@article {pmid40098415,
year = {2026},
author = {Guo, Y and Ge, T and Wang, Q and Liu, TX and Li, Z},
title = {An RPA-CRISPR/Cas12a based platform for rapid, sensitive, and visual detection of Apis mellifera filamentous virus.},
journal = {Insect science},
volume = {33},
number = {3},
pages = {746-758},
doi = {10.1111/1744-7917.70024},
pmid = {40098415},
issn = {1744-7917},
support = {2022JQ-154//Natural Science Basic Research Program of Shaanxi Province/ ; 2452021101//Ministry of Education of the People's Republic of China Chinese Universities Scientific Fund/ ; NWAFU//Ministry of Education of the People's Republic of China Chinese Universities Scientific Fund/ ; },
mesh = {Bees/virology ; Animals ; *CRISPR-Cas Systems ; *DNA Viruses/isolation & purification/genetics ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Recombinases ; *Insect Viruses/isolation & purification/genetics ; },
abstract = {Apis mellifera filamentous virus (AmFV) is an emerging DNA virus significantly affecting honey bee health. AmFV infections weaken bee resistance to other pathogens, and can cause tissue lysis and death. Early, accurate detection of AmFV is crucial for timely intervention and preventing large-scale outbreaks. Current AmFV detection relies largely on polymerase chain reaction (PCR)-based methods. To enable rapid field detection of AmFV, we developed a rapid and ultrasensitive detection platform using recombinase polymerase amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 12a (Cas12a) technology. A CRISPR RNA (crRNA1) specifically targeting the AmFV Bro gene was designed, ensuring no cross-reactivity with other insect DNA viruses or uninfected honey bees. After optimization of the reaction time, the platform generated results within 35 min: 20 min for the RPA reaction and 15 min for CRISPR-mediated cleavage. Two visualization approaches, fluorescence-based and lateral flow dipstick, were used to display the detection results. The detection sensitivity of both approaches was as few as 10 copies of the AmFV genome. Validation with field-collected honey bee samples demonstrated consistency with conventional PCR, revealing widespread latent AmFV infections in the field. Taken together, we successfully developed an RPA-CRISPR/Cas12 platform for rapid, specific, and sensitive detection of AmFV in Apis mellifera and Apis cerana. This platform holds promise as a simple, accurate, and cost-effective tool for point-of-care AmFV diagnosis in the field.},
}

Bees/virology
Animals
*CRISPR-Cas Systems
*DNA Viruses/isolation & purification/genetics
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
Rapid Diagnostic Tests
Recombinases
*Insect Viruses/isolation & purification/genetics

@article {pmid41238927,
year = {2025},
author = {Gundogdu, M and Islek, Z},
title = {Genetic Manipulation Tools in Leishmania: From CRISPR/Cas9 to Vaccine Strategies for Disease Control.},
journal = {Acta parasitologica},
volume = {70},
number = {6},
pages = {217},
pmid = {41238927},
issn = {1896-1851},
abstract = {PURPOSE: Gene editing technologies have emerged as a crucial approach totackling parasitic infections. Recent research underscores the potential of protozoan parasites, including Leishmania, to utilize gene editing strategies, such as the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins)(CRISPR/Cas9) to edit parasite genomes, modify virulence factors, and alter host immune responses. This review aims to explore the potential of CRISPR/Cas9 in the study of Leishmania and Leishmaniasis, focusing on its applications in understanding mechanisms and developing novel editing strategies. METHODS: The immunological response of the mammalian host and the nature of the Leishmania parasites play essential roles in the formation and progression of parasitic diseases. Among alternative treatment strategies, CRISPR/Cas9 has attracted attention as a promising tool for introducing protective or therapeutic mutations in Leishmaniasis. This review will provide recent insights into the role of gene editing, especially CRISPR/Cas9, in host-pathogen interactions, intercellular communication, immunomodulation, and pathogenesis of Leishmania. RESULTS: Current findings reveal that CRISPR/Cas9 enables efficient modification of Leishmania genomes, providing valuable insights into parasite biology, host-pathogen interactions, intercellular communication, immunomodulation, and disease pathogenesis. Additionally, immunotherapeutic models are being investigated to explore the potential applications of CRISPR/Cas9 in theranostics. CONCLUSION: CRISPR/Cas9 has attracted attention as a promising tool for introducing protective or therapeutic mutations in Leishmaniasis. Integrating CRISPR/Cas9 with Leishmania-related research opens new avenues for disease control and understanding, while biologically inspired immunomodulatory strategies highlight its emerging role in next-generation parasite-targeted immunotherapy.},
}

@article {pmid41269653,
year = {2026},
author = {Islam, MM and Han, K and Woo, K and Shin, WS and Oh, MH},
title = {Comprehensive in silico analysis of Acinetobacter isolates from South Korea reveals genomic diversity, antimicrobial resistance, virulence factors, and evolutionary dynamics.},
journal = {Genes & genomics},
volume = {48},
number = {1},
pages = {147-162},
pmid = {41269653},
issn = {2092-9293},
support = {2022R1F1A1071415 and NRF-RS-2023-00275307//Ministry of Education/ ; },
abstract = {BACKGROUND: Multidrug-resistant Acinetobacter strains pose a significant threat to healthcare systems and have become a growing concern. A state-of-the-art understanding of this formidable pathogen—including its antibacterial resistance profile, virulence factors, genomic diversity, and evolutionary dynamics—is crucial for implementing effective responses during a pandemic. OBJECTIVE: To explore the genomic diversity, antimicrobial resistance (AMR) profile, virulence factors, and evolutionary trends of multidrug-resistant Acinetobacter isolates collected in South Korea from 2010 to 2022, we conducted a comprehensive in silico analysis of 74 complete genome sequences. METHODS: 74 Acinetobacter complete genomes were annotated using Prokka (v1.14.6), and a pangenome was constructed using Roary (v3.13). The genomes were systematically analyzed for antibiotic resistance genes (ARGs), virulence factor genes (VFGs), mobile genetic elements (MGEs), CRISPR/cas, and prophage using various in silico tools. Multi-locus sequence typing (MLST) was conducted using the Pasteur scheme via the MLST 2.0 web server. To assess genetic diversity, eBURST and whole-genome SNP-based phylogenetic analyses were employed. We performed a targeted analysis of carbapenem-resistant Acinetobacter strains, investigating the relationship between prophages and CRISPR/Cas systems. RESULT: Pan-genome analysis showed an open genome structure (α = 0.4921) in Acinetobacter, indicating ongoing genetic evolution. A total of 77 unique resistance genes, linked to six resistance mechanisms and 21 drug classes, were identified, including blaOXA-23, efflux pumps (AdeIJK, AdeFGH), and carbapenemases. Additionally, 68 virulence factors associated with adherence, biofilm formation, iron uptake, immune evasion, and serum resistance etc. were prevalent. Mobile genetic elements, such as Tn6207, Tn6209, Tn2006, Tn2008, and ISAba1/blaOXA-23 combinations, were identified, suggesting mechanisms for the spread of resistance genes. Seventeen types of prophage were identified, and a low prevalence of CRISPR sequence implies susceptibility to phage predation. The ST2 genotype was dominant, and SNP-based phylogeny showed significant genomic diversity. CONCLUSION: This study provides a comprehensive understanding of the resistance, virulence, and mobile genetic element profile of Acinetobacter isolates from South Korea, laying the groundwork for future antimicrobial resistance research and intervention strategies, especially the bacteriophage treatment.},
}

@article {pmid41606801,
year = {2026},
author = {Mashreghi, M and Rezazade Bazaz, M and Jaafari, MR},
title = {CRISPR-Cas9 lipid nanoparticles for targeting cyclin-dependent kinases in the tumor microenvironment.},
journal = {Biotechnology progress},
volume = {42},
number = {3},
pages = {e70105},
doi = {10.1002/btpr.70105},
pmid = {41606801},
issn = {1520-6033},
support = {996134//National Institute for Medical Research Development/ ; },
mesh = {*Nanoparticles/chemistry ; Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Tumor Microenvironment/drug effects/genetics ; *Lipids/chemistry ; *Cyclin-Dependent Kinase 4/genetics/antagonists & inhibitors/metabolism ; Cell Line, Tumor ; *Cyclin-Dependent Kinases/metabolism/genetics/antagonists & inhibitors ; Mice ; Apoptosis/drug effects ; Gene Therapy Agents ; Liposomes ; },
abstract = {Due to the significant roles of cyclin-dependent kinases 4 and 6 (CDK4/6) in cancer progression, this study aimed to introduce clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) plasmid DNA (pDNA) encapsulated in lipid nanoparticles (LNPs) as a novel CDK4/6 inhibitor using a gene knock-out strategy for treating cancer. pDNA-LNP was prepared and characterized using a microfluidic system. The results indicated the hydrodynamic diameter of the pDNA-LNP was 90.0 ± 0.1 nm with the PDI of 0.1 and a negative zeta-potential. The cytotoxicity results demonstrated statistically significant differences at doses of 0.250, 500, and 1 μg of pDNA with the capabilities of pDNA-LNP in the induction of apoptosis, as depicted by the Annexin-V-FITC-PI method. Real-time quantitative PCR (qPCR) also indicated a significant reduction in the expression levels of both CDK4 and 6 in the cells that were treated with pDNA-LNP. The in vivo anti-tumor activities of pDNA-LNP have demonstrated that the formulation has the potential to decrease tumor size and improve survival parameters, including median survival time (MST), which was increased from 31 days for the PBS group to 51 days for the pDNA-LNP group at 0.5 μg. On the other hand, the dose of 1 μg had shown signs of toxicity, indicating the need to optimize dosing in future studies. In summary, these findings indicate that CRISPR-Cas9 encapsulated in the LNP can suppress tumor growth and offer a promising strategy for future cancer treatment approaches.},
}

*Nanoparticles/chemistry
Animals
Humans
*CRISPR-Cas Systems/genetics
*Tumor Microenvironment/drug effects/genetics
*Lipids/chemistry
*Cyclin-Dependent Kinase 4/genetics/antagonists & inhibitors/metabolism
Cell Line, Tumor
*Cyclin-Dependent Kinases/metabolism/genetics/antagonists & inhibitors
Mice
Apoptosis/drug effects
Gene Therapy Agents
Liposomes

@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 = {},
number = {},
pages = {},
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 {pmid41794282,
year = {2026},
author = {Xue, Z and Lan, J and Zhao, Y and Yu, P and Liu, L and Lu, B and Yang, F},
title = {A novel rat model harboring two BDNF gene mutations exhibiting autism-like behaviors and cognitive impairments.},
journal = {Neuropharmacology},
volume = {291},
number = {},
pages = {110911},
doi = {10.1016/j.neuropharm.2026.110911},
pmid = {41794282},
issn = {1873-7064},
mesh = {Animals ; *Brain-Derived Neurotrophic Factor/genetics/metabolism ; *Disease Models, Animal ; Male ; Rats ; Social Behavior ; Receptor, trkB/metabolism/genetics ; *Autism Spectrum Disorder/genetics ; Hippocampus/metabolism ; Mutation ; Cognition Disorders/genetics ; *Autistic Disorder/genetics ; Rats, Sprague-Dawley ; Flavones/pharmacology ; Prefrontal Cortex/metabolism ; CRISPR-Cas Systems ; },
abstract = {Autism spectrum disorder (ASD) is a type of neurodevelopmental disorder that occurs most frequently in early childhood, affecting approximately 1% of the global population. Currently, the elusive nature of the pathological mechanisms underlying ASD precludes the existence of a definitive, effective treatment approach. In this study, we have successfully generated a novel ASD rat model utilizing CRISPR/Cas9 technology, offering a promising platform for further investigation and potential therapeutic interventions. The model is characterized by two crucial point mutations occurring at key enzyme cleavage sites of brain-derived neurotrophic factor (BDNF), thereby causing disruptions in enzyme cleavage processes. The phenotypes of this rat model faithfully recapitulate the salient deficits frequently encountered in ASD patients, exhibiting impairments in social behavior, cognition, and anxiety, along with neuronal abnormalities with key brain regions, notably the hippocampus (HPC) and medial prefrontal cortex (mPFC). Through preliminary RNA-seq analysis, we found changes in gene expression patterns related to synapses and neuronal excitability in these areas, providing new insights into the pathogenesis of ASD. Furthermore, our utilization of 7,8-dihydroxyflavone (7,8-DHF), a robust enhancer for the upregulation of both BDNF and TrkB mRNA and simultaneously activates the BDNF-TrkB signaling pathway, appears to strengthen the BDNF-TrkB signaling cascade. This intervention modifies firing patterns of neuronal spikes and synaptic transmission, which may contribute to the amelioration of ASD-like social interaction behavior exhibited in BDNF[met/leu] rats. Our research not only deepens our understanding of the pathogenesis of ASD, but also present encouraging avenue for early intervention strategies and treatments.},
}

Animals
*Brain-Derived Neurotrophic Factor/genetics/metabolism
*Disease Models, Animal
Male
Rats
Social Behavior
Receptor, trkB/metabolism/genetics
*Autism Spectrum Disorder/genetics
Hippocampus/metabolism
Mutation
Cognition Disorders/genetics
*Autistic Disorder/genetics
Rats, Sprague-Dawley
Flavones/pharmacology
Prefrontal Cortex/metabolism
CRISPR-Cas Systems

@article {pmid41814945,
year = {2026},
author = {Lee, S and Kim, S and Chong, J and Kim, MG and Lim, K and Lee, NE and Koo, J and Kang, J and Lee, H},
title = {A Cryoprotectant-Compatible Nanoporous Platform for Stable and Scalable Delivery of Biopharmaceuticals.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {33},
pages = {e10532},
pmid = {41814945},
issn = {1521-4095},
support = {RS-2022-NR072331//National Research Foundation of Korea/ ; RS-2024-00403376//National Research Foundation of Korea/ ; RS-2023-KH135060//Korea Health Industry Development Institute, Ministry of Health & Welfare, Republic of Korea/ ; RS-2024-00438476//Korea Health Industry Development Institute, Ministry of Health & Welfare, Republic of Korea/ ; //KIST Institutional Program/ ; },
mesh = {*Cryoprotective Agents/chemistry ; Animals ; Humans ; Freeze Drying ; *Nanopores ; Gene Editing ; Ribonucleoproteins/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {CRISPR-Cas9 ribonucleoproteins (RNPs) represent a promising class of biopharmaceuticals for treating genetic and complex diseases. However, their clinical translation is limited by instability during storage and delivery. Lyophilization offers a potential solution, though conventional approaches often compromise structural integrity and bioactivity under non-cryogenic conditions. Here, we have developed a nanostructured delivery platform, designated Nano Banker & Blowball (NB[2]), which features a blowball-like architecture and tunable nanoscale pores. These pores are designed to protect RNPs and enable controlled release. The freeze-dried formulation (FNB[2]) integrates optimized cryoprotectants and a surface-engineered nanoparticle design, preserving morphology and function without excessive excipients. FNB[2] exhibits rapid rehydration and retains ∼70% of gene editing activity post-lyophilization, enabling robust functional gene editing in vitro and in vivo. It also maintains long-term stability and supports efficient cellular uptake, enabling administration via multiple routes. FNB[2] represents a scalable and robust platform for genetic therapeutics, vaccines, and biologics, particularly well-suited for resource-limited and emergency medical applications.},
}

*Cryoprotective Agents/chemistry
Animals
Humans
Freeze Drying
*Nanopores
Gene Editing
Ribonucleoproteins/chemistry/metabolism/genetics
CRISPR-Cas Systems

@article {pmid41837831,
year = {2026},
author = {Awinashe, M and Viswaja, K and Pathath, AW and Subair, FP and Varshney, A and Mishra, P and Mulla, M and Mulla, M},
title = {CRISPR-cas9-Mediated Gene Editing to Reverse Oncogenic Mutations in Oral Squamous Cell Carcinoma.},
journal = {Annals of African medicine},
volume = {25},
number = {4},
pages = {805-809},
doi = {10.4103/aam.aam_864_25},
pmid = {41837831},
issn = {0975-5764},
mesh = {Humans ; *Mouth Neoplasms/genetics/pathology/therapy ; *Carcinoma, Squamous Cell/genetics/therapy/pathology ; *Mutation ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Gene Editing/methods ; Cell Proliferation/genetics ; Oncogenes ; },
abstract = {INTRODUCTION: Oral squamous cell carcinoma (OSCC) is a genetically driven malignancy characterized by a high burden of oncogenic mutations that contribute to aggressive tumor behavior, therapeutic resistance, and poor survival outcomes. Conventional treatment modalities largely target downstream molecular pathways without correcting the underlying genetic aberrations, underscoring the need for precision-based therapeutic strategies. This study aimed to assess the feasibility and functional impact of CRISPR-Cas9-mediated gene editing in reversing oncogenic mutations associated with OSCC using an in vitro experimental model.

MATERIALS AND METHODS: A controlled in vitro experimental study was conducted using the established human OSCC cell lines harboring mutations in key oncogenic genes. Cells were divided into control, mock-transfected, and CRISPR-Cas9-edited groups. Target-specific single-guide RNAs were designed to correct oncogenic mutations using advanced CRISPR-based editing platforms. Gene-editing efficiency was validated by molecular assays, while functional outcomes were assessed using cell proliferation analysis. Statistical evaluation was done using the one-way ANOVA with significance set at P < 0.05.

RESULTS: CRISPR-Cas9-edited OSCC cells demonstrated a significant reduction in cell proliferation compared to control and mock-transfected groups (P < 0.001). No significant variation was found between control and mock-transfected cells, confirming minimal procedural influence.

