCR and Sanger sequencing. This SOX17-EGFP hiPSC line retains a normal karyotype and pluripotency and displays specific EGFP expression upon directed definitive endoderm differentiation. This reporter line provides a reliable tool for monitoring SOX17 expression during human endoderm specification.},
}

Humans
*SOXF Transcription Factors/metabolism/genetics
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Green Fluorescent Proteins/metabolism/genetics
Cell Line
Cell Differentiation
Endoderm/cytology/metabolism

@article {pmid41747766,
year = {2026},
author = {Cai, Y and Zhuang, L and Wang, Z and He, L and Li, X and Liu, BF and Li, T and Zhang, G and Zhou, H and Huang, X and Li, Y},
title = {Gravity-Driven Formation of Water-in-Wax Spheres for Efficient One-Pot CRISPR Diagnostics.},
journal = {ACS nano},
volume = {20},
number = {9},
pages = {8055-8067},
doi = {10.1021/acsnano.6c01232},
pmid = {41747766},
issn = {1936-086X},
mesh = {*Water/chemistry ; *Gravitation ; *Waxes/chemistry ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems ; Microspheres ; },
abstract = {Rapid, decentralized molecular diagnostics are urgently needed for effective infectious disease control. Here, we present "Wax-Sphere CRISPR" (WS-CRISPR), a paradigm-shifting platform for CRISPR-based diagnostics centered on a gravity-driven, interfacial phase-change self-encapsulation mechanism. This system fundamentally decouples bioreagent engineering from specific reaction vessels, transforming conventional, labor-intensive manual encapsulation into a standardized, physics-driven assembly process that generates discrete wax microspheres. Guided by fluid mechanics and interfacial thermodynamics, aqueous CRISPR droplets spontaneously traverse air/molten wax/ethanol to self-encapsulate and solidify, enabling standardized, high-throughput fabrication without manual wax handling. Upon temperature modulation, the wax phase change triggers sequential recombinase polymerase amplification (RPA) and CRISPR detection within a sealed, one-pot vessel. As a clinically oriented demonstration, WS-CRISPR enables multiplexed detection and risk stratification of all 14 high-risk HPV genotypes (HPV16/18 vs others) with a detection limit of 1 × 10[-18] M in under 30 min. Integrated with widely accessible devices─including a thermocycler, hand-held fluorescence reader, and microfluidic platform─the system demonstrated 97.4% sensitivity and 100% specificity across 70 clinical samples. By solving the engineering bottlenecks of scalability and universality, WS-CRISPR offers a robust tool for decentralized, large-scale pathogen surveillance.},
}

*Water/chemistry
*Gravitation
*Waxes/chemistry
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems
Microspheres

@article {pmid41748831,
year = {2026},
author = {Moreno, DS and Carvalho, JP and Murray, E and Colombo, NSR and Lamas, A and Cobas, AC and Hill, C and Azeredo, J and Domingues, L},
title = {Evaluation of the delivery of an anti-Listeria endolysin via CRISPR-Cas9 engineered probiotic Saccharomyces boulardii.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {41748831},
issn = {1432-0614},
mesh = {*Endopeptidases/genetics/pharmacology/metabolism ; *Probiotics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Saccharomyces boulardii/genetics/metabolism ; *Listeria monocytogenes/drug effects ; Bacteriophages/genetics/enzymology ; Listeriosis/prevention & control/microbiology ; Saccharomyces cerevisiae/genetics ; },
abstract = {Listeriosis is a foodborne infection caused by Listeria monocytogenes that causes febrile gastroenteritis and central nervous system infections and that can often lead to fatality. Upon consumption of contaminated food, Listeria is able to survive a number of gastrointestinal stressors, including competition with the host microbiota. The emergence of antibiotic-resistant clones of L. monocytogenes, together with the side effects of antibiotic treatment, highlights the need for alternatives or additives for its treatment and prevention. Saccharomyces boulardii is a probiotic yeast that is often used alongside antibiotics to minimize side effects since it is not affected by them as a result of its eukaryotic nature. Furthermore, it can be engineered to produce a wide range of molecules. We previously engineered Saccharomyces cerevisiae through CRISPR-Cas9 integration to produce Ply511, a bacteriophage endolysin active against L. monocytogenes, showing the potential of engineered yeast to produce endolysins for biocontrol. In this study, we extended this approach to the probiotic yeast S. boulardii and directly compared the two yeasts as secretion hosts for Ply511. Using a simulated human gastrointestinal environment, we evaluated their ability to retain endolysin activity and reduce L. monocytogenes levels. We then tested the cell extracts from both yeasts in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI), confirming a specificity for Listeria. Finally, we evaluated their activity in a simulated intestinal fermentation using fecal samples from human donors. Overall, this study demonstrates the potential of delivering endolysins to the gut via engineered probiotic S. boulardii. KEY POINTS: CRISPR-Cas9-engineered S. boulardii and S. cerevisiae were compared, both allowing the expression and activity of endolysin Ply511 against L. monocytogenes. Endolysin Ply511 retained its activity against L. monocytogenes in simulated gastrointestinal digestion and was specific against Listeria in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI). Using fecal samples from human donors, the anti-Listeria effect was reduced potentially due to the lower metabolic activity of S. boulardii and the higher competition with the intestinal microbiome.},
}

*Endopeptidases/genetics/pharmacology/metabolism
*Probiotics/metabolism
Humans
*CRISPR-Cas Systems
*Saccharomyces boulardii/genetics/metabolism
*Listeria monocytogenes/drug effects
Bacteriophages/genetics/enzymology
Listeriosis/prevention & control/microbiology
Saccharomyces cerevisiae/genetics

@article {pmid41750315,
year = {2026},
author = {Schulze, A and Kainz, K and Bauer, MA and Carmona-Gutierrez, D},
title = {Editing Candida: Origins and Advances of CRISPR Tools.},
journal = {Biomolecules},
volume = {16},
number = {2},
pages = {},
pmid = {41750315},
issn = {2218-273X},
support = {10.55776/P37278//FWF Austrian Science Fund/ ; not applicable//University of Graz/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Candida/genetics/pathogenicity ; Humans ; Candidiasis/microbiology ; },
abstract = {Pathogens causing candidiasis encompass a diverse group of ascomycetous yeasts that have become essential models for studying fungal adaptability, pathogenicity, and host-pathogen interactions. Although many candidiasis-promoting species exist as commensals within host microbiota, several have acquired virulence traits that enable opportunistic infections, positioning them as a leading cause of invasive fungal disease in humans. Deciphering the molecular and genetic determinants that underpin the biology of organisms responsible for candidiasis has long been a central objective in medical and molecular mycology. However, research progress has been constrained by intrinsic biological challenges, including noncanonical codon usage and the absence of a complete sexual cycle in diploid species, which have complicated traditional genetic manipulation. CRISPR-Cas9 genome editing has overcome many of these limitations, providing a precise, efficient, and versatile framework for targeted genomic modification. This system has facilitated functional genomic studies ranging from single-gene deletions to high-throughput mutagenesis, yielding new insights into the mechanisms governing virulence, antifungal resistance, and stress adaptation. Since its initial application in Candida albicans, CRISPR-Cas9 technology has been refined and adapted for other clinically and industrially relevant species, including Nakaseomyces glabratus (formerly referred to as Candida glabrata), Candida parapsilosis, and Candida auris. The present work provides an overview of the evolution of genetic approaches employed in research directed against candidiasis-associated species, with a particular focus on the development and optimization of CRISPR-based systems. It highlights how recent advancements have improved the genetic tractability of these pathogens and outlines emerging opportunities for both fundamental and applied studies in fungal biology.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Candida/genetics/pathogenicity
Humans
Candidiasis/microbiology

@article {pmid41751014,
year = {2026},
author = {Wang, Q and Zheng, L and You, G and Dong, H and Chen, S and Wang, S and Chen, S},
title = {Navigating the Complexity: Advancing Diagnostic Strategies for Avian Reovirus in Chinese Poultry.},
journal = {Animals : an open access journal from MDPI},
volume = {16},
number = {4},
pages = {},
pmid = {41751014},
issn = {2076-2615},
support = {XTCXGC2021018, XTCXGC2021012//the '5511' Collaborative Innovation Project of Fujian Academy of Agricultural Sciences, China/ ; },
abstract = {Avian reovirus (ARV) infections pose a significant and evolving threat to China's poultry industry, the world's largest. Diverse farming systems-ranging from modern intensive operations to traditional waterfowl-poultry polyculture-foster a unique ecological niche for ARV, defined by complex serotypic and genotypic diversity, marked regional variations, potential interspecies transmission between chickens and waterfowl, and recurrent co-infections. Collectively, these factors undermine the efficacy of conventional diagnostic approaches. This review systematically outlines the current epidemic landscape of ARV in China, highlighting the molecular characteristics of prevailing strains (particularly those from waterfowl) and their roles in diagnostic evasion. We critically assess the performance and limitations of existing diagnostic techniques (virus isolation, ELISA, PCR/qPCR) within the Chinese epidemiological setting. Furthermore, we discuss innovative technologies-including multiplex qPCR, CRISPR-Cas systems, and next-generation sequencing (NGS)-that offer potential for developing next-generation diagnostics tailored to China's specific challenges. Finally, we propose future directions, with an emphasis on standardization, data sharing, and interdisciplinary collaboration to bridge the gap between cutting-edge innovation and on-farm application for precise ARV control.},
}

@article {pmid41751536,
year = {2026},
author = {Hawkins, V and Rudiger, SR and McLaughlan, CJ and Kelly, JM and Lehnert, K and Jacobsen, JC and Handley, RR and Henare, K and Verma, PJ and Snell, RG},
title = {Foundations of an Ovine Model of Fragile X Syndrome.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751536},
issn = {2073-4425},
support = {3914//Curekids/ ; 20/259//Health Research Council of New Zealand/ ; },
mesh = {Animals ; *Fragile X Syndrome/genetics/pathology ; *Fragile X Messenger Ribonucleoprotein 1/genetics ; *Disease Models, Animal ; Female ; Sheep/genetics ; CRISPR-Cas Systems ; Male ; Gene Knockout Techniques ; Gene Editing ; },
abstract = {BACKGROUND: Fragile X Syndrome (FXS) is an X-linked neurodevelopmental disorder characterised by intellectual disability, developmental delays, anxiety, and social and behavioural challenges. Currently, no effective treatments exist to address the root cause of FXS. Mouse models are the most widely used for studying molecular pathogenesis and conducting preclinical treatment testing. However, therapeutic interventions that show promise in rodent models have yet to succeed in clinical trials. After evaluating the current models, we have developed an ovine model to address this clinical translation gap. We expect this model to more accurately reflect the human condition in brain size, structure, and neurodevelopmental trajectory. We aim to establish this model as a valuable preclinical platform for testing therapies for FXS.

METHODS: To generate the sheep model, we used CRISPR-Cas9 dual-guide editing to knock out the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene in ovine embryos.

RESULTS: Two founder animals were created, one ram (male) and one ewe (female), both of which carried FMR1 gene knockouts. The ewe carries inactivating mutations on both alleles, with the edits in both animals resulting in no detectable Fragile X Messenger Ribonucleoprotein (FMRP) as expected. Both founders have undergone molecular characterisation and basic health checks, with the female founder showing increased joint flexibility, a characteristic of FXS. The ram has been used for breeding, with the successful transmission of the edited allele to his offspring. Importantly, specific lamb cohorts for postnatal treatment testing can be produced efficiently utilising accelerated breeding methods and preimplantation selection.},
}

Animals
*Fragile X Syndrome/genetics/pathology
*Fragile X Messenger Ribonucleoprotein 1/genetics
*Disease Models, Animal
Female
Sheep/genetics
CRISPR-Cas Systems
Male
Gene Knockout Techniques
Gene Editing

@article {pmid41751548,
year = {2026},
author = {Machel Gica, NG and Gica, WT and La, H and Mi, Y and Zhou, Y},
title = {Precision Breeding for a Global Staple Food: A Systematic Review with a Strategic Framework for CRISPR-Cas Applications in Rice (Oryza sativa L.).},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751548},
issn = {2073-4425},
support = {2025GBJ002388//Chinese Government Scholarship Council/ ; },
mesh = {*Oryza/genetics ; *CRISPR-Cas Systems ; *Plant Breeding/methods ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; Genome, Plant ; Crops, Agricultural/genetics ; },
abstract = {Background: Rice is one of the world's main staple crops, and improving its productivity and resilience is important to achieving food security under varying climatic conditions. Objectives: This systematic review synthesizes the existing evidence on the application, technical limitations, and potential of the development of genome editing technologies (CRISPR-Cas) in rice (Oryza sativa L.), as well as presents a novel approach called the CRISPR Trait Prioritization and Readiness Framework (CTPRF). Methods: Peer-reviewed articles that reported applications of genome editing based on the CRISPR-Cas system in the genome of rice for trait improvement or functional genomics were identified through searches fromPubMed, Scopus, Web of Science, and Google Scholar with studies published between 2012 and 2025. Studies were screened on predefined inclusion criteria related to experimental validation, reporting of editing efficiency, and clear phenotypic results. Data on CRISPR systems, target genes, methods of delivery, traits modified, and phenotypic results were extracted and synthesized by comparative analysis. Results: A wide variety of different CRISPR systems have been used in rice, and our results indicate that NHEJ-mediated knockouts are effective in average genotypes with editing efficiencies in the range of 70-90%, but HDR and prime editing are still under 10%. The CTPRF is being introduced as a strategic decision support tool to evaluate traits from four dimensions: technical feasibility, phenotypic predictability, impact potential, and regulatory pathway. We use this framework for case studies in pioneering countries (USA, Japan, China) and show how it can be useful for guiding research investment and policy. Conclusions: CRISPR-Cas technologies have transformed rice breeding, but their introduction requires overcoming genotype-dependent barriers to transformation and negotiating patchwork regulatory environments. The CTPRF offers a roadmap for the acceleration of the development of climate-resilient and nutritious rice varieties for the action plan.},
}

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

@article {pmid41751560,
year = {2026},
author = {Lee, S and Park, S and Bang, H and Kim, SU and Park, YH and Wee, G and Chae, U and Kim, E},
title = {VPS35 Deficiency Markedly Reduces the Proliferation of HEK293 Cells.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751560},
issn = {2073-4425},
mesh = {Humans ; *Vesicular Transport Proteins/genetics/deficiency/metabolism ; *Cell Proliferation/genetics ; HEK293 Cells ; Apoptosis/genetics ; Mitochondrial Dynamics/genetics ; Mitochondria/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Background/Objectives: The retromer protein complex is involved in various physiological processes, especially endosomal trafficking, and its dysregulation has been linked to Alzheimer's disease and Parkinson's disease, as well as VPS35 knockout (KO), causing early embryonic lethality. We aimed to investigate the cellular consequences of VPS35 deficiency. Methods: To investigate the effects of VPS35 loss, we used CRISPR/Cas9 to generate VPS35 KO human embryonic kidney 293 (HEK293) cells. We analyzed changes in retromer component expression, cell proliferation, apoptosis, and mitochondrial dynamics using Western blotting, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and confocal microscopy. Results: VPS35 KO led to a significant reduction in cell proliferation and decreased expression of VPS29 and VPS26, both essential for retromer complex assembly. Consequently, retromer formation was impaired. Compared to control cells, KO cells exhibited elevated levels of cleaved caspase-3, poly(ADP-ribose) polymerase, cytochrome C, and p21, while the expression of Ki-67, CDK4, and cyclin D was reduced. Additionally, VPS35 deletion also promoted mitochondrial fragmentation, associated with increased expression of mitochondrial fission-related proteins. Finally, the rescue experiment using the human VPS35 gene confirmed that the recovery of VPS35 not only led to the recovery of the essential elements constituting the retromer but also the recovery of molecules related to the cell cycle, restoring cell death to a normal level. Conclusions: These findings suggest that VPS35 plays a critical role in cell growth and survival by modulating apoptosis, mitochondrial dynamics, and cell cycle progression.},
}

Humans
*Vesicular Transport Proteins/genetics/deficiency/metabolism
*Cell Proliferation/genetics
HEK293 Cells
Apoptosis/genetics
Mitochondrial Dynamics/genetics
Mitochondria/metabolism/genetics
CRISPR-Cas Systems

