@article {pmid40481608,
year = {2025},
author = {Liu, H and Sun, N and Liu, Z and Li, J and Zhang, X},
title = {Knockout of bcas3 gene causes neurodevelopment defects in zebrafish.},
journal = {Biological research},
volume = {58},
number = {1},
pages = {34},
pmid = {40481608},
issn = {0717-6287},
support = {2023YFC2706302//National Key R&D Program of China/ ; 81000079, 81170165, and 81870959//National Natural Science Foundation of China/ ; 2016QYTD02//Program for HUST Academic Frontier Youth Team/ ; },
mesh = {Animals ; *Zebrafish/genetics ; Neurodevelopment/genetics ; *Zebrafish Proteins/genetics ; Gene Knockout Techniques ; Disease Models, Animal ; *Neurodevelopmental Disorders/genetics ; CRISPR-Cas Systems ; Apoptosis/genetics ; },
abstract = {BACKGROUND: Neurodevelopmental disorders manifest in early childhood and are characterized by cognitive deficits, intellectual disabilities, motor disorders, and social dysfunction. Mutations in BCAS3 gene are associated with syndromic neurodevelopmental disorders in humans, while the detailed pathological mechanism is still unknown. METHODS: CRISPR/Cas9 technology was used to generate a bcas3 knockout zebrafish model. To investigate the effects of bcas3 on development, morphological evaluations were conducted. Locomotor behaviors, including performance in the light-dark test, novel tank test, mirror test, shoaling test, and social test, were assessed through video tracing and quantitative analysis of movement parameters. Transcriptome sequencing analysis was used to identify dysregulated pathways associated with development process. Additionally, Acridine Orange staining was employed to evaluate apoptosis. Western blot and real-time RT-PCR were used to analyze the expression levels of genes. RESULTS: Bcas3 knockout zebrafish exhibited early larval phenotypes resembling clinical features of patients with BCAS3 mutations, including global delayed development at early embryonic development, microcephaly and reduced body length. Behavior analysis revealed abnormal motor dysfunction, such as social impairment, increased anxiety and heightened aggression. Notably, human BCAS3 rescued the developmental defects and motor disorders in bcas3 knockout larvae. Transcriptomic analysis identified substantial downregulation of genes related to embryonic development and startle response, brain development and neuron migration in bcas3 knockout zebrafish, such as rpl10, cyfip2, erbb3b, eya4a, nr2f1b, prkg1b and ackr3b. Additionally, increased apoptosis was observed in bcas3 knockout zebrafish, which was further confirmed by Acridine Orange staining and a decreased Bcl2/Bax ratio in western blot analysis. The increased apoptosis observed in the brain of bcas3 knockout larvae could contribute to the developmental and locomotor deficits. CONCLUSION: The bcas3 knockout zebrafish model recapitulates the clinical features observed in patients with BCAS3 mutations. Our results suggest that increased apoptosis may underlie the developmental deficits and motor disorders in these patients. The bcas3 knockout zebrafish model provides a valuable tool to identify dysregulated molecular targets for therapeutic intervention during the early stages of disease progression.},
}

Animals
*Zebrafish/genetics
Neurodevelopment/genetics
*Zebrafish Proteins/genetics
Gene Knockout Techniques
Disease Models, Animal
*Neurodevelopmental Disorders/genetics
CRISPR-Cas Systems
Apoptosis/genetics

@article {pmid41258590,
year = {2025},
author = {Wang, X and Xia, C and Zhang, X},
title = {Genetic screening identifies Ube2v1 as a suppressor of immunoglobulin class switch recombination in CH12F3 cells.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {101},
pmid = {41258590},
issn = {1573-4978},
support = {31900655//National Natural Science Foundation of China/ ; },
mesh = {*Ubiquitin-Conjugating Enzymes/genetics/metabolism ; Animals ; *Immunoglobulin Class Switching/genetics ; Ubiquitination ; B-Lymphocytes/metabolism/immunology ; CRISPR-Cas Systems ; Mice ; Cell Line ; Genetic Testing/methods ; Humans ; },
abstract = {BACKGROUND: Antibodies are essential mediators of adaptive immunity, providing defense against pathogens and serving as critical tools in therapeutics of infection and cancer. Antibody diversification, including class switch recombination (CSR), is tightly regulated by ubiquitination, a post-translational modification, that modulates protein stability and functional activity. METHODS AND RESULTS: Using CRISPR/Cas9 screening in B cells, we investigated the role of ubiquitin-related enzymes in CSR. Validation experiments with LentiCRISPR-sgAID stable cell lines, which showed significant inhibition of CSR, confirmed that it works in the CSR model CH12F3 cell. Screening of 35 E2 ubiquitin-conjugating enzymes and 85 deubiquitinases (DUBs) identified Ube2v1, an E2 enzyme, as a potent suppressor of CSR. Specifically knockdown of Ube2v1 significantly enhanced CSR efficiency, whereas its overexpression inhibited CSR without affecting germline transcripts (GLTs) or activation-induced cytidine deaminase (AID). Intriguingly, although Ube2v1 canonically functions with Ube2n to mediate polyubiquitination, overexpression and knockdown of Ube2n had no detectable effect on CSR. CONCLUSIONS: Our CRISPR screening identified multiple components of ubiquitin pathway that regulate CSR and established Ube2v1 as a novel inhibitor. Ube2v1 functions independently of expression of GLTs and AID as well as its canonical partner Ube2n, revealing a non-canonical role. These findings underscore the complexity of post-translational regulation of humoral immunity and suggest Ube2v1 as a potential therapeutic target for modulating antibody responses.},
}

*Ubiquitin-Conjugating Enzymes/genetics/metabolism
Animals
*Immunoglobulin Class Switching/genetics
Ubiquitination
B-Lymphocytes/metabolism/immunology
CRISPR-Cas Systems
Mice
Cell Line
Genetic Testing/methods
Humans

@article {pmid41286760,
year = {2025},
author = {Wu, G and Ren, Y and Wang, Y and Zhao, Y and Wu, Y and Sun, H and Sun, Z and Wang, R and Du, Z},
title = {Detection of rifampin-resistant Mycobacterium tuberculosis using CRISPR/Cas14a-enabled molecular techniques.},
journal = {BMC infectious diseases},
volume = {25},
number = {1},
pages = {1813},
pmid = {41286760},
issn = {1471-2334},
mesh = {*Mycobacterium tuberculosis/genetics/drug effects/isolation & purification ; *Rifampin/pharmacology ; Humans ; Bacterial Proteins/genetics ; *Drug Resistance, Bacterial/genetics ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; DNA-Directed RNA Polymerases/genetics ; *Tuberculosis, Multidrug-Resistant/diagnosis/microbiology ; Microbial Sensitivity Tests ; RNA, Ribosomal, 16S/genetics ; *Molecular Diagnostic Techniques/methods ; *Antitubercular Agents/pharmacology ; },
abstract = {BACKGROUND: The emergence of drug-resistant Mycobacterium tuberculosis (MTB) strains highlights the urgent need for precise and timely diagnostic methods to prevent prolonged and complex treatment regimens. This study aims to develops a CRISPR/Cas14a-based assay for accurate identification of MTB and rifampin-resistant MTB (RR-MTB) strains. METHOD: The 16 S rDNA sequence and rifampin resistance-determining region (RRDR) of rpoB gene were chosen for the detection of MTB and RR-MTB, respectively. Several sgRNAs were designed for each target and evaluated for their performance. The platform was then systematic optimized by adjusting the concentrations of different components, followed by the evaluation of its sensitivity for the detection of MTB and RR-MTB. The system’s efficacy was further validated through a double-blind test on 16 clinical MTB isolates, and the results were compared with genomic sequencing. RESULTS: Through a meticulous screening process, we identified optimal single-guide RNAs (sgRNAs) capable of distinguishing MTB from nontuberculous mycobacteria (NTM) and the eight predominant mutation types associated with rifampin resistance. Our refined CRISPR/Cas14a platform demonstrated a remarkable sensitivity, with a limit of detection (LOD) of 200 copies/µL for MTB and 2 copies/µL for RR-MTB, respectively. This platform demonstrated a 100% accuracy rate in identification of RR-MTB using clinical MTB isolates. CONCLUSIONS: The CRISPR/Cas14a-based platform we developed exhibited superior performance for the detection of MTB and RR-MTB, with significant implications for the diagnosis and management of tuberculosis, particularly in regions with high prevalence of drug-resistant strains.},
}

*Mycobacterium tuberculosis/genetics/drug effects/isolation & purification
*Rifampin/pharmacology
Humans
Bacterial Proteins/genetics
*Drug Resistance, Bacterial/genetics
*CRISPR-Cas Systems
Sensitivity and Specificity
DNA-Directed RNA Polymerases/genetics
*Tuberculosis, Multidrug-Resistant/diagnosis/microbiology
Microbial Sensitivity Tests
RNA, Ribosomal, 16S/genetics
*Molecular Diagnostic Techniques/methods
*Antitubercular Agents/pharmacology

@article {pmid41354677,
year = {2025},
author = {Rangaraj, A and Kaur, H and Mejia, L and Hansen, J and Biswas, A and Tuteja, G},
title = {Characterization of a cis-regulatory element upstream of matrix metalloproteinase-9.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {1463},
pmid = {41354677},
issn = {2045-2322},
support = {R01 HD096083/HD/NICHD NIH HHS/United States ; R01HD096083//Eunice Kennedy Shriver National Institute of Child Health and Human Development/ ; },
mesh = {Humans ; *Matrix Metalloproteinase 9/genetics/metabolism ; Female ; *Enhancer Elements, Genetic ; Pregnancy ; Placenta/metabolism/cytology ; Gene Expression Regulation ; Cell Line ; CRISPR-Cas Systems ; *Regulatory Sequences, Nucleic Acid ; },
abstract = {Dysregulated enhancer activity disrupts gene expression, contributing to disease. However, the structural and functional complexity of enhancers hinders their characterization. Here, we investigate a cis-regulatory element upstream of matrix metalloproteinase-9 (MMP9), a gene implicated in cancer, cardiovascular disease, inflammation, and pregnancy complications. Using luciferase assays and CRISPR-Cas9 mediated knockout in a human placental cell line, we define a one kilobase segment that enhances MMP9 expression. Further dissection reveals two activating sub-segments and, unexpectedly, one repressive sub-segment. Molecular assays suggest transcription factors that mediate these opposing effects. This work adds to the growing understanding that enhancers can integrate both activation and repression, revealing a more complex regulatory architecture than previously appreciated. Together, these findings underscore the importance of enhancer dissection for understanding gene regulation across tissues and diseases.},
}

Humans
*Matrix Metalloproteinase 9/genetics/metabolism
Female
*Enhancer Elements, Genetic
Pregnancy
Placenta/metabolism/cytology
Gene Expression Regulation
Cell Line
CRISPR-Cas Systems
*Regulatory Sequences, Nucleic Acid

@article {pmid41392248,
year = {2025},
author = {Liang, J and Liang, T and Wei, C and Li, L and Li, Y and He, S and Liao, Z and Cui, L},
title = {CRISPR/Cas9-engineered Bacillus subtilis chassis for tailored chitooligosaccharide production from marine waste chitosan.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {36},
pmid = {41392248},
issn = {1475-2859},
support = {2023GXNSFAA026505//Natural Science Foundation of Guangxi Province/ ; gxkllst-20241009//Guangxi Key Laboratory of Longevity Science and Technology/ ; },
mesh = {*Bacillus subtilis/genetics/metabolism ; *Chitosan/metabolism ; Glycoside Hydrolases/genetics/metabolism ; *CRISPR-Cas Systems ; *Chitin/analogs & derivatives/biosynthesis ; Fermentation ; Oligosaccharides ; Bacterial Proteins/genetics/metabolism ; },
abstract = {BACKGROUND: This study establishes a sustainable bioprocess for converting chitosan from marine waste into high-value chitooligosaccharides (COSs), offering an eco-friendly alternative to conventional methods that often generate chemical waste. We achieved heterologous production of chitosanase in an engineered Bacillus subtilis chassis by knocking out its endogenous chitosanase, leveraging the dual advantages of this bacterium as a robust synthetic biology platform and an industrial microorganism. RESULTS: The endogenous chitosanase gene (BsCsn) in Bacillus subtilis WB800N was deleted via CRISPR/Cas9-mediated editing, generating the chassis strain B. subtilis WB800N ΔBsCsn. A codon-optimized GH46 chitosanase (CsnA) from Streptomyces coelicolor, fused to the AprE signal peptide, was then expressed in this host. Response surface methodology optimized the fermentation process, enabling a high extracellular CsnA activity of 540.08 ± 6.20 U/mL, in a 5-L bioreactor under DO-stat-controlled fed-batch conditions. This process achieved a productivity of 11.25 U/(mL·h) and a carbon conversion efficiency of 1682.86 U/g glycerol. Furthermore, MALDI-TOF MS analysis confirmed that CsnA produces COSs with defined degrees of polymerization (DP2-DP4). CONCLUSION: This integrated platform enables the upcycling of marine waste into high-value COSs, establishing B. subtilis as an eco-efficient cell factory and providing a valuable framework for the heterologous expression of other chitosanases in this host.},
}

*Bacillus subtilis/genetics/metabolism
*Chitosan/metabolism
Glycoside Hydrolases/genetics/metabolism
*CRISPR-Cas Systems
*Chitin/analogs & derivatives/biosynthesis
Fermentation
Oligosaccharides
Bacterial Proteins/genetics/metabolism

@article {pmid41405764,
year = {2025},
author = {Mansoor, MJ and Al-Taie, SF and Al-Khafaji, ZA and Alkhathami, AG and Renuka, JS and Panigrahi, R and Negi, H and Jassal, P and Mustafa, YF and Hamzah, HF},
title = {Overcoming barriers in CAR-NK immunotherapy: CRISPR-Driven advances in checkpoint editing and allogeneic design.},
journal = {Functional & integrative genomics},
volume = {26},
number = {1},
pages = {4},
pmid = {41405764},
issn = {1438-7948},
mesh = {Humans ; *Killer Cells, Natural/immunology/transplantation ; *Gene Editing/methods ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology/genetics ; *CRISPR-Cas Systems ; Animals ; *Receptors, Chimeric Antigen/genetics/immunology ; Immunotherapy ; Tumor Microenvironment ; },
abstract = {Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells are emerging as an exciting avenue in cancer immunotherapy due to their potent cytotoxicity to malignant cells and lower risk of graft-versus-host disease (GvHD) than conventional T cell therapies. The new technology of CRISPR/Cas9 genome editing has significantly expedited the engineering of CAR-NK cells by enabling easy, multiplex, and precise changes to enhance their efficacy, persistence, and specificity to tumors. This review focuses on the incorporation of CRISPR technology into CAR-NK cell development. It examines uses of knockout of inhibitory checkpoint genes (CISH, PD-1, and TGFBR2), as well as knock-in of CAR into safe genomic locations and multiplex editing of CAR-NK cells to improve cytotoxicity against cancer while resisting suppression from the tumor microenvironment (TME). We further explore immuno-cytokine armoring strategies by knock-in of IL-15 or IL-12, to ensure prolonged proliferation and survival of NK cells, and investigate CRISPR-mediated knockouts of immune inhibitors like NKG2A and TIGIT, to evade immune strategies used by the tumor to evade immune destruction. Furthermore, CRISPR-mediated upregulation of the homing receptor enhances NK cell tumor infiltration, addressing a major obstacle in treating solid tumors. It is significant to mention the progress in generating off-the-shelf products, which is a key step supporting the pursuit of allogeneic therapies. While substantial progress has been made, challenges remain related to optimizing CRISPR delivery, off-target effects, and enhancing in vivo persistence. Future directions of CAR-NK studies will likely capitalize on next-generation genome editing tools and synthetic biology for the development of tunable and logic-gated CAR-NK cells. Overall, this review illustrates the revolutionary capacity of combining CRISPR technology with CAR-NK immunotherapy to develop next-generation programmable and efficacious treatments for hematologic and solid malignancies.},
}

Humans
*Killer Cells, Natural/immunology/transplantation
*Gene Editing/methods
*Immunotherapy, Adoptive/methods
*Neoplasms/therapy/immunology/genetics
*CRISPR-Cas Systems
Animals
*Receptors, Chimeric Antigen/genetics/immunology
Immunotherapy
Tumor Microenvironment

@article {pmid41430619,
year = {2025},
author = {Le Reun, J and Salvioli, Z and Croux, C and Esque, J and André, I and Bordes, F},
title = {A workflow to explore elongase diversity and extend the repertoire of fatty acids produced by Yarrowia lipolytica.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {18},
pmid = {41430619},
issn = {1475-2859},
mesh = {*Yarrowia/metabolism/genetics/enzymology ; *Fatty Acid Elongases/metabolism/genetics ; *Fatty Acids/biosynthesis/metabolism ; Substrate Specificity ; Workflow ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Fatty acids display highly diverse structures that confer these molecules unique chemical properties and distinct physiological functions. Identifying the substrate specificity of enzymes active on fatty acids is crucial, both for understanding their function in natural organisms and for developing efficient cell factories to produce original fatty acids. However, these enzymes are often membrane-bound and/or act on esterified substrates and studying them in vitro is thus challenging. This is why in vivo characterization of these enzymes’ specificity is an interesting approach. Herein, we harness the industrially relevant oleaginous Yarrowia lipolytica as a chassis for characterizing heterologous enzymes active on fatty acids, which can be used to diversify its fatty acid composition. As a case study, we investigated fatty acid elongases (ELO) responsible for the synthesis of very long-chain fatty acids (> 20 carbons), which are specific of given chain lengths and/or unsaturation patterns. Despite their interest, investigation and utilization of these membrane enzymes remain largely underexplored. RESULTS: We developed a workflow for characterizing heterologous elongases in Y. lipolytica, addressing several limitations to increase throughput. First, we set up a strain engineering strategy to easily integrate the ELO cassettes into targeted loci using CRISPR-Cas9, where screening of homologous recombination events is facilitated by fluorescence. We demonstrated that the native elongase YlELO2, responsible for the elongation of saturated and monounsaturated fatty acids up to 26 carbons, has to be inactivated to avoid functional redundancy and finely characterize heterologous elongase specificity. As it is an essential gene, we designed an optimized strategy for YlELO2 Knock-Out by a Knock-In of the elongase cassette. We then miniaturized cultures and fatty acid extraction in 96-well plates format. Using this workflow, we characterized seven human elongases on endogenous fatty acids and on five exogenous polyunsaturated fatty acids in a single series of experiments. CONCLUSION: We have developed tools and methods to characterize elongase specificity, from strain design to fatty acid production and analysis. Applicable to any fatty acid–modifying enzymes, these methodological developments will be useful to expand the repertoire of enzymes usable in Y. lipolytica and pave the way to produce new original fatty acids in this chassis.},
}

*Yarrowia/metabolism/genetics/enzymology
*Fatty Acid Elongases/metabolism/genetics
*Fatty Acids/biosynthesis/metabolism
Substrate Specificity
Workflow
CRISPR-Cas Systems

@article {pmid41508022,
year = {2026},
author = {Fan, T and Zhou, B and Chen, H and Liu, LP and Ni, Y and Zhang, W and Ye, L and Chen, Y and Zhang, D and Yang, S and Bai, Y and Liu, F and Zhi, C and Xu, G and Zheng, B and Lu, S and Qiu, C and Ding, Z and Chen, Y and Jiang, Y},
title = {Novel serum small extracellular vesicle miRNAs with multi-target RCA-CRISPR sensor for liver cancer detection.},
journal = {Journal of translational medicine},
volume = {24},
number = {1},
pages = {144},
pmid = {41508022},
issn = {1479-5876},
support = {21310051//Startup Fund from Shenzhen Bay Laboratory/ ; 2019156//Development and Reform Commission of Shenzhen Municipality/ ; },
mesh = {*MicroRNAs/blood/genetics ; Humans ; *Liver Neoplasms/blood/diagnosis/genetics ; *Extracellular Vesicles/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Biomarkers, Tumor/blood/genetics ; *Biosensing Techniques ; Base Sequence ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Reproducibility of Results ; },
abstract = {BACKGROUND: Detecting liver cancer (LC) remains a significant challenge in clinical practice. Small extracellular vesicle (sEV) miRNAs show promise as non-invasive biomarkers for LC detection, yet their diagnostic potential remains largely unexplored. This study aimed to identify specific sEV miRNA signatures for LC detection and develop a novel synchronized multi-miRNA detection platform to enhance diagnostic efficiency and sensitivity. METHODS: High-throughput sequencing was conducted across four distinct cohorts: normal controls (NC), hepatitis B virus (HBV) patients, liver cirrhosis patients, and LC patients. This sequencing process identified miRNAs with differential expression, followed by RT-qPCR validation in serum sEV miRNAs from LC patients and NC. An innovative detection method, RCA-CRISPR, was introduced, combining rolling circle amplification (RCA) with CRISPR/Cas12a (RCA-CRISPR) for quick and sensitive miRNAs detection. RESULTS: Sequencing results showed a consistent elevation of hsa-miR-203b-5p, hsa-miR-4661-5p, and hsa-miR-219a-2-3p across all cohorts. RT-qPCR validations confirmed significant upregulation of these miRNAs in serum sEVs from LC patients, and the combined three-miRNA panel exhibited high diagnostic accuracy (p = 0.0003; AUC = 0.81). The RCA-CRISPR method demonstrated a detection limit of 3.12 pM for simultaneous multi-target miRNA detection, highlighting its exceptional sensitivity. CONCLUSIONS: Our study identifies hsa-miR-203b-5p, hsa-miR-4661-5p, and hsa-miR-219a-2-3p as promising sEV miRNA biomarkers for LC detection. The developed RCA-CRISPR sensor provides a robust tool for multi-miRNA analysis, potentially advancing non-invasive LC diagnostics. Future validation in larger, prospectively collected cohorts is essential to establish the clinical utility and performance of this biomarker panel and RCA-CRISPR sensor.},
}

*MicroRNAs/blood/genetics
Humans
*Liver Neoplasms/blood/diagnosis/genetics
*Extracellular Vesicles/metabolism/genetics
*CRISPR-Cas Systems/genetics
Biomarkers, Tumor/blood/genetics
*Biosensing Techniques
Base Sequence
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Reproducibility of Results