CONCLUSION: CRISPR-Cas9-mediated correction of oncogenic mutations effectively suppresses malignant cellular proliferation in OSCC, highlighting its promising role as a precision therapeutic strategy in oral cancer management.},
}

Humans
*Mouth Neoplasms/genetics/pathology/therapy
*Carcinoma, Squamous Cell/genetics/therapy/pathology
*Mutation
*CRISPR-Cas Systems
Cell Line, Tumor
*Gene Editing/methods
Cell Proliferation/genetics
Oncogenes

@article {pmid41865101,
year = {2026},
author = {Prakash, S and Mishra, C and Sinha, M and Kumari, H and Kumar, A},
title = {The evolution of next-generation lateral flow assays for bacterial and fungal diagnostics.},
journal = {Mikrochimica acta},
volume = {193},
number = {4},
pages = {},
pmid = {41865101},
issn = {1436-5073},
abstract = {Infectious diseases driven by increasingly resistant bacterial and fungal pathogens demand diagnostics that are faster and more accessible than conventional culture-based methods. This review traces the evolution of lateral flow assays (LFAs) from simple qualitative strips to sophisticated, molecularly enhanced diagnostic platforms. This review synthesizes literature mostly published between 2010 and 2025, identified through PubMed, Scopus, and Web of Science using search terms including 'lateral flow assay,' 'point-of-care diagnostics,' 'CRISPR diagnostics,' 'nanozyme biosensor,' 'antimicrobial resistance,' 'Candida auris,' and 'invasive aspergillosis. It highlights how advances in materials science (including quantum dots and nanozymes), isothermal amplification (RPA, LAMP), and CRISPR/Cas-based recognition have pushed LFAs toward laboratory-comparable sensitivity while preserving their simplicity. The clinical impact of these next-generation LFAs is illustrated using high-threat pathogens such as MRSA, Candida auris, and invasive Aspergillus, where rapid, point-of-care identification improves outcomes and supports antimicrobial stewardship. The review also examines the digital transformation of LFAs through smartphone-based readouts and artificial intelligence, which enable quantitative analysis and real-time epidemiological surveillance, even in remote settings. Despite ongoing challenges, including the hook effect, cross-reactivity, and regulatory fragmentation, the emerging technologies described here suggest that LFAs can help decouple high-quality infectious disease diagnostics from centralized laboratories, supporting a more equitable, global access to precision microbiological testing.},
}

@article {pmid41870761,
year = {2026},
author = {Dhaouadi, S and Titouche, Y and Dhaouadi, F and Akkou, M and Elandoulsi, RB},
title = {Mammaliicoccus sciuri as a sentinel for antimicrobial resistance and virulence: genomic epidemiology, transmission dynamics, and control in the one health era.},
journal = {Veterinary research communications},
volume = {50},
number = {3},
pages = {},
pmid = {41870761},
issn = {1573-7446},
abstract = {Mammaliicoccus sciuri (M. sciuri), a Gram-positive bacterium belonging to the group of coagulase-negative staphylococci (CoNS), has been taxonomically reclassified from the genus Staphylococcus to the newly established genus Mammaliicoccus. Its adaptability across diverse niches supports its relevance to One Health. Widely distributed in livestock and diverse environments, M. sciuri has been increasingly reported as an opportunistic pathogen and genetic reservoir for antimicrobial resistance (AMR) and virulence determinants of relevance to human and veterinary medicine. This review provides a synthesis of existing literature regarding the epidemiology, ecological niches, clinical relevance of the M. sciuri species, as well as the genomic and genetic features that underpin its contribution to the dissemination of resistance and virulence factors. Furthermore, it examined the global spread of methicillin-resistant M. sciuri (MR-M. sciuri) clones, characterized through phylogenetic analyses, sequence types (STs), and their complex transmission dynamics. The molecular basis of its resistance mechanisms is explored, with attention directed toward the genetic environments surrounding the mecA and mecC genes within the SCCmec cassettes, as well as the involvement of mobile genetic elements and heavy metal resistance systems. The genetic repertoire of M. sciuri, including the presence of CRISPR–Cas systems associated with adaptive immunity and genome plasticity, was also examined. Moreover, the review delves into the duality of M. sciuri as it can produce antimicrobials such as bacteriocins, which are active against bacteria and fungi. Finally, this review outlines control strategies, including genomic surveillance and stricter antimicrobial regulations, to address MDR M. sciuri strains in the human-animal-environment interface.},
}

@article {pmid41913088,
year = {2026},
author = {Zhang, S and Sun, W and Xiao, T and Wang, Y and Wu, X and Chen, H and Chen, M and Zhang, J},
title = {TopCas: Topology-Gated Cas12a via DNA-RNA Chimeric Circular crRNA for Amplification-Free Nucleic Acid Detection and Conditional Gene Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {33},
pages = {e75046},
doi = {10.1002/advs.75046},
pmid = {41913088},
issn = {2198-3844},
support = {2025ZD0551200//National Science and Technology Major Project on the Prevention and Treatment of Cancers, Cardiovascular and Cerebrovascular Diseases, Respiratory and Metabolic Diseases/ ; 2022YFC2603800//National Key Research and Development Program of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; RNA/genetics ; *DNA/genetics ; *Bacterial Proteins/genetics ; Endodeoxyribonucleases ; },
abstract = {Controlled activation of CRISPR-Cas12a is critical for achieving conditional gene editing and molecular diagnostics. As an indispensable component for forming an active complex, CRISPR RNA (crRNA) represents a key route to regulate Cas12a activity. Here, we establish TopCas (Topology-gated Cas12a via DNA-RNA Chimeric Circular crRNA) as a platform for preamplification-free nucleic acid detection and conditional gene editing. Within TopCas, the circular crRNA sterically constrains Cas12a's nuclease activity until target-activated complexes trans-cleave the DNA segment of the chimeric crRNA, converting the circular guide into its linear form and initiating an autocatalytic cascade that culminates in fluorophore release and signal amplification. By the same mechanism, the system conditionally activates Cas12a's gene-editing function (cis-cleavage) exclusively in the presence of specific nucleic acid targets (e.g., viral DNA or RNA). We demonstrate that TopCas affords high specificity and sensitivity in nucleic acid detection, supports accurate detection in clinical viral nucleic acid samples, and shows potential for in vivo real-time molecular imaging, while also demonstrating the feasibility of conditional gene editing. This innovative chimeric circular crRNA-Cas12a system not only provides a new tool for precise disease diagnostics but also offers a promising strategy for personalized therapeutic intervention.},
}

*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*CRISPR-Associated Proteins/genetics/metabolism
Humans
RNA/genetics
*DNA/genetics
*Bacterial Proteins/genetics
Endodeoxyribonucleases

@article {pmid41968146,
year = {2026},
author = {Nguyen, LT and Rakestraw, NR and Pizzano, BLM and Iyyappan, R and Young, CB and Huang, Y and Beerensson, KT and Fang, A and Antal, SG and Anamisis, KV and Peggs, CMD and Yan, J and Jing, Y and Lewis, JG and Burdine, RD and Adamson, B and Jiang, Z and Toettcher, JE and Myhrvold, C and Jain, PK},
title = {Efficient genome editing with chimeric oligonucleotide-directed editing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41968146},
issn = {2041-1723},
support = {R35 GM147788/GM/NIGMS NIH HHS/United States ; U01 DK127429/DK/NIDDK NIH HHS/United States ; U01DK127429//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R35GM147788//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01HD113698//U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; R61 AI181016/AI/NIAID NIH HHS/United States ; T32GM148739//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01HD102533//U.S. Department of Health & Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; RM1HG009490//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; T32GM136583//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; T32 GM148739/GM/NIGMS NIH HHS/United States ; R61AI181016//U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 75D30122C15113//U.S. Department of Health & Human Services | Centers for Disease Control and Prevention (CDC)/ ; },
mesh = {*Gene Editing/methods ; Humans ; Animals ; HEK293 Cells ; *Oligonucleotides/genetics ; CRISPR-Cas Systems/genetics ; Mice ; Cattle ; CRISPR-Associated Protein 9/genetics/metabolism ; DNA-Directed DNA Polymerase/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; },
abstract = {Prime editing has emerged as a precise and powerful genome editing tool, offering a favorable gene editing profile compared to other Cas9-based approaches. Here we report several nCas9-DNA polymerase fusion proteins and their engineered versions to create a simple and efficient two-component chimeric oligonucleotide-directed editing (CODE) system. CODE contains a derivative of Bst DNA polymerase engineered for increased thermostability and processivity as well as a chimeric pegRNA (cpegRNA) for programmable search and replace genome editing. Additionally, CODEMax(exo+) features a 5' to 3' exonuclease activity that promotes effective strand invasion and repair outcomes favoring the incorporation of the desired edit. We demonstrate that CODEs can perform small insertions, deletions, and substitutions with improved efficiency compared to PEMax at many loci in HEK293T cells with plasmid- and RNP-based delivery. We also show that CODEMax can successfully modify mouse and bovine embryos with up to 9.3% precise editing. Further optimization of CODEMax systems may enhance editing outcomes in embryos and other challenging contexts. Overall, CODEs complement existing prime editors to expand the toolbox for genome manipulations without double-stranded breaks.},
}

*Gene Editing/methods
Humans
Animals
HEK293 Cells
*Oligonucleotides/genetics
CRISPR-Cas Systems/genetics
Mice
Cattle
CRISPR-Associated Protein 9/genetics/metabolism
DNA-Directed DNA Polymerase/genetics/metabolism
Recombinant Fusion Proteins/genetics/metabolism

@article {pmid41996722,
year = {2026},
author = {Han, K and Qin, Z and Hu, S and Wu, H and Lei, W},
title = {CRISPR/Cas9-mediated generation of a REPS2 knockout human embryonic stem cell line.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {103986},
doi = {10.1016/j.scr.2026.103986},
pmid = {41996722},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Line ; *Gene Knockout Techniques ; Cell Differentiation ; },
abstract = {Ral-binding protein 1-associated Eps domain-containing 2 (REPS2), an Xchromosome-linked gene, is broadly expressed in brain, adrenal gland, and other tissues. REPS2 encodes a protein that forms part of a complex involved in the regulation of growth factor receptor endocytosis, and has been associated with a variety of diseases. We generated a REPS2 knockout human embryonic stem cell line using CRISPR/Cas9 genome editing system, which remained typical stem cell morphology, a normal karyotype, and pluripotency, and demonstrated the capacity to differentiate into all three germ layers. The REPS2 knockout hESC line provides a valuable tool for modeling REPS2-associated pathological process.},
}

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

@article {pmid42000542,
year = {2026},
author = {Kerkhof, LMC and Pepers, BA and van der Graaf, LM and Santiago-Aranda, A and Voesenek, BJB and Reits, EAJ and Buijsen, RAM and van Roon-Mom, WMC},
title = {Generation of an isogenic human induced pluripotent stem cell line for spinocerebellar ataxia type 1.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {103987},
doi = {10.1016/j.scr.2026.103987},
pmid = {42000542},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Spinocerebellar Ataxias/pathology/metabolism/genetics ; Cell Line ; Ataxin-1/genetics/metabolism ; Cell Differentiation ; CRISPR-Cas Systems/genetics ; },
abstract = {Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by an expanded CAG repeat in exon 8 of the ATXN1 gene. In this study, an isogenic human induced pluripotent stem (hiPS) cell SCA1 line was generated using CRISPR/Cas9 genome editing. Characterization revealed an expanded repeat containing 54 CAG repeats in one allele and an unmodified second allele. The isogenic hiPS cell line showed a typical hiPS cell morphology, expressed pluripotency markers and was able to differentiate into all three germ layers.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Spinocerebellar Ataxias/pathology/metabolism/genetics
Cell Line
Ataxin-1/genetics/metabolism
Cell Differentiation
CRISPR-Cas Systems/genetics

@article {pmid42020735,
year = {2026},
author = {White, MT and Wang, K and Zhang, H and Eckhard, U and Hullahalli, K and Chen, J and Wu, S and Geis, AL and Zhang, J and Queen, J and Gomis-Ruth, FX and Waldor, MK and Dong, M and Sears, CL},
title = {A pro-carcinogenic bacterial toxin binds claudin-4 to cleave E-cadherin.},
journal = {Nature},
volume = {654},
number = {8118},
pages = {504-512},
pmid = {42020735},
issn = {1476-4687},
mesh = {*Cadherins/metabolism/chemistry ; *Claudin-4/metabolism/genetics ; Humans ; *Bacterial Toxins/metabolism ; Protein Binding ; Animals ; Bacteroides fragilis/enzymology/pathogenicity/metabolism ; *Proteolysis/drug effects ; *Metalloendopeptidases/metabolism ; CRISPR-Cas Systems/genetics ; Antigens, CD/metabolism ; Colorectal Neoplasms/microbiology/metabolism ; Epithelial Cells/metabolism ; },
abstract = {The human colon is colonized by trillions of bacteria that play substantial roles in human health and disease[1]. Epidemiological and experimental studies suggest that certain colonic bacteria can stimulate the development and progression of colorectal cancer[2]. One such bacterium, enterotoxigenic Bacteroides fragilis, drives colon tumour formation through the action of a single toxin, the B. fragilis toxin (BFT)[3,4]. BFT is a metalloprotease that binds to a colonic epithelial cell receptor and causes cleavage of the E-cadherin ectodomain, leading to epithelial barrier disruption, inflammation and increased cellular proliferation[4-6]. However, the identity of the BFT receptor is unknown and the molecular mechanism of BFT-initiated E-cadherin cleavage is not well understood. Here we identify claudin-4 as a BFT receptor through a genome-wide CRISPR screen and demonstrate that claudin-4 binding promotes BFT-mediated cleavage of cell surface E-cadherin. Our work both sheds light on BFT's mechanism of action and opens avenues for the development of anti-BFT therapies, which may prove useful for colorectal cancer prevention and treatment of acute enterotoxigenic B. fragilis infection.},
}

*Cadherins/metabolism/chemistry
*Claudin-4/metabolism/genetics
Humans
*Bacterial Toxins/metabolism
Protein Binding
Animals
Bacteroides fragilis/enzymology/pathogenicity/metabolism
*Proteolysis/drug effects
*Metalloendopeptidases/metabolism
CRISPR-Cas Systems/genetics
Antigens, CD/metabolism
Colorectal Neoplasms/microbiology/metabolism
Epithelial Cells/metabolism

@article {pmid42033935,
year = {2026},
author = {Yao, F and Ziqing, Z and Jingxi, H and Yuxin, X and Tiancheng, Z and Qi, X and Guangjin, P},
title = {Generation of an IL2 knock-in human induced pluripotent stem cell line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {103996},
doi = {10.1016/j.scr.2026.103996},
pmid = {42033935},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Interleukin-2/genetics/metabolism ; *Gene Knock-In Techniques ; Cell Differentiation ; Cell Line ; Animals ; },
abstract = {Interleukin-2 (IL2) is a critical cytokine that drives T-cell proliferation, activates NK cells, and holds significant therapeutic value. Its established clinical importance in cancer immunotherapy lies in sustaining the expansion, persistence, and effector function of adoptive cell therapies. Here, we established a stable IL2 knock-in human induced pluripotent stem cell (iPSC) line via CRISPR/Cas9 technology. This engineered line constitutively secretes IL2, maintains a normal karyotype, and retains typical pluripotent characteristics, including gene expression and in vivo differentiation potential. It thus provides a reliable model for studying IL2 signaling, immune crosstalk, and therapeutic screening.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*CRISPR-Cas Systems/genetics
*Interleukin-2/genetics/metabolism
*Gene Knock-In Techniques
Cell Differentiation
Cell Line
Animals

@article {pmid42045938,
year = {2026},
author = {Dinçer, C and Fussing, B and Garnett, MJ and Coelho, MA},
title = {BEstimate: a computational tool for the design and interpretation of CRISPR base editing experiments.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {42045938},
issn = {1474-760X},
support = {206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Software ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Computational Biology/methods ; },
abstract = {CRISPR base editors enable scalable targeted DNA mutagenesis and are a powerful tool for analysing the function of variants of uncertain significance and disease modelling. Existing guide RNA (gRNA) design tools lack comprehensive functional annotation of target sequences. Here we developed BEstimate, a flexible computational pipeline that systematically specifies base editor gRNA target sites, generates on-target activity and off-target predictions, and provides functional, structural and clinical annotations of installed variants. BEstimate supports custom gRNA design against variant alleles and reversion of disease variants. BEstimate is a freely available, versatile tool for designing gRNA libraries and analysing base editor screens.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
*Software
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats
*Computational Biology/methods

@article {pmid42070526,
year = {2026},
author = {Jerred, C and Ramachandran, H and Hildebrandt, B and Zink, A and Ventura, N and Rossi, A and Prigione, A},
title = {Generation of an iPSC line IUFi004-A-13 with homozygous NDUFS1 mutation for the study of Leigh syndrome.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {104002},
doi = {10.1016/j.scr.2026.104002},
pmid = {42070526},
issn = {1876-7753},
mesh = {Humans ; *Leigh Disease/genetics/pathology/metabolism ; *NADH Dehydrogenase/genetics/metabolism ; Homozygote ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; Mutation ; *Electron Transport Complex I/genetics ; Cell Differentiation ; CRISPR-Cas Systems ; Mutation, Missense ; },
abstract = {NDUFS1 is a critical component of mitochondrial respiratory chain Complex I (CI). Pathogenic variants of NDUFS1 can cause Leigh syndrome (LS), a severe pediatric mitochondrial disorder. To model NDUFS1-linked LS, we generated an iPSC line with homozygous missense mutations in exon 8 using CRISPR/Cas9. The cell line demonstrated typical morphology, expression of iPSC markers, ability to differentiate into all three germ layers, and genomic integrity. This model will enable the study of LS caused by CI in an isogenic context.},
}