@article {pmid41751614,
year = {2026},
author = {Sun, Q and Guo, Y and Wang, L and Jia, L and Wei, P and Ma, S},
title = {CRISPR-Mediated Silkworm: The Oncoming Agricultural Revolutions and a Rising Model Organism.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751614},
issn = {2073-4425},
support = {32570591//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Bombyx/genetics/growth & development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Insect ; *Agriculture/methods ; Genomics/methods ; },
abstract = {The silkworm (Bombyx mori) is essential to sericulture and is also becoming a key model organism in genomics and agriculture. For decades, genetic studies of the silkworm were limited by inefficient and inflexible genome tools. CRISPR genome editing allows precise and scalable alterations to genes regulating development, physiology, and industrial traits. This review summarizes silkworm genome-editing breakthroughs, highlighting CRISPR's evolution from simple gene knockouts to large-scale genome-wide screening. We highlight how these advancements contribute to disease resistance, higher yields, and the development of new silk-based materials, as well as how they influence the development and growth rate of the sericulture. The creation of high-quality reference genomes, pangenomes, and genome-wide screening systems has made the silkworm a major model for integrating multiple biological datasets and approaches, such as genomic, transcriptomic, and proteomic. By considering the unique biological characteristics of the silkworm, this provides new insights for research on silk biology, piRNA synthetic biology, and hormonal signaling regulation. Finally, we examine new areas at the intersection of CRISPR, pangenomics, and artificial intelligence (AI) and suggest future paths for molecular breeding, pest control, and synthetic biology. Moreover, AI-assisted prediction of CRISPR outcomes is utilized to inform the design of targeted trait modifications, representing an approach to enhancing biomanufacturing efficiency and eco-friendly silk production. Together, these advances have made the silkworm a flexible genetic platform and an important part of sustainable agriculture and biomanufacturing.},
}

Animals
*Bombyx/genetics/growth & development
*CRISPR-Cas Systems
*Gene Editing/methods
Genome, Insect
*Agriculture/methods
Genomics/methods

@article {pmid41751840,
year = {2026},
author = {Begum, SN and Hasan, SK},
title = {Prime Editing Driven Functional Genomics: Bridging Genotype to Phenotype in the Post-Genomic Era.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751840},
issn = {1422-0067},
mesh = {Humans ; *Gene Editing/methods ; *Genomics/methods ; CRISPR-Cas Systems ; Phenotype ; Genotype ; Animals ; },
abstract = {The post-genomic era, defined by large-scale sequencing initiatives, has generated an unprecedented catalogue of human genetic variation. Yet, the vast majority of genetic variants remain classified as variants of uncertain significance or are located within poorly characterized non-coding regions, thereby hindering the effective translation of genomic data into meaningful biological understanding and clinical application. Bridging this genotype-to-phenotype gap requires precise, high-throughput functional genomics. Early CRISPR-Cas9 knockout and CRISPR interference/activation (CRISPRi/a) screens mapped gene-level functions but could not assess single nucleotide variants (SNVs). Bridging this genotype-to-phenotype gap demands precise, high-throughput functional genomics. Multiplexed assays of variant effect (MAVEs), like saturation genome editing, systematically test all possible mutations using CRISPR-Cas9 and donor libraries. Base editors allow targeted single-base changes without double-strand breaks but are limited in scope, while prime editing can introduce any small substitution, insertion, or deletion without double-strand breaks (DSBs) or donor templates. This review traces the evolution of functional screens from gene-level knockouts to saturation genomic editing (SGE), and highlights how prime editing is driving a new paradigm for the systematic functional characterization of thousands of variants across disease-relevant genes. We also detail the architecture, mechanism, and progressive optimization of PE systems and their delivery methods. Collectively, prime editing stands as a transformative platform poised to accelerate precision functional genomics and advance the diagnosis and treatment of genetic diseases.},
}

Humans
*Gene Editing/methods
*Genomics/methods
CRISPR-Cas Systems
Phenotype
Genotype
Animals

@article {pmid41751979,
year = {2026},
author = {Eskildsen, J and Dong, M and Hanak, T and Madsen, CK and Holme, I and Plaszkó, T and Vestergård, M and Nicolaisen, M and Thordal-Christensen, H and Brinch-Pedersen, H},
title = {Novel CRISPR/Cas9-Derived mlo Alleles in Barley: Resistance to Powdery Mildew and Microbiome Implications.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751979},
issn = {1422-0067},
support = {NNF19OC0056580//Novo Nordisk Foundation/ ; BarleyMicroBreed, 101060057//EU Horizon research and innovation/ ; },
mesh = {*Hordeum/genetics/microbiology ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Ascomycota/pathogenicity ; *Plant Proteins/genetics ; *Plant Diseases/microbiology/genetics ; Alleles ; *Microbiota/genetics ; Mutation ; Plant Roots/microbiology/genetics ; },
abstract = {Barley grown in temperate regions is often challenged by powdery mildew disease. An effective solution is mildew resistance locus o (mlo)-based resistance, which is monogenic, durable, and broad-spectrum. While the pleiotropic effects of mlo mutations on above-ground tissues are well documented, their impact on the root-associated microbiome remains underexplored. We utilized CRISPR/Cas9 to generate novel mlo mutant lines and evaluated their resistance to causal fungus Blumeria hordei. We further examined if mlo knockout has any impact on the overall root microbiome diversity and composition under field-like conditions and applied DESeq2 to compare the abundance of microbial taxa between mutants and wild type. We created five novel resistant mlo lines, including the first mutants with amino acid alterations in the protein's extracellular region. Mutant lines showed significantly reduced B. hordei colony formation (0.5-5%). While microbial alpha and beta diversity were not significantly altered, a few microbial taxa displayed time-dependent shifts in abundance. Overall, our study demonstrates the effectiveness of CRISPR/Cas9 in generating mlo-based resistance. Moreover, the study revealed functionally important residues in the protein's extracellular region. Finally, we present the first evidence of limited mlo-associated effects on root microbiome diversity and relative abundance of microbial taxa.},
}

*Hordeum/genetics/microbiology
*CRISPR-Cas Systems
*Disease Resistance/genetics
*Ascomycota/pathogenicity
*Plant Proteins/genetics
*Plant Diseases/microbiology/genetics
Alleles
*Microbiota/genetics
Mutation
Plant Roots/microbiology/genetics

@article {pmid41752145,
year = {2026},
author = {Fayed, S and Amer, S and Badawy, M and Bou Malhab, L and Omran, N and Khoder, G and Ghemrawi, R and Haider, M and Hamoudi, R and Harati, R},
title = {The Role of CRISPR and Its Therapeutic Applications in Glioblastoma.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41752145},
issn = {1422-0067},
support = {210111350//University of Sharjah/ ; 2201110368//University of Sharjah/ ; 23010902146//University of Sharjah/ ; VRI-20-10//ASPIRE Precision Medicine Research Institute Abu Dhabi/ ; },
mesh = {Humans ; *Glioblastoma/genetics/therapy ; *Brain Neoplasms/genetics/therapy ; *Gene Editing/methods ; Animals ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; },
abstract = {Glioblastoma multiforme (GBM) remains the most aggressive and treatment-refractory form of primary brain tumor in adults, characterized by rapid proliferation, intratumoral heterogeneity and resistance to current therapies. Despite therapeutic advancements in surgical resection, radiotherapy and chemotherapy, clinical outcomes remain poor, underscoring the need for innovative molecular strategies. This review examines the therapeutic potential of CRISPR/Cas9 genome-editing technologies in GBM, highlighting their ability to model, dissect and potentially correct the genetic alterations that drive GBM tumorigenesis. Key molecular targets, such as EGFR, PTEN, TP53, NF1 and PIK3CA, are discussed within the context of GBM's mutational and signaling landscape. We further outline emerging CRISPR applications in preclinical models, the current status of CRISPR-based clinical trials and the major barriers hindering translation, including off-target effects, immunogenicity and the challenge of delivering gene-editing systems across the blood-brain barrier. Particular emphasis is placed on delivery technologies, viral and non-viral vectors, including lipid nanoparticles, polymeric systems, inorganic nanocarriers and DNA nanostructures, which are rapidly evolving to improve precision, safety and CNS penetrance. Collectively, this review highlights CRISPR/Cas9 as a powerful tool whose integration with molecular neuro-oncology and precision medicine may ultimately shift GBM treatment toward more personalized and durable therapeutic interventions.},
}

Humans
*Glioblastoma/genetics/therapy
*Brain Neoplasms/genetics/therapy
*Gene Editing/methods
Animals
*CRISPR-Cas Systems
*Genetic Therapy/methods

@article {pmid41752921,
year = {2026},
author = {Panov, J and Elbert, A and Rosenthal, DS and Levi, M and Chumakov, K and Andino, R and Brodsky, L and Kaphzan, H},
title = {Spacio-Linear Screening for Ligand-Docking Cavities in Protein Structures: SLAM Algorithm.},
journal = {Life (Basel, Switzerland)},
volume = {16},
number = {2},
pages = {},
pmid = {41752921},
issn = {2075-1729},
support = {R01 DK127830/DK/NIDDK NIH HHS/United States ; N/A//Tauber Foundation/ ; },
abstract = {Identifying structurally similar ligand-binding sites in unrelated proteins can facilitate drug repurposing, reveal off-target effects, and deepen our understanding of protein function. A number of tools were developed for structural screening, but many of them suffer from limited sensitivity and scalability. Using a data bank of crystallized protein structures, we aimed to discover novel protein targets for a ligand by leveraging a known ligand-binding query protein with a resolved structure. Here, we present SLAM (Spacio-Linear Alignment of Macromolecules), a novel alignment-based algorithm that detects local 3D similarities between ligand-binding cavities or protein-exposed surfaces of query and target proteins. SLAM encodes spatial substructure neighborhoods into short linear sequences of physicochemically annotated atoms, then applies pairwise sequence alignment combined with distance-correlation scoring to identify high-fidelity structural matches. Benchmarking using the Kahraman-36 dataset demonstrated that SLAM outperforms the state-of-the-art ProBiS algorithm in true-positive rate for predicting ligand-docking compatibility. Furthermore, SLAM identifies candidate ligands that may inhibit functionally critical domains of CRISPR-Cas proteins and predicts novel binding partners of toxic per- and polyfluoroalkyl Substance (PFAS) compounds (PFOA, PFOS) with plausible mechanistic links to toxicity. In conclusion, SLAM is a robust computationally efficient and flexible structural screening tool capable of detecting subtle physicochemical compatibilities between protein surfaces, promising to accelerate target discovery in pharmacology and elucidate protein-ligand interactions in environmental toxicology.},
}

@article {pmid41753780,
year = {2026},
author = {Palanisamy, V and Bosilevac, JM and Barkhouse, DA and Velez, SE and Dass, SC},
title = {Unraveling the Coevolutionary Dynamics of Phage and Bacterial Protein Warfare Occurring in the Drains of Beef-Processing Plants.},
journal = {Microorganisms},
volume = {14},
number = {2},
pages = {},
pmid = {41753780},
issn = {2076-2607},
support = {2020-67017-30776//USDA-NIFA/ ; },
abstract = {Phages, the most abundant entities on Earth, exhibit a complex interplay with bacteria, especially within environmental biofilms, resulting in an ecological arms race. This study investigates the interaction between phages and bacteria in the drains of beef-processing plants using high-throughput sequencing and metagenomic analysis. Metagenomic data collected from 75 drain samples from beef-processing plants were analyzed to investigate phage-bacterial interactions. First, assembled contigs were screened to identify viral sequences, which were then taxonomically annotated to determine the viral composition, including phages. Functional annotation of these viral sequences provided information about the viral genes and their roles in bacterial interactions specifically associated with attack and counterattack of bacteria. In parallel, bacterial contigs were examined to identify genes associated with antiphage defense systems, providing insights into the strategies adapted by bacteria to resist phage infection. Taxonomic annotation of viral sequences from the bulk metagenomic data revealed the presence of phages targeting Pseudomonas, Klebsiella, and Enterococcus. The higher abundance of Pseudomonas phages aligns with our previous study, where Pseudomonas was identified as the dominant bacterial genus, suggesting potential copersistence of phages and their hosts. Functional annotation of phage contigs revealed infective and lysis-related genes, highlighting their potential role in bacterial attack. Conversely, bacterial contigs encoded antiphage defense systems, including CRISPR-Cas, restriction-modification, and other defense-related genes. The study also uncovered the presence of anti-CRISPR proteins in phages, suggesting a counterattack on the bacterial defense. These findings provide evidence for phage attack, bacterial defense, and phage counterattack and may showcase the ongoing coevolutionary arms race between phages and bacteria. While this evidence looks promising, these results remain preliminary and further studies are needed to validate these findings. Still, this study provides a foundational understanding of bacteria-phage coexistence in beef-processing plant drains and paves the way for further explorations of these intricate interactions and their possible applications in controlling pathogenic microorganisms within biofilms.},
}

@article {pmid41754535,
year = {2026},
author = {Li, H and Wang, R and Li, J and Duan, W and Liang, Y and Sun, Q and Zhou, J and Zhang, Y},
title = {SHFL Post-Transcriptionally Restricts Coxsackievirus A16 In Vitro and In Vivo.},
journal = {Viruses},
volume = {18},
number = {2},
pages = {},
pmid = {41754535},
issn = {1999-4915},
support = {GJJKJ-2024-ZY//National Disease Control and Prevention Administration Public Health Talent Training Support Project/ ; ZDGWNLJS25-36//National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases (NITFID)/ ; 2021YFC2302003//National Key Research and Development Program of China/ ; },
mesh = {Animals ; Virus Replication ; Mice ; Humans ; *Coxsackievirus Infections/virology/immunology ; Viral Load ; *Host-Pathogen Interactions ; *Enterovirus/physiology/genetics ; Cell Line, Tumor ; },
abstract = {Coxsackievirus A16 (CVA16), a major etiological agent of hand, foot, and mouth disease, is increasingly contributing to neurological complications, with no vaccines or virus-specific antivirals currently available. To identify CVA16-restricting host factors, we investigated the role of the interferon-stimulated gene shiftless (SHFL), previously implicated in the control of other RNA viruses. Using CRISPR-Cas 9, we generated SHFL knockout rhabdomyosarcoma cells and assessed viral replication, cytopathic effects, and replication stage dynamics. We evaluated disease progression and tissue injury in neonatal mice infected with a mouse-adapted CVA16 strain. SHFL expression was strongly induced during CVA16 infection and was inducible by exogenous interferon-β treatment, and its loss markedly increased infectious virus production, accelerated early replication, and exerted severe cytopathic effects. In vivo, SHFL deficiency led to rapid weight loss, pronounced neurological signs, increased viral burden across multiple tissues, and uniform mortality, together with high viral loads and extensive pathological damage in the central nervous system, lungs, and skeletal muscle. Transcriptomic analyses revealed SHFL-dependent modulation of adhesion- and mitogen-activated protein kinase-related pathways. Overall, our results suggest SHFL as a key determinant of host resistance to CVA16, acting mainly at the post-transcriptional stage to limit viral spread and tissue injury, and highlight SHFL-linked pathways as promising host-directed antiviral targets.},
}

Animals
Virus Replication
Mice
Humans
*Coxsackievirus Infections/virology/immunology
Viral Load
*Host-Pathogen Interactions
*Enterovirus/physiology/genetics
Cell Line, Tumor

@article {pmid41754561,
year = {2026},
author = {Wupori, K and Garnett, L and Bello, A and Strong, JE},
title = {CRISPR-Based Detection of Viral Hemorrhagic Fevers at the Point of Care.},
journal = {Viruses},
volume = {18},
number = {2},
pages = {},
pmid = {41754561},
issn = {1999-4915},
support = {8//Genomics Research and Development Initiative/ ; CSSP-2022-CP-2546//Canadian Safety and Security Program/ ; },
mesh = {Humans ; *Hemorrhagic Fevers, Viral/diagnosis/virology ; *Point-of-Care Systems ; Rapid Diagnostic Tests ; Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Ebolavirus/genetics/isolation & purification ; Point-of-Care Testing ; },
abstract = {Viral hemorrhagic fevers (VHFs) are highly lethal diseases that often present non-specific, influenza-like symptoms in their early stages, making clinical recognition and differentiation from other febrile illnesses difficult. This overlap underscores the critical need for diagnostic tests that are both sensitive and specific. Point-of-care (POC) diagnostic tests are an invaluable tool for detecting and controlling the spread of pathogens that threaten public health, such as VHFs, as these require fast, accurate diagnostics to ensure biosafety and appropriate mobilization of resources during outbreaks. Current molecular and serological diagnostic tests, while efficient and effective, lack the characteristics required of a POC test (POCT) to quickly and easily respond to a VHF outbreak while maintaining a low cost. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tests have gained popularity as POCTs due to their inherent attractive qualities, including high sensitivity and specificity, adaptability, low cost, quick turnaround time, and ease of use. However, studies on the development of CRISPR-based POC diagnostic tests for VHFs are limited. This review summarizes the current CRISPR-based POCTs for VHFs, including Ebola virus (EBOV), Lassa virus (LASV), Dengue virus (DENV), and Crimean-Congo hemorrhagic fever virus (CCHF). The isothermal pre-amplification methods commonly paired with CRISPR-based tests, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), are also discussed.},
}

Humans
*Hemorrhagic Fevers, Viral/diagnosis/virology
*Point-of-Care Systems
Rapid Diagnostic Tests
Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Ebolavirus/genetics/isolation & purification
Point-of-Care Testing