@article {pmid41530441,
year = {2026},
author = {Li, W and Sun, Y and Ye, M and Liang, Y and Ouyang, J and Xu, W and Su, Y and Huang, X and Nie, D and Ouyang, S},
title = {Rapid visual detection of Treponema pallidum using the RPA-CRISPR/Cas12a system.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {5120},
pmid = {41530441},
issn = {2045-2322},
mesh = {*Treponema pallidum/genetics/isolation & purification ; *CRISPR-Cas Systems/genetics ; Humans ; *Syphilis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; DNA, Bacterial/genetics ; },
abstract = {Syphilis, caused by Treponema pallidum, is a sexually transmitted infection that has re-emerged globally over the past decade, posing significant public health challenges. Conventional diagnostic methods are limited by lengthy processing times, operational complexity, and moderate sensitivity, highlighting the urgent need for rapid, sensitive, and user-friendly detection strategies. In this study, we developed a visual detection platform for T. pallidum DNA by integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. The assay can be completed within one hour, with results directly interpreted via fluorescence readout. It demonstrated a detection limit as low as 11.34 copies/µL and high specificity, accurately distinguishing T. pallidum without cross-reactivity with common blood-borne pathogens, including HIV, HBV, HCV, and DENV. The clinical sample verification showed a consistency rate of 96.6% with the actual diagnosis. To enhance suitability for point-of-care applications, the RPA-CRISPR/Cas12a system was further adapted to a lateral flow assay (LFA) format, achieving a detection sensitivity of 5.56 × 10[2] copies/µL while minimizing reliance on specialized instrumentation. Overall, this platform provides a rapid, sensitive, and robust approach for point-of-care syphilis diagnosis and offers a reference framework for detecting other pathogenic organisms.},
}

*Treponema pallidum/genetics/isolation & purification
*CRISPR-Cas Systems/genetics
Humans
*Syphilis/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
Rapid Diagnostic Tests
Sensitivity and Specificity
DNA, Bacterial/genetics

@article {pmid41535756,
year = {2026},
author = {Park, HR and Park, S and Jun, JM and Shin, YJ and Hwang, Y and Jeong, KY and Kim, MY and Kim, ST and Park, S and Yoo, YH and Lee, E and Park, G and Kim, SG and Park, SK},
title = {CRISPR/Cas9 editing of β-Conglycinin subunits reduces IgE binding in soybean [Glycine max (L.) Merr.].},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {265},
pmid = {41535756},
issn = {1471-2229},
support = {PJ016784022024//Agricultural Research Program/ ; },
mesh = {*Glycine max/genetics/immunology/metabolism ; *Seed Storage Proteins/genetics/metabolism/immunology ; *Globulins/genetics/metabolism/immunology ; *Soybean Proteins/genetics/metabolism/immunology ; *Immunoglobulin E/metabolism/immunology ; *Antigens, Plant/genetics/metabolism/immunology ; *CRISPR-Cas Systems ; *Gene Editing ; Protein Subunits/genetics ; Food Hypersensitivity/immunology ; Allergens/genetics/immunology ; Plants, Genetically Modified ; },
abstract = {BACKGROUND: Soybean [Glycine max (L.) Merr.] is a major source of plant-based protein, yet the seed storage protein β-conglycinin (7 S globulin) is a prominent allergen. The αʹ, α, and β subunits contain IgE-binding epitopes, and their high sequence similarity enables simultaneous genome editing. The development of soybean lines with reduced β-conglycinin-specific IgE-binding capacity could enhance food safety for individuals with soy allergies. RESULTS: We employed CRISPR/Cas9 to disrupt the αʹ (Glyma.10G246300) and α (Glyma.20G148300, Glyma.20G148400), subunit genes and to target the β subunit genes (Glyma.20G146200, Glyma.20G148200) of β-conglycinin, generating four edited lines: SP1 (αʹ-null), SP2 (αʹα-null), SP3 (β-null), and SP4, which shows an αʹα-edited genotype and a β subunit-null protein phenotype. SDS-PAGE and DNA sequencing confirmed complete or near-complete loss of the targeted proteins across the T0 to T6 generations, demonstrating stable inheritance of the edited seed protein profiles. IgE immunoblotting and inhibition ELISA using pooled sera from soy-allergic individuals revealed distinct IgE-binding inhibition profiles among the edited lines. At the highest inhibitor concentration, SP4 showed the lowest IgE-binding inhibition (70.0%) compared with the wild type (87.7%), whereas SP1-SP3 exhibited inhibition values similar to or only slightly lower than those of the wild type. CONCLUSIONS: CRISPR/Cas9-mediated elimination of β-conglycinin subunits reduces IgE binding to soybean seed proteins and yields lines with stably inherited seed protein phenotypes. These results highlight the potential of targeted genome editing to generate soybean lines with reduced β-conglycinin-specific IgE recognition, supporting the application of precise genome modification in crop improvement for safer soy-based foods.},
}

*Glycine max/genetics/immunology/metabolism
*Seed Storage Proteins/genetics/metabolism/immunology
*Globulins/genetics/metabolism/immunology
*Soybean Proteins/genetics/metabolism/immunology
*Immunoglobulin E/metabolism/immunology
*Antigens, Plant/genetics/metabolism/immunology
*CRISPR-Cas Systems
*Gene Editing
Protein Subunits/genetics
Food Hypersensitivity/immunology
Allergens/genetics/immunology
Plants, Genetically Modified

@article {pmid41547741,
year = {2026},
author = {Wei, Y and Jiang, J and Gao, Y and Zhu, Y and He, W and Wu, F and Xie, H and Chen, L and Cai, Q and Zhang, J},
title = {Accelerated breeding for early-maturing and aromatic glutinous restorer lines with CRISPR/Cas9-mediated targeted editing for hybrid rice.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {284},
pmid = {41547741},
issn = {1471-2229},
support = {2024R1054//the Fundamental Research Project of Fujian Provincial Research Institute for Public Welfare, China/ ; CARS-01-08//the National Rice Industry Technology System of Modern Agriculture for China/ ; XTCXGC2021001//he "5511" Collaborative Innovation project for High-quality Development and Surpasses of Agriculture between Government of Fujian and Chinese Academy of Agricultural Sciences/ ; 2024NZ029027//Key program of Science and Technology in Fujian province, China/ ; },
mesh = {*Oryza/genetics/growth & development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Plant Breeding/methods ; Plants, Genetically Modified ; Edible Grain/genetics ; Mutation ; },
abstract = {Hybrid rice breeding depends on the development of elite parental lines with favorable traits such as grain quality, heading time, and plant architecture. However, improving restorer lines through conventional breeding is time-consuming and labor-intensive. Here, we employed a multiplex CRISPR/Cas9 editing strategy to simultaneously target Wx, Badh2, and Se14 in the elite restorer line FH676, aiming to generate glutinous, aromatic, and early-maturing lines. Through Agrobacterium-mediated transformation, we obtained Wx/Badh2 double mutants (Dm) and Wx/Badh2/Se14 triple mutants (Tm). Grain quality analysis revealed significantly reduced amylose content and enhanced aroma content in the edited lines, consistent with Wx and Badh2 knockouts. The triple mutants also exhibited significantly earlier heading compared to the wild type. The early-maturing Tm lines and the Dm lines achieved grain yields of 39.2 ~ 39.4 g and 42.9 ~ 43.0 g per plant, respectively, both exceeding yields of conventional glutinous cultivars used at present. To evaluate hybrid performance, we crossed Tm and Dm lines with two sterile lines: LX (aromatic) and NX (glutinous). The LX/Tm hybrid headed 4.8 days earlier than its wild-type counterpart, with no reduction in plant height or yield. The NX/Tm hybrid showed an advance of ~ 6.3 days in heading but a 6.2 ~ 7.0% reduction in yield due to decreased grain number per panicle. The Se14 knockout likely relieves repression of RFT1 expression during the floral transition under long-day conditions. Variations in phenotypic response across different maternal backgrounds suggest epistatic interactions affecting the dosage response of Se14. In summary, Se14 is a promising target for engineering early-maturing hybrid rice. The edited FH676 lines provide valuable germplasm resource for developing early-maturing, aromatic, and glutinous hybrids through CRISPR-based genome editing.},
}

*Oryza/genetics/growth & development
*CRISPR-Cas Systems
*Gene Editing/methods
*Plant Breeding/methods
Plants, Genetically Modified
Edible Grain/genetics
Mutation

@article {pmid41549083,
year = {2026},
author = {Khayer, A and Ye, P and Eti, FS and Sakif, TI and Azad, RB and Ali, J and Gupta, DR and Asuke, S and Pan, Q and Moni, MA and Kang, H and Islam, T},
title = {Field pathogenomics and evolutionary conservation unveil CRISPR-targetable susceptibility genes for wheat blast resistance.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {5677},
pmid = {41549083},
issn = {2045-2322},
support = {32261143468//National Natural Science Foundation of China/ ; OFANS project (Project Code No: FT-92-FNS/21)//Krishi Gobeshona Foundation/ ; Grant Code: V0156.01//Bill and Melinda Gates Foundation/ ; },
mesh = {*Triticum/genetics/microbiology ; *Plant Diseases/microbiology/genetics ; *Disease Resistance/genetics ; *Magnaporthe/pathogenicity ; Gene Expression Regulation, Plant ; *CRISPR-Cas Systems ; Genes, Plant ; Evolution, Molecular ; Ascomycota/pathogenicity ; Plant Proteins/genetics ; Transcriptome ; Gene Expression Profiling ; Host-Pathogen Interactions/genetics ; },
abstract = {Wheat blast, caused by Magnaporthe oryzae pathotype Triticum (MoT), threatens global wheat production, yet durable resistance mechanisms remain elusive. Current strategies relying on race-specific resistance genes or fungicides are vulnerable to pathogen evolution and inefficacy. Here, we investigated field-derived transcriptomes from the 2016 Bangladesh wheat blast epidemic, a catastrophic event devastating all local varieties to identify host susceptibility (S) genes co-opted by MoT. By analyzing RNA-seq data from infected and healthy plants across geographically distinct regions, we pinpointed 273 consistently upregulated wheat genes, enriched in defense-related pathways. Ortholog analysis with rice, a model for blast resistance, identified three conserved susceptibility (S)-gene candidates: TaSULTR3-3B (an ortholog of a rice bacterial blight susceptibility gene), TaSTP3-4D (associated with stripe rust), and TaMLO1-5A (a wheat powdery mildew susceptibility gene). While all three candidates exhibited significant expression correlation with M. oryzae Triticum (MoT) effectors in field-derived samples, in planta spike assays revealed distinct expression dynamics. Only TaMLO1-5A was significantly upregulated in the susceptible cultivar BARI Gom 26 following MoT inoculation, with no induction observed in the resistant cultivar S-615 (carrying Rmg8). Conversely, TaSULTR3-3B and TaSTP3-4D did not show significant induction under the specific conditions and time points of the in planta spike assays. This discrepancy potentially arises from tissue-specific regulation (spike vs. leaf), environmental variations, or differences in sampling time points between the field and greenhouse experiments. Disruption of such S genes, validated in other cereals for durable resistance, offers a transformative strategy to engineer non-race-specific wheat blast resilience. Our findings shift the paradigm from transient resistance genes to foundational susceptibility networks, proposing CRISPR-based editing of the candidate gene as an actionable target. This approach, resilient to pathogen evolution, could preempt epidemics in climate-vulnerable regions, safeguarding global wheat security. By bridging field pathogenomics and evolutionary genomics, we provide a roadmap for sustainable disease management in an era of expanding fungal threats.},
}

*Triticum/genetics/microbiology
*Plant Diseases/microbiology/genetics
*Disease Resistance/genetics
*Magnaporthe/pathogenicity
Gene Expression Regulation, Plant
*CRISPR-Cas Systems
Genes, Plant
Evolution, Molecular
Ascomycota/pathogenicity
Plant Proteins/genetics
Transcriptome
Gene Expression Profiling
Host-Pathogen Interactions/genetics

@article {pmid41620642,
year = {2026},
author = {Kumar, S and Murugan, B and Das, M and Sanan-Mishra, N and Sahoo, L},
title = {Geminiviral-CR-gRNA expressed in cowpea efficiently edited MYMV and MYMIV genome to provide resistance against cowpea yellow mosaic disease without hampering plant growth and yield.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {41620642},
issn = {1471-2229},
mesh = {*Vigna/genetics/virology/growth & development ; *Begomovirus/genetics ; *Plant Diseases/virology/genetics ; Plants, Genetically Modified ; *Disease Resistance/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Editing ; *Genome, Viral ; *Geminiviridae/genetics ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Cowpea is an economically important grain legume widely cultivated in Africa, Latin America, and Southeast Asia. In Southeast Asia, two of the most devastating viral diseases affecting cowpea are cowpea golden mosaic and severe leaf curl disease, both caused by Mungbean yellow mosaic India virus (MYMIV). Despite the availability of various molecular breeding strategies to manage viral infections, progress in cowpea improvement remains limited due to the lack of resistant germplasm, the absence of a reliable transformation system, and the restricted availability of efficient tools for viral gene inactivation. RESULTS: In this study, we employed CRISPR/Cas9-mediated genome editing technology to efficiently disrupt the common region (CR) of the single-stranded DNA-A component of legume-infecting geminiviruses, using cowpea as a test system. Transgenic cowpea plants expressing Cas9 and a guide RNA (gRNA) targeting the CR of MYMV/MYMIV were evaluated for resistance to yellow mosaic disease (YMD). Agrobacterium tumefaciens strain EHA105 carrying pXSE901B-Cas9 and CR-gRNA cassettes was used to generate the transgenic plants. PCR and Southern blot analyses confirmed the integration of transgenes into the cowpea genome. Transgenic lines in the T1 and T2 generations were tested for YMD resistance via agroinfiltration using MYMV and MYMIV agroinfectious clones. Accumulation of AV2 and AC2 transcripts was drastically reduced in T2 lines, which also displayed either no or minimal mosaic symptoms. Mutation analysis of the viral genome revealed frameshift mutations near the PAM region of the targeted CR sequence, with editing frequencies of 28%, 34%, 22%, and 33% in MYMV/MYMIV-infected cowpea lines #L2, #L4, #L7, and #L11, respectively. The transgenic cowpea plants exhibited a normal phenotype and did not show any yield reduction under greenhouse conditions. CONCLUSION: To the best of our knowledge, this is the first report of transgenic cowpea plants stably expressing a geminiviral common region (CR)–targeting gRNA via the CRISPR/Cas9 system, leading to efficient editing of the MYMV/MYMIV genome and conferring durable resistance to Yellow Mosaic Disease without adversely affecting plant growth or yield. These findings demonstrate the potential of CRISPR/Cas9 as a precise and robust platform for developing virus-resistant cowpea and other legume crops.},
}

*Vigna/genetics/virology/growth & development
*Begomovirus/genetics
*Plant Diseases/virology/genetics
Plants, Genetically Modified
*Disease Resistance/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Editing
*Genome, Viral
*Geminiviridae/genetics
CRISPR-Cas Systems

@article {pmid41634544,
year = {2026},
author = {Velangani, HG and Ghosh, A and Singh, S and Kiran, S},
title = {Strategies and considerations for the generation of ssDNA-Based HDR templates for CRISPR-based genome editing.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41634544},
issn = {1471-2164},
support = {[EMDR/SG/15/2024-01-03223 (E-File No. 222379)]//Indian Council of Medical Research/ ; FILE NO. CRG/2023/007695//ANRF, India/ ; },
mesh = {*DNA, Single-Stranded/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Recombinational DNA Repair ; },
abstract = {The usefulness of genome editing lies in the ability to induce any desirable change in the target genome. It is most efficiently achieved using ssDNA (single-stranded DNA) as the HDR template and CRISPR-Cas9 targeted DNA breaks. However, the low efficiency of HDR integration is a challenge for achieving efficient edits. Among different HDR templates, ssDNA has the highest efficiency in inducing repair after CRISPR-induced DNA breaks. Several methods are used to generate the ssDNA-HDR template. However, each method has limitations in terms of feasibility for different ssDNA types, efficiency, time required, expenses incurred, etc. Often, these factors are overlooked, confusing users regarding the most appropriate method for generating ssDNA. This study describes and compares methods for generating ssDNA and outlines considerations for designing an efficient ssDNA template. Most frequently used methods are PCR-based, utilizing modified primers (phosphorylation, biotinylation, or phosphorothioate-based) or those utilizing the IVT-RT (In vitro transcription- reverse transcriptase) method. Asymmetric PCR and M13-based methods of ssDNA generation have been used as non-PCR methods. The IVT-RT method is widely adopted as it provides a middle-ground in yield and ease. The advantages and shortcomings of these methods, based on reported studies and our own results, are discussed.},
}

*DNA, Single-Stranded/genetics
*Gene Editing/methods
*CRISPR-Cas Systems
*Recombinational DNA Repair

@article {pmid41701371,
year = {2026},
author = {Yi, F and Li, Z and Jiang, F and Zhang, Z and Chen, Y and Zhao, Z and Deng, X and Chen, H and Xu, S and Tao, Y},
title = {An electrochemiluminescence biosensor governed by a CRISPR-actuated electrostatic gate for ultrasensitive aflatoxin B1 detection.},
journal = {Mikrochimica acta},
volume = {193},
number = {3},
pages = {163},
pmid = {41701371},
issn = {1436-5073},
support = {22264018//National Natural Science Foundation of China/ ; 20232BAB203019, 20232BAB216118//Jiangxi Provincial Natural Science Foundation/ ; 202410412021, 202410412246, 202410412114//the College Students' Innovation and Entrepreneurship Training Program Project of Jiangxi Province/ ; },
mesh = {*Aflatoxin B1/analysis/chemistry ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; Static Electricity ; *CRISPR-Cas Systems ; Limit of Detection ; Aptamers, Nucleotide/chemistry/genetics ; Food Contamination/analysis ; Electrodes ; Nucleic Acid Hybridization ; },
abstract = {Aflatoxin B1 (AFB1), a potent mycotoxin, poses a critical threat to global food safety, demanding analytical methods with exceptional sensitivity. Here, we introduce a homogeneous electrochemiluminescence (ECL) biosensor that operates on a novel CRISPR-actuated electrostatic gating mechanism. The core of our strategy relies on controlling the access of ECL reporters to a positively charged electrode surface (PAH-ITO). In the absence of AFB1, cationic Ru(phen)32+ reporters are electrostatically repelled from the electrode, resulting in a low background signal. The presence of AFB1 triggers a CRISPR/Cas12a enzymatic cascade, which activates its trans-cleavage activity to release a highly anionic hybridization chain reaction (HCR) scaffold from magnetic beads (MB). This scaffold serves as a nanocarrier, capturing the Ru(phen)32+ reporters and, by virtue of its strong negative charge, shuttling them to the electrode through potent electrostatic attraction. This action effectively “opens” the electrostatic gate, switching on a robust ECL signal. By synergistically integrating the high specificity of the aptamer-CRISPR system with the immense signal amplification of the HCR scaffold, all under the control of a charge-dominant switch, our biosensor achieves an outstanding limit of detection of 0.121 fg/mL and a broad linear range from 1 fg/mL to 100 pg/mL. Its successful application in spiked food samples validates its practicality and robustness, presenting a powerful new paradigm for designing minimal-background, high-gain ECL sensors for mycotoxin determination.},
}

*Aflatoxin B1/analysis/chemistry
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*Luminescent Measurements/methods
Static Electricity
*CRISPR-Cas Systems
Limit of Detection
Aptamers, Nucleotide/chemistry/genetics
Food Contamination/analysis
Electrodes
Nucleic Acid Hybridization

@article {pmid41721389,
year = {2026},
author = {Umashankar, P and Choi, B and Nygård, Y},
title = {Towards the development of a CRISPR-Cas9 based kill switch for Saccharomyces cerevisiae.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {41721389},
issn = {1475-2859},
mesh = {*Saccharomyces cerevisiae/genetics/growth & development ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; Saccharomyces cerevisiae Proteins/genetics ; },
abstract = {BACKGROUND: Advancements in synthetic genetic circuits have enabled programmable and condition-dependent control of microbial cell growth. CRISPR-Cas9-based kill switches, genetic systems that program cells to lose viability in response to specific conditions, have recently been demonstrated for bacterial cell factories but not yet in yeast. RESULTS: In this study, we present a foundational demonstration for a CRISPR-based kill switch in Saccharomyces cerevisiae, CRISPR KiSS. The CRISPR KiSS employs inducible CRISPR targeting essential genes to elicit growth inhibition. The activation of the KiSS system is achieved through conditional expression of a guide RNA (gRNA) upon anhydrotetracycline (ATc) induction, thereby activating CRISPR-mediated gene disruption. We demonstrate that targeting the essential genes (ERG13, PGA3, TPI1 or CDC19) leads to severe growth inhibition upon ATc induction. Still, the current set up does not allow complete killing of the cells due to system inactivation, e.g. escape from CRISPR based cutting. We studied reasons for system inactivation and substantially improved the system by simultaneous expression of two different gRNAs. Sequencing escape mutants revealed mutations in both the gRNA sequences and target genes as potential sources of system inactivation. CONCLUSIONS: This work highlights the potential of harnessing a CRISPR-based kill switch in S. cerevisiae. Cells expressing the system were able to escape growth inhibition through mutations and further optimization of the KiSS system is still needed for it to be used in various cell factory applications.},
}

*Saccharomyces cerevisiae/genetics/growth & development
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics
Saccharomyces cerevisiae Proteins/genetics

@article {pmid41739318,
year = {2026},
author = {Joshi, D and Kshatri, P and Tiwari, A and Bhardwaj, U and Patel, K},
title = {CRISPR/Cas9 in Cancer Therapy: Precision Genome Editing Approaches Targeting Hematological Malignancies and Solid Tumors Through Cellular, Biochemical, and Molecular Mechanisms.},
journal = {Cell biochemistry and biophysics},
volume = {84},
number = {2},
pages = {1759-1788},
pmid = {41739318},
issn = {1559-0283},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Neoplasms/therapy/genetics ; *Hematologic Neoplasms/therapy/genetics ; Genetic Therapy/methods ; },
abstract = {Being extremely precise in terms of genetic material modifications, CRISPR/Cas9 technology has very rapidly become the cornerstone in the field of precision oncology. This review is focused on the great potential offered by CRISPR/Cas9 in terms of cancer treatment, emphasizing its cellular, biochemical, and molecular mechanisms of action. We provide a short historical background and description of its operational principles, followed by an in-depth analysis of how CRISPR/Cas9 reprograms oncogenic signaling networks by selectively modifying cancer-associated genes such as KRAS, MYC, BRAF, and EGFR, and restoring the function of tumor suppressors including TP53, RB1, and PTEN. The review further explores its ability to remodel cellular pathways involved in apoptosis, DNA repair, and cell-cycle regulation, alongside its modulation of key biochemical cascades. We analyse the technology’s application to epigenetic modifications and the regulation of non-coding RNAs as arising therapeutic targets. It also considers the deployment of CRISPR/Cas9 across various cancers, including haematological malignancies such as leukaemia and lymphoma and solid tumors such as breast, lung, and colorectal cancer, where it is being contextualized to disease-specific outcomes and limitations. To overcome delivery issues, recent advances in various viral vectors (AAV, lentivirus) and non-viral methods such as lipid nanoparticles, polymeric nanotechnologies, exosomes and magnetic nanoparticles are explained. In addition, findings from completed and ongoing clinical trials that demonstrate the clinical translation of CRISPR are presented. Lastly, important issues are considered, including immune reactions, off-target effects, and integration with precision oncology. When combined, these viewpoints provide a thorough understanding of the current state of CRISPR/Cas9 and its potential to revolutionize cancer treatment in the future.},
}