Humans
*Leigh Disease/genetics/pathology/metabolism
*NADH Dehydrogenase/genetics/metabolism
Homozygote
*Induced Pluripotent Stem Cells/metabolism/cytology
Cell Line
Mutation
*Electron Transport Complex I/genetics
Cell Differentiation
CRISPR-Cas Systems
Mutation, Missense

@article {pmid42103732,
year = {2026},
author = {Barazas, M and van Schendel, R and Tijsterman, M},
title = {A mutational scar-based genome-wide map of DNA double-strand break repair.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42103732},
issn = {2041-1723},
support = {2021-2/13905//KWF Kankerbestrijding (Dutch Cancer Society)/ ; 2024-3/16583//KWF Kankerbestrijding (Dutch Cancer Society)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; Humans ; DNA Polymerase theta ; *DNA Repair/genetics ; *Mutation ; CRISPR-Cas Systems ; DNA End-Joining Repair/genetics ; Tumor Suppressor p53-Binding Protein 1/metabolism/genetics ; Werner Syndrome Helicase/genetics/metabolism ; DNA-Directed DNA Polymerase/genetics/metabolism ; Gene Knockout Techniques ; },
abstract = {Genome alterations arise from inaccurate DNA repair and accumulate as distinct mutational signatures. Here, we systematically interrogate the contribution of every protein-coding gene to double-strand break (DSB) repair by generating high-resolution outcome profiles following gene knockouts. Using a CRISPR/Cas9-based, massively parallel bulk screening approach, we establish a comprehensive catalogue of MUtational Scars of Induced DNA Cleavage (MUSIC) that maps the full landscape of DSB repair factors. Our analysis identifies and validates gene clusters - including nearly all known components and several previously unrecognised factors - associated with non-homologous end-joining, the 53BP1 pathway, homology-directed repair, and polymerase theta (POLQ)-mediated end-joining. By focusing on pathway-specific repair outcomes, we uncover an unexpected role for the WRN helicase in suppressing inverted templated insertions, a poorly understood POLQ-associated mutational signature. Finally, dissection of MUSIC features reveals unanticipated functional distinctions among genes within the same DSB pathway, providing mechanistic insight and enabling further investigation into chromosomal break repair.},
}

*DNA Breaks, Double-Stranded
Humans
DNA Polymerase theta
*DNA Repair/genetics
*Mutation
CRISPR-Cas Systems
DNA End-Joining Repair/genetics
Tumor Suppressor p53-Binding Protein 1/metabolism/genetics
Werner Syndrome Helicase/genetics/metabolism
DNA-Directed DNA Polymerase/genetics/metabolism
Gene Knockout Techniques

@article {pmid42107826,
year = {2026},
author = {Xu, T and Guo, Z and Li, Y and Lyu, H},
title = {Determining optimal sgRNA coverage and screening duration for pooled CRISPR screens: A quantitative framework.},
journal = {Methods (San Diego, Calif.)},
volume = {253},
number = {},
pages = {49-60},
doi = {10.1016/j.ymeth.2026.04.016},
pmid = {42107826},
issn = {1095-9130},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HeLa Cells ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Reproducibility of Results ; Gene Library ; },
abstract = {CRISPR-based loss-of-function screening has emerged as a powerful tool for systematically characterizing gene functions. However, standardized quantification metrics for sgRNA coverage-a critical parameter determining genome-wide screening reliability and resource efficiency, remain undefined. In this study, we first conducted systematic sgRNA coverage tests in HeLa cells to determine the optimal coverage for CRISPR[iBAR] knockout libraries. Furthermore, we incorporated multiple timepoints to monitor sgRNA-mediated gene knockout dynamics. Here, balancing data quality, time efficiency, and cost, we identify 15 days and 800 × coverage as the optimal screening parameters for standard iBAR‑based CRISPR screens. Data from varying coverage levels can also serve as essential references for screening under different experimental conditions. Longitudinal analysis revealed that extending the screening period beyond 15 days maintains stable sgRNA distribution patterns within the cell population. This study establishes key parameter benchmarks to ensure CRISPR knockout screening efficacy and reproducibility, providing a solid foundation for downstream drug screening and target identification.},
}

Humans
*CRISPR-Cas Systems/genetics
HeLa Cells
*RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
Reproducibility of Results
Gene Library

@article {pmid42129543,
year = {2026},
author = {Bilanovic, J and Bortolatto, J and Duan, S and Bjørnsdottir, V and Teetz, AK and Thoms, A and Fischbach, J and Schmidt, F and Nyberg, WA and Hartweger, H and Escolano, A and Thomas, PG and Victora, GD and Bilate, AM and Jacobsen, JT},
title = {One-step generation of T-cell receptor knock-in mice in the TCRβ locus.},
journal = {The EMBO journal},
volume = {45},
number = {12},
pages = {4321-4336},
pmid = {42129543},
issn = {1460-2075},
support = {101042650//EC | European Research Council (ERC)/ ; },
mesh = {Animals ; *Receptors, Antigen, T-Cell, alpha-beta/genetics/metabolism ; *Gene Knock-In Techniques/methods ; Mice ; Mice, Transgenic ; Dependovirus/genetics ; T-Lymphocytes/immunology ; CRISPR-Cas Systems ; Gene Editing/methods ; },
abstract = {Transgenic mouse models expressing predefined T-cell receptors (TCRs) have been instrumental in advancing our understanding of T-cell biology. However, these traditional models rely on random genomic insertion of large constructs, require labor-intensive embryo manipulation, and frequently result in aberrant TCR expression and phenotypes. These limitations render traditional models insufficient to meet the mounting demands for rapid and precise model systems to evaluate TCR specificities. In this study, we developed a streamlined method that uses adeno-associated virus (AAV) and CRISPR/Cas9-mediated genome editing to precisely integrate pre-rearranged TCRα/β sequences into the mouse TCRβ (Trb) locus, enabling the rapid generation of TCR knock-in mice with physiological TCR expression and functional T-cell differentiation upon antigenic challenge. This approach bypasses the need for screening multiple founders for faithful TCR expression, enhancing the versatility and utility of monoclonal TCR mice in basic immunology and preclinical research, such as in the fields of cancer immunotherapy and vaccine development.},
}

Animals
*Receptors, Antigen, T-Cell, alpha-beta/genetics/metabolism
*Gene Knock-In Techniques/methods
Mice
Mice, Transgenic
Dependovirus/genetics
T-Lymphocytes/immunology
CRISPR-Cas Systems
Gene Editing/methods

@article {pmid42185540,
year = {2026},
author = {Oberlin, S and Tay, NQ and Xue, A and Mosadeghi, R and Pimentel, H and McManus, MT},
title = {Multiplexed perturbation enables scalable pooled screens.},
journal = {Nature methods},
volume = {23},
number = {6},
pages = {1163-1173},
pmid = {42185540},
issn = {1548-7105},
support = {5U01CA272546-03//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 188001//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; T32HG002536//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; Intercellular Adhesion Molecule-1/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockdown Techniques ; HEK293 Cells ; },
abstract = {CRISPR-based genetic perturbation screens have revolutionized the ability to link genes to cellular phenotypes with unprecedented precision and scale; however, conventional pooled CRISPR screens require large cell numbers to achieve adequate sgRNA representation, posing technical and financial challenges. Here, we investigate the impact of co-delivery of multiple guide RNAs via high multiplicity of infection (MOI) in pooled CRISPR interference screens as a strategy to enhance screening efficiency while reducing cell numbers. We systematically evaluate screen performance across varying MOIs, assessing the effects of multiplexing on knockdown efficiency, sgRNA representation and potential interference of multiple sgRNA phenotypes. Our data demonstrate that sgRNA multiplexing (MOI 2.5-10) can maintain screen performance while enabling significant reductions in cell number requirements. We further apply these optimized conditions to conduct a genome-wide CRISPR screen for regulators of the intracellular adhesion molecule ICAM-1, successfully identifying new candidates using as few as half a million cells. This study provides a framework for adopting multiplexed sgRNA strategies to streamline CRISPR screening applications in resource-limited settings.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
Intercellular Adhesion Molecule-1/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats
Gene Knockdown Techniques
HEK293 Cells

@article {pmid42208154,
year = {2026},
author = {Crudele, M and Barnaba, NF and Di Cosmo, D and Mascia, T and Lum, KY and Cowled, MS and Larsen, TO and Faretra, F and Coleman, JJ and De Miccolis Angelini, RM},
title = {Characterization of the chloromonilicin biosynthetic gene cluster in the brown rot fungus Monilinia fructicola by a CRISPR/Cas9 transformation system.},
journal = {Microbiological research},
volume = {310},
number = {},
pages = {128558},
doi = {10.1016/j.micres.2026.128558},
pmid = {42208154},
issn = {1618-0623},
mesh = {*Multigene Family ; Polyketide Synthases/genetics/metabolism ; *Ascomycota/genetics/metabolism/pathogenicity ; *CRISPR-Cas Systems ; Plant Diseases/microbiology ; Gene Expression Profiling ; Fruit/microbiology ; Transformation, Genetic ; Biosynthetic Pathways/genetics ; },
abstract = {Monilinia fructicola is a major pathogen responsible for brown rot, causing substantial yield losses, particularly in stone fruit. Genomic analysis identified a type I polyketide synthase gene cluster, approximately 55 kb in length and consisting of twenty-four genes, putatively involved in chloromonilicin biosynthesis. These include a polyketide synthase (PKS), flavin-dependent halogenase, N-acetyltransferases, methyltransferases, a lactamase-like protein, scytalone dehydratase, and genes related to oxidoreduction, transcription factors, and transporters. To elucidate the product of the cluster, targeted gene disruption was achieved using a CRISPR/Cas9 ribonucleoprotein complex and polyethylene glycol-mediated transformation in the reference strain Mfrc123. Mutants with disruption in the ChmN core gene (encoding the PKS) were analysed by sequencing, digital droplet PCR to assess the insertion copy number of the hygromycin resistance cassette, and gene expression profiling. Phenotypic analysis indicated no significant differences between mutants and the WT strain in conidia production, germination rate, colony growth, or virulence on inoculated fruits. Metabolomic profiling using UHPLC-MS/MS demonstrated that the mutants did not produce chloromonilicin or other cluster-associated metabolites, such as chloromonilinic acids C and D, and 4-chloropinselin, indicating that the gene cluster is essential for their biosynthesis. In the WT strain, production of these metabolites increased following co-culture with Penicillium expansum compared to monoculture and remained low after fruit inoculation, suggesting a role in competition rather than pathogenesis. Gene expression analysis further showed upregulation of ChmN in response to various plant-associated microbes, but not to other Monilinia species.},
}

*Multigene Family
Polyketide Synthases/genetics/metabolism
*Ascomycota/genetics/metabolism/pathogenicity
*CRISPR-Cas Systems
Plant Diseases/microbiology
Gene Expression Profiling
Fruit/microbiology
Transformation, Genetic
Biosynthetic Pathways/genetics

@article {pmid42229170,
year = {2026},
author = {Koo, C and Lee, D and Lee, B and Kim, S and Lee, J and Kwon, J},
title = {Base editing reveals context-dependent regulation of adhesion, anoikis, and motility by BAP1 in renal cell models.},
journal = {Biochemical and biophysical research communications},
volume = {827},
number = {},
pages = {154092},
doi = {10.1016/j.bbrc.2026.154092},
pmid = {42229170},
issn = {1090-2104},
mesh = {*Ubiquitin Thiolesterase/genetics/metabolism ; *Tumor Suppressor Proteins/genetics/metabolism ; *Anoikis/genetics ; Humans ; *Cell Movement/genetics ; Cell Adhesion/genetics ; *Kidney Neoplasms/genetics/pathology ; *Carcinoma, Renal Cell/genetics/pathology ; *Gene Editing ; CRISPR-Cas Systems ; *Kidney/cytology/pathology/metabolism ; Cell Line, Tumor ; Cell Line ; Mutation ; },
abstract = {BAP1 is a tumor-suppressive deubiquitinase essential for DNA repair, and missense mutations in BAP1 are common in clear cell renal cell carcinoma (ccRCC). We previously showed that correction of the inactivating Glu31Lys mutation in KMRC-20 ccRCC cells using CRISPR/Cas9 base editing restored BAP1 function, reinstated anchorage dependence, and re-sensitized cells to anoikis. Here, we investigated whether disruption of Glu31 is sufficient to induce anchorage-independent growth and anoikis resistance in normal kidney epithelial cells. Using adenine base editing, we introduced an inactivating Glu31Gly mutation into HK-2 cells, generating two independent isogenic BAP1-mutant clones, and established a BAP1-knockout clone by CRISPR/Cas9 as an additional control. Glu31Gly mutants exhibited complete loss of BAP1 deubiquitinase activity and impaired UV-induced DNA damage repair, comparable to knockout cells. Despite the clear functional inactivation of BAP1, the Glu31Gly and knockout HK-2 cells neither acquired anchorage-independent growth nor anoikis resistance; instead, detached cells displayed increased apoptosis. In KMRC-20 cells, restoration of BAP1 enhanced both migration and invasion, whereas BAP1 inactivation or loss in HK-2 cells increased invasion but reduced migration, indicating distinct context-dependent roles for BAP1 in normal versus malignant renal cells. These findings demonstrate that BAP1 inactivation alone is insufficient to confer anchorage-independent survival in normal kidney epithelial cells and suggest that additional oncogenic alterations are required during kidney tumorigenesis. Our study further highlights the utility of precise base editing for dissecting the functional consequences of clinically relevant cancer mutations.},
}

*Ubiquitin Thiolesterase/genetics/metabolism
*Tumor Suppressor Proteins/genetics/metabolism
*Anoikis/genetics
Humans
*Cell Movement/genetics
Cell Adhesion/genetics
*Kidney Neoplasms/genetics/pathology
*Carcinoma, Renal Cell/genetics/pathology
*Gene Editing
CRISPR-Cas Systems
*Kidney/cytology/pathology/metabolism
Cell Line, Tumor
Cell Line
Mutation

@article {pmid42248117,
year = {2026},
author = {der Auweraer, SV and Roth, MB and Vlahos, K and Howden, SE and Lockhart, PJ and Payne, JM and Brems, H and Bozaoglu, K},
title = {Generation and characterization of four iPSC and isogenic gene-corrected lines from Legius syndrome patients.},
journal = {Stem cell research},
volume = {94},
number = {},
pages = {104026},
doi = {10.1016/j.scr.2026.104026},
pmid = {42248117},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; Cell Differentiation ; Leukocytes, Mononuclear/metabolism/cytology ; CRISPR-Cas Systems ; },
abstract = {Legius syndrome is an autosomal dominant disorder caused by variants in SPRED1. In this study, we generated four induced pluripotent stem cell (iPSC) lines derived from patients with Legius syndrome by reprogramming peripheral blood mononuclear cells. Using CRISPR/Cas9 or prime editing, the pathogenic variants were corrected to generate isogenic control lines. All patient and isogenic control lines exhibited a normal morphology and karyotype, expressed pluripotency markers, and possessed trilineage differentiation potential. This is the first established human iPSC model developed for Legius syndrome and is a valuable resource for investigating the molecular mechanisms underlying this condition.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
Cell Line
Cell Differentiation
Leukocytes, Mononuclear/metabolism/cytology
CRISPR-Cas Systems

@article {pmid42251763,
year = {2026},
author = {Byiringiro, I and Contiliani, DF and Davies, C and Gurel, F and Creste, S and Qi, Y},
title = {Improving iSpyMacCas9 multiplex genome editing in rice by CRISPR-combo-mediated BBM1 activation.},
journal = {The Plant journal : for cell and molecular biology},
volume = {126},
number = {5},
pages = {e70980},
pmid = {42251763},
issn = {1365-313X},
support = {IOS-2132693//National Science Foundation/ ; IOS-2224203//National Science Foundation/ ; 21010111//Foundation for Food and Agriculture Research/ ; 2020/07045-3//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2021/13478-2//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; 2022/11738-0//Fundação de Amparo à Pesquisa do Estado de São Paulo/ ; },
mesh = {*Oryza/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Plant Proteins/genetics/metabolism ; Transcriptional Activation/genetics ; Plants, Genetically Modified ; Genome, Plant/genetics ; Gene Expression Regulation, Plant ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The recently developed CRISPR-Combo technology enables simultaneous targeted mutagenesis and transcriptional activation in plants. However, its reliance on SpCas9 limits its use at AT-rich genomic loci, such as promoter regions commonly targeted for transcription activation. To overcome this limitation, we explored the usage of Cas12b and iSpyMacCas9 in the CRISPR-Combo architecture for simultaneous genome editing and gene activation. We tested these expanded CRISPR-Combo systems for hormone-free regeneration of rice plants by transcriptional activation of a morphogenic gene, OsBBM1, while knocking out the genes of interest. The Cas12b-Combo system induced mild OsBBM1 upregulation (~3-fold), which did not affect the genome editing efficiency. By contrast, iSpyMacCas9-Combo achieved approximately 12-fold OsBBM1 transcriptional activation, supporting hormone-free regeneration at a high rate (42%). As a result, iSpyMacCas9-Combo conferred higher genome editing efficiency, including improved multiplex editing, than the standard iSpyMacCas9 system, either with or without hormones during rice regeneration. Hence, our data prove iSpyMacCas9-Combo to be a more efficient system for genome editing in rice, especially at low-efficiency target sites, when coupled with OsBBM1 transcriptional activation. These findings establish iSpyMacCas9-Combo as a useful addition to the CRISPR-Combo toolkit, expanding its genomic targeting scope and enabling more efficient genome editing by activation of an appropriate endogenous gene such as OsBBM1 in rice.},
}