@article {pmid41755633,
year = {2026},
author = {Aguilar-González, A and Martos-Jamai, I and Ramos-Hernández, I and Molina-Estévez, FJ and Villao, NV and Puig-Serra, P and Rodríguez-Perales, S and Torres, R and Labun, K and Sánchez-Martín, RM and Díaz-Mochón, JJ and Martín, F},
title = {A novel Dual-guide CRISPR-Cas13 strategy improves specificity for single-nucleotide variant detection.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41755633},
issn = {1362-4962},
support = {MCIN)/AEI/10.13039/501100011033//Spanish Ministry of Science and Innovation/ ; PID2022-141065OB-I00//European Union Next Generation/ ; CV20-77741//Consejería de Economía y Conocimiento/Project/ ; PI21/00298//Instituto de Salud Carlos III/ ; PI24/00888//Instituto de Salud Carlos III/ ; RD21/0017/0004//Instituto de Salud Carlos III/ ; RD24/0014/0005//Instituto de Salud Carlos III/ ; PI-0236-2024//Consejería de Salud y Familias/ ; PIP-0004-2025//Consejería de Salud y Familias/ ; GeneHumdi-CA21113//European Cooperation in Science and Technology/ ; FPU22/03455//Spanish Ministry of Science, Innovation and Universities/ ; RHJ-0053-2025//Consejería de Salud y Familias, Junta de Andalucía/ ; //European Social Fund/ ; //Universidad de Granada/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *SARS-CoV-2/genetics ; *Polymorphism, Single Nucleotide/genetics ; *COVID-19/diagnosis/virology/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Proto-Oncogene Proteins p21(ras)/genetics ; Sensitivity and Specificity ; RNA, Viral/genetics ; },
abstract = {The emergence of CRISPR-Cas systems has transformed nucleic acid detection and manipulation. Cas13, a type VI CRISPR effector, targets RNA with high sensitivity through both cis (target RNA) and trans (collateral RNA) cleavage. This property enables the use of fluorescent reporters for sensitive diagnostics. However, Cas13's heightened sensitivity also leads to reduced specificity due to its susceptibility to single-nucleotide mismatches, potentially causing off-target effects. To overcome this limitation, we developed the first Dual-guide RNA system for Cas13 that improves mismatch discrimination and enhances target specificity. This system employs two distinct RNAs-dcrRNA and dtracrRNA-which cooperatively recognize the target and reduce off-target activity. In vitro experiments demonstrated robust cis- and trans-RNase activity, indicating efficient and specific cleavage. The system accurately detected SARS-CoV-2 RNA, distinguished KRAS G12D and G12C mutations, and differentiated mucocutaneous from cutaneous Leishmania sequences in analytical assays, with clinical validation confirming accurate detection of positive and negative samples. These results highlight the Dual-guide Cas13 platform's potential for precise, rapid, and reliable RNA detection. Overall, this approach represents a substantial advance over conventional Cas13 systems, offering improved specificity while maintaining clinically relevant sensitivity, and provides a generalizable tool for next-generation molecular diagnostics and precision RNA targeting and regulation.},
}

*CRISPR-Cas Systems/genetics
Humans
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*SARS-CoV-2/genetics
*Polymorphism, Single Nucleotide/genetics
*COVID-19/diagnosis/virology/genetics
*CRISPR-Associated Proteins/metabolism/genetics
Proto-Oncogene Proteins p21(ras)/genetics
Sensitivity and Specificity
RNA, Viral/genetics

@article {pmid41755634,
year = {2026},
author = {Hu, Z and Liu, Y and Han, Y and Li, M and Deng, K and Lu, X and Huang, Y and Liang, C and Wang, Y and Fu, Y and Xu, A},
title = {CRISPR/Cas9 screening with destabilized bicistronic fluorescent protein reporter revealed PABPN1 as a hub of regulators for alternative polyadenylation.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41755634},
issn = {1362-4962},
support = {2022YFA1103900//National Key Research and Development Program of China/ ; 32470586//National Natural Science Foundation of China/ ; 91942301//National Natural Science Foundation of China/ ; 81430099//National Natural Science Foundation of China/ ; 31930084//National Natural Science Foundation of China/ ; 32500472//National Natural Science Foundation of China/ ; 2023B1212060028//Guangdong Science and Technology Department/ ; },
mesh = {*Polyadenylation ; Genes, Reporter ; Humans ; *CRISPR-Cas Systems ; *Poly(A)-Binding Protein I/metabolism/genetics ; *Luminescent Proteins/genetics/metabolism ; DEAD-box RNA Helicases/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; Cell Proliferation ; HEK293 Cells ; Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics ; },
abstract = {Alternative polyadenylation (APA) is intricately intertwined with diverse biological processes. Efficient approaches for screening the regulatory factors of specific APA events are essential to elucidate their regulation mechanisms. Here, we first engineered a destabilized bicistronic fluorescent protein reporter (dBFPR) to enhance the sensitivity of APA detection. Then, we developed a robust high-throughput screening platform for APA regulators by integrating CRISPR/Cas9, dBFPR, and fluorescence-activated cell sorting. With this method, we successfully screened the library of RNA binding proteins and found that PTBP1, ELAVL1, and DDX3X play significant roles in regulating APA and promoting cell proliferation through interaction with PABPN1, suggesting that PABPN1 is an important hub for APA regulation.},
}

*Polyadenylation
Genes, Reporter
Humans
*CRISPR-Cas Systems
*Poly(A)-Binding Protein I/metabolism/genetics
*Luminescent Proteins/genetics/metabolism
DEAD-box RNA Helicases/metabolism/genetics
RNA-Binding Proteins/metabolism/genetics
Cell Proliferation
HEK293 Cells
Heterogeneous-Nuclear Ribonucleoproteins/metabolism/genetics

@article {pmid41755886,
year = {2026},
author = {Kaniganti, S and Saini, H and Chaitanya, AK and Hegde, N and Shah, P and Magar, ND and Rijal, R and Kaushik, JJ and Nanda, D and Sachan, S and Kumar, A and Bhoite, R and Jamedar, HR},
title = {CRISPR/Cas Genome Editing and Its Applications in Cereal Crop Improvement.},
journal = {Plant-environment interactions (Hoboken, N.J.)},
volume = {7},
number = {2},
pages = {e70133},
pmid = {41755886},
issn = {2575-6265},
abstract = {CRISPR/Cas-based genome editing has emerged as a transformative tool for precise genetic improvement of cereal crops. Recent advances in CRISPR technologies, including Cas9, Cas12, Cas13, base editing, and prime editing, have enabled targeted modification of genes and regulatory elements controlling yield, stress tolerance, and grain nutritional quality in major cereals such as rice, wheat, maize, and barley. This review summarizes current progress in CRISPR-mediated genome editing systems, delivery strategies, and representative applications in cereal crop improvement. Emphasis is placed on how genome editing reprograms enzymatic activities and biological pathways underlying complex agronomic traits rather than acting through single-gene effects. The review also discusses challenges related to trait complexity, regulatory considerations, and prospects for translating genome-edited cereal crops from laboratory research to field-level application. Collectively, this review highlights the potential of CRISPR/Cas genome editing as a powerful approach for developing high-yielding, resilient, and nutritionally improved cereal crops.},
}

@article {pmid41757335,
year = {2026},
author = {Shafiq, T and Khan, N and Kausar, T and Ahmed, W and Zhang, Z and Liang, Y and Duan, L},
title = {Red Blood Cell-Derived Extracellular Vesicles for Gene and RNA Therapeutics: Biological, Engineering, and Translational Challenges.},
journal = {International journal of nanomedicine},
volume = {21},
number = {},
pages = {579975},
pmid = {41757335},
issn = {1178-2013},
mesh = {Humans ; *Erythrocytes/chemistry/cytology ; Animals ; *Genetic Therapy/methods ; *Extracellular Vesicles/chemistry ; *RNA/genetics ; RNA, Messenger/genetics ; CRISPR-Cas Systems ; },
abstract = {Gene therapy has great prospects of DNA/RNA manipulations and protein modulations. Its use in clinic is, however, stifled by risks of immunogenicity, low target specificity, and adverse effects. The red blood cell (RBC-EVs) extracellular vesicles can serve as a solution to this issue since they are biocompatible, long-term stable, and with low immunogenicity. RBC-EVs permit the accurate delivery of therapeutic cargo to space and time, thus minimizing systemic toxicity. This review presents the most recent developments on the expansion of the use of RBC-EVs to encapsulate the components of mRNA and CRISPR-Cas. Through the addition of the means to address these deficiencies, including stimulus-sensitive release mechanisms (eg, pH- or light-activated systems) and tissue-selective targeting approaches, RBC-EVs can be applied to enable the precise application in genetic diseases, inflammatory diseases, and cancer. Such innovations have the potential to overcome the clinical need and enable the biological complexity of mRNA- and CRISPR-Cas-based agents to provide a powerful delivery platform. Moreover, the review also demonstrates the unprecedented benefits of red blood cell EVs, which include immune evasion, scalability, and universal loading capacity, which can establish them as the next-generation delivery vehicles. Red blood cell EVs have the potential to increase the efficacy of precision medicine by increasing its feasibility. Lastly, we note the potential and translational issues in the provision of red blood cell EV-based mRNA and CRISPR-Cas therapeutic delivery of gene therapy.},
}

Humans
*Erythrocytes/chemistry/cytology
Animals
*Genetic Therapy/methods
*Extracellular Vesicles/chemistry
*RNA/genetics
RNA, Messenger/genetics
CRISPR-Cas Systems

@article {pmid41757451,
year = {2026},
author = {Molina-Márquez, A and Kelterborn, S and Hegemann, P and Pérez-Rodríguez, M and Vigara, J and León, R},
title = {Characterization of Phytoene Desaturase Knockout Carotenoid-Deficient Microalgal Mutants Generated by Cas9-Ribonucleoprotein Complexes.},
journal = {Physiologia plantarum},
volume = {178},
number = {2},
pages = {e70811},
pmid = {41757451},
issn = {1399-3054},
support = {2019-110438RB-C22//Agencia Estatal de Investigación-MCIN/AEI/10.13039/501100011033/ ; PID2022-140995OB-C21//Agencia Estatal de Investigación-MCIN/AEI/10.13039/501100011033/ ; 426566805//German Research Foundation (DFG)/ ; //Hertie Foundation/ ; },
mesh = {*Carotenoids/metabolism ; *Oxidoreductases/genetics/metabolism ; *Chlamydomonas reinhardtii/genetics/metabolism ; *Ribonucleoproteins/metabolism/genetics ; *Microalgae/genetics/metabolism ; Gene Knockout Techniques ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Phytoene desaturase (PDS; EC 1.3.5.5) is a key enzyme of the carotenoid biosynthetic pathway, catalyzing the desaturation of phytoene, precursor of all carotenoids. In this study, several PDS-knockout (PDS-KO) transformants of the chlorophyte microalga Chlamydomonas reinhardtii were generated using a reverse genetics strategy. Two single guide RNAs (sgRNA) were designed to target the first exon of the PDS gene, and pre-assembled Cas9 ribonucleoprotein (RNPs) complexes were delivered into microalgal nuclei by electroporation. Multiple white PDS-KO transformants were successfully obtained by this approach, and three independent transformant lines were subsequently characterized. By integrating ultrastructural, pigment and transcriptomic analyses of dark-grown cells of several PDS-KO carotenoid-deficient mutants in comparison with the parental strain, it was demonstrated that carotenoids are indispensable components of multiple cellular architectures. Chromatographic analysis confirmed that the only carotenoid accumulated in these transformants was phytoene, which lacks the critical structural and photoprotective functions of its colored derivatives. Transmission Electron Microscopy (TEM) observations revealed profound ultrastructure alterations, including poorly developed chloroplasts and effects on other cellular structures that were either absent or severely disorganized. Consistently, clustering differentially expressed genes into functional groups revealed downregulation of pathways associated with photosynthesis, chlorophyll and carotenoid biosynthesis, ribosome biogenesis, and vesicle and membrane trafficking in the PDS-KO lines. Conversely, upregulation of regulatory and retrotransposon-inducing genes was observed. These findings underscore the central metabolic role of colored carotenoids in plant cells, highlighting their essential contribution to cellular homeostasis and photosynthetic competence.},
}

*Carotenoids/metabolism
*Oxidoreductases/genetics/metabolism
*Chlamydomonas reinhardtii/genetics/metabolism
*Ribonucleoproteins/metabolism/genetics
*Microalgae/genetics/metabolism
Gene Knockout Techniques
Mutation
CRISPR-Cas Systems

@article {pmid41758321,
year = {2026},
author = {Pathak, A and Singh, J and Swati, and Dwibedi, V},
title = {Deciphering microbial biofilm: mechanism, infection, and advanced approaches for control.},
journal = {Folia microbiologica},
volume = {},
number = {},
pages = {},
pmid = {41758321},
issn = {1874-9356},
abstract = {Microbial biofilms are densely organised microbial communities that adhere to biotic and abiotic surfaces, encased within an extracellular polymeric substance (EPS). Microorganisms within these biofilm structures gain enhanced protection, versatility, and resistance to external stresses, antibiotics, and host immune systems. The biofilm formation follows a series of steps, including initial microbial adherence, microcolony establishment, EPS production, regulation by quorum sensing (QS), and dispersal. This flexibility enables biofilm survival in multiple environments, such as medical devices and natural systems, posing serious challenges in healthcare, agricultural, and industrial sectors. The review focuses on the mechanisms involved in biofilm formation and discusses the role of EPS in promoting biofilm stability and resistance to antimicrobials. It addresses biofilm-associated infections in medical environments, such as chronic wounds, cystic fibrosis, urinary tract infections (UTIs), and complications with implanted medical devices. The capacity of biofilm-forming microorganisms to evade immune responses and persist through extended antibiotic use highlights the urgent demand for novel therapeutic approaches. The discussion includes emerging strategies for biofilm control, including anti-biofilm agents, QS inhibitors, enzymatic treatments, and innovative combination therapies combining antibiotics with biofilm-disrupting agents. Emerging technologies, like antimicrobial peptides (AMPs), CRISPR-Cas systems, nanotechnology, and bioelectric therapies, present innovative biofilm disruption and removal approaches. This paper discusses the effectiveness of natural products, plant-derived compounds, and bacteriophage therapies for mitigating biofilm-associated infections linked to biofilms. The review examines the dynamic challenges posed by biofilms, particularly their role in chronic and device-related infections, which contribute to significant healthcare complications. The study highlights the significance of adopting new approaches to overcome biofilm-induced antimicrobial resistance (AMR) and improve therapeutic outcomes. Furthermore, this paper discusses the promising potential of emerging technologies, such as nanomaterials, QS interference, and biofilm-specific antimicrobial agents, in enhancing biofilm control and prevention measures across clinical, industrial, and environmental domains.},
}

@article {pmid41758644,
year = {2026},
author = {Adami, A and Garza, R and Dorazehi, F and Douse, CH and Jakobsson, J},
title = {Protocol for efficient CRISPRi-mediated silencing of retrotransposons in human pluripotent stem cells.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104398},
pmid = {41758644},
issn = {2666-1667},
mesh = {Humans ; *Gene Silencing ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Retroelements/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Pluripotent Stem Cells/metabolism ; },
abstract = {Here, we present a workflow for transcriptional silencing of transposable elements (TEs) in human induced pluripotent stem cells (hiPSCs). We describe steps for designing guide RNAs (gRNAs) to target TE families or unique TE loci. We also detail procedures for validating the efficiency and specificity of large-scale CRISPRi-based silencing using a multiome approach combining bulk RNA sequencing, CUT&RUN epigenetic profiling, and proteomics. This framework optimizes the performance and interpretation of in vitro functional studies based on transcriptional manipulation of TEs in hiPSC models. For complete details on the use and execution of this protocol, please refer to Adami et al.[1].},
}

Humans
*Gene Silencing
*Induced Pluripotent Stem Cells/metabolism/cytology
*Retroelements/genetics
*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Pluripotent Stem Cells/metabolism

@article {pmid41759295,
year = {2026},
author = {Molina, CE and Knight, AL and Lisi, GP},
title = {Comparative thermodynamic and kinetic properties governing the nucleic acid interactions of CRISPR-Cas9 and Cas12a.},
journal = {Physical biology},
volume = {23},
number = {2},
pages = {},
doi = {10.1088/1478-3975/ae4b7f},
pmid = {41759295},
issn = {1478-3975},
support = {MCB 2143760//NSF/ ; },
mesh = {Thermodynamics ; *CRISPR-Cas Systems ; Kinetics ; *CRISPR-Associated Proteins/metabolism/chemistry ; *DNA/chemistry/metabolism ; *Endodeoxyribonucleases/metabolism/chemistry ; *Bacterial Proteins/metabolism/chemistry ; },
abstract = {Clustered regularly interspaced short palindromic repeat-associated proteins (CRISPR-Cas) biochemistry has been leveraged for genome editing applications in biochemical research and therapeutics. CRISPR-Cas9 and CRISPR-Cas12a are the two most widely used RNA-guided endonucleases and while Cas9 and Cas12a have a shared function, both have unique biophysical properties that alter their specificity and efficiency. The thermodynamic and kinetic properties governing their molecular interactions, recognition and binding of target DNA, and R-loop formation can differ. In some cases, these critical biophysical metrics have not been resolved. Distinctions between Cas9 and Cas12a enzymes are also prevalent in RNA:DNA hybrid binding affinities, DNA localization relative to the preferred PAM site and the DNA cleavage mechanism. In this review, we examine the thermodynamic and kinetic properties of both endonucleases, focused on the nucleic acid interactions that confer specificity and function. Complementing this biophysical overview, we discuss case studies in disparate model organisms that compare the genome editing and fidelity of Cas9 and Cas12a.},
}