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

@article {pmid41742232,
year = {2026},
author = {Deng, H and Wang, J and Meng, F and Ma, C and Li, J and Wang, J and Wang, X and Zhao, C and Zhang, Y and Wang, R and Chen, N},
title = {Combining computer-aided enzyme design and chromosomal integration for plasmid-free biosynthesis of 1,5-pentanediol in Escherichia coli.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {41742232},
issn = {1475-2859},
support = {22JCQNJC01310//the Natural Science Foundation of Tianjin/ ; 2023YFD13000700//the National Key Research and Development Program of China/ ; 2021ZDSYS10//the Key Research and Development Program of Shandong Province/ ; },
mesh = {*Escherichia coli/metabolism/genetics ; *Glycols/metabolism ; Plasmids/genetics ; Nocardia/genetics/enzymology ; *Metabolic Engineering/methods ; Alcohol Dehydrogenase/metabolism/genetics ; CRISPR-Cas Systems ; Computer-Aided Design ; Biosynthetic Pathways ; Pentanes ; },
abstract = {BACKGROUND: 1,5-Pentanediol (1,5-PDO) is a high-value chemical with broad uses in polymer, cosmetic, and pharmaceutical industries. Although diverse biosynthetic pathways have been constructed, current recombinant strains typically rely on plasmid-based overexpression, which necessitates antibiotics and hinders industrial-scale production. RESULTS: We developed a robust, plasmid-free Escherichia coli platform for de novo 1,5-PDO synthesis by integrating pathway genes (davB, davA, gabT, yahK, car, sfp and yqhD) into the chromosome of a lysine-hyperproducing strain via CRISPR/Cas9. Screening of carboxylic acid reductases identified Nocardia iowensis CAR-Ni as the most effective, yielding a base strain (D13) that produced 0.672 g/L 1,5-PDO. Integrated analysis confirmed the alcohol dehydrogenase (ADH)-mediated reduction of 5-hydroxypentanal (5-HP) as an underappreciated bottleneck. We subsequently screened ten endogenous ADHs and selected YjgB for computational optimization. Docking-guided saturation mutagenesis at position E205 yielded the variant YjgB(E205C), which exhibited a 3.34-fold increase in in vitro activity, reduced 5-HP accumulation, and elevated the titer to 0.935 g/L. Enhancing NADPH supply by integrating pntAB further raised the shake-flask titer to 1.5 g/L. In a 5-L fed-batch bioreactor, the final strain (D91) achieved 12.1 g/L 1,5-PDO (yield of 0.225 mol/mol glucose) without antibiotics or inducers. To our knowledge, this is the highest reported 1,5-PDO titer in E. coli. CONCLUSION: This study establishes a scalable, sustainable biosynthetic platform through synergistic metabolic engineering and computational enzyme optimization.},
}

*Escherichia coli/metabolism/genetics
*Glycols/metabolism
Plasmids/genetics
Nocardia/genetics/enzymology
*Metabolic Engineering/methods
Alcohol Dehydrogenase/metabolism/genetics
CRISPR-Cas Systems
Computer-Aided Design
Biosynthetic Pathways
Pentanes

@article {pmid41746436,
year = {2026},
author = {Chen, N and Sun, X and Liang, S and Cao, C and Lai, X and Song, C and Yan, Z and Ge, C},
title = {Single particle mediated CRISPR/Cas13a ultrasensitive direct detection of miRNA-21 at femtomolar levels without nucleic acid amplification.},
journal = {Mikrochimica acta},
volume = {193},
number = {3},
pages = {},
pmid = {41746436},
issn = {1436-5073},
mesh = {*MicroRNAs/blood/genetics ; Humans ; Gold/chemistry ; Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; Limit of Detection ; *Biosensing Techniques/methods ; Nucleic Acid Hybridization ; DNA Probes/chemistry/genetics ; },
abstract = {A novel biosensing platform has been developed that couples the collateral cleavage activity of the CRISPR/Cas13a system with single-particle inductively coupled plasma mass spectrometry (sp-ICP-MS) using gold nanoparticle (AuNP)-DNA reporter probes. In the presence of target microRNA-21, Cas13a is activated and specifically cleaves RNA bases within the DNA-RNA hybrid linkers on AuNPs, preventing their hybridization with biotinylated capture probes on magnetic beads. As a result, the cleaved AuNPs remain in the supernatant and are directly quantified by sp-ICP-MS. The number of detected AuNPs correlates linearly with the concentration of miRNA-21 (y = 0.2537logCmiRNA + 0.2477 with a correlation coefficient of R2 = 0.9978), enabling a detection limit as low as 68 fM with excellent single-base mismatch discrimination. The assay demonstrated high recoveries (97–108%) and reproducibility in spiked human serum samples, confirming its reliability in complex matrices. This amplification-free strategy combines the high specificity of CRISPR/Cas13a with the single-particle sensitivity of sp-ICP-MS, providing a robust, quantitative, and versatile platform for miRNA detection with promising potential for early cancer diagnostics and precision medicine.},
}

*MicroRNAs/blood/genetics
Humans
Gold/chemistry
Metal Nanoparticles/chemistry
*CRISPR-Cas Systems
Limit of Detection
*Biosensing Techniques/methods
Nucleic Acid Hybridization
DNA Probes/chemistry/genetics

@article {pmid41762386,
year = {2026},
author = {Duran, T and Karaselek, MA and Dagdelen, B and Kuccukturk, S and Guner, SN and Keles, S and Reisli, I},
title = {CRISPR-Cas9-based gene editing as a proof-of-concept approach in an inborn error of immunity caused by a DCLRE1C variant.},
journal = {Immunologic research},
volume = {74},
number = {1},
pages = {},
pmid = {41762386},
issn = {1559-0755},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Severe Combined Immunodeficiency/genetics/therapy ; *Endonucleases/genetics ; DNA-Binding Proteins/genetics ; *Nuclear Proteins/genetics ; Interleukin-2 Receptor alpha Subunit/metabolism/genetics ; },
abstract = {Hypomorphic DCLRE1C variants impair T and B cell development, leading to combined immunodeficiency (CID) or leaky severe combined immunodeficiency (SCID). Current treatment options, such as allogeneic hematopoietic stem cell transplantation (aHSCT), are associated with significant risks, highlighting the need for alternative therapeutic strategies. In this study, we report the first a proof-of-concept CRISPR-Cas9–mediated correction of a hypomorphic DCLRE1C variant (c.194 C > T; p.T65I) in CD4 + helper T (Th) cells using CRISPR-Cas9 gene-editing technology. CD4 + Th cells were isolated, and the variant region was edited with sgRNA and donor DNA. Gene editing efficiency was confirmed by Sanger sequencing, revealing successful restoration of the target region to its wild-type sequence. Functional analyses showed a significant increase in CD25 activation and Artemis protein expression post-editing, although DCLRE1C mRNA levels remained unchanged. The approximately 6–8% increase in CD25 expression was statistically significant but did not reach healthy control levels. These findings suggest that CRISPR-Cas9 –mediated gene editing may enable precise correction and induce measurable cellular-level functional changes, supporting biological feasibility rather than therapeutic efficacy. This study provides a foundation for future research on HSCs and underscores the potential role of CRISPR-Cas9–based approaches in the treatment of inborn errors of immunity (IEIs) associated with DCLRE1C variants.},
}

Humans
CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Severe Combined Immunodeficiency/genetics/therapy
*Endonucleases/genetics
DNA-Binding Proteins/genetics
*Nuclear Proteins/genetics
Interleukin-2 Receptor alpha Subunit/metabolism/genetics

@article {pmid41764471,
year = {2026},
author = {Chen, H and Luo, W and Ma, N and Li, S},
title = {CRISPR/Cas9-mediated B2m knockout paves the way for allogeneic basal cell transplantation.},
journal = {Respiratory research},
volume = {27},
number = {1},
pages = {},
pmid = {41764471},
issn = {1465-993X},
support = {2023B111105006//Guangdong Key Area R&D Program Key Project/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/physiology ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Mice ; *beta 2-Microglobulin/genetics/deficiency ; Transplantation, Homologous/methods ; Mice, Knockout ; *Gene Knockout Techniques/methods ; Cells, Cultured ; *Gene Editing/methods ; },
abstract = {BACKGROUND: Autologous transplantation of basal cells (BCs) has shown promise in treating respiratory diseases, but disease-specific subpopulations among BCs probably diminish the treatment efficacy. An alternative approach involves generating universal and healthy BCs, which offers a potentially more efficient and accessible solution for avoiding using abnormal BCs. However, such hypoimmunogenic BCs have not yet been transplanted into the airways of immunocompetent animals. METHODS: Before producing hypoimmunogenic BCs, the predominant transplantation antigen in BCs was explored through RT-qPCR and flow cytometry to identify the key target of CRISPR/Cas9‐mediated editing. The proliferation and expression of specific markers of BCs were evaluated after gene editing by CCK‐8 and RT‐qPCR, respectively. These gene‐edited BCs and wild‐type (WT) BCs, which were both derived from the same BALB/c mouse, were subsequently allogeneically transplanted into C57BL/6 mice with polidocanol‐induced airway injury to evaluate the differentiation and immune response in the recipient mice via immunehistological staining. RESULTS: In the present study, it was demonstrated that major histocompatibility complex class I (MHC-I) is the predominant transplantation antigen in BCs. The hypoimmunogenic BCs were generated through editing beta‐2 microglobulin (B2m) participating in the encoding of MHC‐I. The knockout of B2m in BCs did not affect their proliferation or the expression of specific markers in vitro. Both WT BCs and B2m‐edited BCs (B2m‾ BCs) successfully differentiated into ciliated and secretory cells in the tracheas following allogeneic transplantation and did not elicit an immune response during the 26‐day observation period in the tracheas. However, WT BCs, compared to B2m‾ BCs, induced severe lung injury by provoking an immune response in the lower airways and alveolar regions, as indicated by increased infiltration of CD45+ immune cells, epithelial cell shedding in the bronchi, and obvious alveolar hyperemia with collapse. CONCLUSIONS: The hypoimmunogenic BCs generated through CRISPR/Cas9-mediated B2m gene editing retained their ability to differentiate and maintained viability in the allogeneic respiratory system, supporting the potential application in the cell regeneration therapy of airway diseases.},
}

Animals
*CRISPR-Cas Systems/physiology
Mice, Inbred BALB C
Mice, Inbred C57BL
Mice
*beta 2-Microglobulin/genetics/deficiency
Transplantation, Homologous/methods
Mice, Knockout
*Gene Knockout Techniques/methods
Cells, Cultured
*Gene Editing/methods

@article {pmid41766009,
year = {2026},
author = {Vargas-Reyes, M and Alcántara, R and Alfonsi, S and Peñaranda, K and Petrelli, D and Spurio, R and Pajuelo, MJ and Milon, P},
title = {Versatile and portable Cas12a-mediated detection of antibiotic resistance markers.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41766009},
issn = {2045-2322},
support = {C-004-2021-2//Universidad Peruana de Ciencias Aplicadas/ ; D43TW001140//Forgarty International Center/ ; PE501079419-2022//PROCIENCIA - CONCYTEC/ ; },
mesh = {*Escherichia coli/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; *CRISPR-Cas Systems ; *Drug Resistance, Bacterial/genetics ; Genetic Markers ; Microbial Sensitivity Tests ; *Drug Resistance, Microbial/genetics ; },
abstract = {Antibiotic-resistant bacteria are spreading in clinical, industrial, and environmental ecosystems. The spreading dynamics to and from the environment are unknown, largely due to the lack of appropriate (robust, fast, low-cost) analytical assays. In this study, we developed C12a, a versatile molecular toolbox to detect genetic markers of antibiotic resistance using CRISPR/Cas12a. Biochemical characterization show that the C12a toolbox can detect less than 100 attoMolar of pure DNA fragments from the blaCTX-M15 and floR genes, conferring resistance to b-lactams and amphenicols, respectively important for human and veterinary uses. In microbiological assays, C12a detected less than 102 CFU/mL and high concordance was observed if compared to antibiotic susceptibility tests, PCR, or to whole genome sequencing. Additionally, C12a confirmed a high prevalence of the integrase/integron system in E. coli isolates containing multiple antibiotic resistance genes (ARGs). The C12a toolbox shows equivalent detection performance in diverse laboratory settings, results readout (Fluorescence vs. FLA) or input sample. Altogether, this work presents a comprehensive proof-of-concept, development description, and biochemical characterization of a collection of molecular tools to detect antibiotic resistance markers in a one health setup.},
}

*Escherichia coli/genetics/drug effects
Anti-Bacterial Agents/pharmacology
*CRISPR-Cas Systems
*Drug Resistance, Bacterial/genetics
Genetic Markers
Microbial Sensitivity Tests
*Drug Resistance, Microbial/genetics

@article {pmid41770417,
year = {2026},
author = {Melamed, J and Barnoy, S},
title = {Understanding the public's intention to adopt CRISPR-Cas9: the effect of beliefs, knowledge, and innovativeness.},
journal = {Human genetics},
volume = {145},
number = {1},
pages = {},
pmid = {41770417},
issn = {1432-1203},
mesh = {Humans ; Adult ; *CRISPR-Cas Systems ; Female ; Male ; *Gene Editing ; Young Adult ; Middle Aged ; *Intention ; Surveys and Questionnaires ; *Health Knowledge, Attitudes, Practice ; },
abstract = {CRISPR-Cas9 is a gene editing technology with wide-ranging medical potential and significant ethical implications. This study examined how personality traits, familiarity with CRISPR, knowledge about CRISPR, and beliefs concerning its applications are connected to the public’s willingness to adopt this technology, drawing on Rogers’ Diffusion of Innovations framework. A sample of 500 young adults aged 20–45 completed questionnaires assessing innovativeness as well as familiarity, knowledge, beliefs, and willingness to adopt CRISPR-Cas9 for therapeutic and non-therapeutic purposes. Results showed that only 24% of participants were familiar with CRISPR and that knowledge levels were generally low. Based on Rogers’ typology, 18.8% were identified as innovators, and 26.8% as early adopters, and only 3.2% as laggards. Beliefs were strongly associated with willingness to adopt CRISPR-Cas9 (r = .63, p < .001), moderating the weak associations of personality traits with adoption intentions. These findings suggest that beliefs are an important factor in CRISPR-Cas9 adoption and they appear to have more influence on willingness to adopt CRISPR-Cas9 than knowledge and innovativeness in this sample. Efforts to promote informed public discussion on CRISPR should be made, with an emphasis on ethical aspects alongside scientific information. This is critical for the responsible adoption of this technology.},
}

Humans
Adult
*CRISPR-Cas Systems
Female
Male
*Gene Editing
Young Adult
Middle Aged
*Intention
Surveys and Questionnaires
*Health Knowledge, Attitudes, Practice

@article {pmid41772313,
year = {2026},
author = {Li, Y and Ye, Z and Zhao, C and Tan, Y and Chen, J and Wu, Z and Zhang, Y and Guo, H and Cheng, Y and Wang, R and Wang, J and Wang, D},
title = {Integrated on-site detection of Fusarium temperatum based on a droplet digital CRISPR-based platform.},
journal = {Mikrochimica acta},
volume = {193},
number = {3},
pages = {},
pmid = {41772313},
issn = {1436-5073},
support = {2024A1515011281//Guangdong Basic and Applied Basic Research/ ; 2023C043-5, 2022C044-9//Technology Research and Development Project from the Development and Reform Commission in Jilin Province of China/ ; },
mesh = {*Fusarium/genetics/isolation & purification ; *CRISPR-Cas Systems ; Zea mays/microbiology ; DNA, Fungal/genetics/analysis ; Rapid Diagnostic Tests ; Smartphone ; Microscopy, Fluorescence/methods ; },
abstract = {Fusarium temperatum (F. temperatum) is a fungus whose infection can cause various diseases in maize plants, leading to premature death, and F. temperatum mycotoxin poses a serious threat to human and animal health. Rapid and early on-site detection of F. temperatum infection facilitates the prevention of disease progression, which is an unmet need. In this study, a droplet digital CRISPR-Cas12a-based platform (DD-Cas), combined with a rapid extraction procedure, was developed for amplification-free on-site detection of F. temperatum. The DD-Cas assay can rapidly detect F. temperatum genomic DNA in infected maize samples with high sensitivity (102 CFU/mL) and specificity. Furthermore, we developed a smartphone-based fluorescence microscope integrating the heating module, that could accurately detect infected samples within 30 min, enabling low-cost point-of-care testing (POCT). This platform can avoid cross-contamination and amplification bias, thus having great potential for on field detection of pathogenic bacteria in agriculture.},
}

*Fusarium/genetics/isolation & purification
*CRISPR-Cas Systems
Zea mays/microbiology
DNA, Fungal/genetics/analysis
Rapid Diagnostic Tests
Smartphone
Microscopy, Fluorescence/methods

@article {pmid41775739,
year = {2026},
author = {Li, X and Wang, S and Qiu, Z and Sun, R and Wang, T and Ren, X and Lv, B and Ma, X and Cheng, L and Liu, Y and Jiang, J},
title = {Construction and initial validation of key gene network for progesterone resistance in endometrial cancer based on genome-wide CRISPR screening.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41775739},
issn = {2045-2322},
support = {2023M731847//China Postdoctoral Science Foundation/ ; 2022YFC2704304//National Key Technology Research and Development Programme of China/ ; },
mesh = {Female ; *Endometrial Neoplasms/genetics/drug therapy/pathology ; Humans ; Animals ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Gene Regulatory Networks ; *Drug Resistance, Neoplasm/genetics ; Mice ; Gene Expression Regulation, Neoplastic/drug effects ; Progestins/pharmacology ; *Progesterone/pharmacology ; Apoptosis/drug effects ; Endometrium/abnormalities ; Uterine Diseases ; },
abstract = {Endometrial carcinoma, a prevailing malignancy of the female reproductive system, exhibits escalating incidence and a trend towards early onset. Hormone therapy serves as a primary choice for fertility preservation and is also considered for advanced and recurrent cases. However, a considerable number of patients fail to respond favorably to progestin treatments. We employed CRISPR/Cas9 technology to establish a comprehensive human genome library in the Ishikawa cell line. Subsequent exposure to medroxyprogesterone was followed by high-throughput sequencing, and differential gene expression and enrichment analyses were conducted using Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) Robust Rank Aggregation (RRA) and MAGeCK Maximum-Likelihood Estimation (MLE) algorithms. An iterative data intersection approach was employed, utilizing sequenced data from progestin-resistant cell lines, to identify pivotal genes associated with progestin resistance. The top 10 identified genes were functionally validated in our previously established progestin-resistant cell model through Cell Counting Kit-8 (CCK-8) assays and apoptosis detection. The progestin-resistant gene NNMT and the progestin-sensitive gene SOX17 were validated in vivo in xenograft mouse models. The constructed library exhibited high quality, meeting sequencing standards. Employing RRA and MLE algorithms, we identified 332 and 829 negative selection genes, as well as 3438 and 5098 positive selection genes. Enrichment analysis implicated pathways linked to DNA and RNA synthesis, metabolism, and related processes. After multiple data intersections, we identified a total of 5 genes promoting progestin resistance and 20 genes inhibiting resistance, with functional experiments confirming their roles. Employing CRISPR/Cas9 technology enables the construction of a relatively reliable network of pivotal genes associated with progestin resistance in endometrial carcinoma. Processes involving DNA and RNA synthesis, metabolism, and related mechanisms appear to significantly impact the progestin sensitivity of endometrial carcinoma.},
}

Female
*Endometrial Neoplasms/genetics/drug therapy/pathology
Humans
Animals
*CRISPR-Cas Systems
Cell Line, Tumor
*Gene Regulatory Networks
*Drug Resistance, Neoplasm/genetics
Mice
Gene Expression Regulation, Neoplastic/drug effects
Progestins/pharmacology
*Progesterone/pharmacology
Apoptosis/drug effects
Endometrium/abnormalities
Uterine Diseases

@article {pmid41792204,
year = {2026},
author = {Kim, Y and Jun, Y and Han, J and Choi, S and Kim, H and Lee, M and Kim, SL and Kang, SH and Suh, EJ and Park, SR and Mo, Y},
title = {CRISPR/Cas9-mediated mutagenesis of SMXL4 alters plant height and yield-related traits in rice (cv. Samkwang).},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41792204},
issn = {2045-2322},
support = {RS-2024-00322166//Rural Development Administration/ ; },
mesh = {*Oryza/genetics/growth & development ; *CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Mutagenesis ; Plants, Genetically Modified ; Gene Editing ; Phenotype ; },
abstract = {Recent climate change and frequent extreme weather events during the maturation period of rice exacerbate lodging and threaten stable production. Samkwang, a widely cultivated rice variety in Korea, is particularly vulnerable to lodging due to its tall stature. To improve lodging tolerance while preserving Samkwang’s elite genetic background, we identified an SMXL4-edited line (smxl4) with reduced culm length and stable growth from a CRISPR/Cas9-edited Samkwang population. The biological function of SMXL4, a clade Ⅳ member of the SMXL (SUPPRESSOR OF MAX2 1-LIKE) family, has not been well characterized in rice. Compared to Samkwang, the smxl4 plants showed reduced plant height, internode length, panicle length, grain number per panicle, and grain weight, while panicle number per plant increased. Transcriptome profiling of elongating internodes at booting and heading stages revealed upregulation of genes associated with cell wall remodeling and defense responses in smxl4 relative to Samkwang. These findings highlight the broad involvement of SMXL4 in rice growth and development and provide insights for breeding lodging tolerant rice cultivars.},
}

*Oryza/genetics/growth & development
*CRISPR-Cas Systems
*Plant Proteins/genetics/metabolism
Gene Expression Regulation, Plant
*Mutagenesis
Plants, Genetically Modified
Gene Editing
Phenotype