*Oryza/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Plant Proteins/genetics/metabolism
Transcriptional Activation/genetics
Plants, Genetically Modified
Genome, Plant/genetics
Gene Expression Regulation, Plant
Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid42252785,
year = {2026},
author = {Jiang, J and Yan, Y},
title = {Compositional Optimization of CRISPR/Cas9 Lipid Nanoparticles for Efficient Knockdown of Target Genes.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {27},
number = {11},
pages = {e70413},
doi = {10.1002/cbic.70413},
pmid = {42252785},
issn = {1439-7633},
support = {Grant 22275167//National Natural Science Foundation of China/ ; 22405240//National Natural Science Foundation of China/ ; Grant LZ25B040002//Zhejiang Provincial Natural Science Foundation of China/ ; LTGY24B040001//Zhejiang Provincial Natural Science Foundation of China/ ; },
mesh = {Humans ; *Nanoparticles/chemistry ; *Lipids/chemistry ; *CRISPR-Cas Systems/genetics ; HeLa Cells ; Animals ; *Gene Knockdown Techniques ; Mice ; Gene Editing ; Liposomes ; },
abstract = {Efficient and safe delivery systems remain a major barrier to the clinical translation of CRISPR/Cas9 gene-editing technologies; among these, formulation optimization of lipid nanoparticles (LNPs) is a key approach to improve delivery performance. Here, we constructed an orthogonal formulation library of LNPs using the ionizable lipids 4A2C2C6-A8 and 4A2C2C8-A8 at varied molar ratios and screened for optimal compositions. We identified formulations that combined high editing efficiency with low cytotoxicity; in HeLa-Luc cells, the optimized formulation achieved >80% knockout of the luciferase reporter. Further physicochemical and functional investigations showed that LNPs with relatively high zeta potential, mean diameters near 200 nm, and appropriate internal hydrophobicity-when paired with superior cellular uptake and endosomal escape capabilities-synergistically enhanced delivery efficiency. Using the compositionally optimized LNP to codeliver Cas9 mRNA and an sgRNA targeting HSP47 (whose overexpression has been implicated in fibrosis), we achieved efficient protein-level knockdown of HSP47 in L929 cells. These results provide important guidance for formulation optimization of CRISPR/Cas9 LNPs and support their potential application in antifibrotic therapies.},
}

Humans
*Nanoparticles/chemistry
*Lipids/chemistry
*CRISPR-Cas Systems/genetics
HeLa Cells
Animals
*Gene Knockdown Techniques
Mice
Gene Editing
Liposomes

@article {pmid42258486,
year = {2026},
author = {Pilarski, J and Stadler, T and Seidel, S},
title = {Assessing the inference of single-cell phylogenies and population dynamics from CRISPR lineage recordings.},
journal = {PLoS computational biology},
volume = {22},
number = {6},
pages = {e1014370},
doi = {10.1371/journal.pcbi.1014370},
pmid = {42258486},
issn = {1553-7358},
mesh = {*Phylogeny ; *Cell Lineage/genetics ; Bayes Theorem ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Computer Simulation ; *Single-Cell Analysis/methods ; Computational Biology ; Cell Division/genetics ; Population Dynamics ; Cell Differentiation/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {Multicellular organisms develop from a single cell by repeated rounds of cell division, differentiation, and death, which can be represented as a single-cell phylogenetic tree. Genetic lineage tracing allows us to investigate this development by tracking the ancestry of individual cells as populations grow and change over time. However, accurate reconstruction of the cell phylogeny and quantification of the corresponding phylodynamic parameters - cell division, differentiation, and death rates - from this tracking data remains challenging and needs to be systematically evaluated. We perform simulations and assess, using the Bayesian framework, the joint inference of time-scaled cell phylogenies and phylodynamic parameters from CRISPR lineage recordings with random or sequential edits. Principally, we characterize the inference improvements as the recorder capacity increases. We observe more accurate phylogenetic reconstruction from sequential compared to random recordings, but no substantial improvement in phylodynamic inference when using the additional information contained in the order of edits. Overall, we find that CRISPR lineage recordings carry a strong signal on the rates of cell division when appropriate models are used. However, we detect biases in the inferred rates of cell division and death under phylodynamic model misspecification, i.e., when fitting classic memoryless birth-death processes to synchronous cell divisions. Moreover, for scenarios when cells differentiate into distinct types, we demonstrate that Bayesian phylodynamic analysis of sparse end-point measurements can resolve these cell differentiation trajectories by lineage and time. Under prototypical dynamics, we recover cell type-specific division and death rates, and cell type transition rates in over 80% of simulations. Overall, this simulation study explores how much information on cellular development can be extracted from state-of-the-art genetic lineage tracing data using phylogenetic and phylodynamic methodology.},
}

*Phylogeny
*Cell Lineage/genetics
Bayes Theorem
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Computer Simulation
*Single-Cell Analysis/methods
Computational Biology
Cell Division/genetics
Population Dynamics
Cell Differentiation/genetics
*CRISPR-Cas Systems/genetics

@article {pmid42258532,
year = {2026},
author = {Kammerdiener, EK and Hren, AP and Harrison, R and Charles, S and Klingeman, D and Wiser, TD and Eckert, CA and Alexander, WG},
title = {Empirical evaluation of all unique Cas9 protospacers in E. coli reveal widespread functionality and rules for gRNA design.},
journal = {Nucleic acids research},
volume = {54},
number = {11},
pages = {},
pmid = {42258532},
issn = {1362-4962},
support = {//Center for Bioenergy Innovation/ ; ERKP886//U.S. Department of Energy/ ; //Laboratory Directed Research and Development Program/ ; //Oak Ridge National Laboratory/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; Genome, Bacterial ; *Escherichia coli K12/genetics ; *Escherichia coli/genetics ; *CRISPR-Cas Systems ; },
abstract = {The Cas9 nuclease has become central to modern methods and technologies in synthetic biology, largely due to the ease with which it can be targeted to specific DNA loci via guide RNAs (gRNAs). Reports vary widely on the actual specificity of this targeting, with some studies observing 60% of gRNAs possessing no activity against the genome, yet an assumption persists within the E. coli community that inactive gRNAs are rare. To resolve these contradictions, we evaluated the activity of 463 000 unique gRNAs in the E. coli K12 MG1655 genome. We show that the overwhelming majority (at least 93%) of unique gRNAs are functional while only 0.3% are nonfunctional. These nonfunctional gRNAs exhibit strong spacer self-interaction, which can either be excluded using a simple design rule or "repaired" during library design. Finally, this work provides the greater microbial synthetic biology community both a set of nearly half a million empirically evaluated E. coli gRNAs as well as a thoroughly evaluated experimental procedure, complete with appropriate controls for Cas9 activity, for conducting Cas9 assays in E. coli specifically and bacteria more generally. Lastly, we have produced a webapp to allow users to easily browse and extract gRNA sequences from the E. coli genome, which can be accessed at https://grna.ornl.gov.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism
Genome, Bacterial
*Escherichia coli K12/genetics
*Escherichia coli/genetics
*CRISPR-Cas Systems

@article {pmid42259773,
year = {2026},
author = {Luo, G and Ma, F and Yang, Y and Yang, C and Li, X and Xie, J and Xiong, K and Chen, P and Ma, K and Zhao, Z and Tang, BZ and Duo, Y},
title = {Engineering an AIEgen-based platform integrating CRISPR/Cas9 to remodel the tumor microenvironment and reinforce photo-immunotherapy against glioblastom.},
journal = {Signal transduction and targeted therapy},
volume = {11},
number = {1},
pages = {},
pmid = {42259773},
issn = {2059-3635},
mesh = {Humans ; *Tumor Microenvironment/genetics/drug effects/immunology ; Animals ; *Glioblastoma/genetics/therapy/pathology/immunology ; *Immunotherapy ; *CRISPR-Cas Systems/genetics ; Mice ; Cell Line, Tumor ; *Brain Neoplasms/genetics/therapy/pathology/immunology ; *Photochemotherapy ; Nanoparticles/chemistry ; Blood-Brain Barrier ; *5'-Nucleotidase/genetics ; Neutrophils ; },
abstract = {Glioblastoma remains one of the most lethal brain tumors. Although immunotherapy and other therapeutic modalities has achieved significant therapeutic success in several malignancies, its efficacy in glioblastoma remains limited primarily due to the complex tumor microenvironment (TME) and physiological barriers such as the blood-brain barrier (BBB). In this context, nanomedicine and gene editing have emerged as promising strategies due to their unique ability to cross the BBB and protect therapeutic agents through intrinsic physicochemical properties. To overcome the physiological barriers for better therapeutic outcomes. Here, a novel aggregation-induced emission luminogen (AIEgen), NDA-DPE, was synthesized, exhibiting NIR-I to NIR-II fluorescence and dual photothermal (PTT) and photodynamic (PDT) properties through restricted intramolecular motion. Bone-derived neutrophil-based biomimetic nanoparticles (bNe@AIE/Cas9-CD73) were then prepared by integrating NDA-DPE with CRISPR/Cas9-mediated CD73 gene silencing. The neutrophil encapsulation enabled efficient BBB penetration and targeted accumulation in glioblastoma tissue. CRISPR/Cas9-CD73 downregulated CD73 expression, disrupted the ATP-adenosine axis, and reshped the immunosuppressive TME into an immuno-supportive one, increasing the therapeutic sensitivity of tumor cells. Under NIR-II excitation, bNe@AIE/Cas9-CD73 achieved fluorescence-guided PTT and PDT, inducing immunogenic cell death (ICD), stimulating immune-cell recruitment, and activating systemic antitumor immunity. bNe@AIE/Cas9-CD73 demonstrated a potent gene-photothermal-photodynamic-immune synergistic effect, significantly inhibiting glioblastoma growth and establishing a promising nanoplatform for effective and targeted glioblastoma treatment.},
}

Humans
*Tumor Microenvironment/genetics/drug effects/immunology
Animals
*Glioblastoma/genetics/therapy/pathology/immunology
*Immunotherapy
*CRISPR-Cas Systems/genetics
Mice
Cell Line, Tumor
*Brain Neoplasms/genetics/therapy/pathology/immunology
*Photochemotherapy
Nanoparticles/chemistry
Blood-Brain Barrier
*5'-Nucleotidase/genetics
Neutrophils

@article {pmid42261185,
year = {2026},
author = {Jahangiri-Sisakht, A and Safari, L and Alipanahi, R},
title = {CRISPR-MBTF: a multi-branch transformer fusion framework for CRISPR-Cas9 off-target prediction.},
journal = {Briefings in bioinformatics},
volume = {27},
number = {3},
pages = {},
pmid = {42261185},
issn = {1477-4054},
mesh = {*CRISPR-Cas Systems ; Computational Biology/methods ; Deep Learning ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) is a revolutionary genome editing technology derived from a bacterial adaptive immune system that uses a single guide RNA (sgRNA) to direct the Cas9 enzyme to specific DNA sequences for precise genetic modifications. Its ease of use and efficiency has accelerated advancements in genetic research and therapeutic development. However, unintended cleavage at off-target sites remains a significant concern, limiting the safety and broader applicability of CRISPR-based editing. Accurate computational prediction of off-target locations is therefore essential to mitigate potential risks and improve experimental design. In this study, we introduce CRISPR multi-branch transformer fusion (CRISPR-MBTF), a novel deep learning-based framework employing a multi-branch Transformer architecture combined with an attention-based fusion mechanism to model the intricate biological context influencing CRISPR activity. By capturing subtle sequence patterns and contextual dependencies, our model achieves enhanced predictive performance compared to existing approaches. Additionally, interpretability analyses uncover biologically meaningful patterns and highlight influential sequence regions, offering valuable insights into the determinants of CRISPR specificity. This work presents a robust and interpretable tool to support the design of safer and more effective genome editing strategies.},
}

*CRISPR-Cas Systems
Computational Biology/methods
Deep Learning
RNA, Guide, CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid42261595,
year = {2026},
author = {Saeed, M and Arham, M and Zafar, I and Jamal, A and Hussian, M and Usman, M and Bahwerth, FS and Noman, M and Hossain, MB},
title = {Harnessing Deep Learning Models for Guide RNA Optimization and Off-Target Prediction in CRISPR Systems.},
journal = {Biotechnology journal},
volume = {21},
number = {6},
pages = {e70255},
doi = {10.1002/biot.70255},
pmid = {42261595},
issn = {1860-7314},
mesh = {*Deep Learning ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Recurrent Neural Networks ; Humans ; Predictive Learning Models ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Convolutional Neural Networks ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based genome and transcriptome editing technologies have emerged as powerful tools for therapeutic, agricultural, and industrial applications. However, their broader clinical and translational use remains limited by variable guide RNA (gRNA) or single-guide RNA (sgRNA) efficiency and unintended off-target activity, which may lead to genotoxic effects and major safety concerns. To address these challenges, recent research has increasingly shifted from heuristic scoring approaches and traditional machine learning (ML) methods toward deep learning (DL) models capable of learning complex sequence-function relationships from large-scale experimental datasets generated by assays such as GUIDE-seq (Genome-wide Unbiased Identification of Double-stranded Breaks Enabled by Sequencing), CIRCLE-seq (Circularization for In Vitro Reporting of Cleavage Effects by Sequencing), and CHANGE-seq (Cumulative and Homology-independent Analysis of Nuclease Genome-wide Effects by Sequencing). This review critically examines recent advances in DL approaches for gRNA optimization and off-target prediction in CRISPR systems. We discuss the development of convolutional neural networks (CNNs), recurrent neural networks (RNNs), transformer-based architectures, and foundation models designed to improve prediction accuracy, specificity, and generalizability across diverse biological contexts.},
}

*Deep Learning
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Recurrent Neural Networks
Humans
Predictive Learning Models
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Convolutional Neural Networks

@article {pmid42266926,
year = {2026},
author = {Ji, T and Fang, X and Gao, Y and Yu, K and He, J and Wang, L and Zhu, W and Huang, G and Gao, X},
title = {Visual detection platform based on RPA-CRISPR/Cas12a for Klebsiella pneumoniae and Carbapenem-resistant Klebsiella pneumoniae in clinical and food safety settings.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1817859},
pmid = {42266926},
issn = {2235-2988},
mesh = {*Klebsiella pneumoniae/genetics/isolation & purification/drug effects ; Humans ; *Food Safety ; *Carbapenem-Resistant Enterobacteriaceae/genetics/isolation & purification ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Klebsiella Infections/microbiology/diagnosis ; Rapid Diagnostic Tests ; *Nucleic Acid Amplification Techniques/methods ; beta-Lactamases/genetics ; DNA-Directed RNA Polymerases/genetics ; Carbapenems/pharmacology ; Bacterial Proteins/genetics ; DNA Primers/genetics ; Anti-Bacterial Agents/pharmacology ; },
abstract = {INTRODUCTION: The rise of Klebsiella pneumoniae (KP) and carbapenem-resistant KP (CRKP) poses grave threats to public health and food safety, creating an urgent demand for rapid point-of-care testing (POCT). Traditional detection methods are limited by laboratory barriers, making them unsuitable for POCT implementation.

METHODS: Herein, a one-tube assay integrating recombinant polymerase amplification (RPA) with CRISPR/Cas12a technology was developed for the rapid, sensitive and specific detection of KP and blaOXA-48-carrying CRKP. Specific primers targeting the KP-specific rpoB gene and carbapenem-resistance gene blaOXA-48 were designed, and optimal primer pairs were screened via agarose gel electrophoresis. CrRNA sequences were designed according to RPA amplicons, and the components of the CRISPR/Cas12a reaction were optimized. A two-step reaction system was initially evaluated, followed by the establishment of an integrated one-tube RPA-CRISPR/Cas12a assay. A total of 66 clinical specimens and artificially contaminated food samples were used for method validation, with microbial culture and qPCR as reference methods.

RESULTS: The two-step assay was capable of detecting bacterial suspensions at a concentration of 100 CFU/mL. The one-tube system could be completed within 1 hour at 37 °C. This assay avoided aerosol contamination and allowed visual result readout under blue light. In the validation test, the detection results of the one-tube assay were consistent with those obtained by microbial culture and qPCR.