Thermodynamics
*CRISPR-Cas Systems
Kinetics
*CRISPR-Associated Proteins/metabolism/chemistry
*DNA/chemistry/metabolism
*Endodeoxyribonucleases/metabolism/chemistry
*Bacterial Proteins/metabolism/chemistry

@article {pmid41759376,
year = {2026},
author = {Xu, Y and Wu, Y and Zheng, H and Zhao, J and Chen, J and Liu, S and Han, M and Li, F and Zhou, F and Zhang, X and Cao, Y and Zhang, H and Zhang, C},
title = {CRISPR-based metabolic screening identifies PLCE1 as a pivotal regulator of oncolytic viral antitumor immunity via tumor immune microenvironment remodeling.},
journal = {Biochemical and biophysical research communications},
volume = {810},
number = {},
pages = {153505},
doi = {10.1016/j.bbrc.2026.153505},
pmid = {41759376},
issn = {1090-2104},
mesh = {*Tumor Microenvironment/immunology ; Animals ; *Oncolytic Virotherapy/methods ; *Oncolytic Viruses/immunology/physiology ; Humans ; Cell Line, Tumor ; Mice ; *Colorectal Neoplasms/therapy/immunology/genetics ; *CRISPR-Cas Systems ; },
abstract = {As a promising cancer immunotherapeutic agent, oncolytic viruses (OV) can specifically kill tumor cells and elicit systemic antitumor immune responses. However, the intrinsic resistance of tumors to oncolytic virotherapy severely limits its therapeutic efficacy. This study identified phospholipase C epsilon 1 (PLCE1) as a key negative regulator of OV antitumor effects via CRISPR-Cas9 metabolic gene screening in MC38 colorectal cancer model. PLCE1 inhibitor U-73122 enhanced OV infection efficiency and immunogenic cell death in vitro. In vivo, U-73122 combined with OV synergistically reduced tumor volume and prolonged survival. The combination therapy has been shown to remodel the tumor immune microenvironment, leading to an increase in CD45[+] immune cells and CD8[+] T cells, including naïve subsets, and a decrease in FOXP3[+] Treg cells. This shift promotes T cell activation by modulating relevant genes and signaling pathways. This study provides a novel target for optimizing OV immunotherapy.},
}

*Tumor Microenvironment/immunology
Animals
*Oncolytic Virotherapy/methods
*Oncolytic Viruses/immunology/physiology
Humans
Cell Line, Tumor
Mice
*Colorectal Neoplasms/therapy/immunology/genetics
*CRISPR-Cas Systems

@article {pmid41759529,
year = {2026},
author = {Cao, Z and Yu, S and Peng, J and Barrett, DR and Liu, Y and Sussman, JH and Chen, C and Thadi, A and Liu, L and Alikarami, F and Xu, J and Carroll, MP and Tan, K and Bernt, KM and Shi, J},
title = {CRISPR-based functional genomics for dissecting therapeutic dependency in primary acute myeloid leukemia samples.},
journal = {Molecular cell},
volume = {86},
number = {5},
pages = {968-985.e7},
pmid = {41759529},
issn = {1097-4164},
support = {R01 CA262260/CA/NCI NIH HHS/United States ; U01 CA243072/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/pathology/metabolism/drug therapy ; Animals ; *CRISPR-Cas Systems ; *Genomics/methods ; Mice ; Mutation ; Single-Cell Gene Expression Analysis ; Gene Expression Regulation, Leukemic ; Gene Regulatory Networks ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cancer functional genomics enables high-throughput target discovery and mechanistic investigation, yet its application has remained largely confined to mouse models and established human cancer cell lines. Direct functional interrogation of heterogeneous primary tumors offers a powerful opportunity to evaluate therapeutic targets and uncover cancer dependencies or resistance mechanisms. Here, we developed an optimized CRISPR-based platform for functional genomics in patient-derived xenograft and primary acute myeloid leukemia (AML) samples harboring diverse pathogenic mutations. Integrated in vitro and in vivo CRISPR-Cas9 knockout and CRISPR interference (CRISPRi) dropout screens validated known AML-biased targets and identified cis-regulatory elements essential for leukemic growth. Coupling pooled CRISPR perturbations with single-cell RNA sequencing (Perturb-seq) further resolved the perturbation-induced alterations in regulatory networks, cell cycle states, and cellular hierarchies in primary AML samples. Together, these studies establish a general and robust framework for leveraging CRISPR-based functional genomics to directly dissect cancer dependencies and cellular heterogeneity in primary AML patient samples.},
}

Humans
*Leukemia, Myeloid, Acute/genetics/pathology/metabolism/drug therapy
Animals
*CRISPR-Cas Systems
*Genomics/methods
Mice
Mutation
Single-Cell Gene Expression Analysis
Gene Expression Regulation, Leukemic
Gene Regulatory Networks
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41759621,
year = {2026},
author = {Yan, X and Chen, M and Yang, S and Guo, Y and Dai, Y and Chen, Y and Zhong, H and Ma, T and Zha, D and He, Y and Li, B and Jia, X and Guo, L and Hu, J and Wei, Y and Chen, X},
title = {Mitochondrial genome editing tools: prospects in animal breeding.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgg.2026.02.018},
pmid = {41759621},
issn = {1673-8527},
abstract = {Mitochondria are vital organelles responsible for driving cellular energy metabolism and regulating key biological processes. Their circular mitochondrial DNA (mtDNA) encodes 13 subunits of the respiratory chain proteins but is susceptible to mutations due to high levels of reactive oxygen species and limited repair mechanisms. Mutant phenotypes manifest only when heteroplasmy surpasses a critical threshold. Understanding the consequences of mtDNA mutations has long been hampered by the lack of precise editing tools. Recently, CRISPR-free, protein-only mitochondrial base editors have enabled C·G-to-T·A and A·T-to-G·C transitions. These breakthroughs facilitate the creation of relevant disease models and offer unique opportunities for animal breeding, as specific mtDNA variants are known to influence economically important traits in livestock, including production, reproduction, and stress tolerance. This review summarizes recent advances in mitochondrial genome editing technologies, including CRISPR/Cas-based systems, restriction endonucleases, double-stranded DNA deaminase toxin A (DddA)-based cytosine and adenine base editors, and DddA-free base editors, along with their delivery strategies and optimization avenues. Furthermore, we outline the associations between mtDNA polymorphisms, copy number variation, and economic traits in livestock and poultry. Finally, we discuss the potential applications of mitochondrial genome editing in animal breeding and highlight the critical safety and ethical considerations that require careful attention.},
}

@article {pmid41759757,
year = {2026},
author = {Schmidt, GE and Weaver, EA and Kim, TH},
title = {CRISPR-based functional analysis of chicken IRF9 reveals distinct modulation of dsRNA stimulated innate immune pathways.},
journal = {Developmental and comparative immunology},
volume = {177},
number = {},
pages = {105577},
doi = {10.1016/j.dci.2026.105577},
pmid = {41759757},
issn = {1879-0089},
mesh = {Animals ; *Chickens/immunology/genetics ; Immunity, Innate/genetics ; *Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics/metabolism ; *Avian Proteins/genetics/metabolism ; Interferon Type I/metabolism ; *RNA, Double-Stranded/immunology ; Signal Transduction/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation ; },
abstract = {The chicken immune system is distinct from mammalian models due to its reduced immune gene repertoire, yet it retains the ability to mount a highly effective immune response. In mammals, interferon regulatory factor 9 (IRF9) is a key transcriptional regulator of the type I interferon (IFN) pathway, stimulating the expression of hundreds of antiviral genes. Although IRF9 was previously thought to be absent in chickens, current chicken reference genome annotation (bGalGal1.mat.broiler.GRCg7b) lists a putative chicken IRF9. To investigate the function of this gene in chickens, we utilized a clustered regularly interspace short palindromic repeats (CRISPR) based transcriptional modulation platform to elucidate the role of the putative chicken IRF9 in the innate immune response. We analyzed the transcriptomes of IRF9 repressed cells stimulated with double stranded RNA at 0, 0.5, 1, and 6 h post-stimulation. Gene set enrichment analysis revealed that IRF9 repression resulted in the enrichment of pathways associated with regulating the type I IFN response, including the retinoic acid inducible gene I like (RIG-I like) receptor pathway and the Toll-like receptor pathway. Furthermore, concurrent transcriptional repression of type I IFN modulator IRF7 with transcriptional activation of IRF9 failed to rescue the expression of downstream IFN-stimulated genes. These results suggest chicken IRF9 plays a distinct regulatory role from canonical mammalian IRF9 in the type I IFN response and demonstrate a need for functional evidence-based classification of chicken IRFs.},
}

Animals
*Chickens/immunology/genetics
Immunity, Innate/genetics
*Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics/metabolism
*Avian Proteins/genetics/metabolism
Interferon Type I/metabolism
*RNA, Double-Stranded/immunology
Signal Transduction/genetics
CRISPR-Cas Systems
Gene Expression Regulation

@article {pmid41761908,
year = {2026},
author = {Perry, TN and Mais, CN and Sanchez-Londono, M and Steinchen, W and Plitzko, PA and Randau, L and Pausch, P and Innis, CA and Bange, G},
title = {Structural basis of Cas8-independent Cas3 recruitment in Type I-F2 CRISPR-Cas.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41761908},
issn = {1362-4962},
support = {//Deutsche Forschungsgemeinschaft/ ; 260989694//DFG/ ; 324652314//DFG/ ; 405858511//DFG/ ; 3869//DFG/ ; //Inserm/ ; 5342-2023//Research Council of Lithuania/ ; //Marburg University/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics/ultrastructure ; Cryoelectron Microscopy ; Models, Molecular ; DNA/chemistry/metabolism/genetics ; *DNA Helicases/chemistry/metabolism/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; },
abstract = {CRISPR-Cas systems provide adaptive immunity in prokaryotes by targeting and degrading invasive genetic elements. Among them, the Type I-F2 system represents the most compact Type I CRISPR-Cas variant, distinguished by the complete absence of both large (Cas8) and small (Cas11) subunits. In other Type I systems, Cas8 is essential for protospacer adjacent motif (PAM) recognition and for triggering Cas3 recruitment, while Cas11 stabilizes the Cascade backbone and guides the nontarget DNA strand during R-loop formation. To elucidate how I-F2 executes interference in their absence, we determined the cryo-electron microscopy structure of the I-F2 Cascade bound to target DNA and Cas3. Our structure reveals that Cas5 alone mediates PAM sensing, while Cas7 subunits directly recruit Cas3, which adopts a helicase-loaded conformation compatible with DNA engagement. We show how the helicase and C-terminal domains of Cas3 capture the displaced nontarget strand to initiate directional unwinding and degradation. These findings uncover key mechanistic adaptations that enable efficient interference without canonical large and small subunits and emphasize the mechanistic diversity among closely related Type I systems, including I-E, I-F1, and I-F2. These insights provide a structural basis for engineering the hypercompact I-F2 system for genome editing and biotechnological applications.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/chemistry/metabolism/genetics/ultrastructure
Cryoelectron Microscopy
Models, Molecular
DNA/chemistry/metabolism/genetics
*DNA Helicases/chemistry/metabolism/genetics
*Bacterial Proteins/chemistry/metabolism/genetics

@article {pmid41762821,
year = {2026},
author = {Golla, DA and Sun, C and Haugh, L and Straub, N and Gao, X},
title = {Advances in multiplex precision genome editing in eukaryotic and prokaryotic systems.},
journal = {Current opinion in biotechnology},
volume = {99},
number = {},
pages = {103470},
pmid = {41762821},
issn = {1879-0429},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; Prokaryotic Cells/metabolism ; Animals ; },
abstract = {Multiplex genome editing (MGE) enables coordinated modification of multiple genomic loci and is foundational for engineering complex biological traits. Traditional CRISPR-Cas nuclease-based strategies rely on DNA double-strand breaks (DSBs), which limit precision and pose scaling challenges for incorporating simultaneous edits across different loci. Recent advances in genome editing technologies that operate without generating DSBs have expanded the accuracy and feasibility of multiplexed genomic manipulation. This review focuses on emerging strategies for precise MGE, including base editing, prime editing, and related genome rewriting platforms. We highlight key engineering principles that impact the success of scalable multiplexing, including the choice of editing platform, edit size, and guide RNA architecture, and discuss applications across mammalian, plant, fungal, and bacterial systems. Together, these technologies establish MGE as a versatile framework for precise multigene control in biotechnology and agriculture.},
}

*Gene Editing/methods
CRISPR-Cas Systems
Prokaryotic Cells/metabolism
Animals

@article {pmid41762887,
year = {2026},
author = {Yu, Y and Sun, S and Song, X and Xiong, Z and Song, Z and Peng, C and Zhang, J and Ai, L},
title = {crRNA-engineered CRISPR/Cas12a system coupled with RPA for ultrasensitive detection of Lactiplantibacillus plantarum.},
journal = {Food chemistry},
volume = {509},
number = {},
pages = {148595},
doi = {10.1016/j.foodchem.2026.148595},
pmid = {41762887},
issn = {1873-7072},
mesh = {*CRISPR-Cas Systems ; *Lactiplantibacillus plantarum/genetics/isolation & purification ; Probiotics/analysis ; *RNA, Bacterial/genetics ; Bacterial Proteins/genetics/metabolism ; },
abstract = {The growing probiotic industry requires rapid and precise strain detection methods. Here, a one-pot fluorescence platform integrating RPA with an enhanced CRISPR/Cas12a system (termed RPA-ECas12a) was developed for the detection of Lactiplantibacillus plantarum. Through rational 5'end DNA extension of the crRNA, an optimal variant (5'crRNA10) was identified, which increased the trans-cleavage catalytic efficiency of Cas12a by 33% (3.6 × 10[8] M[-1] s[-1]) compared to the wild-type crRNA. The resulting RPA-ECas12a platform detected L. plantarum with a limit of detection of 1.3 CFU/mL, a linear range from 10[1] to 10[7] CFU/mL, and excellent precision (CVs < 10%). The entire detection was completed within 45 min. The platform demonstrated high selectivity and robustness when applied to commercial probiotic powders, yogurts and other complex food matrices. This work not only provides a sensitive and rapid detection tool for probiotic authentication but also offers a generalizable crRNA-engineering strategy to enhance the performance of CRISPR/Cas12a in diagnostic.},
}

*CRISPR-Cas Systems
*Lactiplantibacillus plantarum/genetics/isolation & purification
Probiotics/analysis
*RNA, Bacterial/genetics
Bacterial Proteins/genetics/metabolism

@article {pmid41762975,
year = {2026},
author = {Guo, L and Cui, K and Yang, Y and Dong, S and Chen, Y and Liu, K and Lei, X and Duan, B and Zhao, Y and Lv, X and Bai, R and Zheng, M},
title = {Field-deployable multiplex RAA-CRISPR/Cas12a platform rapidly and simultaneously detects seven Eimeria species in chickens.},
journal = {Poultry science},
volume = {105},
number = {5},
pages = {106681},
pmid = {41762975},
issn = {1525-3171},
mesh = {Animals ; *Eimeria/isolation & purification/classification ; *Chickens ; *Coccidiosis/veterinary/diagnosis/parasitology ; *CRISPR-Cas Systems ; *Poultry Diseases/diagnosis/parasitology ; Sensitivity and Specificity ; Feces/parasitology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Reproducibility of Results ; Rapid Diagnostic Tests ; },
abstract = {Chicken coccidiosis, caused by infection with one or more of the seven Eimeria spp., is a major challenge in global poultry production. Rapid and accurate identification at the species level is critical for guiding targeted treatment strategies, minimizing antibiotic misuse, and mitigating disease transmission. In this study, we developed a point-of-care testing (POCT) platform, E-MRC12a (Eimeria-Multiplex RAA-CRISPR/Cas12a), which integrates multiplex recombinase-aided amplification (RAA) with CRISPR/Cas12a technology for the simultaneous detection of all seven Eimeria species in chicken fecal samples. Key assay parameters were optimized to balance detection performance and operational cost. The system was comprehensively evaluated for its sensitivity, specificity, reproducibility, and field applicability. E-MRC12a enables visual, one-pot detection of as few as 1 oocyst/μL. The process from sample loading to result interpretation required 1 h, while the total time from initial sample processing to final result readout was approximately 2 h. The assay exhibited high specificity with no cross-reactivity among Eimeria species, and demonstrated 100% concordance with conventional diagnostic methods in clinical validation. This rapid, field-deployable platform provides a species-specific coccidiosis diagnostic solution, supporting epidemiological surveillance and multivalent anticoccidial vaccine development.},
}