@article {pmid41803678,
year = {2026},
author = {Jiang, Q and Zeng, X and Zhang, Q and Yang, F and Lv, T and Zhang, Y and Wang, J and Li, F and Xu, D},
title = {Development and application of a rapid detection system for Aspergillus fumigatus based on ERA/CRISPR-Cas12a.},
journal = {BMC microbiology},
volume = {26},
number = {1},
pages = {},
pmid = {41803678},
issn = {1471-2180},
support = {2024AH051688, 2024AH051225//the University Natural Science Research Key Projects in Anhui Province/ ; },
mesh = {*Aspergillus fumigatus/genetics/isolation & purification ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Humans ; Rapid Diagnostic Tests ; *Aspergillosis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/genetics ; Sputum/microbiology ; Limit of Detection ; },
abstract = {Aspergillus fumigatus (AF) is the predominant pathogen implicated in invasive aspergillosis (IA) in humans; therefore, prompt and accurate detection is critical for the effective prevention and management of IA. This study developed a rapid detection system targeting the AF-specific anxC4 gene by integrating enzymatic recombinase amplification (ERA) with CRISPR/Cas12a. The reaction proceeds at a stable temperature of 37 °C, with amplification and detection systems separately positioned in the tube lid and bottom, respectively, effectively minimizing aerosol contamination typically associated with product transfers. To enhance sensitivity, the One-Pot method was optimized. Consequently, the fluorescence detection limit reached 1 fg/µL, and the sensitivity of the test strip reached 10 fg/µL, with no cross-reactivity observed against other fungi. Detection of AF was completed within 60 min, and results were visually displayed through fluorescence signals and nucleic acid test strips. Clinical practicality was further evaluated using aspergillosis samples, which demonstrated satisfactory performance. Pure culture results confirmed that out of 62 sputum samples, 32 were positive and 30 negative. Evaluation of 62 clinical samples using the One-Pot ERA-CRISPR/Cas12a system demonstrated sensitivity and specificity rates of 93.75% and 93.33%, respectively, via fluorescence detection, and 90.63% sensitivity and 96.67% specificity using lateral flow strips.},
}

*Aspergillus fumigatus/genetics/isolation & purification
*CRISPR-Cas Systems
Sensitivity and Specificity
Humans
Rapid Diagnostic Tests
*Aspergillosis/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
*Recombinases/genetics
Sputum/microbiology
Limit of Detection

@article {pmid41808028,
year = {2026},
author = {Grubben, J and Bijsterbosch, G and Visser, RGF and Schouten, HJ},
title = {Influence of gRNA efficiency and inversion size on the frequency of CRISPR/Cas9-induced chromosomal inversions in tomato protoplasts.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {41808028},
issn = {1471-2229},
support = {GSGT.2019.016//Nederlandse Organisatie voor Wetenschappelijk Onderzoek/ ; TU18048//Top Sector Agri and Food/ ; },
mesh = {*Solanum lycopersicum/genetics ; *Protoplasts/metabolism ; *Chromosome Inversion/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 enables induction of chromosomal inversions from hundreds of base pairs to millions of base pairs, but the factors influencing inversion frequency are not well understood. Prior reports differ in species, detection methods, and delivery strategies, making direct comparisons difficult. We addressed this by introducing a normalisation strategy based on a reference guide RNA (gRNA) as an internal standard and testing inversion sizes spanning kilobases to tens of megabases in tomato protoplasts. RESULTS: Tomato (Solanum lycopersicum) protoplasts were transfected with constructs encoding a fixed “reference” gRNA and a second “variable” gRNA positioned at increasing genomic distances, creating potential inversions from 1 kilobase to 37.5 megabases. Using the reference gRNA to normalise across samples, we found that up to ~ 1 megabase, inversion frequency tracked the cutting efficiency of the less efficient gRNA, consistent with gRNA activity being a major contributor within the chromosome tested. For these intervals, the inversion frequencies reached up to 1.24% when both gRNAs were efficient. Above ~ 1 megabase, inversion frequencies declined sharply despite efficient cutting, suggesting a size-dependent barrier to inversion formation; for example, 37.5 megabase inversions occurred at substantially lower frequency (up to 0.18%) despite efficient gRNAs. Because each interval corresponds to a distinct genomic location. Inversions were only observed when both gRNAs were active, and large deletions were more frequent than inversions when dual breaks were induced. CONCLUSIONS: In our experiments, the gRNA cutting efficiency was a major determinant of inversion frequency in our experiment up to ~ 1 megabase, while locus-specific genomic context may also contribute., Larger inversions may be limited by an additional, size-dependent constraint. These findings inform the design of edits aimed at reverting breeding-relevant inversions (for example, those linked to resistance loci) and suggest that achieving high efficiency for multi-megabase inversions will require strategies that overcome spatial or repair-related constraints. The internal reference gRNA normalises sample-to-sample variability in DNA delivery and Cas9 activity, enabling direct comparison of the performance of different gRNAs across samples on a shared, ratio-based scale. This ratio-to-reference strategy may likewise be used to benchmark gRNA performance and edit yields (inversions, deletions, translocations, and base/prime edits) across transfections in plant protoplasts, and may be extended to additional cell systems beyond plants.},
}

*Solanum lycopersicum/genetics
*Protoplasts/metabolism
*Chromosome Inversion/genetics
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41817895,
year = {2026},
author = {Prins, TJ and Lai, TJ and Li, T and Fisher, A and Eldred, BSC and Mostafavi, R and Liau, LM and Chong, RA and Nghiemphu, PL and Cloughesy, TF and Nathanson, DA and Lai, A},
title = {MGMT downregulation by CRISPR/Cas13 RNA-guided RNA targeting enhances glioma cell sensitivity to TMZ chemotherapy.},
journal = {Journal of neuro-oncology},
volume = {177},
number = {1},
pages = {},
pmid = {41817895},
issn = {1573-7373},
support = {P50 CA211015/CA/NCI NIH HHS/United States ; P50 CA211015-01A1/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; Temozolomide ; *Tumor Suppressor Proteins/metabolism/genetics ; *Glioma/drug therapy/metabolism/genetics ; *Antineoplastic Agents, Alkylating/pharmacology ; *DNA Modification Methylases/metabolism/genetics ; *DNA Repair Enzymes/metabolism/genetics ; *Brain Neoplasms/drug therapy/metabolism/genetics ; Down-Regulation ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Dacarbazine/analogs & derivatives/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; Drug Resistance, Neoplasm ; RNA, Messenger/metabolism ; Cell Survival/drug effects ; },
abstract = {BACKGROUND: Current standard of care for glioblastoma involves fractionated radiotherapy administered with Temozolomide (TMZ), a DNA-alkylating agent. Inhibition of the DNA repair enzyme, O[6]-methylguanine-DNA methyltransferase (MGMT), promotes sensitivity to TMZ, particularly in tumors that repress MGMT mRNA transcription through promoter methylation. Novel strategies to inhibit MGMT are a promising avenue to improve therapeutic outcomes to TMZ. We hypothesized that CRISPR-Cas13-mediated RNA regulatory silencing of MGMT mRNA enhances response of immortalized and primary patient-derived gliomaspheres to TMZ in vitro. METHODS: We utilized the Cas13x and Cas13d variants to target MGMT mRNA in the MGMT-expressing LN18 glioma cell line and in two patient-derived gliomasphere lines (GS104, GS081). Cas13-guide RNA ribonucleoproteins were delivered via lipofection, and stable knockdown was achieved using a lentiviral all-in-one system. MGMT mRNA and protein downregulation were assessed by RT-PCR and Western blot, respectively. Cell viability and chemosensitivity to TMZ were evaluated using MTT assays. RESULTS: Both Cas13x and Cas13d systems, directed by specific guide CRISPR RNAs, achieved rapid and potent knockdown of MGMT mRNA and protein in all tested cell lines. This downregulation of MGMT expression led to an increase in the cytotoxic effects of TMZ, sensitizing previously resistant glioma cells and patient-derived gliomaspheres to standard chemotherapy. The lentiviral Cas13d system established stable chemosensitization in gliomasphere models. CONCLUSION: CRISPR-Cas13-mediated targeting of MGMT mRNA is an effective strategy for overcoming TMZ resistance in in vitro glioblastoma models. This RNA regulatory editing approach offers a proof-of-principle for CRISPR mediated therapeutics in patients with MGMT unmethylated gliomas.},
}

Humans
Temozolomide
*Tumor Suppressor Proteins/metabolism/genetics
*Glioma/drug therapy/metabolism/genetics
*Antineoplastic Agents, Alkylating/pharmacology
*DNA Modification Methylases/metabolism/genetics
*DNA Repair Enzymes/metabolism/genetics
*Brain Neoplasms/drug therapy/metabolism/genetics
Down-Regulation
Cell Line, Tumor
RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
*Dacarbazine/analogs & derivatives/pharmacology
Gene Expression Regulation, Neoplastic/drug effects
Drug Resistance, Neoplasm
RNA, Messenger/metabolism
Cell Survival/drug effects

@article {pmid41839945,
year = {2026},
author = {Yanguas-Casás, N and Pedrosa, L and Horcajo, B and Gómez, S and Garcia-Grande, A and Muñoz-Viana, R and Fernández-Miranda, I and Pérez-Aguilera, M and Torres-Ruiz, R and Rodríguez-Perales, S and Sánchez-Beato, M},
title = {Splice-site mutations in POU2AF1 are associated with B-cell lymphomagenesis and therapeutic response.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41839945},
issn = {2045-2322},
support = {POSTD18029SANC//Fundación Científica de la Asociación Española Contra el Cáncer/ ; iPFIS predoctoral fellowship (IFI18/0004)//ISCIII-MINECO AES-FEDER, Plan Estatal I+D+I 2014-2020/ ; PEJ-2020-TL/BMD-19530//Plan de Empleo Juvenil de la CM/ ; PEJ-2023-AI/SAL-GL-28806//Plan de Empleo Juvenil de la CM/ ; B2017/BMD-3778//Dirección General de Universidades e Investigación de la Consejería de Educación e Investigación de la Comunidad de Madrid (CM)/ ; PI17/00272, PI20/00591, PI23/01587//Spanish Ministry of Economy and Competence (MINECO) and Instituto de Salud Carlos III (ISCIII), ISCIII-MINECO AES-FEDER/ ; },
mesh = {Humans ; Cell Line, Tumor ; *RNA Splice Sites/genetics ; *Lymphoma, B-Cell/genetics/pathology/drug therapy ; Cell Proliferation/genetics ; *Mutation ; Cell Movement/genetics ; *Trans-Activators/genetics ; Point Mutation ; CRISPR-Cas Systems ; },
abstract = {BOB.1, encoded by POU2AF1, is one of many factors regulating physiological B-cell maturation in the germinal center. Recently, several studies have described recurrent mutations in a three-nucleotide region in the POU2AF1 splice site in the two most common B-cell non-Hodgkin lymphomas: diffuse large B-cell lymphoma and, more frequently, follicular lymphoma. In this study, we introduced a C→G mutation at the + 1 position of the POU2AF1 splice site in two B-cell lymphoma cell lines (WSU-NHL and SUDHL4) using CRISPR/Cas9 gene editing. Our results demonstrate how point mutations in the POU2AF1 splice site decreased BOB.1 expression levels. The mutation did not produce significant changes in cell proliferation, migration, or invasiveness, but did affect cell morphology, aggregation, and cell survival in a cell-line-dependent manner. Lastly, we found that the POU2AF1 mutation c.16 + 1G > C increased BCR activation, especially in SUDHL4 cells, downregulated oxidative phosphorylation (OxPhos) metabolism, and modified therapy sensitivities in both cell lines. Mutated B-cells were more sensitive to the BTK inhibitor ibrutinib. In conclusion, mutations in the POU2AF1 splice site impact B-cell lymphomagenesis at multiple levels and represent a potential therapeutic target for patients with tumors harboring this mutation.},
}

Humans
Cell Line, Tumor
*RNA Splice Sites/genetics
*Lymphoma, B-Cell/genetics/pathology/drug therapy
Cell Proliferation/genetics
*Mutation
Cell Movement/genetics
*Trans-Activators/genetics
Point Mutation
CRISPR-Cas Systems

@article {pmid41839984,
year = {2026},
author = {Taguchi, YH and Turki, T},
title = {Gene and cell line efficiency of CRISPR computed by tensor decomposition in genome-wide CRISPR-Cas9 knockout screens.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41839984},
issn = {2045-2322},
support = {Deanship of Scientific Research, IPP: 70-611-2025//King Abdulaziz University/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Cell Line ; Algorithms ; },
abstract = {Genome-wide CRISPR-Cas9 knockout screens are often used to experimentally evaluate gene function. However, the efficacy of individual sgRNAs targeting unique genes varies and is difficult to integrate. In this study, tensor decomposition (TD) was used to integrate multiple sgRNAs and sgRNA profiles simultaneously. Thus, TD can discriminate between essential and non-essential genes with the performance comparative to that of Joint analysis of CRISPR/Cas9 knockout screens (JACKS), a type of SOTA that previously outperformed various other SOTA. In addition, although TD uses simple linear algebra, it can achieve good performance even without control samples, without which JACKS cannot be performed. Moreover, because raw and logarithmic values can achieve similar performances through TD for the largest dataset among the tested datasets, taking logarithmic values as has been done frequently, which is questioned. In conclusion, TD is the first method that can integrate multiple sgRNAs attributed to single a target and sgRNA profiles at the beginning simultaneously and can achieve a performance comparable to that of JACKS.},
}

*CRISPR-Cas Systems
*Gene Knockout Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Humans
Cell Line
Algorithms

@article {pmid41888883,
year = {2026},
author = {Yuan, G and Deng, S and Dai, Z and Hofstad, BA and Pomraning, KR},
title = {Expanding the genetic toolkit: adenine and cytosine base editors for gene disruption in Aspergillus niger.},
journal = {Microbial cell factories},
volume = {25},
number = {1},
pages = {},
pmid = {41888883},
issn = {1475-2859},
mesh = {*Aspergillus niger/genetics ; *Cytosine/metabolism ; *Gene Editing/methods ; *Adenine/metabolism ; CRISPR-Cas Systems ; },
abstract = {Despite revolutionizing fungal genetic engineering, conventional CRISPR/Cas9-mediated knockouts rely on DNA double-strand breaks (DSBs), which can cause unwanted insertions and deletions, chromosomal abnormalities, and cytotoxicity. Base editors such as adenine base editors (ABEs), which convert A‧T to G‧C, and cytosine base editors (CBEs), which convert C‧G to T‧A, offer a safer alternative by enabling predictable, target-specific single-nucleotide changes without introducing DSBs. To overcome the limitations of traditional genome editing in filamentous fungi, we developed efficient base-editing systems in Aspergillus niger. For the first time, we constructed an ABE in A. niger, achieving up to 80% editing efficiency and inducing predictable A-to-G mutations at the intended intron sites, disrupting gene function through mRNA mis-splicing. We also developed a highly efficient CBE system, capable of introducing premature stop codons with 50–100% efficiency. To broaden the editing scope, we implemented a Cas9-NG variant recognizing a relaxed PAM sequence requiring only a single guanine (G), enabling editing at start codons and splice sites. Leveraging this expanded scope, we established gene disruption approaches by targeting start codons via ABE-mediated A-to-G conversions (ATG-to-GTG and ATG-to-ACG) and CBE-mediated C-to-T conversion (ATG-to-ATA). Additionally, our base-editing systems enable multiplex gRNA delivery and marker-free editing of multiple genes. Collectively, the scope-expanding strategies increase the number of genes targetable for disruption by base-editing in A. niger by 26.3% and enable near-complete coverage of 96% of the coding genes. Overall, this work demonstrates the potential of ABE and CBE systems as versatile, efficient, and safer alternatives to DSBs-based gene disruption in filamentous fungi.},
}

*Aspergillus niger/genetics
*Cytosine/metabolism
*Gene Editing/methods
*Adenine/metabolism
CRISPR-Cas Systems

@article {pmid41961403,
year = {2026},
author = {Sun, J and Hu, Z and Yang, Y},
title = {An ATMT-CRISPR/Cas9 system for genome editing in Monascus purpureus.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {4},
pages = {},
pmid = {41961403},
issn = {1573-0972},
mesh = {*Monascus/genetics/metabolism ; *CRISPR-Cas Systems ; *Agrobacterium tumefaciens/genetics ; *Gene Editing/methods ; Transformation, Genetic ; Homologous Recombination ; Genome, Fungal ; Gene Knockout Techniques ; Protoplasts ; Biosynthetic Pathways/genetics ; Fungal Proteins/genetics ; },
abstract = {Monascus purpureus is a filamentous fungus of significant economic value in the food and pharmaceutical industries, capable of producing a diverse array of secondary metabolites. Although CRISPR/Cas9 systems have been extensively utilized in filamentous fungi, predominantly employing protoplasts as recipients, the genetic manipulation of M. purpureus remains challenging due to the inherent difficulties associated with protoplast preparation. In addition, it has been reported that the Cas9 protein may demonstrate toxicity to cells. To overcome this limitation, this study developed a CRISPR/Cas9 gene-editing system based on Agrobacterium tumefaciens -mediated transformation (ATMT). This system utilizes M. purpureus spores as recipients and employs a homologous recombination strategy to achieve stable, site-specific integration of the Cas9 expression cassette into non-coding regions of the host genome, thereby avoiding the complexity of protoplast preparation and the uncertainty of random integration events. System evaluations indicate that the stable expression of Cas9 protein has no significant adverse effects on the nutritional growth, reproductive development, or characteristic pigment biosynthesis of M. purpureus. The system demonstrated high efficacy in single-gene editing, achieving a knockout efficiency of 74% for the key pigment biosynthetic pathway gene pksPT. Although efficiency decreased when performing double-gene and triple-gene editing (4.8% and 1.7%, respectively), this study successfully validated the system’s potential for multi-gene genome engineering. The ATMT-CRISPR/Cas9 system established herein demonstrates the feasibility of genetic manipulation of M. purpureus, providing a methodological foundation with the potential to facilitating functional genomic studies and the targeted regulation of secondary metabolism in this industrially important fungus.},
}

*Monascus/genetics/metabolism
*CRISPR-Cas Systems
*Agrobacterium tumefaciens/genetics
*Gene Editing/methods
Transformation, Genetic
Homologous Recombination
Genome, Fungal
Gene Knockout Techniques
Protoplasts
Biosynthetic Pathways/genetics
Fungal Proteins/genetics

@article {pmid42000863,
year = {2026},
author = {Piñeiro-Silva, C and Bermejo-Álvarez, P and García-Purriños, FJ and Gadea, J},
title = {Gene editing of the GJB2 locus in porcine embryos using CRISPR/Cas9 and cytosine base editors: toward a model of congenital deafness.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42000863},
issn = {2045-2322},
support = {22065/PI/22//Fundación Séneca-Agencia de Ciencia y Tecnología de la Región de Murcia/ ; 22545/PDC/24//Fundación Séneca-Agencia de Ciencia y Tecnología de la Región de Murcia/ ; 23031/GERM/25 supported by FSRM/10.13039/100007801//Fundación Séneca-Agencia de Ciencia y Tecnología de la Región de Murcia/ ; R-496/2022//Universidad de Murcia predoctoral fellowship/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Connexin 26/genetics ; *Cytosine/metabolism ; Swine ; *Deafness/genetics/congenital ; *Connexins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Female ; Mutation ; Disease Models, Animal ; Oocytes/metabolism ; },
abstract = {Mutations in the GJB2 gene, which encodes Connexin 26 (Cx26), are responsible for the majority of cases of non-syndromic congenital hearing loss in humans. While murine GJB2 knockout models have provided mechanistic insight, anatomical and physiological differences limit their translational relevance. Pigs represent a valuable large-animal model because their auditory anatomy and maturation closely resemble those of humans. This study compared two genome-editing approaches to disrupt GJB2 in porcine oocytes before fertilization: (1) electroporation with CRISPR/Cas9 ribonucleoprotein and (2) microinjection with cytosine base editor (BE3) and single-guide RNAs (sgRNAs). Electroporation produced high mutation rates (70–90%) across three concentrations of Cas9/sgRNA but yielded mostly heterozygous or mosaic blastocysts, with limited homozygous knockouts (< 4%). BE3 achieved precise cytosine-to-thymine conversions that introduced premature stop codons, reaching up to 47% total editing and 20% homozygous nonsense alleles. However, blastocyst formation declined at higher component concentrations. Overall, BE3 produced more predictable mutations than conventional CRISPR/Cas9, although embryo developmental competence was dose-dependent. Both methods effectively targeted GJB2 and demonstrated feasibility of pre-fertilization genome editing in porcine oocytes. These findings establish the groundwork for generating GJB2-deficient pigs as translational models of Cx26-related congenital deafness and for future evaluation of gene-therapy strategies in a large-animal system.},
}

Animals
*CRISPR-Cas Systems
*Gene Editing/methods
*Connexin 26/genetics
*Cytosine/metabolism
Swine
*Deafness/genetics/congenital
*Connexins/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Female
Mutation
Disease Models, Animal
Oocytes/metabolism

@article {pmid42012544,
year = {2026},
author = {Shen, W and Xiao, R and Li, J and Zheng, S and Wang, C and Hao, R and Yin, J},
title = {CRISPR-based dual-mode lateral flow assay driven by magnetic SERS tags for highly sensitive detection of respiratory viruses.},
journal = {Mikrochimica acta},
volume = {193},
number = {5},
pages = {},
pmid = {42012544},
issn = {1436-5073},
support = {82372348//National Natural Science Foundation of China/ ; },
mesh = {*Spectrum Analysis, Raman/methods ; *SARS-CoV-2/isolation & purification/genetics ; Humans ; *Influenza A Virus, H1N1 Subtype/isolation & purification/genetics ; Gold/chemistry ; Colorimetry/methods ; Rapid Diagnostic Tests ; Limit of Detection ; CRISPR-Cas Systems ; COVID-19/diagnosis/virology ; },
abstract = {Effective epidemic control hinges on rapid point-of-care detection of respiratory viruses, but the sensitivity of current screening technologies remains inadequate. Here, we developed a CRISPR-activated, colorimetric Surface-Enhanced Raman Scattering (SERS) dual-mode nucleic acid lateral flow assay (LFA) that enables highly sensitive, flexible, and simultaneous detection of two common respiratory viruses, influenza A (H1N1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). By coupling recombinase polymerase amplification with CRISPR for sensitive amplification and specific target recognition, viral targets activate Cas13 trans-cleavage to efficiently cleave reporter probes. In parallel, a dual-functional magnetic SERS tag (Fe3O4@Au/Au-SA) was introduced, which captures two biotinylated reporter molecules through streptavidin modification, and generates strong and stable SERS signals through built-in hotspot effects. The resulting CRISPR-magnetic SERS-LFA enables rapid qualitative screening of H1N1 and SARS-CoV-2 through reduced colorimetric signal intensity on two test lines and accurate quantification via SERS signal changes. The establish method achieves a detection limit of 7–9 copies/µL for the two target viruses and shows good agreement with quantitative reverse transcription polymerase chain reaction (qRT-PCR) in validation with 74 clinical samples. Both the sensitivity and specificity for clinical samples reach 100%, highlighting its potential for field deployment.},
}