DISCUSSION: This study constructed a dual-target RPA-CRISPR/Cas12a platform for the visual detection of KP and blaOXA-48-positive CRKP under blue light. This assay reduces reliance on sophisticated equipment and professional personnel. It can serve as a promising POCT tool for clinical diagnosis and food safety surveillance, and provides evidence for the timely formulation of rational antimicrobial treatment strategies.},
}

*Klebsiella pneumoniae/genetics/isolation & purification/drug effects
Humans
*Food Safety
*Carbapenem-Resistant Enterobacteriaceae/genetics/isolation & purification
Sensitivity and Specificity
*CRISPR-Cas Systems
*Klebsiella Infections/microbiology/diagnosis
Rapid Diagnostic Tests
*Nucleic Acid Amplification Techniques/methods
beta-Lactamases/genetics
DNA-Directed RNA Polymerases/genetics
Carbapenems/pharmacology
Bacterial Proteins/genetics
DNA Primers/genetics
Anti-Bacterial Agents/pharmacology

@article {pmid42267671,
year = {2026},
author = {Brown, RA and Dangel, AW and Saini, A and Collins, PL and Colonna, M and Oltz, EM},
title = {CRISPRi screening identifies SON and MAP4K1 as regulators of type III cytokine expression in innate lymphoid cells.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {215},
number = {6},
pages = {},
doi = {10.1093/jimmun/vkag110},
pmid = {42267671},
issn = {1550-6606},
support = {P30 CA016058/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Protein Serine-Threonine Kinases/genetics/metabolism ; *Immunity, Innate ; Interleukin-22 ; *Lymphocytes/immunology/metabolism ; Interleukins/genetics/metabolism ; *DNA-Binding Proteins/genetics/metabolism ; Interleukin-17/genetics/metabolism ; Gene Expression Regulation ; *Cytokines/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {The cytokines interleukin (IL)-22 and IL-17 are secreted by innate and adaptive immune cells to drive "type III" responses that protect against extracellular pathogens, promote mucosal barrier integrity, and foster microbiota homeostasis. However, dysregulation of IL-22 and/or IL-17 contributes to autoimmunity, chronic inflammation, and malignancy. Thus, a deeper understanding of mechanisms regulating type III cytokine production could provide new therapeutic targets for a spectrum of immune-mediated diseases. Toward this goal, we performed a genome-wide CRISPR inhibition (CRISPRi) screen to identify factors that regulate IL-22/IL-17 expression in a murine type III innate lymphoid cell (ILC3) model, MNK3, following stimulation with IL-23 and IL-1β. In addition to previously known regulators of type III cytokines, including IL-23 receptor components IL23R and IL12Rβ1, the screen identified a large set of new factors that either potentiate or attenuate expression of IL-22 and/or IL-17. A subset of these novel factors was chosen for validation, from which two were selected for further study. Knockdown of nuclear protein, SON, which binds both DNA and RNA, impaired expression of IL12Rβ1 at the levels of de novo transcription and RNA processing. The second, MAP4K1 (HPK1), is a serine/threonine kinase that is required for IL-22 but not IL-17 expression. Depletion of MAP4K1 in MNK3 also enhanced expression of the type I cytokine, IFN-γ, which was co-expressed with IL-17, a phenotype reminiscent of pathogenic Th17 cells. Together, results from the CRISPRi screen broaden our understanding of the factors involved in type III immune responses and offer new targets for modulating IL-22/17 expression.},
}

Animals
Mice
*Protein Serine-Threonine Kinases/genetics/metabolism
*Immunity, Innate
Interleukin-22
*Lymphocytes/immunology/metabolism
Interleukins/genetics/metabolism
*DNA-Binding Proteins/genetics/metabolism
Interleukin-17/genetics/metabolism
Gene Expression Regulation
*Cytokines/metabolism/genetics
CRISPR-Cas Systems

@article {pmid42268478,
year = {2026},
author = {Khajouei, F and Ghaemi, A and Abnous, K and Taghdisi, SM and Ramezani, M and Alibolandi, M},
title = {CRISPR-Based Gene Therapy for Brain Disease.},
journal = {Molecular neurobiology},
volume = {63},
number = {1},
pages = {},
pmid = {42268478},
issn = {1559-1182},
mesh = {Humans ; *Genetic Therapy/methods ; Animals ; *Brain Diseases/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Neurological disorders are complex and often very challenging for patients. Many of these conditions result from mutations in genes that are essential for normal function. Most existing treatments only alleviate symptoms, highlighting the urgent need for more effective therapeutic strategies. In the current drug development landscape, gene therapy offers hope as a promising approach. Specifically, CRISPR-Cas9 technology enables precise gene editing across diverse cell types and organisms. An increasing number of research groups are investigating innovative therapies and the molecular mechanisms behind neurological diseases. This review highlights the use of CRISPR-based gene therapies for various brain diseases, including multiple sclerosis, Alzheimer's, Parkinson's disease, epilepsy, stroke, and brain tumors. It consistently recognizes significant challenges in clinical applications, including overcoming the blood-brain barrier (BBB), managing off-target effects, ensuring efficient delivery, and addressing immunogenicity and ethical concerns.},
}

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

@article {pmid42269591,
year = {2026},
author = {Christensen, OP and Markham, A and Kang, H and Gabriel, E and Pers, TH},
title = {Causal effect estimation from trans-regulatory single-cell CRISPR screens.},
journal = {Cell genomics},
volume = {6},
number = {6},
pages = {101251},
pmid = {42269591},
issn = {2666-979X},
mesh = {*Single-Cell Analysis/methods ; Humans ; Single-Cell Gene Expression Analysis ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Animals ; Transcriptome/genetics ; },
abstract = {Recent advances in single-cell transcriptomics and CRISPR-based genome editing have enabled large-scale perturbation experiments with genome-wide expression readouts. Single-cell CRISPR screens offer the opportunity to move beyond correlation and estimate causal effects of genetic perturbations on gene expression at scale. These approaches promise to substantially deepen insights into cellular functions and disease mechanisms. However, interpreting statistical associations as causal effects requires additional assumptions beyond those needed for standard statistical analyses. In this minireview, we introduce key concepts and principles for causal effect estimation in trans-regulatory single-cell CRISPR studies. We describe a set of assumptions under which estimates from existing statistical methods admit a causal interpretation and provide a concise overview of these approaches. Finally, through an illustrative example, we demonstrate how violations of these assumptions can bias estimated effects.},
}

*Single-Cell Analysis/methods
Humans
Single-Cell Gene Expression Analysis
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*CRISPR-Cas Systems/genetics
Gene Editing/methods
Animals
Transcriptome/genetics

@article {pmid42275032,
year = {2026},
author = {Yuan, S and Tan, D and Zhu, D and Balcazar, JL and Wang, H and Friman, VP and Sun, M and Hu, F},
title = {Global transmission and distribution of phage-encoded cholera toxin genes constrained by toxin-repression genes and anti-phage defense systems.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
doi = {10.1093/ismejo/wrag139},
pmid = {42275032},
issn = {1751-7370},
abstract = {Cholera is a severe diarrheal disease caused by toxigenic Vibrio cholerae, whose virulence depends on lysogenic infection by CTXφ bacteriophages encoding the cholera toxin genes (ctxA and ctxB) and associated accessory genes (ace and zot). However, the global distribution and transmission dynamics of phage-encoded cholera toxin genes across environments remain poorly understood. To address this, we performed a large-scale bioinformatic analysis of publicly available whole genomes. We show that both phages and bacteria carrying toxin genes are globally distributed across human-associated, freshwater, fish, and mammalian habitats, with Vibrio and Aeromonas being the dominant bacterial taxa and Inoviridae is the most prevalent phage family. Phage-mediated horizontal gene transfer (HGT) of toxin genes occurred in both Vibrio and non-Vibrio species, with the highest transfer between Inoviridae and V. cholerae occuring predominantly among bacteria from the same habitat. Temporal analysis revealed an increase in candidate HGT events after 2000, peaking at 377845 events during 2010-2019. HGT events negatively correlated with the presence of CRISPR-Cas system and toxin-repression genes (hns, hapR, and tsrA) in host bacteria. Experimental validation indicated that H-NS and HapR inhibit phage infection by repressing phage release. Together, our results suggest that CRISPR-Cas phage defense system and toxin-repression mechanisms could constrain the spread of toxin-carrying phages, with potential implications for the occurrence and severity of cholera outbreaks worldwide.},
}

@article {pmid42276271,
year = {2026},
author = {Hiya-Kawaguchi, U and Kashiwakura, Y and Baatartsogt, N and Lodoi, K and Togashi, T and Sato, T and Tsuchida, K and Batjargal, K and Hayakawa, M and Sato, R and Sato, Y and Ohmori, T},
title = {Non-viral delivery of a base editor enables personalized correction of hemophilia B nonsense variants in a mouse model.},
journal = {Journal of thrombosis and haemostasis : JTH},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jtha.2026.06.003},
pmid = {42276271},
issn = {1538-7836},
abstract = {BACKGROUND: Hemophilia B is caused by loss-of-function variants in the F9 gene encoding factor IX (FIX). While adeno-associated virus (AAV) vector-based gene therapy can restore FIX expression for over a decade, it does not restore the pathogenic variants and faces limitations related to immunogenicity and re-dosing.

AIM: To develop a variant-specific, non-viral base editing strategy using lipid nanoparticles (LNPs) for the precise personalized correction of pathogenic nonsense variations in F9.

METHODS: Editing efficiency was evaluated in HEK293 cells harboring each F9 variant treatable by A•T to G•C base editing. Functional and molecular restoration in vivo was assessed in hemophilia B model mice created with AAV8 vectors expressing F9 variants. LNPs harboring adenine base editor (Cas9 nickase conjugated with ABE8e) mRNA and the corresponding guide RNA were intravenously injected into the mice.

RESULTS: We created HEK293 stably expressing six correctable nonsense variants: c.1067G>A, c.1068G>A, c.1222C>T, c.1292G>A, c.1358G>A, and c.1359G>A. Transfection of the cells with a base editor consisting of SpRY, an engineered SpCas9 with broader proximal protospacer adjacent motif compatibility, and ABE8e, together with the guide RNA sequence, successfully induced A>G conversion at all target sites. The administration of LNPs harboring the base editor mRNA and guide RNA to the hemophilia B model restored the pathological variants (62.8% ± 14.4% for c.1068G>A, 35.9% ± 3.8% for c.1222C>T, and 70.6% ± 2.7% for c.1292G>A) and increased plasma FIX activity.

CONCLUSION: The variant-specific, non-viral base editing platform represents a truly curative intervention for severe hemophilia B caused by single-nucleotide variants.},
}

@article {pmid42276705,
year = {2026},
author = {Radoua, A and Alrustom, B and Wang, J and Bordessoules, M and Chluba, J and Plenchette, S and Micheau, O},
title = {Efficient generation of isogenic FADD[-/-], RIPK1[-/-] and Caspase-8[-/-] cells using a the ptARgenOM non-viral CRISPR-Cas9 system.},
journal = {Methods in cell biology},
volume = {208},
number = {},
pages = {115-147},
doi = {10.1016/bs.mcb.2026.02.005},
pmid = {42276705},
issn = {0091-679X},
mesh = {*Caspase 8/genetics ; Animals ; *CRISPR-Cas Systems/genetics ; *Fas-Associated Death Domain Protein/genetics/deficiency ; *Receptor-Interacting Protein Serine-Threonine Kinases/genetics/deficiency ; *Gene Knockout Techniques/methods ; Mice ; Humans ; Green Fluorescent Proteins/genetics ; Apoptosis/genetics ; Genetic Vectors ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; Transfection ; },
abstract = {The generation of isogenic knockout (KO) cell lines for intracellular proteins using non-viral CRISPR-Cas9 approaches has long been technically demanding and time-consuming. Here, we describe a streamlined and cost-effective method based on ptARgenOM, an all-in-one mammalian expression vector designed for efficient delivery of the CRISPR-Cas9 system. This vector co-expresses the guide RNA (gRNA) and Cas9 endonuclease, which is fused to a ribosomal skipping peptide sequence followed by the enhanced green fluorescent protein (EGFP) and the puromycin N-acetyltransferase. This design enables transient, expression-dependent antibiotic selection and fluorescence-based enrichment of successfully transfected cells, facilitating the rapid generation of isogenic KO populations or clones. The method is particularly well-suited, though not limited, to functional studies involving intracellular components of the cell death machinery, including both the extrinsic and intrinsic apoptotic signaling pathways. We illustrate the utility of this system by targeting and deleting FADD, Caspase-8, and RIPK1. This approach can be easily adapted to any intracellular target protein, offering a robust platform for gene function analysis in mammalian cells.},
}

*Caspase 8/genetics
Animals
*CRISPR-Cas Systems/genetics
*Fas-Associated Death Domain Protein/genetics/deficiency
*Receptor-Interacting Protein Serine-Threonine Kinases/genetics/deficiency
*Gene Knockout Techniques/methods
Mice
Humans
Green Fluorescent Proteins/genetics
Apoptosis/genetics
Genetic Vectors
Cell Line
RNA, Guide, CRISPR-Cas Systems/genetics
Transfection

@article {pmid41955489,
year = {2026},
author = {Jiang, B and Zhang, T and Lu, Y and Zhou, S and Xiao, W and Pan, S and Shi, N and Sheng, Y and Hu, J},
title = {CRISPR/Cas13a: Compensatory Target Activation Mechanism.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {32},
pages = {e24156},
pmid = {41955489},
issn = {2198-3844},
support = {22576127//National Natural Science Foundation of China/ ; 2023YFC2417200//National Key Research and Development Program of China/ ; 25ZR1401116//Sci-Tech Innovation Initiative Science and Technology Commission of Shanghai Municipality/ ; 24ZR1423000//Sci-Tech Innovation Initiative Science and Technology Commission of Shanghai Municipality/ ; 2025-YJRC-03//Putuo Hospital, Shanghai University of Traditional Chinese Medicine Start-up Grant/ ; 2019QN01Y725//Guangdong Provincial Pearl River Talents Program/ ; 202206010108//Science and Technology Program of Guangzhou/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; MicroRNAs/genetics ; Humans ; },
abstract = {CRISPR/Cas13a is a powerful RNA-targeting platform for molecular diagnostics, but conventional single-effector systems typically require contiguous RNA targets longer than ∼20-28 nt, limiting sensitivity and target flexibility. CRISPR/Cas13a-CTAM is presented as a compensatory target activation mechanism that facilitates synergistic Cas13a activation through two independently programmable short RNA effectors. By functionally decoupling allosteric activation and binding stabilization, CRISPR/Cas13a-CTAM supports robust activation by ultra-short RNA targets as short as 13 nt, substantially expanding the detectable target range. Compared with traditional single-effector Cas13a assays, CRISPR/Cas13a-CTAM achieves a detection limit of 1 fM for a 13-nt RNA target, representing an approximately tenfold sensitivity improvement. Notably, a single-nucleotide mismatch within the 13-nt target induces up to a 35-fold reduction in apparent cleavage rate, corresponding to a sevenfold enhancement in mismatch discrimination. The dual-effector architecture further enables simultaneous dual-target detection, demonstrated by dual miRNA profiling related to COVID-19 and combined detection of exosome membrane proteins. Moreover, the weakly activating effector was utilized as an anchoring module to achieve the first functional immobilization of Cas13a on a sensing surface, enabling in situ electrochemical miRNA detection. By overcoming the reliance on long RNA targets, CRISPR/Cas13a-CTAM provides a sensitive, programmable platform for RNA diagnostics and integrated biosensor development.},
}

*CRISPR-Cas Systems/genetics
MicroRNAs/genetics
Humans

@article {pmid42202397,
year = {2026},
author = {Han, S and Lin, X and Lei, Y and Liu, Z and Li, J and Ou, X},
title = {Universal primer-based RPA combined with parallel CRISPR/Cas12a decoding for rapid multi-species meat authentication.},
journal = {Food chemistry},
volume = {520},
number = {},
pages = {148905},
doi = {10.1016/j.foodchem.2026.148905},
pmid = {42202397},
issn = {1873-7072},
mesh = {*Meat/analysis/classification ; Animals ; *Food Contamination/analysis ; DNA Primers/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Swine ; },
abstract = {Rapid meat authentication is critical for food safety. Here, we report a two-step assay employing universal primer-based RPA amplification followed by species-specific CRISPR/Cas12a detection for the identification of 11 common meat species. A single universal primer pair enables broad amplification, while specific crRNAs allow parallel single-target CRISPR reactions. The assay takes approximately 40 min at constant temperature, costing ∼$4 per test with dual fluorescence and lateral flow strips. High specificity without cross-reactivity was observed, and detection limits ranged from 10[0] to 10[4] copies/μL. In binary meat mixtures, the fluorescence assay achieved adulteration detection limits of 0.05-0.5% (w/w), while the lateral flow format showed 0.05-5% (w/w) depending on the species. The method was verified using commercially processed products. This laboratory-validated strategy simplifies primer design and provides a promising platform for the qualitative screening of multiple meat targets, while further validation is required to assess its field robustness.},
}

*Meat/analysis/classification
Animals
*Food Contamination/analysis
DNA Primers/genetics
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Bacterial Proteins/genetics
Swine

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

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

@article {pmid41935970,
year = {2026},
author = {Chen, YW and Marpaung, DSS and Chen, YY and Singuru, MMR and Chuang, MC},
title = {Mismatch-Driven CRISPR/Cas12a Biosensing of UV-Induced DNA Lesions for Environmental Solar Exposure Surveillance.},
journal = {Environmental science & technology},
volume = {60},
number = {22},
pages = {15930-15939},
doi = {10.1021/acs.est.5c12461},
pmid = {41935970},
issn = {1520-5851},
mesh = {*Ultraviolet Rays ; *Biosensing Techniques ; *CRISPR-Cas Systems ; *DNA Damage ; Sunlight ; },
abstract = {Monitoring environmentally relevant ultraviolet (UV) radiation is critical for understanding its biological impacts on ecosystems and human health. However, conventional UV dosimeters lack the molecular sensitivity to detect DNA-level damage that initiates such effects. Here, we present a CRISPR/Cas12a-based biosensing platform capable of quantifying solar UV exposure through the detection of UV-induced thymine dimers in DNA activators. This system harnesses mismatch-driven suppression of Cas12a activity, enabling a reduction in the fluorescence signal in response to UV-induced molecular lesions. The impact of thymine arrangement and the dimerization position of the activators on sensitivity were investigated. UV-induced diminution in Cas12a's trans-cleavage efficiency (kcat/Km) was also characterized, revealing a 1.67-fold decrease as the UVB dose increased from 0 to 2 J/cm[2]. Under optimized conditions, the sensor achieved a detection limit of 0.029 J/cm[2] for UVB and demonstrated high sensitivity to UVC. Field validation under natural sunlight showed a strong correlation with reference radiometric measurements, validating the biosensor's accuracy and environmental relevance. The system's sensitivity to low lesion densities, straightforward mechanism, and simple operation highlights its potential for environmental surveillance, human health risk assessment, and ecological monitoring in response to solar UV radiation.},
}

*Ultraviolet Rays
*Biosensing Techniques
*CRISPR-Cas Systems
*DNA Damage
Sunlight

@article {pmid41962729,
year = {2026},
author = {Liu, Y and Chen, S and Zhang, C and Xue, H and Abubakar, MU and Yin, S and Yang, X and Fan, B and Tai, P and Xiong, M and Li, J and He, B},
title = {Asy-RPA/PCR combined with One-crRNA-CRISPR/Cas12a for simultaneous detection of multiple Clarithromycin resistance mutations in Helicobacter pylori.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {74},
number = {},
pages = {102942},
doi = {10.1016/j.nano.2026.102942},
pmid = {41962729},
issn = {1549-9642},
mesh = {*Helicobacter pylori/genetics/drug effects ; *Clarithromycin/pharmacology ; *CRISPR-Cas Systems/genetics ; *Mutation ; *Drug Resistance, Bacterial/genetics ; *Polymerase Chain Reaction/methods ; Humans ; Anti-Bacterial Agents/pharmacology ; Polymorphism, Single Nucleotide ; Rapid Diagnostic Tests ; Helicobacter Infections/microbiology/drug therapy/genetics ; },
abstract = {METHODS: Genetic testing for Clarithromycin resistance-associated single-nucleotide variations (SNVs) in H. pylori could be applied for formulating individual eradication plan. In this study, we integrated asymmetric recombinase polymerase amplification (Asy-RPA) with a single crRNA for CRISPR/Cas12a-designated the ARoRC system-to circumvent protospacer adjacent motif (PAM) dependency.