Animals
*Eimeria/isolation & purification/classification
*Chickens
*Coccidiosis/veterinary/diagnosis/parasitology
*CRISPR-Cas Systems
*Poultry Diseases/diagnosis/parasitology
Sensitivity and Specificity
Feces/parasitology
*Nucleic Acid Amplification Techniques/veterinary/methods
Reproducibility of Results
Rapid Diagnostic Tests

@article {pmid41762997,
year = {2026},
author = {Yu, D and Ren, H and He, P and Li, W and Tang, Q and Huang, L and Wei, J and Zhang, K and Liao, X},
title = {Stage-aware quantification of the SARS-CoV-2 3CL[pro] biomarker via CsPbBr3@COF-LZU1@AuNP electrochemiluminescence and Cas13a amplification.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129593},
doi = {10.1016/j.talanta.2026.129593},
pmid = {41762997},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; Humans ; *SARS-CoV-2/isolation & purification/genetics ; Electrochemical Techniques/methods ; Gold/chemistry ; Luminescent Measurements/methods ; *COVID-19/diagnosis/virology ; Metal Nanoparticles/chemistry ; Metallocenes/chemistry ; CRISPR-Cas Systems ; Ferrous Compounds/chemistry ; Titanium/chemistry ; Limit of Detection ; Oxides/chemistry ; Biomarkers/analysis ; Nucleic Acid Amplification Techniques/methods ; Calcium Compounds ; },
abstract = {Direct, activity-based quantification of the SARS-CoV-2 main protease (3CL[pro]) remains challenging in complex matrices. Here we report a water-compatible electrochemiluminescent (ECL) biosensor that integrates a CsPbBr3@COF-LZU1 emitter, a peptide-DNA conformational switch, and CRISPR/Cas13a-assisted amplification to convert protease activity into a robust optical "turn-on" signal. The covalent organic framework physically stabilizes perovskite nanocrystals in aqueous media and, together with a sparse Au nanoparticle layer, supports assembly of ferrocene-terminated reporters that impose an ultralow baseline via near-field/redox quenching. Target-specific cleavage unlocks an initiator that drives entropy-mediated T7 promoter formation and transcription, producing RNA activators that switch on Cas13a collateral cleavage; removal or distancing of ferrocene from the emitter restores photon output. Under optimized conditions the sensor exhibits a broad log-linear response from 10 to 10[8] aM with an ultralow detection limit of 4.31 aM, high analytical selectivity against common interferents, tight fabrication-to-fabrication precision (inter-electrode RSD ∼3%), and practical robustness (≈97% signal retention over 12 h, ≥90% after 7 days at 4 °C, and ∼92% after 120 ECL cycles). Stepwise ECL, cyclic voltammetry, and impedance analysis confirm layer-by-layer assembly and the intended mechanism of ferrocene-mediated quenching and Cas13a-driven recovery. Applied directly to minimally processed pharyngeal swab eluates, the platform resolves cohort-level differences across disease stages and captures the expected attenuation of 3CL[pro] activity in late-stage specimens, supporting stage-aware quantification in real clinical samples. The modular design-reprogrammable at the protease-cleavable motif, promoter template, and crRNA-points to a general route for sensitive, selective, and water-stable ECL assays of enzymatic activity with translational potential.},
}

*Biosensing Techniques/methods
Humans
*SARS-CoV-2/isolation & purification/genetics
Electrochemical Techniques/methods
Gold/chemistry
Luminescent Measurements/methods
*COVID-19/diagnosis/virology
Metal Nanoparticles/chemistry
Metallocenes/chemistry
CRISPR-Cas Systems
Ferrous Compounds/chemistry
Titanium/chemistry
Limit of Detection
Oxides/chemistry
Biomarkers/analysis
Nucleic Acid Amplification Techniques/methods
Calcium Compounds

@article {pmid41763219,
year = {2026},
author = {Aravind, KM and Del Vecchio, D},
title = {Resource competition shapes CRISPR-mediated gene activation.},
journal = {Cell systems},
volume = {17},
number = {3},
pages = {101511},
doi = {10.1016/j.cels.2025.101511},
pmid = {41763219},
issn = {2405-4720},
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Transcriptional Activation/genetics ; Humans ; },
abstract = {CRISPR-mediated gene activation (CRISPRa) allows concurrent transcriptional control of many genes and is widely used in genome-wide screening, bioproduction, and therapeutics. Multi-gene control is possible due to the sequence specificity by which guide RNAs (gRNAs) recruit dCas9 and an activator protein to target genes. Still, the optimization of CRISPRa systems remains difficult. Here, we show that, despite sequence specificity, different gRNAs interfere with each other by competing for dCas9 and the activator protein. This competition breaks modularity and hinders CRISPRa. We also discover that gene activation is biphasic, wherein increased level of a gRNA leads to target repression instead of activation. We introduce a chemical reaction-network model that captures these effects and use it for improving the dynamic range of CRISPRa. Our results demonstrate that CRISPRa is not as modular or scalable as previously thought and establish a predictive modeling tool that enables systematic design and optimization of multi-gRNA CRISPRa systems. A record of this paper's transparent peer review process is included in the supplemental information.},
}

*CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Transcriptional Activation/genetics
Humans

@article {pmid41763555,
year = {2026},
author = {Shi, Y and Yuan, Y and Qin, L and Zhou, F and Wu, G and Li, B and Yao, P and Shi, M and Ma, L and Wang, Y and Zhang, Y and Wang, C and Wang, X and Huang, B and Chen, J and Xiang, Z and Lin, Q and Huang, J},
title = {Optimizing linker length of base editors for precise crop breeding and gene therapy.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {53},
number = {6},
pages = {1125-1137},
doi = {10.1016/j.jgg.2026.02.021},
pmid = {41763555},
issn = {1673-8527},
mesh = {Humans ; *Oryza/genetics ; *Genetic Therapy/methods ; CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; *Plant Breeding ; *Gene Editing/methods ; },
abstract = {Base editing enables efficient nucleotide conversions without inducing DNA double-strand breaks (DSBs) or requiring exogenous donor DNA templates. However, its broader editing window often causes bystander editing, increasing the risk of unintended mutations. In this study, we find that linker length significantly influences the editing window, and base editors with a 7-amino-acid linker reduce bystander editing by an average of 54.4% across 13 endogenous genomic sites in both rice and human cell lines. We further develop an optimized strategy by modulating the linker length between various deaminases and Cas9 nickases, which effectively reduces bystander editing across multiple applications, including functional studies, precise crop breeding, and correction of pathogenic variants. Our work reveals that shortening the linker enhances the specificity of base editing, addressing a key safety concern for its agricultural and therapeutic applications.},
}

Humans
*Oryza/genetics
*Genetic Therapy/methods
CRISPR-Cas Systems/genetics
*Crops, Agricultural/genetics
*Plant Breeding
*Gene Editing/methods

@article {pmid41763755,
year = {2026},
author = {Ma, C and French, N and Wu, X and Gupta, SK and Gupta, TB},
title = {Molecular detection of Clostridium and Bacillus species in foods: recent advances and applications.},
journal = {Food research international (Ottawa, Ont.)},
volume = {229},
number = {},
pages = {118370},
doi = {10.1016/j.foodres.2026.118370},
pmid = {41763755},
issn = {1873-7145},
mesh = {*Clostridium/isolation & purification/genetics ; *Food Microbiology/methods ; *Bacillus/isolation & purification/genetics ; Nucleic Acid Amplification Techniques ; Food Contamination/analysis ; Humans ; DNA, Bacterial/genetics ; CRISPR-Cas Systems ; Animals ; },
abstract = {Spore-forming bacteria, especially Clostridium spp. and Bacillus spp., are ubiquitous in food systems, and their ingestion can cause serious diseases in humans and animals. Their persistence in diverse food matrices and resistance to conventional treatments make rapid and accurate detection essential for effective monitoring and control. Traditional culture-based and biochemical assays remain the standard for identifying these bacteria but are often time-consuming, labor-intensive and limited in sensitivity. In contrast, nucleic acid-based methods provide rapid, specific and sensitive alternatives by directly targeting genetic markers of pathogenic or spoilage strains. This review summarizes how nucleic acid methods, including PCR, FISH, LAMP, RPA, WGS, and the emerging CRISPR/Cas systems, have been applied specifically to detect Clostridium spp. and Bacillus spp. in food systems. Each method offers unique advantages and limitations. PCR-based methods enable accurate quantification but require thermal cycling. FISH-based methods are simple but require microscopy and have limited validation in food. WGS-based methods provide strain-level characterization but depend on informatics and specialized equipment. Isothermal techniques such as LAMP- and RPA-based methods allow rapid field detection but involve complex primer design or poor discrimination of closely related genes. CRISPR/Cas-based platforms further enhance simplicity, specificity, sensitivity for on-site detection, though the validation for spore-forming bacteria remains limited. Overall, this review provides an overview of gene targets, methodological adaptations, and analytical performance of nucleic acid-based assays for detecting Clostridium spp. and Bacillus spp., highlighting current progress and future opportunities for improving food safety monitoring.},
}

*Clostridium/isolation & purification/genetics
*Food Microbiology/methods
*Bacillus/isolation & purification/genetics
Nucleic Acid Amplification Techniques
Food Contamination/analysis
Humans
DNA, Bacterial/genetics
CRISPR-Cas Systems
Animals

@article {pmid41764730,
year = {2026},
author = {D'Souza, LJ and Young, JN and Coffman, H and Petrow, EP and Bhattacharya, D},
title = {A genome-wide CRISPR screen reveals novel determinants of long-lived plasma cell secretory capacity.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {215},
number = {2},
pages = {},
doi = {10.1093/jimmun/vkaf354},
pmid = {41764730},
issn = {1550-6606},
support = {R01AI129945//National Institutes of Health (NIH)/ ; P30CA023074//Research, Innovation & Impact (RII) of the University of Arizona and National Cancer Institute/ ; S10 OD028466/GF/NIH HHS/United States ; },
mesh = {Animals ; *Plasma Cells/immunology/metabolism ; Mice ; Humans ; CRISPR-Cas Systems ; Myeloid Differentiation Factor 88/genetics/metabolism ; *Multiple Myeloma/immunology/genetics ; Vacuolar Proton-Translocating ATPases/genetics/metabolism ; Cell Line, Tumor ; },
abstract = {Plasma cell subsets vary in their lifespans and ability to sustain humoral immunity. We conducted a genome-wide CRISPR-Cas9 screen in myeloma cells for factors that promote surface expression of CD98, a marker of longevity in mouse plasma cells. A large fraction of genes found to promote CD98 expression in this screen are involved in secretory and other vesicles, including subunits of the V-type ATPase complex. Genetic ablation and chemical inhibition of V-type ATPases in myeloma cells and primary plasma cells, respectively, reduced antibody secretion. Mouse and human long-lived plasma cells had greater numbers of acidified vesicles than their short-lived counterparts, and this correlated with increased antibody secretory capacity. The screen also revealed a requirement for the signaling adapter MYD88 in CD98 expression. Plasma cell-specific deletion of Myd88 led to reduced survival and antibody secretion by antigen-specific cells in vivo and unresponsiveness to BAFF and APRIL ex vivo. These data reveal novel regulators that link plasma cell secretory capacity and lifespan.},
}

Animals
*Plasma Cells/immunology/metabolism
Mice
Humans
CRISPR-Cas Systems
Myeloid Differentiation Factor 88/genetics/metabolism
*Multiple Myeloma/immunology/genetics
Vacuolar Proton-Translocating ATPases/genetics/metabolism
Cell Line, Tumor

@article {pmid41766140,
year = {2026},
author = {Hartig, AM and Dai, W and Zhang, K and Rottinghaus, AG and Moon, TS and Parker, KM},
title = {Genetic Markers Remain Detectable in Genetically Engineered Microbes Biocontained with a CRISPR Kill Switch.},
journal = {Environmental science & technology},
volume = {60},
number = {10},
pages = {7983-7994},
doi = {10.1021/acs.est.6c00321},
pmid = {41766140},
issn = {1520-5851},
mesh = {*Escherichia coli/genetics ; Genetic Markers ; *CRISPR-Cas Systems ; *Microorganisms, Genetically-Modified/genetics ; Genetic Engineering ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Biocontainment strategies, such as kill switches, have been developed to avoid the unintended proliferation of genetically engineered microbes (GEMs) intended for open-release environmental applications. However, the presence of GEM DNA after successful biocontainment presents new environmental risks and challenges for monitoring. In this study, we investigated whether biocontainment using a CRISPR-Cas9 kill switch, which causes double-strand breaks in target genes essential for GEM growth, could resolve this challenge in a model Escherichia coli GEM. Surprisingly, the escape rates of the GEM as determined by CRISPR-targeted gene abundances were as high as 10[-1.6] to 10[-1.0] in LB media, despite the escape rates measured by colony forming units (cfu) being only 10[-6.2] under the same condition. This discrepancy suggested that the CRISPR-Cas9 kill switch prevents colony growth while still leaving a large fraction of target genes intact for detection by molecular methods. Within 1 h after biocontainment, these target genes remained predominantly inside an intact cell membrane and were resistant to degradation by DNase, though degradation was observed in river water over multiple days. Overall, a detailed understanding of the impact of the biocontainment mechanism on both the GEM and its DNA is needed to minimize unintended environmental risks.},
}

*Escherichia coli/genetics
Genetic Markers
*CRISPR-Cas Systems
*Microorganisms, Genetically-Modified/genetics
Genetic Engineering
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41766608,
year = {2026},
author = {Yin, J and Wen, H and Zeng, J and Sun, Y and Chen, X and Jin, L and Song, Y and Xia, S},
title = {CRISPR-based genome editing in human embryos: a review of efficiency, safety, and ethical implications.},
journal = {Biology of reproduction},
volume = {114},
number = {6},
pages = {1775-1790},
doi = {10.1093/biolre/ioag056},
pmid = {41766608},
issn = {1529-7268},
mesh = {Humans ; *Gene Editing/ethics/methods ; Animals ; *CRISPR-Cas Systems ; *Embryo, Mammalian ; },
abstract = {Programmable gene editing tools, particularly CRISPR/Cas9 and its advanced derivatives (base and prime editors), have revolutionized biomedical research and offer unprecedented potential for studying human embryogenesis and correcting monogenic diseases. This review systematically examines the evolution and challenges of these technologies in human embryos and mammalian models. We trace key methodological advancements, from initial studies hampered by low efficiency and mosaicism to refined strategies like RNP delivery and base editing that improved precision. A critical shift occurred with the discovery that CRISPR/Cas9 can cause severe on-target damage, such as large deletions and chromosomal loss, redirecting the field's focus toward safety. We present a comparative analysis of editing efficiencies across species (human, mouse, primate, pig, cow, and rabbit) and tools (Cas9, BEs, and PEs), consistently demonstrating the superiority of RNP for precise editing. Fundamental barriers to clinical translation are discussed, including the trade-off between efficiency and mosaicism, persistent off-target effects, and profound ethical concerns. The review concludes that while somatic gene therapy advances rapidly, heritable genome editing remains premature due to unresolved risks. Future progress depends on developing safer editors, understanding on-target consequences, and adhering to rigorous ethical standards.},
}

Humans
*Gene Editing/ethics/methods
Animals
*CRISPR-Cas Systems
*Embryo, Mammalian

@article {pmid41766888,
year = {2026},
author = {Li, X and Zhao, Y and Guo, X and Bai, Y and Wang, J},
title = {Characterization and diversity of defense systems in Providencia pathogen.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1755933},
pmid = {41766888},
issn = {1664-3224},
mesh = {*Providencia/genetics/immunology/pathogenicity ; Phylogeny ; Genome, Bacterial ; CRISPR-Cas Systems ; *Enterobacteriaceae Infections/microbiology/immunology ; },
abstract = {INTRODUCTION: Providencia species are emerging opportunistic pathogens associated with multidrug-resistant infections, yet their molecular defense mechanisms against phage or mobile genetic elements remain poorly characterized.

METHODS: We present a comprehensive pan-genomic analysis of antiviral defense systems across 73 complete genomes (or chromosomes) of Providencia stuartii (n = 31) and Providencia rettgeri (n = 42), using DefenseFinder and CRISPRCasFinder. We further expanded analysis of contig/scaffold assemblies to confirm conservation of core defense profiles across assembly types. BacMGEnet was employed to derive spacer-MGE interaction networks. Phylogenetic reconstruction and gene gain and loss modeling were performed to assess evolutionary patterns. To validate functionality, we experimentally tested the anti-phage activity of Gabija and Septu in heterologous E. coli assays, including point mutation analysis of conserved residues.