*Spectrum Analysis, Raman/methods
*SARS-CoV-2/isolation & purification/genetics
Humans
*Influenza A Virus, H1N1 Subtype/isolation & purification/genetics
Gold/chemistry
Colorimetry/methods
Rapid Diagnostic Tests
Limit of Detection
CRISPR-Cas Systems
COVID-19/diagnosis/virology

@article {pmid42015157,
year = {2026},
author = {Yang, P and Ma, R and Zeng, J and Li, L and Peng, J and Zhou, M and Qu, M and Li, X and Lai, T and Zhou, W and Wu, Y and Lu, Y and Zhang, Y},
title = {Simple and rapid profiling of tumor EVs for differential diagnosis of NSCLC via orthogonal barcode-driven CRISPR/Cas12a.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {},
pmid = {42015157},
issn = {1477-3155},
support = {82172374//National Natural Science Foundation of China/ ; 2023GDRC005//Chongqing Science and technology joint major funding program/ ; 2022CDJYGRH-010//Natural Science Foundation Project of Chongqing, Chongqing Science and Technology Commission/ ; 2021M693730//China Postdoctoral Science Foundation/ ; },
mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung/diagnosis ; *Lung Neoplasms/diagnosis ; *CRISPR-Cas Systems/genetics ; *Extracellular Vesicles/metabolism ; Biomarkers, Tumor/blood ; Diagnosis, Differential ; Cell Line, Tumor ; },
abstract = {Tumor-derived extracellular vesicles (tEVs), a class of nanoscale vesicles actively released by malignant cells, have emerged as attractive biomarkers for non-invasive cancer diagnosis. However, their clinical translation remains challenging due to low abundance, molecular heterogeneity, and the requirement for multiplexed surface marker discrimination. Here, we report a dual aptamer-mediated CRISPR/Cas12a-assisted sensing platform (DA-CAS) for rapid and orthogonally programmable dual-marker profiling of tEVs, enabling differential diagnosis of non-small cell lung cancer (NSCLC) using only 10 µL of plasma within 100 min. The DA-CAS system integrates proximity ligation-based dual-marker recognition with hyperbranched rolling circle amplification (HRCA) to generate programmable DNA barcodes, which selectively trigger Cas12a trans-cleavage in an orthogonal manner. Using EpCAM and PD-L1 as representative surface markers, the platform achieves subtype-specific detection of NSCLC-derived tEVs with minimal background activation and a detection limit as low as 75 particles/mL. Moreover, a portable lateral flow readout enables accurate, instrument-free visual detection at concentrations down to 406 particles/mL. Under the condition of free-ultracentrifugation, clinical validation using a cohort of 45 plasma samples demonstrated a sensitivity of 97%, specificity of 88%, and overall diagnostic accuracy of 96%, outperforming conventional ELISA assays and multi-marker serum panels in both analytical sensitivity and subtype resolution. In addition, this platform demonstrated preliminary potential for discriminating between benign and malignant lung diseases and for dynamically monitoring radiotherapeutic responses. The orthogonal barcode design effectively eliminates inter-channel crosstalk and enzymatic interference, enabling orthogonal dual-target recognition with high subpopulation specificity. Overall, DA-CAS provides a robust, rapid, and point-of-care-compatible strategy for tEV-based cancer diagnostics, offering strong translational potential for non-invasive tumor profiling and dynamic immune status monitoring.},
}

Humans
*Carcinoma, Non-Small-Cell Lung/diagnosis
*Lung Neoplasms/diagnosis
*CRISPR-Cas Systems/genetics
*Extracellular Vesicles/metabolism
Biomarkers, Tumor/blood
Diagnosis, Differential
Cell Line, Tumor

@article {pmid42031935,
year = {2026},
author = {Hao, D and Xu, X and Li, P and Liu, Y and Liu, G and Deng, S},
title = {A method for CRISPR/Cas9-induced genetic barcoding and lineage tracing in sheep.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {42031935},
issn = {2045-2322},
support = {2023-PT180-01//Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Sheep/genetics/embryology ; *Cell Lineage/genetics ; DNA Transposable Elements/genetics ; *Gene Editing/methods ; *DNA Barcoding, Taxonomic/methods ; Embryonic Development/genetics ; },
abstract = {Sheep development involves continuous dynamic processes in which cells propagate, differentiate and orchestrate. However, the method for tracing cell fate during sheep (Ovis aries) embryogenesis, cell differentiation and tissue regeneration remains largely undeveloped. Here, we developed a CRISPR/Cas9-based lineage barcode recording method that directly acts on target cells. With this method, several contiguous CRISPR/Cas9 targeting arrays were synthesized and introduced into multiple loci in the sheep genome using the PiggyBac transposon vector to form the barcode region. Cas9 integrated at the Rosa26 gene locus was used to generate edits in the barcode region at multiple timepoints during early sheep embryonic development. We detected multiple integrated barcodes (intBCs) that were stably inherited in the developing embryos, confirming that the method can generate heritable clonal markers. The method could enable lineage tracing in sheep when combined with single-cell sequencing technologies. Our method establishes a foundation for ruminant lineage tracing technology by combining PiggyBac transposons and CRISPR/Cas9 gene editing tools, providing a new platform for analyzing sheep embryonic development, organ regeneration, and disease mechanisms.},
}

Animals
*CRISPR-Cas Systems
Sheep/genetics/embryology
*Cell Lineage/genetics
DNA Transposable Elements/genetics
*Gene Editing/methods
*DNA Barcoding, Taxonomic/methods
Embryonic Development/genetics

@article {pmid42050530,
year = {2026},
author = {Liao, X and Zhou, J and Shan, Y and Li, X and Peng, Y},
title = {CRISPR/Cas12a-assisted visual and on-site detection of porcine circovirus type 2.},
journal = {BMC veterinary research},
volume = {22},
number = {1},
pages = {},
pmid = {42050530},
issn = {1746-6148},
support = {2024NSFSC0761//the Science and Technology Foundation of Sichuan Province, China/ ; 2021R52041//High-level Talents Special Support Plan of Zhejiang Province/ ; ZYGD23027//the 1·3·5 Project for Disciplines of Excellence, West China Hospital of Sichuan University/ ; },
mesh = {*Circovirus/isolation & purification/genetics ; Animals ; Swine ; *CRISPR-Cas Systems ; *Circoviridae Infections/veterinary/diagnosis/virology ; *Swine Diseases/diagnosis/virology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; DNA, Viral ; },
abstract = {BACKGROUND: Porcine circovirus type 2 (PCV2) is a globally prevalent viral pathogen that causes substantial economic losses in the swine industry. Rapid and accurate on-site diagnosis is critical for controlling the spread of PCV2. In recent years, RNA-guided CRISPR/Cas12a nucleases combined with recombinase polymerase amplification (RPA) have emerged as a promising approach for nucleic acid detection. This study aimed to develop a novel RPA-CRISPR-based method for the rapid and sensitive detection of PCV2 in field settings. RESULTS: We designed and optimized CRISPR RNAs (crRNAs) targeting conserved regions of the PCV2 Cap and Rep genes. Upon recognition of the target sequence, the Cas12a nuclease was activated to cleave a single-stranded DNA-fluorophore quencher (ssDNA-FQ) reporter, generating a fluorescent signal detectable either by a fluorescence detector or via visual readout. The entire procedure was performed at 37 °C and completed within one hour. The assay achieved a detection limit as low as 10 copies/µL and showed no cross-reactivity with other major porcine viruses. Furthermore, a rapid-release reagent was used to replace conventional DNA extraction from serum samples, facilitating efficient on-site detection. The assay was validated using clinical samples, and the results showed strong concordance with those obtained by PCR. CONCLUSIONS: The RPA-CRISPR-based assay developed in this study is highly sensitive and specific, enabling detection of PCV2 within one hour. Its simplicity, rapidity, and ease of use in the field offer significant practical advantages, making it a valuable tool for the on-site diagnosis of PCV2. This method represents a promising alternative for the early and rapid detection of PCV2 infections and holds potential for contributing to the prevention and control of the disease in the swine industry.},
}

*Circovirus/isolation & purification/genetics
Animals
Swine
*CRISPR-Cas Systems
*Circoviridae Infections/veterinary/diagnosis/virology
*Swine Diseases/diagnosis/virology
*Nucleic Acid Amplification Techniques/veterinary/methods
Rapid Diagnostic Tests
Sensitivity and Specificity
DNA, Viral

@article {pmid40506596,
year = {2025},
author = {Sajjad, MW and Muzamil, F and Naqvi, RZ and Amin, I},
title = {QBEmax redefines the precise base editing in crop plants.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {127},
pmid = {40506596},
issn = {1438-7948},
mesh = {*Crops, Agricultural/genetics ; *CRISPR-Cas Systems ; Genome, Plant ; Cytidine Deaminase/genetics/chemistry/metabolism ; *Gene Editing ; },
abstract = {Hu et al.‘s new study, published in Nature Biotechnology, introduces QBEmax; a tiny, conformationally sound editing tool with a cytidine deaminase buried within a looping permuted Cas9 (cpCas9). Supported by molecular dynamics models and AlphaFold3 structural predictions, this unique internal fusion creates a structurally protected complex that improves editing accuracy and lowers typical artifacts such as indels and impure base conversions (Hu et al. Nature Biotechnology:1-7, 2025). High precision editing (up to 99.8% purity), lower indels, and lower off target impacts well suit imminent plant transformation events. Its tiny, stable architecture and wider editing window at PAM sites increase its ability for precise and adaptable trait change in complex plant genomes.},
}

*Crops, Agricultural/genetics
*CRISPR-Cas Systems
Genome, Plant
Cytidine Deaminase/genetics/chemistry/metabolism
*Gene Editing

@article {pmid40999513,
year = {2025},
author = {Lee, S and Yu, Y and Kim, DH and Bock, M and Kim, Y and An, SB and Choi, H and Shin, HE and Hwang, DY and Han, I},
title = {Enhanced disc regeneration through CRISPR/Cas9-mediated SOX9 and TGFβ1 coexpression in tonsil-derived mesenchymal stromal cells.},
journal = {Stem cell research & therapy},
volume = {16},
number = {1},
pages = {501},
pmid = {40999513},
issn = {1757-6512},
support = {RS-2024-00347403//National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT: Ministry of Science and ICT)/ ; RS-2023-KH141187//Korean Cell-Based Artificial Blood Project funded by the Korean government (The Minis-try of Science and ICT, The Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety)/ ; },
mesh = {*SOX9 Transcription Factor/genetics/metabolism ; *Transforming Growth Factor beta1/genetics/metabolism ; *Mesenchymal Stem Cells/metabolism/cytology ; Animals ; Rats ; Humans ; *Intervertebral Disc Degeneration/therapy/genetics/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Regeneration ; Rats, Sprague-Dawley ; *Palatine Tonsil/cytology/metabolism ; *Intervertebral Disc/physiology/metabolism ; Male ; Cell Differentiation ; Chondrogenesis ; },
abstract = {BACKGROUND: Intervertebral disc (IVD) degeneration, a primary cause of chronic low back pain, currently lacks treatments that target its underlying pathological mechanisms. Tonsil-derived mesenchymal stromal cells (ToMSCs) have shown promise for IVD regeneration; however, their therapeutic potential is limited by the harsh microenvironment of degenerated discs. This study investigated whether ToMSCs engineered to co‐overexpress SOX9 and TGFβ1 using a tetracycline‐off (Tet‐off) regulatory system could enhance extracellular matrix (ECM) restoration and reduce inflammation in degenerative IVDs. METHODS: We used CRISPR/Cas9 technology to generate ToMSCs that express SOX9, TGFβ1, or both factors under Tet-off regulation. Gene expression was confirmed by Western blot and qRT-PCR analyses. In vitro studies assessed chondrogenic differentiation capacity, while in vivo assessments were performed using a rat tail needle puncture model of IVD degeneration. After administering the CRISPR-engineered ToMSCs, we monitored mechanical allodynia with the von Frey test over six weeks. Therapeutic outcomes were evaluated through T2‐weighted MRI and histological analysis. RESULTS: In vitro experiments showed that ToMSCs co-expressing SOX9 and TGFβ1 exhibited superior chondrogenic differentiation compared to cells expressing a single factor. In vivo studies demonstrated that dual-factor expressing ToMSCs significantly improved disc hydration (as confirmed by MRI), enhanced ECM synthesis—particularly aggrecan and type II collagen—and reduced inflammation compared to single-factor treatments. These improvements were accompanied by reduced mechanical allodynia, indicating functional recovery. CONCLUSION: Our study demonstrates that ToMSCs engineered to co-express SOX9 and TGFβ1 effectively promote IVD regeneration by enhancing ECM production and reducing inflammation. This dual-factor approach represents a promising therapeutic strategy for treating degenerative disc disease and warrants further investigation for clinical application.},
}

*SOX9 Transcription Factor/genetics/metabolism
*Transforming Growth Factor beta1/genetics/metabolism
*Mesenchymal Stem Cells/metabolism/cytology
Animals
Rats
Humans
*Intervertebral Disc Degeneration/therapy/genetics/pathology/metabolism
*CRISPR-Cas Systems/genetics
*Regeneration
Rats, Sprague-Dawley
*Palatine Tonsil/cytology/metabolism
*Intervertebral Disc/physiology/metabolism
Male
Cell Differentiation
Chondrogenesis

@article {pmid41003970,
year = {2026},
author = {Kanafi, MM and Moazzami, R},
title = {Leveraging CRISPR/Cas9 To Overcome Hypoxic Barriers in Regenerative Dentistry.},
journal = {Stem cell reviews and reports},
volume = {22},
number = {1},
pages = {82-86},
pmid = {41003970},
issn = {2629-3277},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Dental Pulp/cytology/metabolism ; *Stem Cells/metabolism/cytology ; Animals ; *Dentistry/methods ; Cell Hypoxia ; *Regeneration ; *Regenerative Medicine/methods ; Oxidative Stress ; Gene Editing/methods ; },
abstract = {Dental pulp stem cells (DPSCs) have gained increasing attention as a valuable cell source for regenerative dentistry owing to their accessibility, high proliferative potential, and capacity for multilineage differentiation. Despite these advantages, their therapeutic efficacy is substantially compromised by pathological hypoxia, a common feature of injured or poorly vascularized oral tissues. Hypoxic stress not only impairs DPSC survival but also diminishes their regenerative capacity, creating a major barrier to effective clinical translation. Addressing this limitation is therefore essential to harness the full therapeutic potential of DPSCs. Recent advances in genome-editing technologies, particularly the CRISPR/Cas9 system, have created novel opportunities to enhance the resilience of DPSCs against hypoxic stress. By enabling precise genetic modifications, CRISPR offers a powerful platform to reprogram cellular pathways associated with oxygen deprivation, oxidative stress, and apoptosis. Current preclinical investigations have focused on key targets such as HIF1α, PHD2, NRF2, BAX, and VEGF, exploring their modulation through CRISPR-mediated activation, inhibition, or knockout strategies. Upregulation of HIF1α and VEGF has demonstrated the ability to enhance angiogenesis and promote cell survival in oxygen-deficient microenvironments. Similarly, activation of NRF2 improves antioxidant defense mechanisms and mitigates oxidative damage, while suppression of pro-apoptotic genes such as BAX increases overall viability. Collectively, these strategies represent a multifaceted approach to strengthening DPSC performance in adverse conditions. The integration of CRISPR/Cas9 into regenerative dentistry represents a paradigm shift in addressing hypoxia-induced barriers to stem cell therapy. While early findings are promising, several critical challenges remain, including the potential for off-target effects, the need for stable and long-term genetic modifications, and concerns regarding biosafety and ethical considerations. Robust preclinical validation and carefully designed translational studies will be required before CRISPR-engineered DPSCs can be considered for clinical application. In summary, CRISPR/Cas9-based modulation of hypoxia-responsive pathways offers a transformative strategy to enhance the therapeutic efficacy of DPSCs. By improving survival, stress tolerance, and angiogenic potential under hypoxic conditions, this approach may significantly expand the clinical applicability of stem cell–based interventions in dentistry. Continued research is essential to ensure the safety, reliability, and long-term benefits of this promising therapeutic avenue.},
}

Humans
*CRISPR-Cas Systems/genetics
*Dental Pulp/cytology/metabolism
*Stem Cells/metabolism/cytology
Animals
*Dentistry/methods
Cell Hypoxia
*Regeneration
*Regenerative Medicine/methods
Oxidative Stress
Gene Editing/methods

@article {pmid41171582,
year = {2026},
author = {Feger, M and Tsapara, A and Hülße, S and Rausch, S and Barholz, M and Stournaras, C and Föller, M},
title = {Chorein Regulates Key Osteoblast Genes in UMR-106 Cells.},
journal = {Cell biochemistry and biophysics},
volume = {84},
number = {1},
pages = {1245-1252},
pmid = {41171582},
issn = {1559-0283},
mesh = {*Osteoblasts/metabolism/cytology ; Humans ; *Vesicular Transport Proteins/genetics/metabolism/antagonists & inhibitors ; Cell Line ; Fibroblast Growth Factor-23 ; Fibroblast Growth Factors/metabolism/genetics ; PHEX Phosphate Regulating Neutral Endopeptidase/metabolism/genetics ; Cell Differentiation ; CRISPR-Cas Systems ; Gene Knockdown Techniques ; Animals ; *Gene Expression Regulation ; },
abstract = {Chorein is an endoplasmic reticulum protein expressed in many cell types. Loss-of-function mutations of the gene encoding chorein (VPS13A) are the cause of chorea-acanthocytosis, a rare and severe neurodegenerative disease with chorea-like movements, loss of mental function, progressive muscle weakness and misshaped erythrocytes (acanthocytes). Chorein regulates diverse cellular functions including the cytoskeleton, apoptosis, Ca2+ entry, or autophagy. Since its role in bone is enigmatic, we aimed to explore the function of chorein in osteoblasts. To this end, we generated UMR-106 osteoblast-like cells with stable chorein knockdown (KD) using a CRISPR/Cas9-based approach and compared them to cells undergoing CRISPR/Cas9 with a non-targeting sequence (NT). Gene expression was assessed by qPCR and protein by Western blotting and ELISA. Gene and protein expression of chorein and fibroblast growth factor 23 (FGF23), an osteoblast-derived hormonal regulator of phosphate metabolism, were decreased in KD compared to NT cells. Moreover, FGF23 regulator Phex was down- and Galnt3 was up-regulated in KD compared to NT cells. The expression of further genes regulating osteoblast and osteoclast differentiation was affected by chorein knockdown. Taken together, chorein is expressed in UMR-106 osteoblasts and modulates the expression of various genes affecting osteoblast and osteoclast differentiation and function.},
}

*Osteoblasts/metabolism/cytology
Humans
*Vesicular Transport Proteins/genetics/metabolism/antagonists & inhibitors
Cell Line
Fibroblast Growth Factor-23
Fibroblast Growth Factors/metabolism/genetics
PHEX Phosphate Regulating Neutral Endopeptidase/metabolism/genetics
Cell Differentiation
CRISPR-Cas Systems
Gene Knockdown Techniques
Animals
*Gene Expression Regulation

@article {pmid41250247,
year = {2025},
author = {Qian, Y and Hui, F and Niu, W and Wang, D and Hao, Y and Meng, Q and Ren, S and Kong, D and Gong, H and Wu, J and Chen, K and Alariqi, M and Gao, J and Li, Z and Jin, S},
title = {Double-stranded DNA deaminase DddA[E1347A] can increase the efficiency and targeting range of cytidine base editors.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {391},
pmid = {41250247},
issn = {1474-760X},
support = {2024M761134//The China Postdoctoral Fund/ ; KY2020YC0002//The China Tobacco Hunan Industrial Co., Ltd. Research Project/ ; 2023YFF1000204//The National Key R&D Program of China/ ; 32272128//The National Science Foundation of China/ ; 2021hszd013//Hubei Hongshan Laboratory/ ; },
mesh = {*Cytidine/metabolism/genetics ; *Gene Editing/methods ; *Cytidine Deaminase/genetics/metabolism ; Humans ; CRISPR-Cas Systems ; *Cytosine Deaminase/genetics/metabolism ; DNA ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {BACKGROUND: Cytidine base editors (CBEs) consist of a single-strand specific cytidine deaminase fused to Cas9 nickase, enabling efficient C-to-T conversion across diverse organisms. Enhancing editing range and efficiency of these tools is essential for expanding their applications. RESULTS: In this study, we report that fusing a double-stranded DNA-specific cytosine deaminase DddAE1347A to CBEs significantly improves editing activity and broadens the editing window in cell lines, embryos, tobacco, and cotton. Compared to BE4max, the optimized DddAE1347A-BE4max exhibits up to a 93- fold increase in editing efficiency, achieving up to 52% efficiency at C14 and C15 in cell lines. Further investigation reveals that DddAE1347A is compatible with various Cas9 variants (SpCas9, SpaCas9, and Nme2Cas9) and deaminase variants (rA1, A3G, and A3A). Additionally, we demonstrate that cytosine deaminases with single-stranded DNA activity fail to enhance the CBE system. In contrast, various DddA variants can improve CBE editing activity at PAM-proximal cytosine positions, highlighting the modularity of fusion between DddAs and CBEs. CONCLUSIONS: These findings suggest that the double-stranded DNA-specific cytosine deaminase protein can act as an engineered fusion module in the CBE system, altering the performance (window/efficiency) of CBEs.},
}

*Cytidine/metabolism/genetics
*Gene Editing/methods
*Cytidine Deaminase/genetics/metabolism
Humans
CRISPR-Cas Systems
*Cytosine Deaminase/genetics/metabolism
DNA
CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid41381576,
year = {2025},
author = {Palavesam, A and Karthik Raj, BN and Madan, N and Sri, SSL and Babitha, R and Tirumurugaan, KG},
title = {CRISPR-Cas12a-based rapid detection of Babesia gibsoni and Ehrlichia canis in dogs using fluorometer platform.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {402},
pmid = {41381576},
issn = {2045-2322},
mesh = {Animals ; Dogs ; *Ehrlichia canis/genetics/isolation & purification ; *Babesia/genetics/isolation & purification ; *Dog Diseases/diagnosis/microbiology/parasitology ; *Ehrlichiosis/diagnosis/veterinary/microbiology ; *Babesiosis/diagnosis/parasitology ; *CRISPR-Cas Systems/genetics ; *Fluorometry/methods ; Rapid Diagnostic Tests ; Sensitivity and Specificity ; },
abstract = {Canine babesiosis and canine monocytic ehrlichiosis are important tick-borne diseases caused by Babesia gibsoni (B. gibsoni) and Ehrlichia canis (E. canis) in dogs. Early diagnosis is important for effective clinical management, as these infections can result in other complications if untreated. This study aimed to develop a CRISPR-Cas12a-based detection assay for B. gibsoni and E. canis in a fluorometer platform. Custom designed guide RNAs (gRNAs) were synthesised with the spacer sequence targeting the B. gibsoni18S rRNA and E. canis p43 genes, located 20 bp downstream of the PAM site (5’-TTTV- 3’). Following PCR amplification of the short fragments encompassing the above target regions, the specific gRNA binding the LbaCas12a-gRNA complex initiated the collateral cleavage of FAM- labelled AT rich ssDNA probe for detection using a fluorometer and Biotin-digoxigenin (DIG) labelled GT-rich ssDNA probe for visual detection in LFA.The limit of detection (LOD) using the fluorometer based detection platform was 6 × 108and 6 × 109 for B. gibsoni and E. canis respectively. The LOD was comparable to that of real-time PCR, but more sensitive than point-of-care methods such as LFA, indicating its potential applicability in veterinary clinical settings.},
}