RESULTS: The ARoRC platform detected all targeted mutations with 100% agreement compared to Sanger sequencing. Assay sensitivity was determined as follows: A2143G (10[-2] ng/μL), A2142C (2.58 × 10[-3] ng/μL), A2142G (2.49 × 10[-3] ng/μL), and A2142G + A2143G (2.39 × 10[-3] ng/μL), enabling Asy-PCR-CRISPR/Cas12a detection suitable for fecal samples. The assay achieved visual results within 1 h using lateral flow strips, with no cross-reactivity to WT or non-target sequences.

DISCUSSION: We developed a rapid, ultrasensitive, and portable assay for detecting Clarithromycin resistance-associated mutations in H. pylori. The robustness of the platform in complex matrices such as feces, along with its dual readout capability (fluorescence and lateral flow), supports its potential for point-of-care (POC) application.},
}

*Helicobacter pylori/genetics/drug effects
*Clarithromycin/pharmacology
*CRISPR-Cas Systems/genetics
*Mutation
*Drug Resistance, Bacterial/genetics
*Polymerase Chain Reaction/methods
Humans
Anti-Bacterial Agents/pharmacology
Polymorphism, Single Nucleotide
Rapid Diagnostic Tests
Helicobacter Infections/microbiology/drug therapy/genetics

@article {pmid41974708,
year = {2026},
author = {Lai, S and Keller, MP and Zhang, J and Fang, Z and Xie, Y and Weng, C and Zhang, S and Zhang, S and Gao, P and Ke, L and Wang, Y and Mitok, KA and Clark, L and Schueler, KL and Liu, H and Hatipoglu, B and Hatzoglou, M and Chen, Y and Shalev, A and Jin, F and Attie, AD and Li, Y},
title = {Proinsulin regulators identified with CRISPR screen and in vivo mouse QTL mapping.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41974708},
issn = {2041-1723},
support = {R01 DK131437/DK/NIDDK NIH HHS/United States ; R01HG012384//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01 HG012384/HG/NHGRI NIH HHS/United States ; R01DK131437//U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes & Digestive & Kidney Diseases)/ ; P30 AG092586/AG/NIA NIH HHS/United States ; R01CA267872//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; R01HG009658//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01DK113185//U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes & Digestive & Kidney Diseases)/ ; R01 DK113185/DK/NIDDK NIH HHS/United States ; UG3NS132061//U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Animals ; *Quantitative Trait Loci/genetics ; *Proinsulin/metabolism/blood/genetics ; Mice ; Golgi Apparatus/metabolism ; Humans ; Protein Disulfide-Isomerases/genetics/metabolism ; Insulin-Secreting Cells/metabolism ; Insulin/metabolism ; Chromosome Mapping ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Exocytosis ; Cell Line ; Secretory Vesicles/metabolism ; },
abstract = {Altered proinsulin levels in β-cells and bloodstream are hallmarks of diabetes and other diseases, but our knowledge about the proinsulin regulators remains limited. Here we perform a genome-wide CRISPR screen to identify 84 proinsulin regulators that alter intracellular proinsulin/insulin ratio in a mouse β-cell line. The proinsulin regulators are distinct from the insulin regulators from a previous orthogonal CRISPR screen. Functional annotation of the proinsulin regulators highlights Golgi as the primary organelle for proinsulin storage and regulation. Trafficking towards the Golgi increases the intra-cellular proinsulin/insulin ratio, while trafficking away from the Golgi, including exocytosis and Golgi-to-ER retrograde transport, decreases the intracellular proinsulin levels. We also map mouse quantitative trait loci (QTLs) associated with plasma proinsulin levels and use the CRISPR screen results to pinpoint the causal genes within the QTL loci. Interestingly, protein disulfide isomerase Pdia6 is the strongest hit from both CRISPR screen and the in vivo QTL mapping. Knocking down Pdia6 significantly reduce proinsulin accumulation in Golgi and secretory granules. Intriguingly, Pdia6-depletion in both human and mouse β-cells does not affect the folding status of proinsulin but causes significantly impaired proinsulin production through a UPR-independent mechanism. Taken together, our genetic profiles provide mechanistic insights into the regulation of proinsulin/insulin homeostasis.},
}

Animals
*Quantitative Trait Loci/genetics
*Proinsulin/metabolism/blood/genetics
Mice
Golgi Apparatus/metabolism
Humans
Protein Disulfide-Isomerases/genetics/metabolism
Insulin-Secreting Cells/metabolism
Insulin/metabolism
Chromosome Mapping
CRISPR-Cas Systems
Clustered Regularly Interspaced Short Palindromic Repeats
Exocytosis
Cell Line
Secretory Vesicles/metabolism

@article {pmid42010284,
year = {2026},
author = {Zhao, J and Zhang, J and Gao, M and Miao, Z and Zhang, Y and Guo, Y and Fan, Z and Tian, J and Yang, L and Jiang, N and Ma, J and Jiao, J and Pan, J and Ma, X},
title = {Photoactivatable CRISPR/Cas13d via upconversion nanoparticles for deep tissue RNA engineering and orthopedic therapy.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42010284},
issn = {2041-1723},
support = {82572860//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82102639//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82302347//National Natural Science Foundation of China (National Science Foundation of China)/ ; 25JCYBJC01510, 22JCQNJC00850//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; 25YDTPJC00330//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; },
mesh = {Animals ; Mice ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; Humans ; *Tissue Engineering/methods ; Polyethyleneimine/chemistry ; *RNA/genetics ; Blue Light ; Bone and Bones ; Osteocytes/metabolism ; },
abstract = {Spatiotemporal control of RNA therapeutics remains a fundamental challenge limiting clinical translation. Here, we develop a photoactivatable CRISPR/Cas13d (paCas13d) system that enables non-invasive, light-controlled RNA manipulation in deep tissues. Through structure-guided engineering, we identify optimal split sites within RfxCas13d and create light-switchable fragments using CRY2PHR/CIBN optogenetic dimerization. To overcome the limited tissue penetration of blue light, we engineer polyethylenimine-functionalized upconversion nanoparticles (UCNPs-PEI) that serve dual roles as gene carriers and photon transducers, converting tissue-penetrating near-infrared (NIR) to blue light. The UCNPs-PEI@paCas13d system achieves precise spatiotemporal control of RNA targeting within bone tissue in vivo. In a murine steroid-associated osteonecrosis model, NIR-activated paCas13d achieves robust TET3 knockdown, disrupting the TET3-5hmC-PTEN axis that drives glucocorticoid-induced osteocyte apoptosis. This targeted intervention prevents bone deterioration, with treated mice showing preserved trabecular architecture, enhanced bone volume, and favorable shifts in bone turnover markers, while maintaining systemic glucocorticoid efficacy. Our platform combines the programmability of CRISPR/Cas13d with non-invasive optical control, offering a versatile approach for treating diseases requiring localized RNA modulation while minimizing systemic effects.},
}

Animals
Mice
*Nanoparticles/chemistry
*CRISPR-Cas Systems/genetics
Humans
*Tissue Engineering/methods
Polyethyleneimine/chemistry
*RNA/genetics
Blue Light
Bone and Bones
Osteocytes/metabolism

@article {pmid42080370,
year = {2026},
author = {Wang, Z and Li, J and Yue, Z and He, B and Zhang, W and Fang, Z and Xia, Q and Liu, Y and Li, Y},
title = {A Modular and Programmable Cas13d Platform for RNA Single Nucleotide Variant Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {32},
pages = {e23680},
pmid = {42080370},
issn = {2198-3844},
support = {32471528//National Natural Science Foundation of China/ ; 82404929//National Natural Science Foundation of China/ ; zz-RCPY-23-25//State Key Laboratory of Systems Medicine for Cancer/ ; zz-94-25-22//State Key Laboratory of Systems Medicine for Cancer/ ; SB2023-13//State Key Laboratory of Systems Medicine for Cancer/ ; zz-GZR-25-09//State Key Laboratory of Systems Medicine for Cancer/ ; 23QC1400600 to Y.L//Shanghai Rising-Star Program/ ; DFYC-LYJ2022 to Y.L//Shanghai Oriental Talent Youth Program/ ; 10000015Z155080000004//National Clinical Key Specialty Construction Project/ ; YG2023QNA09//Shanghai Jiao Tong University Star Program Medical, Industrial Cross Research Fund/ ; QiankeherencaiXKBF[2025]025)//Guizhou Provincial Science and Technology Projects/ ; XK202401//Jiading district medical key discipline construction project/ ; },
mesh = {Humans ; *Polymorphism, Single Nucleotide/genetics ; *CRISPR-Cas Systems/genetics ; *RNA/genetics ; },
abstract = {CRISPR-based nucleic acid diagnostics have shown broad potential, yet reliable single-nucleotide variant (SNV) discrimination remains limited by flanking sequence requirements that constrain targetability, and an inherent specificity-sensitivity trade-off where mismatch designs used to suppress wild type recognition often penalize enzymatic activity. Here we develop a scenario-guided Cas13d framework that supports pre-defined operating modes tailored to distinct analytical goals. Leveraging the minimal protospacer flanking site constraints of Cas13d, we first map mismatch-sensitive windows to derive rule-based crRNA designs that improve allelic discrimination. We then restore assay performance through structure-guided engineering of a miniaturized Cas13d scaffold by internally inserting auxiliary RNA binding domains (RBDs). Systematic benchmarking across representative oncology hotspots delineates two practical regimes comprising an ultra-sensitive, amplification-free mode in which a dual-RBD variant paired with optimized mismatched crRNAs achieves ∼0.6% variant allele fraction (VAF) detection, and a robust amplified mode incorporating optional loop-mediated isothermal amplification coupling that favors simpler architectures to balance performance and background across broader low-VAF ranges. In an evaluation of 45 clinical tumor RNA specimens spanning pancreatic, cholangiocarcinoma, and colorectal cancers, the assay correctly classified mutation status with full concordance for KRAS G12D, IDH1 R132C and BRAF V600E, with a subset of positive cases corroborated by orthogonal RT-ddPCR. A prospective IDH1 R132C clinical-matrix spike-in further supported sub-1% detection without pre-amplification. Collectively, this work establishes a configurable Cas13d toolkit and a rule-guided strategy for deploying CRISPR-based RNA SNV diagnostics with application-specific performance objectives.},
}

Humans
*Polymorphism, Single Nucleotide/genetics
*CRISPR-Cas Systems/genetics
*RNA/genetics

@article {pmid42172545,
year = {2026},
author = {Jiang, Y and Wen, H and Xu, J and Peng, W and Zhou, J and Mao, H and Gu, Z and He, Y},
title = {Dual-Gene CRISPR Editing via Peptide Dendrimers Regulates Redox Balance for Diabetic Wound Repair.},
journal = {Biomacromolecules},
volume = {27},
number = {6},
pages = {3647-3661},
doi = {10.1021/acs.biomac.6c00110},
pmid = {42172545},
issn = {1526-4602},
mesh = {Animals ; *Wound Healing/genetics/drug effects ; Mice ; *Dendrimers/chemistry/pharmacology ; Oxidation-Reduction ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Oxidative Stress ; Kelch-Like ECH-Associated Protein 1/genetics/antagonists & inhibitors ; NF-E2-Related Factor 2/genetics/metabolism ; *Peptides/chemistry ; *Diabetes Mellitus, Experimental/genetics ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; Nanoparticles/chemistry ; Genetic Therapy/methods ; Hypoxia-Inducible Factor-Proline Dioxygenases/genetics ; },
abstract = {The management of chronic diabetic wounds, plagued by persistent oxidative stress, remains a major clinical challenge. We devised a CRISPR/Cas9-based gene therapy to fundamentally reprogram this pathological microenvironment. A single system was engineered for the simultaneous knockdown of Keap1 and PHD2, key negative regulators of the Nrf2 and HIF-1α pathways, respectively. This payload was delivered by multifunctional peptide-modified lysine dendrimers (MsRNPs), which self-assembled into stable, positively charged nanoparticles that effectively complexed with DNA. The MsRNPs showed excellent biocompatibility and mediated efficient cellular uptake and gene editing in vitro, leading to reduced ROS levels. Consequently, a single topical application of the polyplexes in a diabetic mouse model robustly accelerated wound closure, enhanced collagen deposition, and promoted angiogenesis, driven by the synergistic activation of Nrf2 and HIF-1α. This study establishes a novel combinatorial gene-editing strategy and a versatile nanoplatform for treating oxidative stress-related pathologies.},
}

Animals
*Wound Healing/genetics/drug effects
Mice
*Dendrimers/chemistry/pharmacology
Oxidation-Reduction
*Gene Editing/methods
*CRISPR-Cas Systems
Oxidative Stress
Kelch-Like ECH-Associated Protein 1/genetics/antagonists & inhibitors
NF-E2-Related Factor 2/genetics/metabolism
*Peptides/chemistry
*Diabetes Mellitus, Experimental/genetics
Humans
Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism
Nanoparticles/chemistry
Genetic Therapy/methods
Hypoxia-Inducible Factor-Proline Dioxygenases/genetics

@article {pmid42190792,
year = {2026},
author = {Zhang, YH and Yuan, Y and Chen, BT and Yang, N and Chen, TJ and Lin, YT and Na, XM and Wang, SH and Xiong, YN and Zhu, MX and Chen, LZ and Mokwatlo, SC and Ouyang, P and Ling, C},
title = {Engineering complex phenotypes in Halomonas bluephagenesis TD01 via large-fragment manipulation and multiplex base editing.},
journal = {Metabolic engineering},
volume = {96},
number = {},
pages = {405-419},
doi = {10.1016/j.ymben.2026.05.008},
pmid = {42190792},
issn = {1096-7184},
mesh = {*Halomonas/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Metabolic Engineering/methods ; Polyhydroxyalkanoates/biosynthesis/genetics ; },
abstract = {Halomonas bluephagenesis is a representative platform strain of next generation industrial biotechnology (NGIB), enabling contamination-resistant open fermentation due to inherent tolerance to high salinity and alkalinity. However, progress in strain development has been constrained by limited genome engineering tools, particularly for large-fragment manipulation and multiplex base editing. Herein, we developed a counterselection marker-based single-plasmid system (pHaloFM) that leverages native homologous recombination to enable sequential insertion of fragments up to 8 kb and deletion of regions up to 50 kb. Additionally, we re-engineered a CRISPR/nCas9-assisted cytidine base editor system (pHaloBE) through host-specific adaptations, achieving multiplex editing of nine target sites. These tools were applied to engineer cellular morphology in one step, and successively construct polyhydroxyalkanoate (PHA) copolymers P34HB and PHBV biosynthetic pathways. This integrated toolkit resolves long-standing genetic manipulation bottlenecks in H. bluephagenesis and provides a systematic framework for engineering complex phenotypes in other non-model organisms.},
}

*Halomonas/genetics/metabolism
*Gene Editing/methods
*CRISPR-Cas Systems
*Metabolic Engineering/methods
Polyhydroxyalkanoates/biosynthesis/genetics