RESULTS: We reveal a diverse and complex defense repertoire dominated by restriction-modification systems and CRISPR-Cas Class 1 Type I-F, with significant contributions from toxin-antitoxin, GAPS2, PsyrTA, and Mokosh systems. Notably, defense genes are non-randomly distributed, often clustering into genomic islands suggestive of horizontal acquisition. Expanded analysis confirms conservation of core defense profiles across assembly types, supporting the utility of lower-quality data when complete genomes are scarce. Comparative analysis uncovers species-specific differences, with P. rettgeri harboring a higher abundance of non-CRISPR systems. BacMGEnet-derived spacer-MGE interaction networks further highlight species-specific dynamics, dense, hub-driven networks in P. stuartii versus sparser networks in P. rettgeri. Correlation analysis indicates potential associations between specific defense systems and virulence or antibiotic resistance genes. Phylogenetic reconstruction and gene gain and loss modeling further highlight dynamic evolutionary patterns. Both Gabija and Septu systems conferred robust, phage-specific protection; point mutations in conserved residues (GajA E465K and PtuB H53K) abolished defense.

DISCUSSION: Our findings unveil a multi-layered, modular immune architecture in Providencia, providing crucial insights into its genome plasticity, phage resistance, and adaptation in clinical environments. This work establishes a foundation for understanding the role of defense systems in the evolution and pathogenicity of the Providencia genus.},
}

*Providencia/genetics/immunology/pathogenicity
Phylogeny
Genome, Bacterial
CRISPR-Cas Systems
*Enterobacteriaceae Infections/microbiology/immunology

@article {pmid41769381,
year = {2026},
author = {Mandal, S and Baloch, AR and Yuan, X and Chen, J and Saribas, AS and Zhu, Y and Zhang, D and Jaijyan, D and Xu, J and Hossain, R and Sisto, I and Wang, H and Yang, X and Li, Q and Hu, W},
title = {Bipolar CD4-targeted dual-DARPin-55/57 lipid nanoparticle enables efficient CRISPR/Cas-mediated HIV-1 DNA excision and reactivation blockade in latent CD4 T cell lines.},
journal = {Materials today. Bio},
volume = {37},
number = {},
pages = {102939},
pmid = {41769381},
issn = {2590-0064},
support = {R01 AI145034/AI/NIAID NIH HHS/United States ; R01 AI174301/AI/NIAID NIH HHS/United States ; },
abstract = {The persistence of HIV-1 latent reservoirs remains the principal barrier to a cure, as viral rebound occurs upon interruption of antiretroviral therapy. CRISPR/Cas genome editing offers a promising strategy to excise proviruses from host genome; however, the absence of a targeted and clinically viable delivery platform has hindered its translational application. Here, we report a chemistry-driven, CD4-targeted lipid nanoparticle (LNP) delivery platform employing a unique bipolar conjugation strategy to decorate dual CD4-targeted Designed Ankyrin Repeat Proteins (DARPins-55 and -57) on LNP (dual-DARPin-LNP). The N- and C-terminally modified DARPin-55/57 was thiolated stepwise, then bipolar maleimide-thiol coupling conjugated the thiolates to the maleimide-functionalized LNP surface. This coupling strategy ensured DARPin proper orientation on the LNP surface for efficient uptake by resting CD4 T cells. This dual-DARPin-LNP system was engineered for selective and efficient co-delivery of spCas9-GFP mRNA (Sp9m) and HIV-1-specific single-guide RNAs (sgRNAs) targeting LTR and Gag (LGsg) into HIV-1 latently infected CD4 T cells. In widely used HIV-1 latency models with defined proviral modifications (J-Lat 10.6 and 2D10 cell lines), dual-DARPin-LNP loaded with Sp9m/LGsg efficiently excised integrated HIV-1 proviral DNA, as confirmed by standard PCR genotyping, absolute digital PCR quantification, confocal microscopy, and flow cytometry. Importantly, proviral excision functionally blocked HIV-1 reactivation following stimulation with latency-reversing agents suberoylanilide hydroxamic acid (SAHA) and TNFα. Together, these findings establish a modular, non-viral, receptor-guided delivery platform for CD4 T cell targeting and provide proof-of-concept for precise HIV-1 DNA excision and reactivation blockade in established latency models. This new strategy represents a step toward next-generation curative interventions against persistent HIV-1 infection.},
}

@article {pmid41771837,
year = {2026},
author = {Schoger, E and Kim, R and Bleckwedel, F and Peralta, TM and Priesmeier, L and Fischer, JA and Stengel, L and Rocha, C and Santos, GL and Lutz, S and Boileau, E and Baumgarten, N and Schulz, MH and Dieterich, C and Müller, OJ and Cyganek, L and Cabrera-Orefice, A and Eberl, H and Maack, C and Streckfuss-Bömeke, K and Pavez-Giani, MG and Doroudgar, S and Sossalla, S and Zelarayán, LC},
title = {Enhancing KLF15 activity in cardiomyocytes: a novel approach to prevent pathological reprogramming and fibrosis via nuclease-deficient dCas9VPR.},
journal = {Signal transduction and targeted therapy},
volume = {11},
number = {1},
pages = {},
pmid = {41771837},
issn = {2059-3635},
mesh = {*Myocytes, Cardiac/metabolism/pathology ; *Kruppel-Like Transcription Factors/genetics/metabolism ; Humans ; Animals ; Fibrosis/genetics/pathology ; CRISPR-Cas Systems/genetics ; *Cellular Reprogramming/genetics ; *Transcription Factors/genetics ; Fibroblasts/metabolism/pathology ; Dependovirus/genetics ; Mice ; },
abstract = {Transcriptional activity perturbation holds promise for selectively modulating harmful transcriptional networks, but its therapeutic potential remains largely unexplored. We employed a network-based analysis of single-cell heart transcriptomes to identify transcription factor activities linked to pathological cardiomyocytes in vivo. This analysis revealed that transcriptional activity of Krüppel-like factor 15 (KLF15) exhibited the most significant change in pathological cardiomyocytes, characterized by less effective repression of disease-associated genes in stressed hearts, which correlated with reduced KLF15 expression. To restore KLF15 activity, we utilized CRISPR/nuclease-dead (d)Cas9-based transcriptional enhancement (CRISPRa) in cardiomyocytes, which effectively abolished fetal reprogramming by simultaneously suppressing pathological gene expression and restoring metabolic homeostasis under sustained stress conditions. Furthermore, we identified a novel cell-nonautonomous anti-fibrotic effect mediated by cardiomyocyte-fibroblast crosstalk, and revealed the contribution of KLF15-dependent Alpha-2-glycoprotein 1, zinc-binding (AZGP1) regulation in this process. We also elucidated the upstream mechanisms of KLF15 regulation, highlighting its role as a cell-specific downstream target of the broad TGF-β canonical signaling pathway, along with its downstream-dependent mechanisms in human cardiomyocytes. Finally, to enhance the therapeutic potential of this approach, we engineered and validated an adeno-associated viral (AAV) vector with a small CRISPRa system for endogenous regulation in human cardiomyocytes suitable for clinical applications. Overall, we elucidated a regulatory circuit involving TGF-β, KLF15, and AZGP1, which coordinates critical pathological responses through cellular crosstalk between cardiomyocytes and fibroblasts. Importantly, we demonstrated the efficacy of CRISPRa as an epigenetic intervention restoring a critical transcriptional function disrupted in non-genetic heart failure. This approach provides a promising blueprint for future adaptation targeting additional non-hereditary pathologies.},
}

*Myocytes, Cardiac/metabolism/pathology
*Kruppel-Like Transcription Factors/genetics/metabolism
Humans
Animals
Fibrosis/genetics/pathology
CRISPR-Cas Systems/genetics
*Cellular Reprogramming/genetics
*Transcription Factors/genetics
Fibroblasts/metabolism/pathology
Dependovirus/genetics
Mice

@article {pmid41771871,
year = {2026},
author = {Becerra, B and Wittibschlager, S and Patel, ZM and Kutschat, AP and Delano, J and Che, E and Tauber, A and Wu, T and Starrs, M and Horstmann, CS and Müller, S and Whittaker, MN and Sylvander, E and Lehner, M and Love, MI and Kleinstiver, BP and Jankowiak, M and Bauer, DE and Seruggia, D and Pinello, L},
title = {Nucleotide-resolution mapping of regulatory elements via allelic readout of tiled base editing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41771871},
issn = {2041-1723},
mesh = {Alleles ; Humans ; Antigens, CD19/genetics/metabolism ; PAX5 Transcription Factor/genetics/metabolism ; CRISPR-Cas Systems ; *Regulatory Sequences, Nucleic Acid/genetics ; Mutation ; Trans-Activators/genetics/metabolism ; Nucleotides/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR tiling screens have enabled the characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing and enrichment analysis. This study introduces an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. As a proof of concept, we studied a putative CD19 enhancer, an immunotherapy target in leukemia, and identified alleles and single nucleotides crucial for CD19 regulation. Our visualization tools revealed transcription factor motifs corresponding to the top-ranked nucleotides. Validation experiments confirmed that mutations in MYB, PAX5, and EBF1 binding sites reduce CD19 expression. Critically, editing MYB and PAX5 motifs conferred resistance to CD19 CAR-T cell therapy, revealing how non-coding variants can drive immunotherapy escape. Taken together, this approach achieves nucleotide-resolution genotype-phenotype mapping at regulatory elements beyond conventional gRNA-based screens.},
}

Alleles
Humans
Antigens, CD19/genetics/metabolism
PAX5 Transcription Factor/genetics/metabolism
CRISPR-Cas Systems
*Regulatory Sequences, Nucleic Acid/genetics
Mutation
Trans-Activators/genetics/metabolism
Nucleotides/genetics
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41772232,
year = {2026},
author = {Tinoco, AI and Henderson, CF and Meier, EK and Swinhoe, N and Cleves, PA},
title = {Efficient genome editing using CRISPR-Cas9 in reef-building corals.},
journal = {Nature protocols},
volume = {21},
number = {6},
pages = {2851-2879},
pmid = {41772232},
issn = {1750-2799},
support = {2128073//NSF | BIO | Division of Integrative Organismal Systems (IOS)/ ; },
mesh = {Animals ; *Anthozoa/genetics ; *CRISPR-Cas Systems ; Coral Reefs ; RNA, Guide, CRISPR-Cas Systems/genetics ; Microinjections ; },
abstract = {Coral reefs are one of the most biodiverse and productive ecosystems on Earth. However, corals are currently under threat from increasing ocean temperatures driven by climate change. Despite the known importance of these fragile ecosystems, our understanding of the molecular mechanisms driving ecologically important traits has been constrained by a lack of genetic tools for functional characterization. To address this limitation, we have developed straightforward and efficient methods to genetically modify corals and study gene function throughout various life history stages using CRISPR-Cas9-based mutagenesis. In this protocol, we first describe how to spawn and collect gametes from the coral Acropora millepora during seasonal spawning events. Next, we describe a method for microinjection of one-cell coral zygotes with CRISPR-Cas9 reagents. We include considerations about effective single-guide RNA design, methods for identifying successfully injected animals, strategies for rearing mutant larvae and juveniles, and methods for the detection and quantification of genomic modifications. This protocol is currently the only way to perform gene editing in corals and takes ~2-4 weeks to complete and has been successfully applied to study genes controlling heat tolerance in coral larvae and skeleton formation in coral juveniles. These technical advances set the foundation for a new field using reverse genetics to study ecologically important traits in corals, such as the establishment of symbiosis and its breakdown upon heat stress.},
}

Animals
*Anthozoa/genetics
*CRISPR-Cas Systems
Coral Reefs
RNA, Guide, CRISPR-Cas Systems/genetics
Microinjections

@article {pmid41772360,
year = {2026},
author = {Guo, Y and Yu, Z and Fan, S and Zhu, M and Ci, L and Yang, X and Chen, Y and Li, Q and Wang, N and Wang, J and Ye, S and Wang, J and Sun, R and Shen, R},
title = {A Bioluminescence Reporter Mouse Strain for In Vivo Imaging of IFNγ Cell Localization and Function.},
journal = {Immunology},
volume = {178},
number = {3},
pages = {428-438},
doi = {10.1111/imm.70127},
pmid = {41772360},
issn = {1365-2567},
support = {24141901100//Science and Technology Innovation Plan of Shanghai Science and Technology Commission/ ; 24YF2732000//Science and Technology Innovation Plan of Shanghai Science and Technology Commission/ ; 2025NS04//Shanghai Laboratory Animal Research Center/ ; 2025NS05//Shanghai Laboratory Animal Research Center/ ; },
mesh = {Animals ; *Interferon-gamma/genetics/metabolism/immunology ; Mice, Inbred C57BL ; *Luminescent Measurements/methods ; Mice, Transgenic ; Mice ; *Genes, Reporter ; Luciferases/genetics ; Disease Models, Animal ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; Humans ; Promoter Regions, Genetic ; },
abstract = {Interferon gamma (IFNγ) is a pivotal inflammatory mediator and immune regulator, but its in vivo spatiotemporal dynamics and functional roles in inflammation and carcinogenesis remain incompletely understood. Here, we developed a C57BL/6J- Ifng-2A-luciferase knock-in mouse strain using CRISPR/Cas9-mediated homology-directed repair, enabling real-time bioluminescence imaging (BLI) of IFNγ-expressing cells by inserting a luciferase cassette under the endogenous Ifng promoter. The validation confirmed that this model is capable of directly detecting Poly(I:C) -induced transient IFNγ, enhancing intratumoral IFNγ signals upon anti-PD-1/CTLA-4 therapy, and dynamically tracking IFNγ expression during imiquimod-induced psoriasis. This transgenic mouse model provides a powerful tool for non-invasive, longitudinal tracking of IFNγ-expressing cells, offering novel insights into IFNγ-mediated immune regulation in inflammation and cancer. It holds promise for identifying IFNγ-related therapeutic targets and predicting responses to immunotherapies.},
}

Animals
*Interferon-gamma/genetics/metabolism/immunology
Mice, Inbred C57BL
*Luminescent Measurements/methods
Mice, Transgenic
Mice
*Genes, Reporter
Luciferases/genetics
Disease Models, Animal
CRISPR-Cas Systems
Gene Knock-In Techniques
Humans
Promoter Regions, Genetic

@article {pmid41772759,
year = {2026},
author = {Donega, S and Gorospe, M and Harries, LW and Ferrucci, L},
title = {Loss of Splicing Homeostasis as a Hallmark of Aging.},
journal = {Molecular and cellular biology},
volume = {},
number = {},
pages = {1-19},
doi = {10.1080/10985549.2026.2627235},
pmid = {41772759},
issn = {1098-5549},
abstract = {Alternative splicing is a fundamental mechanism that ensures accurate gene expression, supports cellular adaptability, and expands protein diversity beyond the limits of a fixed gene pool. With aging, splicing fidelity weakens, contributing to decline in RNA homeostasis and disrupting essential cellular functions, including mitochondrial oxidative phosphorylation, genome stability, and immune regulation, and in turn accelerating tissue and organ dysfunction. Evidence from senescent cells, aged tissues, and model organisms shows that altered levels of splicing factors and increased RNA polymerase II elongation rates impair co-transcriptional splicing and promote mis-spliced isoforms that reinforce senescence and drive pathology. Dysfunction of RNA-binding proteins further contributes to aberrant splicing, linking splicing defects to age-related diseases such as atherosclerosis, osteoarthritis, sarcopenia, and neurodegenerative disorders like Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Therapeutic strategies to correct splicing defects, such as antisense oligonucleotides, RNA interference, CRISPR-Cas systems, ADAR-mediated editing, and RNA aptamers, can restore a homeostatic balance of mRNA isoforms. However, major challenges remain, including distinguishing adaptive physiological from pathological splicing 'noise' and achieving targeted delivery to tissues. Despite these obstacles, RNA splicing dysregulation represents a promising avenue to extend health span by reestablishing homeostatic RNA programs, and reinforces the idea that "transcriptomic instability" is a hallmark of aging.},
}

@article {pmid41773016,
year = {2026},
author = {Park, H and Yun, J and Lee, K and Kim, JH and Park, JH and Park, YJ and Park, JH and Lee, H and Kim, MG},
title = {Functional decoupling of crRNA enables customizable CRISPR diagnostics.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41773016},
issn = {1362-4962},
support = {//National Research Foundation of Korea/ ; RS-2025-16063091//National Research Council of Science and Technology/ ; RS-2024-00411137//National Research Council of Science and Technology/ ; CRC22024-500//National Research Council of Science and Technology/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {One-pot CRISPR-based diagnostics have transformed nucleic acid testing, yet their design customizability remains constrained. Because target programming and cis-cleavage activity are simultaneously determined during CRISPR RNA (crRNA) design, optimizing cleavage activity to match isothermal amplification inevitably requires altering the programmed crRNA sequence. This requirement fundamentally constrains the range of compatible target sequences, imposing limitations on the flexible design of diagnostic assays. Here, we establish a customizable one-pot system by decoupling the dual functions inherent in crRNA design to enable their independent control. In this strategy, target programming remains defined by the crRNA sequence, whereas cis-cleavage activity is regulated by the reaction energy barrier. We selectively modulate this energy barrier through the introduction of a crRNA-complementary RNA oligonucleotide, achieving cleavage regulation without altering the crRNA sequence. Consequently, this approach ensures that cis-cleavage activity matches isothermal amplification conditions independent of the programmed target sequence, thereby realizing a customizable CRISPR diagnostic system. We validated the clinical applicability of this system using 120 patient-derived samples, achieving sensitivity and specificity comparable to quantitative polymerase chain reaction. Collectively, this work resolves a fundamental constraint of CRISPR diagnostics and establishes a customizable and clinically deployable platform for next-generation nucleic acid testing.},
}