Animals
Dogs
*Ehrlichia canis/genetics/isolation & purification
*Babesia/genetics/isolation & purification
*Dog Diseases/diagnosis/microbiology/parasitology
*Ehrlichiosis/diagnosis/veterinary/microbiology
*Babesiosis/diagnosis/parasitology
*CRISPR-Cas Systems/genetics
*Fluorometry/methods
Rapid Diagnostic Tests
Sensitivity and Specificity

@article {pmid41437324,
year = {2025},
author = {Jun, Y and Han, J and Kim, Y and Choi, S and Kim, SL and Oh, JH and Suh, EJ and Kang, SH and Park, HR and Jeong, HC and Park, SR and Mo, Y},
title = {Phenotypic and transcriptomic characterization of OsSWEET14-edited rice (cv. Samkwang) with enhanced bacterial blight resistance.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1771},
pmid = {41437324},
issn = {1471-2229},
support = {RS-2022-RD010034//Rural Development Administration/ ; RS-2024-00322166//Rural Development Administration/ ; },
mesh = {*Oryza/genetics/microbiology/immunology ; *Xanthomonas/physiology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; Phenotype ; *Transcriptome ; Plants, Genetically Modified ; *Plant Proteins/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; },
abstract = {BACKGROUND: Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) poses a serious threat to rice production. The pathogen promotes infection by targeting effector binding elements in the promoter regions of rice susceptibility genes such as SWEET (Sugars Will Eventually be Exported Transporters) genes. Previous studies have shown that natural or induced variations in SWEET genes can effectively enhance BB resistance in rice. However, the effects of variations in SWEET genes on disease resistance and agronomic performance vary depending on the Xoo strains and the genetic background of rice cultivars, highlighting the need for precise evaluations in breeding applications. RESULTS: In this study, we developed a CRISPR/Cas9-edited OsSWEET14 knockout line (SK-s14) in the background of the elite Korean rice cultivar Samkwang. The SK-s14 line exhibited enhanced resistance against the Xoo strain KACC10859. Transcriptomic analysis revealed that defense-related genes, including pathogenesis-related genes and salicylic acid signaling genes, were downregulated in SK-s14 relative to Samkwang under Xoo inoculation. These results suggest that the enhanced resistance may result from pathogen starvation due to restricted sugar efflux to apoplast, rather than from classical defense activation. Field evaluations showed that SK-s14 plants headed 1–7 days earlier, had longer panicles (+ 8.0%), and exhibited modest reductions in grain weight (-3.0%) and grain fertility (-4.4%) compared to Samkwang. CONCLUSIONS: Our results show that knocking out OsSWEET14 in the Samkwang background enhances BB resistance against the KACC10859 strain, likely through a pathogen starvation mechanism. Despite a modest grain yield reduction (-7.2%) observed under field conditions, SK-s14 represents a promising breeding material for disease-prone rice growing regions.},
}

*Oryza/genetics/microbiology/immunology
*Xanthomonas/physiology
*Disease Resistance/genetics
*Plant Diseases/microbiology/genetics/immunology
Phenotype
*Transcriptome
Plants, Genetically Modified
*Plant Proteins/genetics/metabolism
Gene Editing
CRISPR-Cas Systems
Gene Expression Profiling
Gene Expression Regulation, Plant

@article {pmid41513714,
year = {2026},
author = {Antunes, M and Moura, F and Sebastian, IR and Alves, P and Gomes-Alves, P and Escandell, JM},
title = {Streamlined rAAV HeLaS3 producer cell line generation via GS selection.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {4658},
pmid = {41513714},
issn = {2045-2322},
support = {UID/04462: iNOVA4Health//Fundação para a Ciência e a Tecnologia/ ; 10.55776/W1224//Austrian Science Fund/ ; },
mesh = {*Dependovirus/genetics ; *Glutamate-Ammonia Ligase/genetics/metabolism ; *Genetic Vectors/genetics ; Humans ; Animals ; CRISPR-Cas Systems ; HeLa Cells ; Cricetulus ; CHO Cells ; Gene Knockout Techniques ; },
abstract = {The high cost and complexity of manufacturing recombinant adeno-associated virus vectors continue to limit the broader application of gene therapies, which offer life-changing potential for individuals affected by genetic diseases. Although stable producer cell lines represent a scalable and cost-effective alternative to transient transfection methods, their development is often delayed by inefficient selection strategies and extended timelines. In this study, we present a novel application of the glutamine synthetase-based selection system - commonly used in CHO cells - to a HeLaS3-based rAAV production platform. By generating glutamine synthetase-knockout HeLaS3 cells via CRISPR-Cas9 and applying glutamine deprivation under serum-free conditions, we significantly streamlined the PCL generation process, reducing the timeline to approximately two months while maintaining rAAV productivity (>1x1011 vg/mL) and product quality (~70% full capsids). This work establishes a robust and scalable workflow for rAAV manufacturing, with the potential to enhance accessibility and reduce viral vector production costs for applications in gene therapy.},
}

*Dependovirus/genetics
*Glutamate-Ammonia Ligase/genetics/metabolism
*Genetic Vectors/genetics
Humans
Animals
CRISPR-Cas Systems
HeLa Cells
Cricetulus
CHO Cells
Gene Knockout Techniques

@article {pmid41746183,
year = {2026},
author = {Liu, S and Ding, Z and Lu, X and Liu, Z and Ma, W and Xu, H and Zhang, H and Dai, X and Shen, M and Huang, Y and Gao, M and Bao, J and Chen, M},
title = {Deoxyribonucleic Acid Activator-Triggered Entropy-Driven Catalysis-Modulated CRISPR/Cas12a-Based Portable Biosensor for Simultaneous Detection of Multiple Pathogenic Bacteria.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4311-4324},
doi = {10.1021/acssensors.5c03012},
pmid = {41746183},
issn = {2379-3694},
mesh = {*Biosensing Techniques/methods/instrumentation ; *CRISPR-Cas Systems/genetics ; Pseudomonas aeruginosa/isolation & purification ; Bacterial Proteins/genetics ; beta-Lactamases/genetics/analysis/metabolism ; *Methicillin-Resistant Staphylococcus aureus/isolation & purification ; Catalysis ; *Klebsiella pneumoniae/isolation & purification ; Lab-On-A-Chip Devices ; *DNA/chemistry ; Rapid Diagnostic Tests ; Limit of Detection ; },
abstract = {Rapid and sensitive detection of antibiotic-resistant bacteria (ARB) remains a critical challenge in clinical and public health settings. This study describes the successful construction of a portable DNA activator-triggered entropy-driven catalysis-modulated CRISPR/Cas12a-based sensor (PSDA) for the ultrasensitive and rapid detection of multiple ARB. This PSDA platform utilizes a CRISPR/Cas12a-mediated signal transduction strategy, in which a target-specific DNA activator initiates an entropy-driven dynamic DNA network for signal amplification. To further enhance detection performance, a 3D-printed microfluidic chip device with a smartphone-based readout system has been integrated into the sensor, using green-emitting Zn2GeO4:Mn persistent luminescent nanoparticles as a novel molecular beacon for fluorescence enhancement. This platform enables the simultaneous detection of methicillin-resistant Staphylococcus aureus, carbapenem-resistant Pseudomonas aeruginosa, and Klebsiella pneumoniae carbapenemase 2 (KPC-2)-expressing Klebsiella pneumoniae (KPC-2 KP) with a broad dynamic range (1-10[7] CFU/mL), an ultralow detection limit (1 CFU/mL), and rapid analysis (∼45 min). The assay results are also highly consistent with those of conventional plate counting methods (95.48-115.15%). Overall, this study presents a cost-effective, rapid-response biosensing platform for the simultaneous detection of multiple ARB, with direct applications in clinical diagnostics, food safety monitoring, and environmental surveillance.},
}

*Biosensing Techniques/methods/instrumentation
*CRISPR-Cas Systems/genetics
Pseudomonas aeruginosa/isolation & purification
Bacterial Proteins/genetics
beta-Lactamases/genetics/analysis/metabolism
*Methicillin-Resistant Staphylococcus aureus/isolation & purification
Catalysis
*Klebsiella pneumoniae/isolation & purification
Lab-On-A-Chip Devices
*DNA/chemistry
Rapid Diagnostic Tests
Limit of Detection

@article {pmid42136104,
year = {2026},
author = {Purman, C and Lu, C and Modi, A and Vijaykumar, V and Flister, MJ and den Hollander, AI and Kadri, S and Stender, JD},
title = {Optimization of Genome-Wide CRISPR Screens Using Dual-Guide RNA Infection with Cas9 Electroporation (DICE).},
journal = {The CRISPR journal},
volume = {9},
number = {3},
pages = {141-150},
doi = {10.1177/25731599261448141},
pmid = {42136104},
issn = {2573-1602},
mesh = {Humans ; *Electroporation/methods ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Lentivirus/genetics ; *Gene Editing/methods ; HEK293 Cells ; THP-1 Cells ; *CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Single-guide RNA (sgRNA) lentiviral infection with Cas9 protein electroporation (SLICE) enables CRISPR screening in primary cell types that require transient Cas9 expression, yet is limited by scalability and robustness. Here, we introduce dual guide RNA infection with Cas9 electroporation (DICE), which expresses two guides from the same lentiviral construct that target the same gene. In genome-wide screens, DICE outperformed SLICE in defining essential genes and modulators of PD-L1 expression in Interferon-gamma-activated THP1 cells. Collectively, these data demonstrate that DICE can be utilized for reduced-scale CRISPR screens in cell types with transient Cas9 protein expression without sacrificing screening quality.},
}

Humans
*Electroporation/methods
*CRISPR-Cas Systems/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Lentivirus/genetics
*Gene Editing/methods
HEK293 Cells
THP-1 Cells
*CRISPR-Associated Protein 9/genetics/metabolism

@article {pmid42160542,
year = {2026},
author = {Liu, S and Hu, Y and Zheng, M and Zhang, F and Yu, Z and Sun, Q and Tang, D and Weng, Z and Ye, Z},
title = {A CRISPR/Cas12a-MXene Nanozyme Platform for Universal Detection of Trace DNA.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4345-4356},
doi = {10.1021/acssensors.5c03702},
pmid = {42160542},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; Colorimetry/methods ; *Biosensing Techniques/methods ; Proto-Oncogene Mas ; Methicillin-Resistant Staphylococcus aureus/genetics ; Humans ; *CRISPR-Associated Proteins/genetics/chemistry ; Limit of Detection ; Bacterial Proteins/genetics ; *DNA, Bacterial/analysis/genetics ; *Endodeoxyribonucleases/genetics/chemistry ; *DNA/analysis ; Nitrites ; Transition Elements ; },
abstract = {The instrument-free detection of specific DNA sequences is critical for point-of-care diagnostics yet remains challenging. To address this, we developed a universal colorimetric biosensing platform integrating the CRISPR/Cas12a system with MXene nanozymes. Target recognition by the Cas12a/crRNA complex triggers trans-cleavage of single-stranded DNA inhibitors, restoring the peroxidase-like activity of MXenes to produce a visible signal. The platform achieved a detection limit of 132 copies/μL for the methicillin-resistant Staphylococcus aureus mecA gene. By incorporating an exponential circular DNA (CirDNA) amplification strategy and rationally designed crRNAs for single-base discrimination, sensitivity was further enhanced, enabling detection of the B-raf proto-oncogene V600E mutation down to 109 aM with 0.5% variant allele frequency. The platform also showed excellent reproducibility and high recovery rates in simulated clinical samples. This work provides a low-cost, label/instrument-free, and highly sensitive universal approach through the synergistic combination of CRISPR/Cas12a programmability, MXene nanozyme activity, and CirDNA amplification.},
}

*CRISPR-Cas Systems/genetics
Colorimetry/methods
*Biosensing Techniques/methods
Proto-Oncogene Mas
Methicillin-Resistant Staphylococcus aureus/genetics
Humans
*CRISPR-Associated Proteins/genetics/chemistry
Limit of Detection
Bacterial Proteins/genetics
*DNA, Bacterial/analysis/genetics
*Endodeoxyribonucleases/genetics/chemistry
*DNA/analysis
Nitrites
Transition Elements

@article {pmid42163774,
year = {2026},
author = {Urbaitis, T and Trinkuniene, L and Lenkaite, I and Petrauskyte, M and Krasauskas, R and Stitilyte, M and Sabaliauskas, M and Sasnauskas, G and Tamulaitiene, G and Young, JK and Siksnys, V and Gasiunas, G},
title = {A Potent CRISPR-Cas12l Double-Strand Break Gene Editor.},
journal = {The CRISPR journal},
volume = {9},
number = {3},
pages = {126-140},
doi = {10.1177/25731599261448428},
pmid = {42163774},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA Breaks, Double-Stranded ; *Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Endonucleases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Recently, a new family of CRISPR-Cas12 endonucleases from an unexplored phylum of bacteria, Armatimonadota, was discovered. Named Cas12l, they are compact (800-900 aa), recognize a 5' C-rich protospacer adjacent motif, and present an N-terminal domain that stretches from the beginning to the end of the ribonucleoprotein-bound DNA target site, effectively locking it in place. Here, structure-guided rational design supplemented with AI-based large protein language model predictions was used to improve rates of DNA target cleavage of a family member, Asp2Cas12l. Compared to the wild-type, engineered variants exhibited an approximately 10-fold increase in double-strand break (DSB) editing efficiency in human cells with less target-to-target variation. Moreover, frequencies of editing were comparable to those of SpCas9 at overlapping target sites, and their DSBs efficiently corrected by homology-directed repair (39-56% of editing outcomes). Altogether, this study extends our understanding of CRISPR-Cas12 protein engineering and offers a potent new alternative for DSB-mediated genome editing in human cells.},
}

*CRISPR-Cas Systems/genetics
Humans
*DNA Breaks, Double-Stranded
*Gene Editing/methods
*CRISPR-Associated Proteins/genetics/metabolism
Endonucleases/genetics/metabolism
Bacterial Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42213084,
year = {2026},
author = {Wang, XY and Wang, JX and Li, QN and Pang, XZ and Yang, QF and Zhu, LN and Kong, DM},
title = {A Review of Activator Strand Engineering Strategies for Smart CRISPR/Cas12a Diagnostics.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4224-4244},
doi = {10.1021/acssensors.6c00469},
pmid = {42213084},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; DNA/genetics ; Biosensing Techniques/methods ; *Genetic Engineering/methods ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; },
abstract = {The CRISPR/Cas12a system has emerged as a transformative tool in molecular diagnostics and biosensing, leveraging its high-efficiency DNA-targeting and unique trans-cleavage activity. However, its practical deployment is hindered by persistent challenges such as elevated background signals, constrained target versatility, and insufficient controllability. The activator strand (AS), serving as the molecular trigger for Cas12a activation, presents a promising engineering target to systematically enhance system performance. This review comprehensively summarizes recent advances in AS-driven regulation of the CRISPR/Cas12a system, focusing on four core engineering strategies: terminal modification engineering, split activator design, PAM (protospacer adjacent motif) engineering and regulation, and topological conformation engineering. By redesigning AS architecture, introducing allosteric control, and refining spatial assembly, these approaches significantly improve detection sensitivity, specificity, and versatility. AS engineering has effectively mitigated background interference, expanded target scope to include non-nucleic acid analytes, and enabled precise conditional activation of Cas12a. We further discuss current challenges and future directions, aiming to guide the development of next-generation CRISPR diagnostic systems with enhanced robustness, programmability, and adaptability for real-world applications.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/genetics/metabolism
DNA/genetics
Biosensing Techniques/methods
*Genetic Engineering/methods
*Endodeoxyribonucleases/genetics/metabolism
Bacterial Proteins

@article {pmid42215315,
year = {2026},
author = {Zhang, J and Liang, S and Sun, Y and Zhan, C and Xue, G and Zhou, X},
title = {Glycine/PVP-Enabled One-Pot CRISPR/Cas13a Detection of Vibrio parahaemolyticus by Tetrahedron-Mediated Electrochemistry.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4980-4990},
doi = {10.1021/acssensors.6c00902},
pmid = {42215315},
issn = {2379-3694},
mesh = {*Vibrio parahaemolyticus/isolation & purification/genetics ; *Electrochemical Techniques/methods ; *Glycine/chemistry ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Povidone/chemistry ; Limit of Detection ; },
abstract = {Vibrio parahaemolyticus is a major foodborne pathogen widely distributed in aquatic environments and seafood supply chains, necessitating rapid and ultrasensitive detection strategies adaptable to diverse testing scenarios. Here, we present a glycine/ PVP- and tetrahedron-integrated one-pot CRISPR sensing platform (termed GPT-CRISPR) for robust and ultrasensitive nucleic acid detection. The platform introduces chemical regulation into a multienzyme one-pot RAA-CRISPR/Cas13a network, where glycine may help reduce nonspecific Cas13a background activity, possibly through weak competitive interactions, while polyvinylpyrrolidone (PVP) may enhance reaction compatibility through macromolecular crowding and spatial shielding. This coordinated microenvironment enables stable amplification and CRISPR activation within a single closed vessel while minimizing background interference. Upon target recognition, activated Cas13a cleaves uracil-containing, surface-immobilized DNA tetrahedra, translating molecular recognition into amplified electrochemical signals. This transduction strategy enables quantitative detection with a linear dynamic range of 1.5 to 3 × 10[3] copies μL[-1] and a limit of detection of 0.38 copies μL[-1]. The same chemically regulated one-pot CRISPR framework remains compatible with fluorescence and lateral flow readouts. The assay operates under isothermal conditions and delivers results within 30 min without complex sample preparation. Validation across real-world samples demonstrates robustness in complex matrices. Collectively, GPT-CRISPR integrates chemical stabilization of a one-pot CRISPR framework with electrochemical transduction, defining a robust sensing architecture with adaptable readout capability.},
}

*Vibrio parahaemolyticus/isolation & purification/genetics
*Electrochemical Techniques/methods
*Glycine/chemistry
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Povidone/chemistry
Limit of Detection

@article {pmid42263201,
year = {2026},
author = {Sun, Y and Wang, R and Sun, H and Chen, J and Liang, C and Zhang, Z and Feng, Y and Chen, L and Wang, X},
title = {Modular CRISPR-Cas12a-Activated Gold Nanoparticle Assay for Rapid Visual Detection of Hepatocellular Carcinoma-Related miRNAs.},
journal = {ACS sensors},
volume = {11},
number = {6},
pages = {4991-5002},
doi = {10.1021/acssensors.6c00916},
pmid = {42263201},
issn = {2379-3694},
mesh = {*MicroRNAs/genetics/analysis ; *Gold/chemistry ; *Metal Nanoparticles/chemistry ; *Carcinoma, Hepatocellular/genetics/diagnosis ; Humans ; *Liver Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; Colorimetry/methods ; *CRISPR-Associated Proteins/metabolism ; Rapid Diagnostic Tests ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; },
abstract = {MicroRNAs (miRNAs) are emerging biomarkers for early hepatocellular carcinoma (HCC) detection, but most CRISPR-Cas12a assays rely on reverse transcription and preamplification and often lack simple visual readouts. Here, we develop a reverse transcription-free, cleavage-guided strategy that converts miRNA recognition into visual outputs. In the presence of the target miRNA, a scaffold RNA forms an active crRNA that triggers Cas12a trans-cleavage using a preformed dsDNA activator, enabling two readout formats. In a homogeneous non-crosslinking colorimetric assay (mC-NCA), Cas12a cleavage regulates π-π-stacking-mediated gold nanoparticle (AuNP) aggregation to produce a rapid visual colorimetric response. In a heterogeneous versatile lateral flow assay (mC-vLFA), the cleavage of partially hybridized DNA-magnetic bead probes generates a target-dependent test line while maintaining a built-in control line. Using miRNA-21 and miRNA-122 as targets, mC-NCA shows calculated limits of detection (LODs) of 1.62 and 1.64 fM with a linear range of 50 fM-500 pM, whereas mC-vLFA shows calculated LODs of 1.59 and 1.40 fM with a linear range of 10 fM-10 nM. In a preliminary clinical evaluation, both formats show good agreement with reverse-transcription quantitative polymerase chain reaction (RT-qPCR) and clinical assessment while enabling faster detection with minimal instrumentation. Overall, this dual-readout platform couples RT-free CRISPR-Cas12a miRNA recognition with cleavage-directed visual signal transduction, enabling rapid and low-instrument-dependence CRISPR diagnostics for miRNAs.},
}

*MicroRNAs/genetics/analysis
*Gold/chemistry
*Metal Nanoparticles/chemistry
*Carcinoma, Hepatocellular/genetics/diagnosis
Humans
*Liver Neoplasms/genetics/diagnosis
*CRISPR-Cas Systems/genetics
Colorimetry/methods
*CRISPR-Associated Proteins/metabolism
Rapid Diagnostic Tests
*Endodeoxyribonucleases/metabolism/genetics
Bacterial Proteins

@article {pmid42272131,
year = {2026},
author = {Peng, CL and Kamau, WS and Freeman, J and Hill, ZJ and Esvelt, KM},
title = {Increasing the Effective Gene Drive Homing Rate by Targeting the Haploinsufficient Spermatogenesis Gene Klhl10.},
journal = {The CRISPR journal},
volume = {9},
number = {3},
pages = {167-176},
doi = {10.1177/25731599261452112},
pmid = {42272131},
issn = {2573-1602},
mesh = {Animals ; *Spermatogenesis/genetics ; Male ; Mice ; *Haploinsufficiency/genetics ; CRISPR-Cas Systems ; Frameshift Mutation ; Female ; DNA End-Joining Repair/genetics ; Spermatozoa/metabolism ; *Gene Targeting/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR-based gene drives represent a powerful new technology for limiting disease transmission and controlling invasive populations. These systems rely on homology-directed repair (HDR) to "drive" a genetic element through a population. However, mammals tend to favor non-homologous end joining (NHEJ), which generates mutations that halt further drive propagation. Here, we describe the experimental characterization of a putative target locus for a gene drive system targeting the haploinsufficient spermatogenesis gene Klhl10 in the laboratory mouse. Using a newly designed "coding sequence cassette," we introduce downstream guide RNAs within the gene, ensuring that sperm undergoing NHEJ are selectively removed from the population. As a proof of principle, we demonstrate that targeting Klhl10 with constitutively expressed LbCas12a results in strong selection against frameshift-containing sperm, validating the core purification mechanism required for this drive strategy. Unexpectedly, we also observed that female offspring lacked most frameshift mutations, suggesting a previously unrecognized role for Klhl10 in oogenesis or early embryonic development.},
}