@article {pmid42202254,
year = {2026},
author = {Liu, Z and Zhang, R and Chang, C and Xu, D and Mao, D and Zhu, X and Xu, H},
title = {Detection of Ultralow-Frequency ctDNA Mutations Using a Dual Hairpin-Competition CRISPR/Cas14a System.},
journal = {Analytical chemistry},
volume = {98},
number = {22},
pages = {16682-16693},
doi = {10.1021/acs.analchem.6c02495},
pmid = {42202254},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Circulating Tumor DNA/genetics/blood ; *Mutation ; Polymerase Chain Reaction ; },
abstract = {Circulating tumor DNA (ctDNA) mutation profiling is essential for guiding targeted therapy and monitoring cancer recurrence, yet its clinical adoption is constrained by overwhelming wild-type DNA background and the limited sensitivity of existing platforms. Here, we introduce a dual hairpin-competition CRISPR/Cas14a (DHCC) system that integrates two sequential layers of hairpin competition: selective enrichment of mutant DNA during asymmetric PCR, followed by suppression of nonspecific sgRNA binding during Cas14a detection. This design dramatically enhances mutant-wild-type discrimination, elevating the discrimination factor from 2.48 to 145─a 58-fold improvement. While previous Cas14a methods achieve detection limits of 0.5-0.1% variant allele frequency (VAF), DHCC delivers a 250-fold sensitivity gain, routinely detecting four clinically relevant mutations (EGFR T790M, L858R, G719A, and NRAS Q61K) at VAFs as low as 0.002%. In multiplexed format, sensitivities of 0.005-0.01% VAF are maintained. Clinical validation using 22 plasma ctDNA samples demonstrated 100% concordance with droplet digital PCR for EGFR L858R detection. Compared to ddPCR and next-generation sequencing, DHCC substantially reduces turnaround time and cost while operating on standard qPCR instruments, eliminating the need for specialized infrastructure. By combining ultrahigh sensitivity, PAM independence, multiplexing preamplification capability, and practical affordability, DHCC provides an accessible platform for ctDNA-based liquid biopsy in clinical settings.},
}

Humans
*CRISPR-Cas Systems/genetics
*Circulating Tumor DNA/genetics/blood
*Mutation
Polymerase Chain Reaction

@article {pmid42212930,
year = {2026},
author = {Zhang, Y and Hao, L and Li, Q and Zhou, Z and Liu, D and Qiu, H and Yang, W and Zhang, B},
title = {Ultrasensitive Wash-Free Homogeneous CRISPR Assay Using Spatial Proximity Chemiluminescence Reporter.},
journal = {ACS nano},
volume = {20},
number = {22},
pages = {16001-16015},
doi = {10.1021/acsnano.5c22763},
pmid = {42212930},
issn = {1936-086X},
mesh = {*Luminescent Measurements/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Reactive Oxygen Species/metabolism ; Limit of Detection ; Luminescence ; },
abstract = {CRISPR-based diagnostics are promising platforms for point-of-care (POC) testing, but are often hindered by limited sensitivity and complex workflows. Here, we present a spatial proximity chemiluminescence (SPC) reporter that converts CRISPR-Cas12a trans-cleavage activity into a highly sensitive, excitation-free signal. Mechanistically, the intact SPC reporter ensures efficient intramolecular reactive oxygen species (ROS) transfer by spatially confining a catalytic donor and a luminescent acceptor. Upon target-activated Cas12a cleavage, this spatial proximity is disrupted, drastically attenuating ROS transfer and reducing oxidized luminescent acceptors for chemiluminescence. The SPC-CRISPR exhibits an attomolar-level limit of detection with an optimal nearly 50,000-fold sensitivity enhancement, and features an 8-log dynamic range suitable for target quantification. This platform exhibits robust resistance to matrix interference, ensuring high accuracy while requiring only minimal sample preprocessing. We demonstrate SPC-CRISPR is compatible with recombinase polymerase amplification to develop a single-tube reaction with a sensitivity of 1 copy/μL. Extensive clinical validation demonstrated 99.2% overall accuracy for HPV16 identification in 126 cervical swabs, alongside 86.4% accuracy for miR-19a profiling in 22 bladder cancer plasma samples. Furthermore, this wash-free homogeneous workflow is embedded in a portable and sealed microfluidic-based device for sample-to-result diagnostics, showing 100% concordance with qPCR. SPC-CRISPR integrates enhanced sensitivity and simplified operation, holding great potential for POC molecular diagnostics.},
}

*Luminescent Measurements/methods
*CRISPR-Cas Systems/genetics
Humans
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Reactive Oxygen Species/metabolism
Limit of Detection
Luminescence

@article {pmid42214852,
year = {2026},
author = {Xiao, S and Song, J and Chen, H and Yin, W and Yan, X and Hu, K and Shi, J and Yang, M},
title = {Digital droplet microfluidics integrating DNA walkers and CRISPR-Cas13a for simultaneous surface protein and miRNA profiling in single exosomes.},
journal = {Biosensors & bioelectronics},
volume = {310},
number = {},
pages = {118854},
doi = {10.1016/j.bios.2026.118854},
pmid = {42214852},
issn = {1873-4235},
mesh = {Humans ; *Exosomes/chemistry/genetics ; *MicroRNAs/genetics/isolation & purification/analysis ; *Erb-b2 Receptor Tyrosine Kinases/genetics/isolation & purification ; CRISPR-Cas Systems/genetics ; *Biosensing Techniques/instrumentation ; *Epithelial Cell Adhesion Molecule/genetics/isolation & purification/analysis ; DNA/chemistry ; Equipment Design ; },
abstract = {Tumor-derived exosomes carry multi-scale molecular signatures (e.g., surface proteins and nucleic acids) that reflect tumor heterogeneity, yet simultaneously profiling these biomarkers in single intact vesicles remains technically challenging. Herein, we developed a digital droplet microfluidic platform that integrates a DNA walker and a CRISPR/Cas13a system for the simultaneous detection of surface proteins (EpCAM, HER2) and miRNA (miR-21) at the single exosome level. This platform employed engineered liposome nanoprobes (eLipo-NPs) with EpCAM aptamers and hairpin probes (HPs) functionalized on their outer membranes, and encapsulated a CRISPR/Cas13a system within their lumen. Upon co-encapsulation with single exosomes into droplets, EpCAM-mediated membrane fusion redistributed HPs across the hybrid membrane and delivered CRISPR/Cas13a into the exosomes. The membrane-anchored DNA walker then bound HER2 and drove cyclic DNAzyme cleavage of HPs to restore red fluorescence. At the same time, crRNA-guided Cas13a recognized miR-21 and triggered trans-cleavage of reporters to generate green fluorescence. Digital counting of dual-positive droplets enabled quantitative single-exosome analysis with a limit of detection (LOD) of 10 particles/μL and a detection time of 60 min. Clinical validation using plasma-derived exosomes from 24 breast cancer patients and 14 healthy donors demonstrated distinct distributions among HER2-positive, HER2-negative, and healthy control groups, with the percentage of dual-positive droplets significantly correlated with clinical HER2 status, highlighting the platform's potential for liquid biopsy and precision oncology.},
}

Humans
*Exosomes/chemistry/genetics
*MicroRNAs/genetics/isolation & purification/analysis
*Erb-b2 Receptor Tyrosine Kinases/genetics/isolation & purification
CRISPR-Cas Systems/genetics
*Biosensing Techniques/instrumentation
*Epithelial Cell Adhesion Molecule/genetics/isolation & purification/analysis
DNA/chemistry
Equipment Design

@article {pmid42217942,
year = {2026},
author = {Chen, B and Yang, H and Zhao, J and Wang, Y and Li, H and Wang, C and Guo, L and Xu, J},
title = {Template-independent poly-adenine elongation enables multivalent CRISPR/Cas12a activation for amplified lateral flow biosensing.},
journal = {Talanta},
volume = {309},
number = {},
pages = {130054},
doi = {10.1016/j.talanta.2026.130054},
pmid = {42217942},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; *DNA Nucleotidylexotransferase/metabolism/analysis ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Associated Proteins/metabolism ; *Poly A/metabolism/chemistry ; Limit of Detection ; Rapid Diagnostic Tests ; *Endodeoxyribonucleases/metabolism ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Terminal deoxynucleotidyl transferase (TdT) is a template-independent DNA polymerase that plays a critical role in immune system development and serves as an important biomarker for acute lymphoblastic leukemia. However, current methods for TdT activity analysis often rely on sophisticated instrumentation and lack simple and portable detection formats. Herein, we report a TdT-enabled multivalent CRISPR/Cas12a lateral flow assay for sensitive and instrument-free detection of TdT activity. In this strategy, TdT-catalyzed poly-adenine (poly-A) extension converts enzymatic activity into adenine-rich DNA scaffolds, which recruit multiple crRNA molecules to trigger multivalent activation of Cas12a. This design effectively bridges TdT activity with CRISPR/Cas12a signal amplification. The activated Cas12a subsequently induces trans-cleavage of a reporter probe, and the cleavage event is translated into a visual signal on a lateral flow strip. The proposed assay enables sensitive detection of TdT with a limit of detection of 0.016 U/mL and a visual detection limit of 0.05 U/mL. In addition, the assay exhibits high specificity toward TdT over other polymerases and demonstrates satisfactory performance in human serum samples with recoveries ranging from 98.8% to 103.7%. This work expands the applicability of CRISPR/Cas12a systems to enzyme activity sensing and provides a simple and practical platform for point-of-care detection of TdT.},
}

*CRISPR-Cas Systems
*DNA Nucleotidylexotransferase/metabolism/analysis
*Biosensing Techniques/methods
Humans
*CRISPR-Associated Proteins/metabolism
*Poly A/metabolism/chemistry
Limit of Detection
Rapid Diagnostic Tests
*Endodeoxyribonucleases/metabolism
*Bacterial Proteins/metabolism/genetics

@article {pmid42229577,
year = {2026},
author = {Saberian, M and Roosta, A and Afrisham, R},
title = {CRISPR-dCas9 epigenetic reprogramming in cancer: platforms, immuno-modulation and delivery challenges.},
journal = {Gene},
volume = {1005},
number = {},
pages = {150246},
doi = {10.1016/j.gene.2026.150246},
pmid = {42229577},
issn = {1879-0038},
mesh = {Humans ; *Neoplasms/genetics/therapy/immunology ; *CRISPR-Cas Systems/genetics ; Epigenome Editing ; *Epigenesis, Genetic ; Immunotherapy/methods ; Animals ; *Gene Editing/methods ; Cellular Reprogramming/genetics ; },
abstract = {CRISPR-dCas9 (catalytically dead Cas9) has revolutionized targeted epigenetic editing, offering locus-specific modulation of gene expression without altering DNA sequence. Beyond conventional approaches, novel strategies are rapidly emerging. These include combinatorial epigenetic reprogramming (co-recruiting multiple chromatin modifiers to a single locus), precision enhancer targeting (modulating oncogenic cis-regulatory elements), epigenetic modulation of immune pathways (reprogramming tumor or immune cells to boost anti-tumor immunity), and next-generation delivery systems for dCas9-based tools. This review synthesizes peer-reviewed literature (2015-2025) to highlight promising, yet still preclinical, advances in combinatorial reprogramming, enhancer targeting, immune-modulatory epigenetic approaches and delivery strategies, and to identify gaps that must be addressed prior to clinical translation. We highlight multi-effectors platforms (e.g. SunTag-like arrays, SSSavi modular docking, CRISPRoff memory writers) that amplify and diversify chromatin modifications. Precision enhancer editing systems (e.g. enCRISPRa/enCRISPRi) enable direct activation or silencing of distal regulatory elements in cancer cells. Epigenetic immunotherapy approaches use dCas9-activators to upregulate NK/T-cell ligands (MICA/MICB) and antigen-presentation genes (MHC I/II) in tumor cells. Finally, we survey innovations in dCas9 delivery that address in vivo challenges. Our review critically evaluates these advances, identifies gaps (off-target effects, context-dependence), and outlines future directions toward precision epigenetic therapies for diverse cancers.},
}

Humans
*Neoplasms/genetics/therapy/immunology
*CRISPR-Cas Systems/genetics
Epigenome Editing
*Epigenesis, Genetic
Immunotherapy/methods
Animals
*Gene Editing/methods
Cellular Reprogramming/genetics

@article {pmid42242804,
year = {2026},
author = {Qian, J and Lu, J and Chen, X and Shen, H and Lu, F},
title = {Programmable Fc-encoded DNA tile-cube capture enables a thrombin-activated ratiometric ECL/SERS biosensor via a PAM-engineered toehold switch and CRISPR/Cas12a cleavage.},
journal = {Analytica chimica acta},
volume = {1415},
number = {},
pages = {345709},
doi = {10.1016/j.aca.2026.345709},
pmid = {42242804},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *Thrombin/analysis ; *CRISPR-Cas Systems/genetics ; Spectrum Analysis, Raman ; Electrochemical Techniques/methods ; *DNA/chemistry ; Humans ; Luminescent Measurements ; Electrodes ; *CRISPR-Associated Proteins/metabolism/chemistry ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; Dendrimers ; },
abstract = {BACKGROUND: Accurate thrombin detection is important for coagulation-related assessment, but reliable quantification at ultralow levels remains challenging because matrix interference, electrode-to-electrode variation, and single-channel signal drift can compromise analytical accuracy. Herein, we developed a thrombin-responsive ratiometric electrochemiluminescence/surface-enhanced Raman scattering (ECL/SERS) biosensor integrating Fc-encoded DNA tiles, a DNA-cube capture scaffold, and a PAM-engineered toehold-switch-regulated CRISPR/Cas12a module on a Ti3C2/CsPbBr3@PDA@Au-modified electrode.

RESULTS: An intentionally cleavable linker probe (LP) serves as the bridge for retaining Fc-rich DNA tiles near the electrode. Without thrombin, intact LP enables tile capture, causing ECL quenching and strong Fc SERS output. With thrombin, split-aptamer proximity assembly activates the toehold switch and Cas12a/crRNA, leading to LP cleavage, Fc-tile depletion, ECL recovery, and SERS attenuation. The anti-correlated signals were integrated as Q = IECL/ISERS. The biosensor showed a detection range from 1 × 10[-7] to 1 × 10[-1] nM and a detection limit of approximately 0.064 fM. Synthetic cleaved LP standards confirmed that LP cleavage can be directly converted into ratiometric ECL/SERS switching. Serum spike-recovery tests gave recoveries of 96.8%-104.0%.

SIGNIFICANCE: This work establishes a programmable capture-release strategy that converts thrombin recognition into CRISPR/Cas12a-mediated LP cleavage and deterministic interfacial reconfiguration, providing a sensitive, internally referenced, and extensible platform for protein biosensing.},
}

*Biosensing Techniques/methods
*Thrombin/analysis
*CRISPR-Cas Systems/genetics
Spectrum Analysis, Raman
Electrochemical Techniques/methods
*DNA/chemistry
Humans
Luminescent Measurements
Electrodes
*CRISPR-Associated Proteins/metabolism/chemistry
Aptamers, Nucleotide/chemistry
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
Dendrimers

@article {pmid41709520,
year = {2026},
author = {Kulshreshtha, A and Ramasamy, M and Irigoyen, S and Padilla, CS and Tu, CK and Laughlin, K and Borneman, J and Mandadi, KK},
title = {High-efficiency genome-editing, transgene evaluation, and antimicrobial efficacy testing using Citrus medica L. hairy roots.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {4},
pages = {e70745},
pmid = {41709520},
issn = {1365-313X},
support = {2025-70029-44033//National Institute of Food and Agriculture/ ; 2024-51181-43464//National Institute of Food and Agriculture/ ; 2021-70029-36056//National Institute of Food and Agriculture/ ; 2019-70016-29066//National Institute of Food and Agriculture/ ; HATCH TEX09621//National Institute of Food and Agriculture/ ; TEX0-7790//National Institute of Food and Agriculture/ ; //Texas A&M AgriLife Institute for Advancing Health Through Agriculture/ ; //Texas A&M Vegetable and Fruit Improvement Center/ ; 124190-96210//Texas A&M AgriLife Research Insect-vectored Disease Seed Grants/ ; },
mesh = {*Citrus/genetics/microbiology ; *Plant Roots/genetics/microbiology ; Plants, Genetically Modified/genetics ; *Plant Diseases/microbiology ; *Gene Editing/methods ; Transformation, Genetic ; Transgenes/genetics ; Disease Resistance/genetics ; Liberibacter ; CRISPR-Cas Systems ; Anti-Infective Agents/pharmacology ; Rhizobiaceae ; },
abstract = {Huanglongbing (HLB) disease, associated with the fastidious bacterium Candidatus Liberibacter asiaticus (CLas), has a significant impact on citrus production worldwide. Conventional biochemical and genetic evaluation studies to identify potential disease resistance strategies have been mainly hindered due to the inability to culture CLas in a defined medium and the general recalcitrance of Citrus cultivars (grapefruits and oranges) to Agrobacterium-mediated plant transformation. We previously demonstrated the utility of plant hairy roots to co-cultivate CLas. In this study, we developed a hairy root transformation system using citron (Citrus medica L.), which is highly amenable to Rhizobium-mediated hairy root transformation. The explant survival and hairy root transformation efficiencies were up to 100% and 73%, respectively, and transgenic roots can be attained in as little as 30-60 days. We demonstrate the utility of this citron-based hairy root transformation for rapid CRISPR/Cas9-mediated gene editing, transgene evaluation, and antimicrobial efficacy testing. The citron-based hairy root transformation system will significantly help the research community to speed-track the assessment of potential HLB disease resistance strategies.},
}

*Citrus/genetics/microbiology
*Plant Roots/genetics/microbiology
Plants, Genetically Modified/genetics
*Plant Diseases/microbiology
*Gene Editing/methods
Transformation, Genetic
Transgenes/genetics
Disease Resistance/genetics
Liberibacter
CRISPR-Cas Systems
Anti-Infective Agents/pharmacology
Rhizobiaceae

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

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

@article {pmid41865336,
year = {2026},
author = {An, H and Kim, H and Kim, DY and Yoon, HJ and Lee, JY and Eo, WK and Kim, MY and Kim, KH and Cha, HJ},
title = {Transcriptomic analysis of zonula occludens-1 (ZO-1) knockout in ovarian cancer cell lines.},
journal = {Genes & genomics},
volume = {48},
number = {6},
pages = {911-921},
pmid = {41865336},
issn = {2092-9293},
mesh = {Humans ; Female ; *Zonula Occludens-1 Protein/genetics/metabolism ; *Ovarian Neoplasms/genetics/metabolism/pathology ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Epithelial-Mesenchymal Transition/genetics ; *Transcriptome/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Gene Expression Profiling ; },
abstract = {BACKGROUND: Zonula occludens-1 (ZO-1) is a crucial tight junction protein that regulates intercellular permeability and adhesion, thereby preserving the integrity of epithelial and endothelial barriers. ZO-1 is associated with tumorigenesis and the progression of epithelial-mesenchymal transition (EMT), invasion, and metastasis. In our previous study, knockout (KO) of ZO-1 using clustered regularly interspaced short palindromic repeats (CRISPR) reduced proliferation but increased migration and invasion, suggesting that ZO-1 may have a dual role. Therefore, this study aimed to elucidate the role of ZO-1 in ovarian cancer by analyzing transcriptomic changes associated with ZO-1.