*CRISPR-Cas Systems
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats
Nucleic Acid Amplification Techniques/methods

@article {pmid41773018,
year = {2026},
author = {Yang, T and Tang, M and Xu, L and Jiang, L and Jiang, L and Zou, Y and Wang, J and Liu, Z and Chen, F and Ban, Y and Ren, W and Cheng, W},
title = {A tailored phosphorothioate coordinator enables CRISPR/Cas in-situ amplification.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41773018},
issn = {1362-4962},
support = {U24A20751//National Natural Science Foundation of China/ ; 82372334//National Natural Science Foundation of China/ ; 82502827//National Natural Science Foundation of China/ ; CSTB2023NSCQ-LZX0022//Chongqing Education Commission/ ; CSTB2024NSCQ-QCXMX0006//New Chongqing Youth Innovative Talents Project/ ; //National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Phosphorothioate Oligonucleotides/chemistry ; Human papillomavirus 18/genetics ; Human papillomavirus 16/genetics ; RNA, Messenger/genetics/metabolism ; HeLa Cells ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {The CRISPR/Cas system is a powerful tool for molecular diagnostics, but its reliance on linear amplification constrains sensitivity, particularly for in situ imaging. Here, we discovered that phosphorothioate (PS)-modified activators can modulate Cas enzyme conformation via hydrophobic anchoring. By adjusting the PS modification sites, we achieved precise control over Cas activation and trans-cleavage resistance. Guided by this mechanism, we proposed a tailored design strategy featuring a "scattered" PS modification to engineer a linear "Coordinator" probe. This design effectively decouples Cas enzyme activation from substrate trans-cleavage resistance, enabling the construction of a Scattered PS Nucleic Acid-driven Cas Autocatalytic system (SACA). SACA achieves exponential amplification without external enzymes, enhancing Cas12a and Cas13a sensitivity by 50 000-fold and 10 000-fold, respectively. Furthermore, the superior biostability and structural simplicity of these linear probes endow SACA with excellent compatibility, facilitating precise in situ imaging of HPV16 and HPV18 mRNA in cervical cancer cells. This study not only advances the understanding of Cas enzyme regulation by chemically modified nucleic acids but also establishes a new paradigm for precise and efficient molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
Humans
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
*Phosphorothioate Oligonucleotides/chemistry
Human papillomavirus 18/genetics
Human papillomavirus 16/genetics
RNA, Messenger/genetics/metabolism
HeLa Cells
*Nucleic Acid Amplification Techniques/methods

@article {pmid41773916,
year = {2026},
author = {Sui, Z and Chen, B and Zhao, J and Deng, R and Xu, J},
title = {Pronounced Fluorescence Polarization Enhancement Driven by RPA-CRISPR/Cas12a Induced Nucleoprotein Assembly for Salmonella Analysis in Animal-Derived Food Matrices.},
journal = {Analytical chemistry},
volume = {98},
number = {10},
pages = {7822-7831},
doi = {10.1021/acs.analchem.5c08279},
pmid = {41773916},
issn = {1520-6882},
mesh = {Animals ; *Fluorescence Polarization/methods ; *Salmonella/isolation & purification/genetics ; *Food Microbiology ; *CRISPR-Cas Systems/genetics ; *Nucleoproteins/metabolism/chemistry ; *Bacterial Proteins/metabolism/genetics ; Nucleic Acid Amplification Techniques/methods ; Food Contamination/analysis ; *CRISPR-Associated Proteins/metabolism/genetics ; Limit of Detection ; Endodeoxyribonucleases ; },
abstract = {Salmonella is one of the most hazardous foodborne pathogens, posing a serious threat to public health and food safety worldwide. Conventional recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection assays predominantly rely on the trans-cleavage of fluorescent reporters; however, such signal-generation modes are inherently susceptible to photobleaching, signal drift, and fluctuation, thereby compromising quantitative accuracy and long-term signal stability in practical pathogen detections. To overcome these limitations, we developed a trans-cleavage-independent fluorescence polarization (FP) sensing platform for the rapid and quantitative detection of Salmonella. Unlike conventional reporter-cleavage-based readouts, the proposed system exploits target-induced nucleoprotein assembly to achieve direct, physical signal amplification. In this design, a FAM-labeled forward primer serves as an intrinsic molecular reporter, while exonuclease I (Exo I) selectively degrades unincorporated primers, effectively suppressing background interference. Upon recognition of Salmonella genomic DNA, RPA produces rigid double-stranded amplicons that restrict fluorophore rotational freedom, and subsequent crRNA-guided Cas12a binding further increases molecular size and hydrodynamic volume, resulting in a stepwise enhancement of FP signals. The assay exhibits excellent linearity over a concentration range of 3 × 10[1]-3 × 10[6] CFU mL[-1], with an ultralow detection limit of 5 CFU mL[-1]. In addition, it demonstrates outstanding photostability, reproducibility, and high specificity against nontarget bacteria. Importantly, reliable Salmonella detection was achieved in complex food matrices, including meat, eggs, and dairy products, with consistently high recoveries and strong tolerance to matrix interference, offering a promising alternative to conventional fluorescence-intensity-based CRISPR diagnostics in complex food systems.},
}

Animals
*Fluorescence Polarization/methods
*Salmonella/isolation & purification/genetics
*Food Microbiology
*CRISPR-Cas Systems/genetics
*Nucleoproteins/metabolism/chemistry
*Bacterial Proteins/metabolism/genetics
Nucleic Acid Amplification Techniques/methods
Food Contamination/analysis
*CRISPR-Associated Proteins/metabolism/genetics
Limit of Detection
Endodeoxyribonucleases

@article {pmid41774834,
year = {2026},
author = {Chen, Z and Lin, H and Yoon, C and Huang, H and Kim, Y and Meng, C and Jang, H and Xie, Z and Li, L and Liu, Y and Kim, JS and Zhang, H},
title = {Bismuthene-Based Nanoplatform for Synergistic Thermogenetic CRISPR and Photothermal Cancer Therapy.},
journal = {Nano letters},
volume = {26},
number = {10},
pages = {3407-3416},
doi = {10.1021/acs.nanolett.5c06006},
pmid = {41774834},
issn = {1530-6992},
mesh = {*Photothermal Therapy/methods ; Animals ; Humans ; *CRISPR-Cas Systems ; Female ; Cell Line, Tumor ; *Nanostructures/chemistry ; *Triple Negative Breast Neoplasms/therapy/genetics/pathology ; Mice ; Hyperthermia, Induced ; },
abstract = {Overcoming tumor thermotolerance within clinically safe temperature ranges remains a central limitation of photothermal therapy (PTT). Here we report a closed-loop therapeutic nanoplatform that integrates topologically enhanced photothermal conversion with thermally gated CRISPR/Cas9 regulation. Rationally engineered hexagonal bismuthene nanodiscs exhibit strong near-infrared responsiveness, enabling mild hyperthermia (∼45 °C) that activates a heat-sensitive CRISPR switch targeting CDK7. The resulting disruption of the CDK7-HSP70 stress axis lowers the thermal resistance threshold and reprograms tumor adaptation, thereby amplifying photothermal efficacy and promoting immunogenic cell death. In triple-negative breast cancer models, this gene-thermal feedback achieves >93% tumor inhibition with minimal systemic toxicity. This work establishes a genetically programmable, thermogenetic nanomaterial paradigm that links material design with gene logic for next-generation precision cancer therapy.},
}

*Photothermal Therapy/methods
Animals
Humans
*CRISPR-Cas Systems
Female
Cell Line, Tumor
*Nanostructures/chemistry
*Triple Negative Breast Neoplasms/therapy/genetics/pathology
Mice
Hyperthermia, Induced

@article {pmid41776280,
year = {2026},
author = {Shirai, Y and Kao, JA and Kumar, T and Matsuda, N and Nakagawa, R and Nureki, O and Extavour, CG and Daimon, T},
title = {HUH-tagged Cas9 as a platform for efficient ssODN-mediated knock-in via embryo and adult injection in insects.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41776280},
issn = {2399-3642},
support = {20H02999, 20K21311, 22K19179, 24H00511, 24K21869//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21J20658//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; Overseas Research Fellow//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; Long-Term Fellow//Human Frontier Science Program (HFSP)/ ; JPJ008000//Cabinet Office, Government of Japan/ ; IOS-2220747//NSF | BIO | Division of Integrative Organismal Systems (IOS)/ ; },
mesh = {Animals ; *Gene Knock-In Techniques/methods ; *Tribolium/genetics/embryology ; *CRISPR-Cas Systems ; DNA End-Joining Repair ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Gene Editing/methods ; *Oligodeoxyribonucleotides/genetics ; DNA, Single-Stranded/genetics ; },
abstract = {Recent advances in adult injection-based insect genome editing have enabled genetic manipulation of a wide range of insect species, including those previously considered difficult or even impervious to genetic modification. However, achieving efficient knock-in remains a significant challenge with this approach. Here, we demonstrate that fusing a HUH endonuclease tag to Cas9 significantly enhances both non-homologous end joining (NHEJ)-mediated knockout and homology-directed repair (HDR)-mediated knock-in via adult injection. This fusion increased knockout efficiency by up to fivefold in the beetle Tribolium castaneum through adult injection, likely due to its previously unrecognized nuclear localization activity. It also improved single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in efficiency, which we attribute to its characteristic ssDNA-tethering activity. To evaluate its versatility, we tested the HUH-tagged Cas9 in conventional embryo injection, which significantly enhanced HDR-mediated knock-in of an epitope tag in cricket and milkweed bug embryos. Our findings establish the HUH-tag as a versatile platform for improving both NHEJ- and HDR-based genome editing, providing a robust framework to advance genetic engineering across a broad spectrum of arthropods.},
}

Animals
*Gene Knock-In Techniques/methods
*Tribolium/genetics/embryology
*CRISPR-Cas Systems
DNA End-Joining Repair
*CRISPR-Associated Protein 9/genetics/metabolism
*Gene Editing/methods
*Oligodeoxyribonucleotides/genetics
DNA, Single-Stranded/genetics

@article {pmid41777069,
year = {2026},
author = {Zamperin, G and Palumbo, E and Castellan, M and Marciano, S and Fusaro, A and Monne, I},
title = {Metagenomic sequencing of zoonotic viruses: evaluation of a CRISPR-Cas-based rRNA depletion system.},
journal = {Veterinaria italiana},
volume = {62},
number = {2},
pages = {},
doi = {10.12834/VetIt.3908.38985.2},
pmid = {41777069},
issn = {1828-1427},
mesh = {Animals ; *CRISPR-Cas Systems ; *Metagenomics/methods ; *RNA, Ribosomal/genetics ; *Zoonoses/virology ; Genome, Viral ; RNA, Viral/genetics ; },
abstract = {Pathogen-agnostic diagnostics are crucial for the early detection of emerging viruses. Shotgun metagenomic sequencing enables unbiased detection of viral genomes but is frequently constrained by the abundance of host and microbial ribosomal RNA (rRNA), which reduces sensitivity and increases sequencing costs. CRISPR-Cas9-based rRNA depletion has emerged as an alternative to enzymatic methods; however, its performance for the characterization of zoonotic viruses across diverse animal hosts and tissues remains underexplored. We compared CRISPR-Cas9 (Jumpcode CRISPRclean™ Plus) and RNase H-based enzymatic depletion (Ribo-Zero Plus, Illumina) using 12 samples positive for rabies lyssavirus, influenza A virus, West Nile virus or norovirus, from multiple host species and tissues, including both high-quality and degraded RNA. CRISPR-Cas9 efficiently reduced rRNA content (14.5%) but recovered fewer viral reads than Ribo-Zero, which achieved up to 60.7× enrichment. Both methods produced complete viral consensus genomes when RNA quality and viral load were sufficient. However, based on the data generated here, enzymatic depletion currently remains more efficient and cost-effective for viral metagenomics. Further optimization of CRISPR-Cas9 workflows could enhance its utility for viral surveillance and diagnostics.},
}

Animals
*CRISPR-Cas Systems
*Metagenomics/methods
*RNA, Ribosomal/genetics
*Zoonoses/virology
Genome, Viral
RNA, Viral/genetics

@article {pmid41779781,
year = {2026},
author = {Ju, X and Dong, L and Liu, T and Zhang, F and Sun, X and Schwoerer, MP and Ren, W and Gong, M and Ploss, A and Qin, W and Wu, X and Wang, L and Ding, Q},
title = {EIF4H and YBX1 are essential host factors for hepatitis E virus replication and pathogenesis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2529289123},
pmid = {41779781},
issn = {1091-6490},
support = {2023YFC2306900//National Key Research and Development Plan of China/ ; 82341084//MOST | National Natural Science Foundation of China (NSFC)/ ; 82272302//MOST | National Natural Science Foundation of China (NSFC)/ ; 82522053//MOST | National Natural Science Foundation of China (NSFC)/ ; 20251080029//Tsinghua University Dushi Program/ ; Not applicable//SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine/ ; Not applicable//High Meadows Environmental Institute/ ; T32 GM007388/GM/NIGMS NIH HHS/United States ; },
mesh = {*Virus Replication ; Humans ; *Hepatitis E virus/physiology/pathogenicity/genetics ; *Y-Box-Binding Protein 1/metabolism/genetics ; Animals ; *Hepatitis E/virology/metabolism/pathology/genetics ; *Eukaryotic Initiation Factors/metabolism/genetics ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; },
abstract = {Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Despite the availability of a vaccine in China, no direct-acting antivirals are approved, and host factors required for HEV replication remain poorly defined. Here, using a genome-wide CRISPR/Cas9 knockout screen in a replicon system, we identified Eukaryotic Translation Initiation Factor 4H (EIF4H) and Y-Box Binding Protein 1 (YBX1) as essential host factors for HEV replication and pathogenesis. Knockout of either factor markedly impaired replication of HEV genotypes 1, 3, and 4, as well as HEV infection and production in hepatocellular carcinoma cells and human induced pluripotent stem cell-derived hepatocyte-like cells, while leaving SARS-CoV-2, hepatitis B virus, hepatitis C virus, and Zika virus unaffected, underscoring their HEV-specific roles. Mechanistically, EIF4H interacts with ORF1 via its methyltransferase-Y-papain-like protease region, and EIF4H deficiency alters the composition of the ORF1-associated replication complex. By contrast, YBX1 is dispensable for ORF1 translation and RNA binding but is specifically required for ORF1 proteolytic processing, a prerequisite for assembling a functional replication machinery. EIF4H knockout rats and liver-specific YBX1 knockout rats were largely resistant to rat HEV-C1 infection, showing profound reductions in viral shedding, suppressed hepatic and intestinal viral loads, and protection from liver pathology. Together, our findings establish EIF4H and YBX1 as essential host factors for HEV infection and pathogenesis and reveal potential targets for antiviral intervention.},
}

*Virus Replication
Humans
*Hepatitis E virus/physiology/pathogenicity/genetics
*Y-Box-Binding Protein 1/metabolism/genetics
Animals
*Hepatitis E/virology/metabolism/pathology/genetics
*Eukaryotic Initiation Factors/metabolism/genetics
Host-Pathogen Interactions
CRISPR-Cas Systems

@article {pmid41781609,
year = {2026},
author = {Xiao, R and Hoffmann, FT and Xie, D and Wiegand, T and Palmieri, AI and Sternberg, SH and Chang, L},
title = {Structural basis of RNA-guided transcription by a dCas12f-σ[E]-RNAP complex.},
journal = {Nature},
volume = {653},
number = {8113},
pages = {288-296},
pmid = {41781609},
issn = {1476-4687},
mesh = {*Bacterial Proteins/chemistry/metabolism/ultrastructure ; *CRISPR-Associated Proteins/metabolism/chemistry/ultrastructure ; CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; DNA/metabolism/chemistry ; *DNA-Directed RNA Polymerases/metabolism/chemistry/ultrastructure ; Gene Expression Regulation, Bacterial ; Holoenzymes/chemistry/metabolism/ultrastructure ; Models, Molecular ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; *Sigma Factor/chemistry/metabolism/ultrastructure ; *Transcription Initiation, Genetic ; Transcription, Genetic ; },
abstract = {In both natural and engineered biological systems, RNA-guided proteins have emerged as critical transcriptional regulators by modulating RNA polymerase (RNAP) and its associated factors[1-3]. In bacteria, diverse clades of repurposed TnpB and CRISPR-associated proteins repress gene expression by blocking transcription initiation or elongation, enabling non-canonical modes of regulatory control and adaptive immunity[1,4,5]. A distinct class of nuclease-dead Cas12f homologues (dCas12f) instead activates gene expression through its association with unique extracytoplasmic function sigma factors (σ[E])[6], although the molecular basis has remained elusive. Here we reveal a new mode of RNA-guided transcription initiation by determining the cryo-electron microscopy structures of the dCas12f-σ[E] system from Flagellimonas taeanensis. We captured multiple conformational and compositional states, including the DNA-bound dCas12f-σ[E]-RNAP holoenzyme complex, revealing how RNA-guided DNA binding leads to σ[E]-RNAP recruitment and nascent mRNA synthesis at a precisely defined distance downstream of the R-loop. Rather than following the classical paradigm of σ[E]-dependent promoter recognition, these studies show that recognition of the -35 element is largely supplanted by CRISPR-Cas targeting, whereas the melted -10 element is stabilized through unusual stacking interactions rather than insertion into the typical recognition pocket. Collectively, this work provides high-resolution insights into an unexpected mechanism of RNA-guided transcription, expanding our understanding of bacterial gene regulation and opening new avenues for programmable transcriptional control.},
}