Animals
*Spermatogenesis/genetics
Male
Mice
*Haploinsufficiency/genetics
CRISPR-Cas Systems
Frameshift Mutation
Female
DNA End-Joining Repair/genetics
Spermatozoa/metabolism
*Gene Targeting/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid42290195,
year = {2026},
author = {Michalski, MN and Diegel, CR and Zhong, ZA and Marshall, ME and Foxa Wiartalla, GE and Stevens, PD and Suino-Powell, K and Blazer, LL and Adams, JJ and Melcher, K and Sidhu, SS and Angers, S and Williams, BO},
title = {Clarifying Frizzled 2 function in development through genetically validated mouse models.},
journal = {Disease models & mechanisms},
volume = {19},
number = {7},
pages = {},
doi = {10.1242/dmm.052410},
pmid = {42290195},
issn = {1754-8411},
support = {K08DE031039/DE/NIDCR NIH HHS/United States ; K08DE031039//National Institute of Health/ ; /DE/NIDCR NIH HHS/United States ; //Van Andel Research Institute/ ; },
mesh = {Animals ; *Frizzled Receptors/metabolism/genetics/deficiency ; Phenotype ; Gene Deletion ; Mice, Knockout ; Mice ; Reproducibility of Results ; Integrases/metabolism ; *Embryonic Development/genetics ; CRISPR-Cas Systems/genetics ; Extremities/embryology ; Base Sequence ; Embryo, Mammalian/metabolism ; Models, Animal ; },
abstract = {Wnt receptors of the Frizzled (Fzd) family are widely considered to exhibit substantial functional redundancy, complicating efforts to therapeutically target individual receptors. Fzd2 was believed to be functionally redundant with Fzd1 and Fzd7, based on previously published global knockout mouse studies. By contrast, homozygosity for a Fzd2 global knockout mouse allele developed by the International Mouse Phenotype Consortium (IMPC) has been reported to cause embryonic lethality, suggesting that Fzd2 is critical for early embryonic development. If global deletion of Fzd2 leads to early lethality, conditional deletion models are necessary to identify tissue-specific phenotypes. We found that a previously published Fzd2 conditional deletion model does not eliminate Fzd2. We have generated a new conditional model to address the contradictory previous studies and allow tissue-specific studies of Fzd2. We successfully inserted two loxP sites around the Fzd2 gene and confirmed that subsequent Cre-mediated recombination creates a Fzd2 null allele. Global deletion of Fzd2 in this model does not cause embryonic lethality while limb-specific deletion causes limb shortening. This work supports the hypothesis that Fzd2 regulates limb development and emphasizes the importance of thoroughly validating newly generated mouse models.},
}

Animals
*Frizzled Receptors/metabolism/genetics/deficiency
Phenotype
Gene Deletion
Mice, Knockout
Mice
Reproducibility of Results
Integrases/metabolism
*Embryonic Development/genetics
CRISPR-Cas Systems/genetics
Extremities/embryology
Base Sequence
Embryo, Mammalian/metabolism
Models, Animal

@article {pmid42299032,
year = {2026},
author = {Porenta, D and Benne, N and Sijts, A and Broere, F and Mastrobattista, E},
title = {Induction of SpCas9-Directed Immune Responses Using Lipid Nanoparticles and Identification of SpCas9-Derived T Cell Epitopes in C57BL/6 Mice.},
journal = {The CRISPR journal},
volume = {9},
number = {3},
pages = {151-166},
doi = {10.1177/25731599261456887},
pmid = {42299032},
issn = {2573-1602},
mesh = {Animals ; Mice, Inbred C57BL ; Mice ; *Epitopes, T-Lymphocyte/immunology/genetics ; *Nanoparticles/chemistry/administration & dosage ; Lipids/chemistry ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/immunology/genetics/metabolism ; Gene Editing/methods ; Female ; Interferon-gamma ; Liposomes ; },
abstract = {A major goal of clinically oriented CRISPR-Cas9-based applications is safe and effective in vivo gene editing (knockout or correction) with precise targeting. Substantial efforts have been devoted to the preclinical development of novel drug delivery platforms that enable efficient, targeted delivery. However, the immune responses induced by CRISPR-Cas9 treatment are often overlooked. Preexisting immunity to clinically relevant Cas9 proteins has already been established as a consequence of natural exposure to Cas9-bearing bacteria, which may implicate the safety and efficacy of CRISPR-Cas9-based therapies. Naturally, CRISPR-Cas9 therapies should be nonimmunogenic to avoid amplifying existing Cas9-specific immunity, especially cytotoxic T cell responses. Nonviral delivery systems, such as lipid nanoparticles (LNPs), are widely regarded as less immunogenic than more traditionally used viral vectors, even though LNPs are suitable as a vaccination platforms. In this study, we investigate the induction of SpCas9-directed immunity in C57BL/6 mice upon repeated dosing of LNPs encapsulating Cas9-coding mRNA in two different settings: (1) a vaccination-resembling setting using intramuscularly administered adjuvanted LNPs, and (2) a therapy-resembling setting using intravenously injected, liver-targeting LNPs. In both settings, Cas9-specific T cell responses were detected by evaluating increased total IFN-γ levels upon ex vivo restimulation of isolated splenocytes. However, undetectable Cas9-reactive antibodies induced in the therapeutic setting emphasize the discrepancy between humoral and cellular responses. To improve future monitoring of Cas9-specific T cell responses, we report six Cas9-derived epitopes recognized by CD8[+] T cells, as well as a CD4[+] T cell polypeptide carrying one of the CD8[+] T cell epitopes that induced strong IFN-γ production ex vivo. This work is intended to facilitate the preclinical monitoring of Cas9-specific T cell responses in C57BL/6 mice and support the development of safe CRISPR-Cas9-based therapies.},
}

Animals
Mice, Inbred C57BL
Mice
*Epitopes, T-Lymphocyte/immunology/genetics
*Nanoparticles/chemistry/administration & dosage
Lipids/chemistry
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/immunology/genetics/metabolism
Gene Editing/methods
Female
Interferon-gamma
Liposomes

@article {pmid42328786,
year = {2026},
author = {Xu, C and Yang, Q and Niu, X and Ke, A},
title = {Structure basis for single-strand nucleic acid targeting by IscB and variants.},
journal = {Nucleic acids research},
volume = {54},
number = {12},
pages = {},
pmid = {42328786},
issn = {1362-4962},
support = {R35GM118174/NH/NIH HHS/United States ; S10OD023603/NH/NIH HHS/United States ; //Brookhaven National Laboratory─Laboratory for BioMolecular Structure/ ; KP1607011//DOE Office of Biological and Environmental Research/ ; //Yale CryoEM Resource/ ; },
mesh = {*DNA, Single-Stranded/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; Gene Editing/methods ; Models, Molecular ; Nucleic Acid Conformation ; Humans ; },
abstract = {Transposon-encoded IscB was defined as the evolutionary ancestor of CRISPR-Cas9. This compact RNA-guided endonuclease has since been engineered for genome-editing applications. We previously repurposed IscB and related Cas9s as efficient RNA editors by removing their double-stranded DNA (dsDNA) recognition module, the target-adjacent motif (TAM)/protospacer adjacent motif-interacting domain. Here, we report four cryo-electron microscopy structures of IscB, with or without TAM-interaction domain (TID), in complex with single-stranded nucleic acid (ssNA) targets. Structures reveal that, regardless of TID presence, IscB engages ssNA using the same mechanism. IscB initially facilitates formation of a 10-nt seed duplex with ssNA; further base-pairing is blocked by an alternatively positioned HNH nuclease that acts as a roadblock. In this intermediate state, neither HNH nor RuvC is competent for target cleavage. Only upon full duplex formation is the HNH roadblock dislodged by the duplex extension between guide RNA and ssNA. HNH and RuvC nuclease active sites become exposed as the result. A similar set of conformational rearrangements likely governs IscB activity during dsDNA target interrogation. Guided by the structural and mechanistic insights, we introduced mutations to either improve ssNA binding or ease HNH dislodging. Both approaches improved the RNA-targeting efficiency of IscB in vitro and in human cells.},
}

*DNA, Single-Stranded/chemistry/metabolism/genetics
Cryoelectron Microscopy
CRISPR-Cas Systems/genetics
RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism
Gene Editing/methods
Models, Molecular
Nucleic Acid Conformation
Humans

@article {pmid42329468,
year = {2026},
author = {Ahmad, S and Aftab, MN and Ghalib, M and Aslam, MS},
title = {Overcoming cellular secretion bottlenecks: advanced secretion engineering and molecular tailoring for next-generation microbial α-amylases with enhanced industrial performance.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {7},
pages = {},
pmid = {42329468},
issn = {1573-0972},
mesh = {Bacillus subtilis/genetics/enzymology/metabolism ; *alpha-Amylases/metabolism/genetics/chemistry ; *Metabolic Engineering/methods ; Aspergillus niger/genetics/enzymology/metabolism ; Escherichia coli/genetics/metabolism ; Recombinant Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Industrial Microbiology ; Pichia/genetics/enzymology ; Carbohydrate Binding Modules ; Protein Engineering ; Synthetic Biology ; Saccharomycetales ; },
abstract = {α-amylases are indispensable industrial biocatalysts, yet their recombinant production faces significant biochemical and cellular bottlenecks. Recent scientific advancement shifts the paradigm from traditional cloning toward a design-parameter framework, where host selection predominantly Bacillus subtilis, Pichia pastoris, and Aspergillus niger is dictated by secretion capacity, folding landscapes, and metabolic compatibility. While Escherichia coli remains a common host, its lack of efficient extracellular secretion often leads to inclusion body formation and metabolic stress. Advanced strategies are being employed to overcome these limits, including signal peptide optimization, chaperone co-expression, and the fusion of carbohydrate-binding modules (CBMs) to enhance raw-starch degradation. Furthermore, how rational design, aided by artificial intelligence and molecular dynamics simulations, enables the engineering of hyper-thermostable and alkaline-tolerant variants capable of withstanding extreme industrial processing conditions. The advent of CRISPR-Cas technology has further revolutionized the field, allowing for precise genome editing and metabolic rewiring to achieve record-breaking enzyme titers, such as 102,893 U/mL in engineered B. subtilis. By balancing transcriptional levels with enhanced secretion pathways and stress-mitigation systems, modern synthetic biology provides the tools to tailor α-amylases for specific needs, ranging from biofuel production to high-purity malto-oligosaccharide synthesis. Therefore, this comprehensive analysis underscores that reconciling host biology with enzyme biochemistry is essential for meeting global industrial demands.},
}

Bacillus subtilis/genetics/enzymology/metabolism
*alpha-Amylases/metabolism/genetics/chemistry
*Metabolic Engineering/methods
Aspergillus niger/genetics/enzymology/metabolism
Escherichia coli/genetics/metabolism
Recombinant Proteins/metabolism/genetics
CRISPR-Cas Systems
Industrial Microbiology
Pichia/genetics/enzymology
Carbohydrate Binding Modules
Protein Engineering
Synthetic Biology
Saccharomycetales

@article {pmid42330666,
year = {2026},
author = {Ling, H and Su, N and Huang, L and Wang, M and Ren, L},
title = {Cleavage-responsive DNA/AgNCs enable accelerated Cas12a trans-cleavage for rapid multigene methylation diagnosis.},
journal = {Biosensors & bioelectronics},
volume = {311},
number = {},
pages = {118929},
doi = {10.1016/j.bios.2026.118929},
pmid = {42330666},
issn = {1873-4235},
abstract = {DNA methylation, particularly 5-methylcytosine (5 mC), is a key epigenetic modification involved in the regulation of gene expression and genomic stability, and has emerged as a promising biomarker for early cancer screening and molecular stratification. CRISPR-Cas12a systems have been increasingly exploited to convert methylation-associated sequence information into detectable signals owing to their programmability and collateral cleavage-mediated signal amplification. However, many CRISPR-based assays remain constrained by the high background and relatively slow response kinetics of conventional fluorophore-quencher reporters. To overcome these limitations, we developed a cleavage-responsive DNA-templated silver nanocluster (DNA/AgNC) reporter that translates Cas12a trans-cleavage activity into a green-to-red ratiometric fluorescence shift. In this design, the AgNC-templating DNA scaffold itself serves as an enzymatically cleavable signal transducer, rather than relying on a terminal fluorophore-quencher pair. Compared with a representative F-ssDNA-Q reporter, the DNA/AgNC reporter exhibited stronger apparent association with Cas12a and an approximately two-fold improvement in apparent catalytic efficiency. When incorporated into an MSRE-RPA-Cas12a workflow, the platform achieved a limit of detection of 74.5 aM, while completing the Cas12a reporting step within 30 min. Coupling this assay with a miniaturized optoelectronic device further enabled spatially resolved profiling of five genomic loci, with relative errors of approximately 5%. Overall, this strategy establishes a ratiometric reporter format for CRISPR-based DNA methylation profiling and offers potential for point-of-care epigenetic biosensing.},
}

@article {pmid42333809,
year = {2026},
author = {Hou, S and Bi, W and Liu, W and Nie, H and Jiang, J and Zeng, L},
title = {Beyond DNA editing: how Cas13 redefined programmable RNA manipulation and what still limits its therapeutic promise.},
journal = {Nucleic acids research},
volume = {54},
number = {12},
pages = {},
pmid = {42333809},
issn = {1362-4962},
support = {2024ZD0541500//Noncommunicable Chronic Diseases-National Science and Technology Major Project/ ; 82222038//National Natural Science Foundation of China/ ; 01-SWKJYCJJ06//Outstanding Young Talents of National Defense Biotechnology/ ; 2023XRC01//High-Level Talent Program Cultivation Project of Army Medical University/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *RNA Editing/genetics ; *RNA/genetics/metabolism ; *Gene Editing/methods ; Animals ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {The Type VI Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas13 system, evolved from prokaryotic immunity, has become a versatile, programmable RNA-targeting platform with broad biotechnological potential. Guided by CRISPR RNA (crRNA), Cas13 cleaves single-stranded RNA via higher eukaryotic and prokaryotic nucleotide-binding domains, exerting specific (cis) and nonspecific (trans) collateral cleavage, which enables ultrasensitive nucleic acid detection while introducing cytotoxicity risks in eukaryotic cells. Diversification of Cas13 subtypes, including compact variants, enhances targetability and delivery compatibility, and inhibitory strategies (anti-CRISPR proteins, crRNA mimicry/degradation) enable activity modulation for improved safety. Building on mechanistic foundations, Cas13 is repurposed for targeted RNA knockdown, nucleic acid diagnostics, live-cell RNA imaging with catalytically inactive variants, programmable RNA base editing through deaminase fusions, splicing regulation, epitranscriptomic editing of multiple RNA chemical marks, interaction mapping of RNA-protein and RNA-RNA networks, and translational control, with preliminary clinical translation in antiviral therapies, pathogenic transcript correction, and cancer therapy. Furthermore, Cas13-integrated diagnostics and functional genomics are accelerating biomarker discovery and personalized treatment. Nevertheless, successful clinical translation hinges on overcoming critical bottlenecks, including tissue-specific delivery, mitigation of collateral cytotoxicity, and management of host immunogenicity. This review synthesizes Cas13 classification, structure-function principles, regulatory inhibitors, application modalities, and translational challenges to inform next-generation engineering and responsible deployment of RNA-targeted technologies.},
}

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

@article {pmid42334452,
year = {2026},
author = {Lin, Y and Liou, B and Fannin, V and Adler, S and Mayhew, CN and Hammonds, JE and Hu, YC and Tchieu, J and Zhang, W and Zhao, X and Beres, RL and Setchell, KDR and Kaynak, A and Qi, X and Feldman, RA and Sun, Y},
title = {Patient-specific midbrain organoids with CRISPR correction recapitulate neuronopathic Gaucher disease phenotypes and enable evaluation of novel therapies.},
journal = {eLife},
volume = {15},
number = {},
pages = {},
pmid = {42334452},
issn = {2050-084X},
support = {R21HD1027881//National Institute of Health/ ; R21OD033660//National Institute of Health/ ; R01NS138309//National Institute of Health/ ; R01NS103931//National Institute of Health/ ; Center of Pediatric Genomics Award//Cincinnati Children's Hospital Medical Center/ ; Research Innovation Pilot Funding Program Award//Cincinnati Children's Hospital Medical Center/ ; 2UL1TR001425-05A1, CHMC-CTSA 00003827/NH/NIH HHS/United States ; },
mesh = {*Gaucher Disease/genetics/therapy/pathology ; Humans ; *Organoids/pathology/metabolism ; *Mesencephalon/pathology ; *Glucosylceramidase/genetics/metabolism ; Phenotype ; *CRISPR-Cas Systems ; Mutation ; Induced Pluripotent Stem Cells ; Dopaminergic Neurons ; },
abstract = {Neuronopathic Gaucher disease (nGD) is a lysosomal storage disorder caused by GBA1 mutations, leading to defective acid β-glucosidase (GCase) and accumulation of glycosphingolipid substrates, causing inflammation and neurodegeneration. Patients with nGD manifest severe neurological symptoms, but current animal models fail to fully recapitulate the human condition, posing a major barrier to the development of effective therapies targeting the brain. To bridge this gap, we have developed midbrain-like organoids (MLOs) from human induced pluripotent stem cells of nGD patients with GBA1[L444P/P415R] and GBA1[L444P/RecNcil] mutations to model nGD brain pathogenesis. These nGD MLOs exhibited GCase deficiency, resulting in diminished enzymatic function, accumulation of lipid substrates, widespread transcriptomic changes, and impaired dopaminergic neuron differentiation, mirroring nGD pathology. GBA1 mutation correction mediated by CRISPR/Cas9 restored GCase activity, normalized lipid substrate levels, and rescued dopaminergic neuron function, confirming the causal role of GBA1 mutations during early brain development. Using this novel platform, we further evaluated therapeutic strategies, including SapC-DOPS nanovesicles delivering GCase, AAV9-GBA1 gene therapy, and substrate reduction therapy with GZ452, a glucosylceramide synthase inhibitor currently under clinical investigation. These treatments either restored GCase activity, reduced lipid substrate accumulation, improved autophagic and lysosomal abnormalities, or ameliorated dysregulated genes involved in neural development. These patient-specific, 3D neural models offer a transformative, physiologically relevant platform for unraveling disease mechanisms and accelerating the discovery of therapies for patients with nGD.},
}

*Gaucher Disease/genetics/therapy/pathology
Humans
*Organoids/pathology/metabolism
*Mesencephalon/pathology
*Glucosylceramidase/genetics/metabolism
Phenotype
*CRISPR-Cas Systems
Mutation
Induced Pluripotent Stem Cells
Dopaminergic Neurons

@article {pmid42334517,
year = {2026},
author = {Zhang, XL and Luo, JN and Cao, Y and Zhao, JX and Zhang, Y and Xu, SF and Zhuang, LN and He, J},
title = {Generation of transgenic pigs with targeted insertion of a wildtype copy of human PKD2 gene.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42334517},
issn = {1573-4978},
support = {2021YFA0805902//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Animals, Genetically Modified/genetics ; Swine/genetics ; *TRPP Cation Channels/genetics/metabolism ; Humans ; CRISPR-Cas Systems ; *Polycystic Kidney, Autosomal Dominant/genetics/metabolism ; Female ; Disease Models, Animal ; Kidney/metabolism/pathology ; },
abstract = {BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD), primarily driven by PKD1 or PKD2 mutations, is a prevalent hereditary nephropathy for which large-animal models capturing human renal anatomy and disease tempo remain urgently needed. This study aimed to establish a site-specific human PKD2 (hPKD2) transgenic porcine model as a versatile platform capable of supporting two temporally distinct applications: longitudinal evaluation of whether sustained PKD2 overexpression is sufficient to induce renal pathology, and-should overexpression prove phenotypically silent-future crossbreeding-based functional rescue of PKD2-knockout lines.

METHODS AND RESULTS: A CRISPR/Cas9-mediated, homology-recombination-independent strategy was employed to target full-length hPKD2 cDNA into the porcine pH11 safe harbor locus. Somatic cell nuclear transfer yielded five F0 transgenic founders, and natural mating of two founders with wild-type sows produced four F1 transgenic offspring, confirming stable germline transmission. Quantitative real-time PCR and whole-genome sequencing validated single-copy, site-specific transgene integration at the designated locus. Robust hPKD2 mRNA and FLAG-tagged polycystin-2 (PC-2) expression were detected in renal and other tissues across both generations. During the initial 12-month monitoring period, serum blood urea nitrogen and creatinine levels remained within normal ranges and no gross histological abnormalities were evident. However, given that these biomarkers are insensitive to early-stage renal impairment, extended observation with more comprehensive phenotyping is required before definitive conclusions regarding renal function can be drawn.