*Bacterial Proteins/chemistry/metabolism/ultrastructure
*CRISPR-Associated Proteins/metabolism/chemistry/ultrastructure
CRISPR-Cas Systems/genetics
Cryoelectron Microscopy
DNA/metabolism/chemistry
*DNA-Directed RNA Polymerases/metabolism/chemistry/ultrastructure
Gene Expression Regulation, Bacterial
Holoenzymes/chemistry/metabolism/ultrastructure
Models, Molecular
*RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry
*Sigma Factor/chemistry/metabolism/ultrastructure
*Transcription Initiation, Genetic
Transcription, Genetic

@article {pmid41781627,
year = {2026},
author = {Hoffmann, FT and Wiegand, T and Palmieri, AI and Glass-Klaiber, J and Xiao, R and Tang, S and Le, HC and Meers, C and Lampe, GD and Chang, L and Sternberg, SH},
title = {Exapted CRISPR-Cas12f homologues drive RNA-guided transcription.},
journal = {Nature},
volume = {653},
number = {8113},
pages = {277-287},
pmid = {41781627},
issn = {1476-4687},
mesh = {*Escherichia coli/genetics/enzymology/metabolism ; *CRISPR-Cas Systems/genetics ; Sigma Factor/metabolism/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics ; Promoter Regions, Genetic/genetics ; *Transcription, Genetic ; Gene Expression Regulation, Bacterial ; Transcriptional Activation ; Escherichia coli Proteins/metabolism/genetics ; Transcription Initiation, Genetic ; },
abstract = {Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP)[1]. Although the seven σ factors in Escherichia coli have been extensively characterized[2], Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ[E]) whose precise roles are unknown, pointing to additional layers of regulatory potential[3]. Here we uncover a mechanism of RNA-guided gene activation involving the coordinated action of σ[E] factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically linked dCas12f and σ[E] homologues in E. coli using RNA and chromatin immunoprecipitation experiments, revealing systems that exhibit robust guide RNA enrichment and DNA target binding with a minimal 5'-G target-adjacent motif. Recruitment of σ[E] was dependent on dCas12f and guide RNA, suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σ[E] ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σ[E] complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription that embodies natural features reminiscent of CRISPR activation (CRISPRa) technology[4,5].},
}

*Escherichia coli/genetics/enzymology/metabolism
*CRISPR-Cas Systems/genetics
Sigma Factor/metabolism/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Associated Proteins/metabolism/genetics
Promoter Regions, Genetic/genetics
*Transcription, Genetic
Gene Expression Regulation, Bacterial
Transcriptional Activation
Escherichia coli Proteins/metabolism/genetics
Transcription Initiation, Genetic

@article {pmid41782368,
year = {2026},
author = {Chuecos, MA and Park, SH and Bhakta, MM and Too-Chiobi, U and Betancourth, D and Cao, M and De Giorgi, M and Walkey, CJ and Tiwari, A and Godin, B and Assini, JM and Palmer, DJ and Ng, P and Boffa, MB and Koschinsky, ML and Bao, G and Lagor, WR},
title = {Cytosine base editing of LPA in transgenic mice averts large deletions.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {6},
pages = {3334-3352},
pmid = {41782368},
issn = {1525-0024},
support = {R01 HL169761/HL/NHLBI NIH HHS/United States ; R01 HL132840/HL/NHLBI NIH HHS/United States ; P30 CA125123/CA/NCI NIH HHS/United States ; P30 DK056338/DK/NIDDK NIH HHS/United States ; S10 OD030414/OD/NIH HHS/United States ; U42 OD026645/OD/NIH HHS/United States ; P50 HD103555/HD/NICHD NIH HHS/United States ; R01 HG011459/HG/NHGRI NIH HHS/United States ; R01 DK124477/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Mice, Transgenic ; Mice ; *Gene Editing/methods ; *Lipoprotein(a)/genetics ; Genetic Vectors/genetics/administration & dosage ; Dependovirus/genetics ; *Cytosine/metabolism ; Adenoviridae/genetics ; Humans ; *Sequence Deletion ; CRISPR-Cas Systems ; },
abstract = {Lipoprotein(a) (Lp(a)) is a genetically determined causal risk factor for cardiovascular disease, with approximately 20% of the population exhibiting elevated levels. While there are promising drugs in development, there are currently no approved therapies specifically designed to lower Lp(a) levels. For high-risk individuals with extreme levels of Lp(a), liver-directed genome editing could be an effective one-time solution. Genome editing approaches such as CRISPR and TALENs can reduce Lp(a) in LPA-transgenic mouse models, but they frequently induce large and potentially harmful genomic deletions. Here, we report the first application of TadA-derived cytosine base editing (CBE), delivered via helper-dependent adenovirus (HDAdV) and adeno-associated virus (AAV) vectors, to introduce premature stop codons into LPA. This strategy produced robust and durable lowering of circulating apolipoprotein(a) (apo(a)) in LPA-transgenic mice. Using SMRT-seq with single-molecule unique molecular identifiers, we quantified deletion events and found that CBE did not induce large deletions when targeting a single LPA site and produced only a small fraction (<4%) of large deletions when editing across multiple sites. In contrast, CRISPR-Cas9 cutting of LPA resulted primarily in large deletions. These findings demonstrate that CBE enables sustained reduction of circulating apolipoprotein(a) in an LPA-transgenic mouse model while largely preserving genomic integrity.},
}

Animals
Mice, Transgenic
Mice
*Gene Editing/methods
*Lipoprotein(a)/genetics
Genetic Vectors/genetics/administration & dosage
Dependovirus/genetics
*Cytosine/metabolism
Adenoviridae/genetics
Humans
*Sequence Deletion
CRISPR-Cas Systems

@article {pmid41783940,
year = {2026},
author = {Berti, M and Ceriotti, S and Santi, L and Alberti, G and Beretta, S and Degl'Innocenti, S and Ruatti, C and Savoia, EO and Jofra-Hernandez, R and De Ponti, G and Bolamperti, S and Villa, I and Galeotti, F and Romano, A and Visigalli, I and Norata, R and Rocchi, M and Cristofori, P and Cossutta, M and Consiglieri, G and Tucci, F and Santorelli, L and Grumati, P and Ronfani, L and D'Adamo, P and Giustina, A and Angelozzi, M and Settembre, C and Mortellaro, A and Scala, S and Sanvito, F and Volpi, N and Aiuti, A and Bernardo, ME and Crippa, S},
title = {Development and characterization of a model of mucopolysaccharidosis type IVA for evaluating therapies targeting bone disease.},
journal = {Disease models & mechanisms},
volume = {19},
number = {2},
pages = {},
pmid = {41783940},
issn = {1754-8411},
support = {20228H9T82//Ministero dell'Istruzione, dell'Università e della Ricerca/ ; P20223MF7X_001//Ministero dell'Istruzione, dell'Università e della Ricerca/ ; CN_00000041 - CUP G83C22000270001//NextGenerationEU/ ; TTAGTXEKFA//Else Kröner-Fresenius-Zentrum für Ernährungsmedizin/ ; TELE-MB//Fondazione Telethon/ ; TELE-AA//Fondazione Telethon/ ; //Ospedale San Raffaele/ ; },
mesh = {Animals ; Disease Models, Animal ; *Mucopolysaccharidosis IV/therapy/complications/pathology/blood ; *Bone Diseases/therapy/complications/pathology/blood ; Glycosaminoglycans/metabolism ; Chondroitinsulfatases/metabolism/deficiency/genetics ; Keratan Sulfate/urine/metabolism ; Chondroitin Sulfates/urine/metabolism ; CRISPR-Cas Systems/genetics ; Biomarkers/metabolism/blood ; Bone and Bones/pathology ; Humans ; },
abstract = {Mucopolysaccharidosis type IVA (MPSIVA) is a lysosomal storage disease (LSD) caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), which causes the accumulation of keratan sulphate (KS) and chondroitin sulphate (CS). Patients with MPSIVA typically present with severe skeletal and joint disorders, which are not addressed by conventional therapies. Currently, no animal model accurately replicates the human disease, hindering the development of novel therapeutic interventions. To overcome this limitation, we established, by CRISPR-Cas9 technology, a Galns-/- mouse model that expresses a non-functional enzyme and accumulates CS and KS in the urine, plasma and distinct tissues, and glycosaminoglycans in the spleen. The mice exhibit shortened long bones, trabecular bone alterations and skeletal abnormalities in the growth plate. Additionally, we observed increased levels of inflammatory and oxidative markers in visceral organs and plasma. Our newly developed model of MPSIVA demonstrates clear and quantifiable signs of skeletal alterations, providing novel means of assessment of the safety and efficacy of innovative therapies, including hematopoietic stem and progenitor cell gene therapy, which has recently been shown to provide a beneficial effect on skeletal alterations in Hurler syndrome.},
}

Animals
Disease Models, Animal
*Mucopolysaccharidosis IV/therapy/complications/pathology/blood
*Bone Diseases/therapy/complications/pathology/blood
Glycosaminoglycans/metabolism
Chondroitinsulfatases/metabolism/deficiency/genetics
Keratan Sulfate/urine/metabolism
Chondroitin Sulfates/urine/metabolism
CRISPR-Cas Systems/genetics
Biomarkers/metabolism/blood
Bone and Bones/pathology
Humans

@article {pmid41784267,
year = {2026},
author = {Wang, Z and Wu, Y and Wang, Z and Zhang, S and Liu, H and Nie, Y and Chen, K and Huang, Y and Zhou, Y and Cao, Y and Sun, L and Hao, R},
title = {crRNA scaffold remodeling controls CRISPR-Cas12a activity for enhanced performance.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41784267},
issn = {1362-4962},
support = {//Prevention and Control of Emerging/ ; 2025ZD01903403//National Science and Technology Major Project/ ; L234051//Beijing Municipal Natural Science Foundation/ ; L246011//Beijing Municipal Natural Science Foundation/ ; 2024-03-18//Training Plan for High-Level Public Health Technical Talents of Beijing Municipal Health Commission/ ; 2025ZD01903403//Prevention and Control of Emerging and Major Infectious Diseases-National Science/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; Mycobacterium tuberculosis/genetics/isolation & purification ; *Endodeoxyribonucleases/metabolism/genetics ; Klebsiella pneumoniae/genetics ; Nucleic Acid Conformation ; Bacterial Proteins ; },
abstract = {CRISPR-Cas12a has transformative potential in molecular diagnostics owing to its robust signal amplification, but its sustained activity state severely limits temporal programmability and precise nuclease control in complex detection workflows. Here, we demonstrate that the conserved crRNA scaffold secondary structure itself can be repurposed as a reversible and programmable conformational switch to regulate Cas12a activity. By introducing short complementary DNA blockers of tunable length, we achieved length-dependent disruption and remodeling of scaffold secondary structure, shifting LbCas12a into an inactive conformation. Scaffold structure was subsequently reinstated through either single or cooperative strand displacement activation, enabling time-resolved and on-demand restoration of Cas12a activity. The conserved scaffold ensures intrinsic assay universality, while its programmable rewiring markedly improves SNVs discrimination and enables compatibility with one-pot isothermal amplification assays, delivering analytical sensitivity comparable to conventional two-step assays. This regulatory framework was further demonstrated in the detection of Klebsiella pneumoniae and Mycobacterium tuberculosis. By validating the crRNA scaffold as a practical and programmable switch for Cas12a activity control, this work establishes a universal and reversible framework for scaffold rewiring to modulate CRISPR nucleases and offers mechanistic insight to guide future assay engineering.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/metabolism/genetics
Mycobacterium tuberculosis/genetics/isolation & purification
*Endodeoxyribonucleases/metabolism/genetics
Klebsiella pneumoniae/genetics
Nucleic Acid Conformation
Bacterial Proteins

@article {pmid41784340,
year = {2026},
author = {Zhou, C and Cheng, T and Zhou, J and Zhang, B and Liu, L and Jiang, G and Li, W and Wang, C},
title = {Synthetic Biofilms for Green Membranes: Engineering Low-Energy Filtration Systems.},
journal = {Environmental science & technology},
volume = {60},
number = {12},
pages = {9357-9366},
doi = {10.1021/acs.est.5c15661},
pmid = {41784340},
issn = {1520-5851},
mesh = {*Biofilms ; *Membranes, Artificial ; Filtration ; Biofouling ; Water Purification ; Ultrafiltration ; },
abstract = {Membrane filtration is a key technology to modern water purification, yet its sustainability is compromised by biofouling, which increases energy consumption and ecological impacts. Conventional control strategies often struggle to balance efficacy and environmental footprint. In this study, an inducible, engineered quorum-quenching (QQ) bacterium was constructed via a genomic integration strategy, thereby achieving control over the biofilm structure in membrane filtration biofouling layers. By using the clustered regularly interspaced short palindromic repeat (CRISPR-Cas) targeted gene editing technology, the engineered bacteria that were constructed to express aiiO under l-ribose induction have achieved the regulation of biofilms. Validation using a gravity-driven membrane ultrafiltration system (UF-GDM) model showed that the engineered bacterium effectively reduced extracellular polymeric substances (EPS) components, increased the hydrophilic porosity of the residual biofilm, and decreased its stickiness. This approach reduced transmembrane pressure by 64.5%, increased total organic carbon (TOC) removal by 13.2%, and extended membrane lifespan by 16.1%. A technical-economic analysis indicates that the 100,000 m[3]/day treatment plant achieves an annual net profit increase of 31.52%, reaching 1.55 × 10[7] CNY, while reducing its net carbon footprint by 27.43%, with an annual net reduction of 2.96 × 10[5] kg CO2eq. This study provides a novel solution strategy for achieving biofouling resistance and sustainable, low-energy operation in membrane filtration processes, which contributes to the broader application and adoption of this technology.},
}

*Biofilms
*Membranes, Artificial
Filtration
Biofouling
Water Purification
Ultrafiltration

@article {pmid41785043,
year = {2026},
author = {Xu, X and Zhang, R and Hai, G and Wang, Y and Zheng, H and Cui, P and Kou, B and Jin, X and Peng, J},
title = {DNA Logic-Gated CRISPR/Cas13a and PNTs-Hemin Biomimetic Nanozyme for Ratiometric Detection of BRCA1 and circROBO1.},
journal = {Analytical chemistry},
volume = {98},
number = {10},
pages = {7580-7589},
doi = {10.1021/acs.analchem.5c07475},
pmid = {41785043},
issn = {1520-6882},
mesh = {*Hemin/chemistry ; *BRCA1 Protein/analysis/genetics ; *Biomimetic Materials/chemistry ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Nanotubes, Peptide/chemistry ; Biomimetics ; Gold/chemistry ; *DNA/chemistry ; Electrochemical Techniques ; Logic ; },
abstract = {Although nanozyme-based biosensors show great promise for the early diagnosis of cancer, their application is often limited by poor catalytic activity at neutral pH and susceptibility to matrix interference. This study involved the construction of a biomimetic nanozyme through coordination-driven self-assembly on peptide nanotubes. This rigid framework enables the periodic arrangement of histidine residues to achieve precise axial coordination with the iron center of the hemin molecule, effectively mimicking the active site and catalytic microenvironment of natural horseradish peroxidase. Consequently, PNTs-hemin exhibits peroxidase activity 2.7 times that of free hemin under near-physiological conditions. To ensure detection specificity, an AND logic gate design was integrated, triggering CRISPR/Cas13a-mediated trans-cleavage only when both targets (BRCA1 and circROBO1) are present simultaneously. Furthermore, combining the nanozyme with graphdiyne-supported gold nanoparticles resulted in the formation of a cascade catalytic system that produced a ratiometric electrochemical reading (IFc/Ihemin). This effectively corrects for environmental fluctuations and false positive signals. This study presents an effective strategy that combines the specificity of molecular logic gates with biomimetic catalysis. This opens up new avenues for the precise diagnosis of multiple targets in complex biological samples.},
}

*Hemin/chemistry
*BRCA1 Protein/analysis/genetics
*Biomimetic Materials/chemistry
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
Humans
Nanotubes, Peptide/chemistry
Biomimetics
Gold/chemistry
*DNA/chemistry
Electrochemical Techniques
Logic