CONCLUSIONS: A transgenic porcine model with stable, single-copy hPKD2 integration at the pH11 safe harbor locus was successfully generated and shown to permit germline transmission. This platform provides a foundation for long-term investigation of PKD2 overexpression pathophysiology and, alternatively, for functional complementation of PKD2-deficient models, thereby advancing both mechanistic and translational ADPKD research.},
}

Animals
*Animals, Genetically Modified/genetics
Swine/genetics
*TRPP Cation Channels/genetics/metabolism
Humans
CRISPR-Cas Systems
*Polycystic Kidney, Autosomal Dominant/genetics/metabolism
Female
Disease Models, Animal
Kidney/metabolism/pathology

@article {pmid42337238,
year = {2026},
author = {Shi, LD and Kolody, BC and Wang, S and Valentin-Alvarado, LE and Lei, S and Sachdeva, R and Banfield, JF},
title = {Jumbo circular extrachromosomal elements of methane-oxidizing archaea with variably extensive metabolic and defense gene repertoires.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-74423-z},
pmid = {42337238},
issn = {2041-1723},
support = {DE-AC02-05CH11231//U.S. Department of Energy (DOE)/ ; INV-037174/GATES/Gates Foundation/United States ; },
abstract = {Archaeal extrachromosomal elements (ECEs) are arguably the least well understood of all genetic elements, and few have >200 kbp (jumbo) genomes. Here, we report circular, jumbo ECEs with genomes of up to 535 kbp in length that associate with anaerobic methane-oxidizing Methanoperedens archaea. Notably, a 409-kbp genome related to jumbo ECEs is integrated into a subset of the ~4.2 Mbp Methanoperedens chromosomes at the tRNA-Asp genes. This represents the largest integrative element in Archaea and supports the jumbo ECE-host association. Multiple genome alignments and phylogenetic analyses suggest that the large ECE sizes developed by extensive DNA acquisition from Methanoperedens. The newly identified ECEs encode, and in some cases express, metabolic genes such as tetrahydromethanopterin S-methyltransferase exclusively involved in methane metabolism, and genes for nitrogen and sulfur compound transformations. Also encoded are defense systems, some of which are absent in hosts, such as hybrid Type I/Type III-A CRISPR-Cas systems. In contrast to viruses and plasmids, they have host-like replication machinery and occur at stable copy ratios of 1.44 ± 0.24:1 to the host. Overall, our results reveal a spectrum of jumbo ECEs of Methanoperedens, ranging from plasmid-like to minichromosome-like.},
}

@article {pmid42339381,
year = {2026},
author = {Bashir, T and Rashid, R and Malik, AR and Sundouri, AS and Bhat, KM and Mir, MA and Rehman, MU and Masood, M and Tehleelah, },
title = {Reframing fruit biofortification for sustainable micronutrient security: from crop innovation to nutritional impact.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1853898},
pmid = {42339381},
issn = {1664-462X},
abstract = {Micronutrient malnutrition, commonly referred to as "hidden hunger, " remains a persistent global health challenge, particularly in regions with limited dietary diversity. Although agricultural intensification has substantially improved caloric availability, it has not ensured adequate micronutrient density in food systems, highlighting the urgent need for nutrition-sensitive crop improvement strategies. Biofortification has emerged as a sustainable and cost-effective approach to enhance the micronutrient content of food crops through agronomic, genetic, and biotechnological interventions. While biofortification research has predominantly focused on staple cereals and legumes, horticultural fruit crops have received comparatively limited attention despite their widespread consumption, high consumer acceptance, and natural richness in bioactive compounds. This review advances beyond existing overviews by providing a critical and comparative evaluation of agronomic, conventional breeding, and biotechnological approaches for fruit crop biofortification, with particular emphasis on their effectiveness, scalability, limitations, and translational potential. Current advances aimed at enhancing iron, zinc, iodine, selenium, and provitamin A concentrations are comprehensively synthesized. Special attention is given to the physiological and molecular mechanisms regulating micronutrient uptake, transport, accumulation, and storage in fruit tissues. In addition, advanced biotechnological tools, including CRISPR/Cas-mediated genome editing, are critically assessed in relation to biosafety, regulatory considerations, and practical applicability. Evidence from major fruit crops, including apple, banana, mango, pomegranate, strawberry, and papaya, demonstrates that integrated biofortification strategies can improve micronutrient density while maintaining fruit yield and quality. Importantly, this review addresses a major knowledge gap by linking crop-level nutrient enhancement with micronutrient bioavailability and human nutritional outcomes, emphasizing the influence of food matrix interactions and nutrient absorption efficiency. Key constraints, including genotype × environment interactions, postharvest nutrient instability, climate-driven variability, and limited clinical validation, are also discussed. Finally, a systems-level framework integrating plant science, human nutrition, postharvest biology, and policy perspectives is proposed to support the large-scale adoption of nutrition-sensitive fruit biofortification. Collectively, fruit crop biofortification represents a promising strategy for improving global micronutrient security and advancing sustainable food systems.},
}

@article {pmid42340480,
year = {2026},
author = {Guo, Z and Yang, Z and Liu, Z and Zheng, G and Zou, S},
title = {Fgf8, a gene knockout that leads to intermuscular bones-reduced of crucian carp (Carassius auratus), acts as a potentially regulatory factor in osteogenic development.},
journal = {Fish physiology and biochemistry},
volume = {52},
number = {4},
pages = {},
pmid = {42340480},
issn = {1573-5168},
support = {2023YFD2400300//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Carps/genetics/growth & development ; *Fibroblast Growth Factor 8/genetics/metabolism ; *Osteogenesis/genetics/physiology ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; },
abstract = {Although fibroblast growth factor 8 (FGF8) is a critical regulator of skeletal morphogenesis in vertebrates, its specific role in the formation and development of intermuscular bones (IBs) in teleost fish remains insufficiently characterized. In this study, we generated double mutants (fgf8a[+/-] + fgf8b[+/-]) in diploid Chongming crucian carp (Carassius auratus) using CRISPR/Cas9-mediated gene editing. Compared to wild-type fish, the double mutants exhibited significantly reduced IB number (p < 0.01), demonstrating a synergistic role of fgf8a and fgf8b in IB formation. Notably, reduced number of IBs did not compromise overall growth, muscle architecture, or reproductive performance. Integrated transcriptomic and metabolomic analyses revealed that the reduced IB phenotype was linked to modifications in relevant signaling pathways and a concomitant upregulation of metabolites beneficial for muscle quality and health. Our findings highlight the crucial role of fgf8 in regulating IB formation and development in crucian carp, providing insights into the genetic mechanisms underlying this process in teleosts.},
}

Animals
*Carps/genetics/growth & development
*Fibroblast Growth Factor 8/genetics/metabolism
*Osteogenesis/genetics/physiology
Gene Knockout Techniques
CRISPR-Cas Systems
Gene Expression Regulation, Developmental

@article {pmid42342668,
year = {2026},
author = {Mitsuda, Y and Sugaya, M and Ishikawa, J and Nagahata, N and Okazaki, S and Hiraizumi, M and Kato, K and Gootenberg, JS and Abudayyeh, OO and Osawa, T and Yamashita, K and Nishimasu, H},
title = {Structural mechanism of SAM-AMP and SAM-AMP2 synthesis by the type III-D2 CRISPR effector complex.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42342668},
issn = {2041-1723},
support = {25H00436//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; *S-Adenosylmethionine/metabolism/chemistry ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Escherichia coli/metabolism/genetics/virology ; Adenosine Triphosphate/metabolism ; Escherichia coli Proteins/metabolism/genetics/chemistry ; },
abstract = {The type III-D2 CRISPR-Cas system comprises multiple Cas subunits and a CRISPR RNA, and is likely an evolutionary intermediate between the well-studied type III-A and III-E systems. Here we show that the type III-D2 complex synthesizes two distinct second messengers, SAM-AMP and SAM-AMP2, from S-adenosylmethionine (SAM) and ATP in response to target RNA recognition. We determined cryo-electron microscopy structures of the type III-D2 effector complex in different functional states, providing mechanistic insights into target RNA cleavage and second messenger synthesis. The structures reveal how SAM and ATP are recognized by the Cas10 subunit within the effector complex. Furthermore, our biological data suggest that both SAM-AMP and SAM-AMP2 act on the CorA ancillary effector, inducing growth arrest of infected bacterial cells and thereby conferring immunity. Thus, our study establishes the type III-D2 system as a unique anti-phage defense mechanism that employs both SAM-AMP and SAM-AMP2 as second messengers, expanding the repertoire of second messenger strategies in bacterial defense systems and highlighting the remarkable functional diversity of CRISPR-Cas systems.},
}

*CRISPR-Cas Systems/genetics
Cryoelectron Microscopy
*S-Adenosylmethionine/metabolism/chemistry
*CRISPR-Associated Proteins/metabolism/chemistry/genetics
Escherichia coli/metabolism/genetics/virology
Adenosine Triphosphate/metabolism
Escherichia coli Proteins/metabolism/genetics/chemistry

@article {pmid42342872,
year = {2026},
author = {Van Der Straeten, D and Bulut, M and Cao, D and Aharoni, A and Bouis, H and Granell, A and Gruissem, W and Lindberg Møller, B and Martin, C and Puchta, H and Sreenivasulu, N and Tissier, A and Tripathi, L and Van Montagu, M and Fernie, AR},
title = {Genetic technologies to enhance crop nutritional value under climate change.},
journal = {Nature},
volume = {654},
number = {8120},
pages = {877-891},
pmid = {42342872},
issn = {1476-4687},
mesh = {Humans ; *Biofortification/methods ; *Climate Change ; CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics/metabolism/chemistry ; *Gene Editing/methods ; Metabolic Engineering ; Micronutrients/deficiency/analysis ; Nutritive Value ; },
abstract = {At present, more than 700 million people live with caloric hunger, and more than two billion suffer from micronutrient deficiencies, known as 'hidden hunger'. From an agricultural viewpoint, three major objectives need to be worked towards simultaneously to achieve zero hunger (the United Nations Sustainable Development Goal 2): (1) enhanced yield; (2) higher vitamin and mineral density to sustain recommended daily intake (multi-biofortification); and (3) enhanced climate-change resilience. Although the Green Revolution increased global calorie production, it exacerbated hidden hunger by prioritizing high yield over nutritional quality. Stress from global climate change has been shown to reduce the densities of several micronutrients. CRISPR-Cas, which allows genome editing with extremely high precision, has emerged as a groundbreaking breeding technology that has already been adopted by many countries. Here we examine how CRISPR-Cas-based approaches could be used to achieve biofortification targets by enhancing micronutrient densities to the levels necessary to alleviate dietary vitamin and mineral deficiencies. Given the limited time frame available to achieve zero hunger, we argue that CRISPR-Cas technologies should be combined with metabolic engineering based on transformation and other technologies. We also consider untapped resources beyond metabolic pathways and current CRISPR-Cas methodologies to address one of the most important societal issues of the twenty-first century.},
}

Humans
*Biofortification/methods
*Climate Change
CRISPR-Cas Systems/genetics
*Crops, Agricultural/genetics/metabolism/chemistry
*Gene Editing/methods
Metabolic Engineering
Micronutrients/deficiency/analysis
Nutritive Value

@article {pmid42342925,
year = {2026},
author = {Gong, Q and Liu, H and Nie, Q and Tong, J and Li, W and Pak, M and Mei, Y and He, K and Wang, D and Wang, NN},
title = {GmENDO-like 1, a structurally divergent homolog of S1/P1-type endonuclease, modulates the vegetative-to-reproductive phase transition in soybean.},
journal = {Plant cell reports},
volume = {45},
number = {7},
pages = {},
pmid = {42342925},
issn = {1432-203X},
support = {24JCQNJC01940//Natural Science Foundation of Tianjin/ ; 2023ZD040710207//Biological Breeding-National Science and Technology Major Project/ ; 32070317//National Natural Science Foundation of China, Grant/ ; 32270359//National Natural Science Foundation of China/ ; },
mesh = {*Glycine max/genetics/enzymology/physiology/growth & development ; *Plant Proteins/genetics/metabolism/chemistry ; Amino Acid Sequence ; Gene Expression Regulation, Plant ; *Endonucleases/genetics/metabolism/chemistry ; Plant Leaves/genetics/physiology/growth & development ; Gene Knockout Techniques ; Plants, Genetically Modified ; Flowers/genetics/physiology ; Plant Senescence/genetics ; Reproduction/genetics ; Phylogeny ; CRISPR-Cas Systems ; Phenotype ; Mutation ; },
abstract = {GmENDO-like 1, a structurally divergent S1/P1 endonuclease homolog, modulates soybean vegetative-to-reproductive transition; its knockout delays flowering and accelerates leaf senescence. Soybean is a monocarpic crop in which the transition from vegetative to reproductive growth triggers the onset of senescence in vegetative tissues. In this study, we identified a soybean homolog of the S1/P1-type endonuclease gene, GmENDO-like 1, which was significantly upregulated during leaf senescence. Sequence analysis revealed that GmENDO-like 1 has substantial deletions at both the N- and C-termini, resulting in the loss of key residues essential for the active center of canonical S1/P1-type nucleases. Specifically, among the nine conserved amino acids responsible for metal ion coordination, one tryptophan (Trp), two histidines (His), and one aspartic acid (Asp) residue are absent. Although these deletions result in a significant rearrangement of the trinuclear metal-binding center, GmENDO-like 1 remarkably retains intrinsic catalytic activity. Using CRISPR/Cas9-mediated genome editing, we generated two knockout mutant lines of the GmENDO-like 1 gene. Phenotypic analysis revealed a markedly delayed flowering time and an obviously precocious leaf senescence in the GmENDO-like 1 mutant plants. These observations were further corroborated by the altered expression levels of flowering- and senescence-associated marker genes. Both the 100-seed weight and per-plant yield of the mutants were significantly decreased. Collectively, these findings establish GmENDO-like 1 as a functional, evolutionarily divergent nuclease and an essential modulator regulating the vegetative-to-reproductive phase transition in soybean.},
}

*Glycine max/genetics/enzymology/physiology/growth & development
*Plant Proteins/genetics/metabolism/chemistry
Amino Acid Sequence
Gene Expression Regulation, Plant
*Endonucleases/genetics/metabolism/chemistry
Plant Leaves/genetics/physiology/growth & development
Gene Knockout Techniques
Plants, Genetically Modified
Flowers/genetics/physiology
Plant Senescence/genetics
Reproduction/genetics
Phylogeny
CRISPR-Cas Systems
Phenotype
Mutation

@article {pmid42343762,
year = {2026},
author = {Han, N and Xiao, Y and Zhang, J and Guo, L and Li, Q and Yang, P and Li, X and Lin, X and Xu, L and Zhang, H},
title = {[Research progress of the CRISPR-Cas system in the detection and elimination of antibiotic resistance genes].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {5},
pages = {2051-2068},
doi = {10.13345/j.cjb.250847},
pmid = {42343762},
issn = {1872-2075},
support = {2023-YJ-zd1//the Key Research and Development Program of Health and Healthcare of Fuzhou First General Hospital/ ; 2024-YJ-ZK04//the Fujian Province Clinical Key Specialty Construction Research Project/ ; 2024J011257//the General Program of Fujian Provincial Natural Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Drug Resistance, Bacterial/genetics ; *Bacteria/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; *Gene Editing/methods ; },
abstract = {The abuse of antibiotics has led to increasingly severe antibiotic resistance, which poses a serious threat to global public health security. Therefore, the rapid detection and effective elimination of antibiotic resistance genes are crucial for controlling the spread of drug-resistant bacteria. Conventional methods for detecting antibiotic resistance genes, such as PCR, quantitative real-time PCR, and isothermal amplification, have shortcomings including time-consuming procedures, labor intensiveness, high costs, and poor specificity. Therefore, there is an urgent need to find new solutions to combat bacterial resistance, making the development of novel and efficient technologies for detecting and eliminating antibiotic resistance genes. In recent years, gene editing technology has become a research hotspot. The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas proteins) consisted of the CRISPR-Cas system. As one of the adaptive immune systems in prokaryotes, it possesses unique sequence targeting ability and high sensitivity, which enable it to accurately recognize and cleave target nucleic acid sequences. Thus, it shows great potential in the detection and elimination of antibiotic resistance genes. This review summarizes the classification, structures, and action mechanisms of CRISPR-Cas systems, elaborates on the application of different CRISPR-Cas systems in the detection and elimination of antibiotic resistance genes, and finally discusses the challenges faced by this technology and its future development directions, aiming to provide new ideas for addressing antibiotic resistance in pathogenic bacteria.},
}

*CRISPR-Cas Systems/genetics
*Drug Resistance, Bacterial/genetics
*Bacteria/genetics/drug effects
Anti-Bacterial Agents/pharmacology
*Gene Editing/methods

@article {pmid42343773,
year = {2026},
author = {Chen, Y and Jin, W and Ma, X and Feng, R and Yang, Y},
title = {[YTHDF1 enhances the expression of exogenous proteins in the CHO-K1-GS[‒/‒] cell line].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {5},
pages = {2219-2232},
doi = {10.13345/j.cjb.250823},
pmid = {42343773},
issn = {1872-2075},
support = {2025-2-177//the Lanzhou Science and Technology Program/ ; 26YFFA026//the Gansu Science and Technology Program/ ; 31920250053//the Fundamental Research Funds for the Central Universities/ ; 2025CXZX-242//the "Innovation Star" Project for Postgraduate Students in Gansu Province/ ; 25FNNA002//the Gansu Province Research-Production Integration Sci-Tech Project Empowerment Initiative/ ; },
mesh = {Animals ; CHO Cells ; Cricetulus ; *Glutamate-Ammonia Ligase/genetics ; *RNA-Binding Proteins/genetics/metabolism ; Cricetinae ; Recombinant Proteins/genetics/biosynthesis ; Humans ; *RNA Splicing Factors/genetics ; CRISPR-Cas Systems ; },
abstract = {YTH domain family protein 1 (YTHDF1) is an RNA-binding protein and belongs to the reader modified by N[6]-methyladenosine (m[6]A). It recognizes and binds to m[6]A modifications on RNA through specific domains, thereby performing corresponding biological functions. This study aims to explore the effects of YTHDF1 on the expression levels of exogenous proteins in the Chinese hamster ovary (CHO) cell line and evaluate its application potential in the optimization of the expression system of CHO cells. Firstly, the glutamine synthetase (GS) gene of CHO-K1 cells was knocked out by CRISPR/Cas9 to obtain the CHO-K1-GS[‒/‒] cell line. The enhancement effects of YTHDF1 overexpression on the expression of exogenous proteins in CHO-K1-GS[‒/‒] cells were further analyzed. Western blotting, qPCR, and fluorescence microscopy observations showed that the overexpression of YTHDF1 significantly up-regulated the expression levels of human serum albumin, single-chain antibody, and green fluorescent protein in cells. Moreover, this process did not have a significantly negative impact on the long-term proliferation and survival rate of the cells. In addition, the experiment with the translation inhibitor cycloheximide (CHX) confirmed that YTHDF1 mainly enhanced the protein expression by promoting translation in cells. This study demonstrates that YTHDF1 can enhance the cell's ability to synthesize exogenous proteins by promoting the translation process of mRNA. This strategy provides a new theoretical basis and technical direction for constructing efficient cell lines for producing biopharmaceuticals.},
}

Animals
CHO Cells
Cricetulus
*Glutamate-Ammonia Ligase/genetics
*RNA-Binding Proteins/genetics/metabolism
Cricetinae
Recombinant Proteins/genetics/biosynthesis
Humans
*RNA Splicing Factors/genetics
CRISPR-Cas Systems

@article {pmid42343789,
year = {2026},
author = {Han, C and Li, Y and Zhang, H},
title = {[Advances in genome editing technologies in Komagataella phaffii and their applications in biomanufacturing].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {6},
pages = {2414-2438},
doi = {10.13345/j.cjb.250908},
pmid = {42343789},
issn = {1872-2075},
support = {1012050205238420//the Postgraduate Research & Practice Innovation Program of Jiangsu Province/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Saccharomycetales/genetics/metabolism ; Recombinant Proteins/genetics/biosynthesis ; Metabolic Engineering/methods ; },
abstract = {Komagataella phaffii is widely recognized as a premier host for the production of recombinant proteins and value-added metabolites, owing to its low background secretion of endogenous proteins, strong capacity for heterologous protein secretion, and robust growth and metabolic performance under industrially relevant fermentation conditions. In recent years, rapid progress in genome editing technologies and synthetic biology toolkits has markedly improved the precision and efficiency of gene function interrogation, metabolic pathway reconstruction, and dynamic regulation in K. phaffii, thereby continuously strengthening its performance as a microbial cell factory. Consequently, beyond its established roles in producing recombinant proteins, industrial enzymes, and vaccine antigens, K. phaffii has also demonstrated substantial potential for the biosynthesis of natural products, biopharmaceutical molecules, and emerging biomaterials. This review systematically summarizes the evolution of genome engineering technologies in K. phaffii, spanning the transition from conventional recombination-based methods to next-generation precision editing tools. We highlight recent advances, optimization strategies, and engineering practices of CRISPR/Cas and related systems in this host. Moreover, in light of emerging research trends, we discuss key challenges and opportunities associated with improving editing efficiency, enabling rapid assembly of complex metabolic pathways, and accelerating industrial translation, thereby providing a reference for the rational engineering of Komagataella phaffii and its broader applications in synthetic biology and biomanufacturing.},
}

*Gene Editing/methods
CRISPR-Cas Systems
*Saccharomycetales/genetics/metabolism
Recombinant Proteins/genetics/biosynthesis
Metabolic Engineering/methods

@article {pmid42343816,
year = {2026},
author = {Wang, B and DU, C and Gege, R and Zhao, Q and Wang, H and Li, B and Wang, J and Mei, J and Zhang, S and Bao, F},
title = {[CRISPR/Cas9-mediated knockout of the Soc gene in T4 bacteriophage and mutant construction].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {6},
pages = {2839-2848},
doi = {10.13345/j.cjb.250870},
pmid = {42343816},
issn = {1872-2075},
support = {32260893//the National Natural Science Foundation of China/ ; 2022MS03019//the Natural Science Foundation of Inner Mongolia Autonomous Region/ ; NJYT23094//the Program for Young Talents of Science and Technology in Universities of Inner Mongolia Autonomous Region/ ; 2023YFXZ0002//the Science and Technology Project of Inner Mongolia Autonomous Region/ ; BR250101//the Basic Scientific Research Fund of Directly Affiliated Universities in Inner Mongolia Autonomous Region/ ; },
mesh = {*Bacteriophage T4/genetics ; *CRISPR-Cas Systems ; Gene Knockout Techniques ; Escherichia coli/genetics/metabolism ; Mutation ; },
abstract = {To address the antigen display limitations of current vaccine carriers, we engineered the T4 bacteriophage into a high-capacity platform. The T4 bacteriophage has advantages such as structural stability, high loading capacity, and easy production. However, the presence of high-copy endogenous Soc protein on its capsid surface severely restricts the effective display of exogenous proteins. To overcome the aforementioned spatial limitations, we employed the CRISPR/Cas9 system to precisely knockout the Soc gene of T4 bacteriophage. We successfully achieved the knockout of the Soc gene by co-transferring the three plasmid systems-pCas, pTargetF-sgRNA, and pMD19-T-Soc-arm-into Escherichiacoli TG1, inducing the expression of Cas9 with L-arabinose, and then infecting the engineered bacteria with the wild-type T4 bacteriophage (named T4 WT). The PCR, SDS-PAGE, and sequencing results confirmed a Soc gene-deficient T4 bacteriophage mutant strain (named T4ΔSoc) was successfully constructed. This mutant strain had comparable growth, thermal stability, and pH stability to the wild type, and maintained complete infectivity. After continuous passage for five generations, it remained stable in terms of genome, protein composition, and phage plaque phenotype, with no occurrence of revertant mutations. In conclusion, T4ΔSoc is stable both genetically and phenotypically, effectively breaking through the limitation of the restricted display space of T4 WT. This study provides a reliable vector tool and technical foundation for constructing an efficient and stable antigen display and vaccine delivery platform and has good application potential in vaccine research and the construction of targeted delivery systems.},
}

*Bacteriophage T4/genetics
*CRISPR-Cas Systems
Gene Knockout Techniques
Escherichia coli/genetics/metabolism
Mutation

@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