OBJECTIVE: This study aims to elucidate the impact of ZO-1 KO on gene expression in ovarian cancer cells by performing comparative RNA sequencing (RNA-seq) analysis on two distinct ZO-1 KO ovarian cancer cell lines, SKOV3 and SNU119.

METHODS: ZO-1 was knocked out in SKOV3 and SNU119 cells using CRISPR-Cas9 technology. After identifying differentially expressed genes (DEGs) through RNA sequencing, Gene Ontology (GO) and pathway enrichment analyses were performed. The selected targets were subsequently validated using reverse transcription quantitative PCR (RT-qPCR) and Western blot analysis to assess both transcript- and protein-level expression changes.

RESULTS: Transcriptomic analysis revealed over 400 DEGs in each cell line. Of these, 14 genes were consistently upregulated in both cell lines, while 24 genes were consistently downregulated. The common DEGs were visualized using a heatmap, and a subset of these genes was further validated by RT-qPCR and Western blot analyses. TGFB2 expression was consistently altered at both the mRNA and protein levels following ZO-1 KO in both cell lines. Similar expression patterns were observed for THBS1, VCAN, ITGB8, SEMA3A, and GAS6. The concordant changes observed in transcriptomic and protein analyses suggest a consistent association between ZO-1 KO and TGFB2 expression.

CONCLUSION: ZO-1 KO in ovarian cancer cells induces substantial transcriptional reprogramming, particularly affecting genes associated with extracellular matrix organization and signaling pathways. Multiple candidate genes showed consistent alterations at both the mRNA and protein levels, supporting the robustness of the observed transcriptional changes. These findings provide a framework for understanding ZO-1-associated regulatory networks in ovarian cancer.},
}

Humans
Female
*Zonula Occludens-1 Protein/genetics/metabolism
*Ovarian Neoplasms/genetics/metabolism/pathology
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
Epithelial-Mesenchymal Transition/genetics
*Transcriptome/genetics
Gene Knockout Techniques
CRISPR-Cas Systems
Gene Expression Profiling

@article {pmid42023429,
year = {2026},
author = {Lin, TM and Chang, HF and Lin, TC and Lin, CH and Sun, YL and Lin, CS},
title = {Gene Therapy and Gene Editing in Type 1 Diabetes: CRISPR-Based β-Cell Replacement and Treg Immune Modulation Approaches.},
journal = {Diabetes, obesity & metabolism},
volume = {28},
number = {7},
pages = {5476-5491},
doi = {10.1111/dom.70800},
pmid = {42023429},
issn = {1463-1326},
support = {//Ministry of Education (MOE), Taiwan/ ; NSTC 114-2321-B-A49-001//National Science and Technology Council (NSTC), Taiwan/ ; },
mesh = {*Diabetes Mellitus, Type 1/therapy/immunology/genetics ; Humans ; *Genetic Therapy/methods ; *Insulin-Secreting Cells/immunology/transplantation ; *Gene Editing/methods ; *T-Lymphocytes, Regulatory/immunology ; Animals ; *CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease marked by the destruction of pancreatic β-cells, resulting in lifelong dependence on exogenous insulin. Despite advances in insulin delivery and glucose monitoring technologies, patients remain at risk for acute and long-term complications, underscoring the need for curative strategies. Gene therapy and gene-editing technologies are emerging as transformative approaches capable of restoring β-cell function, modulating immune responses and potentially achieving durable remission.

METHOD: This review synthesizes basic science foundations and clinical trial evidence, focusing on five key protocols (NCT03162237, NCT05210530, NCT05241444, NCT05565248 and NCT06938334).

RESULTS: Strategies include immune modulation (PD-L1, FOXP3), β-cell replacement (CRISPR-edited progenitors, xenotransplantation) and combination approaches. Early-phase clinical trials have demonstrated feasibility and safety; however, long-term efficacy, durability and scalability remain uncertain. Critical challenges include potential off-target effects in CRISPR editing, risks of insertional mutagenesis, safety concerns in xenotransplantation and achieving a balance between immune tolerance and protective immunity. Future directions emphasize combination therapies, personalized medicine and next-generation editing tools such as base and prime editing.

CONCLUSION: Together, these efforts represent a paradigm shift from symptomatic insulin replacement toward curative interventions, while highlighting the considerable translational hurdles that must be overcome before routine clinical application.},
}

*Diabetes Mellitus, Type 1/therapy/immunology/genetics
Humans
*Genetic Therapy/methods
*Insulin-Secreting Cells/immunology/transplantation
*Gene Editing/methods
*T-Lymphocytes, Regulatory/immunology
Animals
*CRISPR-Cas Systems

@article {pmid42119166,
year = {2026},
author = {Huang, D and Sun, D and Ou, C and Zhang, X and Luo, R and Kou, X and Li, X and Li, Y and Le, H and Li, W and You, Y and Gong, C},
title = {A hierarchical self-adjuvanted nanoCRISPR-based vaccine restores endogenous immune recognition and surveillance to amplify adaptive immune responses.},
journal = {Biomaterials},
volume = {334},
number = {},
pages = {124285},
doi = {10.1016/j.biomaterials.2026.124285},
pmid = {42119166},
issn = {1878-5905},
mesh = {Animals ; *Adaptive Immunity ; *Cancer Vaccines/immunology ; Humans ; *Nanoparticles/chemistry ; B7-H1 Antigen/genetics/immunology ; Nanovaccines ; Cell Line, Tumor ; CRISPR-Cas Systems ; Adjuvants, Immunologic ; Mice ; Female ; Mice, Inbred C57BL ; },
abstract = {Tumor vaccines are considered a promising approach in immunotherapy, designed to boost the immune system's capacity to identify tumor-associated antigens and subsequently trigger immune responses against tumors. However, the inherent genetic instability of tumor cells frequently results in decreased expression or loss of antigen and/or major histocompatibility complex (MHC) expression and upregulation of immune checkpoint molecule PD-L1, thus evading endogenous immune recognition and surveillance. Herein, we developed a hierarchical self-adjuvanted nanoCRISPR-based vaccine (HEDERA) loaded with LSD1/PD-L1 dual-editing CRISPR/Cas9 system, seeking to reinstate the endogenous immune detection and monitoring mechanisms to enhance adaptive immune reactions. Knockdown of LSD1 increases the presence of tumor-specific antigens and major histocompatibility complex class I molecules on the surface of cancer cells, thereby restoring immune recognition. Simultaneously, silencing PD-L1 alleviates the "exhaustion" of T cells and reactivates their cytotoxic activity. Moreover, LSD1 knockdown activates the type I interferon pathway to induce a self-adjuvant effect that enhances innate immune responses and thereby strengthens T cell-mediated adaptive immunity. This dual strategy achieves unprecedented efficacy, with 90% primary tumor inhibition, and demonstrates an 87.3% and 90.6% inhibition rate for post-surgical metastatic and recurrent tumors, respectively. Overall, HEDERA overcomes the single-action constraint of traditional tumor vaccines, and avoids combined medication-related poor patient compliance, delivering a more efficient, convenient integrated tumor immunotherapy solution.},
}

Animals
*Adaptive Immunity
*Cancer Vaccines/immunology
Humans
*Nanoparticles/chemistry
B7-H1 Antigen/genetics/immunology
Nanovaccines
Cell Line, Tumor
CRISPR-Cas Systems
Adjuvants, Immunologic
Mice
Female
Mice, Inbred C57BL

@article {pmid42186741,
year = {2026},
author = {Li, J and Ji, C and Yang, W and Han, Y and Zhao, P and Cai, X and Tian, S and Zhu, W and Zhang, J and Xu, J and Yang, W and Li, F and Liu, P},
title = {Engineered CRISPR/Cas12a2 Nanoprobe Imaging in Living Cells for Precise Tumor Diagnosis.},
journal = {Small methods},
volume = {10},
number = {11},
pages = {e70727},
pmid = {42186741},
issn = {2366-9608},
support = {U25C2029//National Natural Science Foundation of China/ ; 32371468//National Natural Science Foundation of China/ ; 22474077//National Natural Science Foundation of China/ ; 23ZR1461400//Shanghai Municipal Natural Science Foundation/ ; 22ZR1459600//Shanghai Municipal Natural Science Foundation/ ; YG2023ZD07//Medical-Engineering Joint Funds from the Shanghai Jiao Tong University/ ; YG2024QNB09//Medical-Engineering Joint Funds from the Shanghai Jiao Tong University/ ; 20234Y0201//Foundation of Shanghai Municipal Health Commission/ ; 2022JC002//Foundation of Shanghai Municipal Health Commission/ ; TMSK-2024-203//National Key Scientific Infrastructure for Translational Medicine (Shanghai)/ ; 10000015Z155080000004//2024 National Clinical Key Specialty Construction Project/ ; XK202401//Jiading District Medical Key Discipline Construction Project/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Animals ; RNA, Messenger/genetics/metabolism ; Mice ; Cell Line, Tumor ; Glutathione ; CRISPR-Associated Proteins/genetics ; Molecular Imaging/methods ; *Neoplasms/diagnosis/diagnostic imaging ; },
abstract = {Messenger RNA (mRNA) imaging in tumor cells plays a crucial role in monitoring the occurrence and development of tumors. However, achieving highly specific and sensitive mRNA imaging remains a significant challenge due to the complex intracellular environment and high background signal. Here, we engineered a CRISPR/Cas12a2 system with an RNA blocking strand that binds to CRISPR RNA (crRNA). After glutathione (GSH) stimulation, the RNA blocking strand is cleaved, allowing the release of crRNA and restoring the capability of CRISPR/Cas12a2 ribonucleoprotein (RNP). Furthermore, we developed a nanoprobe (termed eRNP-FHR) by converging engineered Cas12a2 RNP (eRNP) with framework-hotspot reporters (FHR). FHR features four vertices that modify the sgc8 aptamer to specifically target the protein tyrosine kinase 7 receptor on the surface of tumor cell membranes, link to the eRNP by hybridizing with crRNA, and incorporate fluorescence quenching groups. The eRNP-FHR precisely targets tumor cells through aptamer-mediated endocytosis, specifically recognizes mRNA upon GSH stimulation, and simultaneously cleaves FHR to release a significant fluorescent signal. Excitingly, eRNP-FHR successfully achieved imaging of baculoviral IAP repeat-containing 5 mRNA in pancreatic tumor cells, accurately distinguishing pancreatic tumor cells from normal cells. In a murine pancreatic tumor model, eRNP-FHR exhibited excellent mRNA imaging, highlighting significant potential for precise tumor diagnosis.},
}

Humans
*CRISPR-Cas Systems/genetics
Animals
RNA, Messenger/genetics/metabolism
Mice
Cell Line, Tumor
Glutathione
CRISPR-Associated Proteins/genetics
Molecular Imaging/methods
*Neoplasms/diagnosis/diagnostic imaging

@article {pmid42189082,
year = {2026},
author = {Wu, Y and Jin, R and Lei, T and Liu, J and Chang, Y and Zhang, Z and Li, J and Liu, M},
title = {Aptamer-Coupled Droplet CRISPR/Cas12a Enables Ultrasensitive sPD-L1 Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {22},
pages = {16296-16305},
doi = {10.1021/acs.analchem.6c00779},
pmid = {42189082},
issn = {1520-6882},
mesh = {*Aptamers, Nucleotide/chemistry ; Humans ; *CRISPR-Cas Systems/genetics ; *B7-H1 Antigen/blood ; Limit of Detection ; Lung Neoplasms/blood/diagnosis ; },
abstract = {Ultrasensitive detection of soluble programmed death-ligand 1 (sPD-L1) in peripheral blood is essential for early cancer diagnosis and immunotherapy monitoring. Conventional enzyme-linked immunosorbent assays lack the requisite sensitivity, whereas PCR quantifies nucleic-acid surrogates rather than the immunologically active protein. Here we report an aptamer-coupled droplet CRISPR/Cas12a (ADC) platform that integrates a structure-switching aptamer with picolitre droplet microfluidics to achieve femtomolar quantification of sPD-L1 within 70 min. Target binding with aptamer displaces a blocking sequence that activates Cas12a trans-cleavage, generating fluorescent droplets without preamplification. Confinement in picolitre droplets accelerates reaction kinetics through elevated local reagent concentrations and suppresses background fluorescence, collectively enhancing sensitivity. The assay exhibits a 0.5 pM limit of detection for sPD-L1, a dynamic range spanning 3 orders of magnitude, and 100% diagnostic accuracy in blinded plasma from lung cancer patients and healthy donors. The modular ADC architecture is readily adaptable to other protein biomarkers, offering a universal strategy for rapid, ultrasensitive liquid-biopsy analysis.},
}

*Aptamers, Nucleotide/chemistry
Humans
*CRISPR-Cas Systems/genetics
*B7-H1 Antigen/blood
Limit of Detection
Lung Neoplasms/blood/diagnosis

@article {pmid42217941,
year = {2026},
author = {Liu, D and Ma, G and Bai, L and Guo, K and Wang, T and Jiang, Z and Qian, F and Wang, Y and Pang, Y and Zou, W and Wang, R},
title = {STAR-CRISPR: a one-pot ultraspecific CRISPR strategy for rapid, visualized SNV detection and genotyping in point-of-care diagnostics.},
journal = {Talanta},
volume = {309},
number = {},
pages = {130036},
doi = {10.1016/j.talanta.2026.130036},
pmid = {42217941},
issn = {1873-3573},
mesh = {Humans ; *Point-of-Care Systems ; *Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems ; *Pancreatic Neoplasms/genetics/diagnosis ; *Leukemia, Myeloid, Acute/genetics/diagnosis ; *Genotyping Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Rapid Diagnostic Tests ; Genotype ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Single nucleotide variation (SNV), as a key biomarker for disease diagnosis and personalized treatment, faces challenges in rapid and accurate detection. This study developed a single-tube accelerated recognition of SNVs strategy named STAR-CRISPR, which could accomplish SNV detection within only 20 min. This method integrated isothermal amplification and CRISPR/Cas12b cleavage system in one pot, and results could be directly identified by the naked eye. This method could accurately distinguish single-base differences, and could detect as low as 1% mutations against high background interference. We verified the proposed method by testing 70 clinical samples of idiopathic chronic pancreatitis, pancreatic cancer and acute myeloid leukemia. Results showed 100% consistency with next-generation sequencing results, demonstrating good accuracy and reliability of the proposed method. To further facilitate point-of-care diagnosis, we developed integrated miniature microfluidic chips, which greatly simplified sample identification and enabled logical interpretation of results. The combined STAR-CRISPR and microfluidic platform not only identifies SNVs but also supports simultaneous visual genotyping of wild-type, homozygous, and heterozygous mutations. Consequently, the proposed strategy is accurate, rapid, and versatile, holding significant potential for next-generation molecular diagnostics.},
}

Humans
*Point-of-Care Systems
*Polymorphism, Single Nucleotide
*CRISPR-Cas Systems
*Pancreatic Neoplasms/genetics/diagnosis
*Leukemia, Myeloid, Acute/genetics/diagnosis
*Genotyping Techniques/methods
Nucleic Acid Amplification Techniques/methods
Rapid Diagnostic Tests
Genotype
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41707424,
year = {2026},
author = {Zhao, L and Liu, Z and Ding, G and Zhu, Y and Wang, H and Liu, R and Qu, F and Ao, Q and Zhu, X and Zhang, Y and Yang, G and Wang, Z},
title = {Dual-readout aptasensor based on CRISPR/Cas12a and nanozyme for accurate detection of KIM-1 and its application in kidney transplant prognosis.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118533},
doi = {10.1016/j.bios.2026.118533},
pmid = {41707424},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Hepatitis A Virus Cellular Receptor 1/isolation & purification ; *Aptamers, Nucleotide/chemistry ; CRISPR-Cas Systems ; *Kidney Transplantation ; Limit of Detection ; Prognosis ; Metal Nanoparticles/chemistry ; Silver/chemistry ; Colorimetry/methods ; Metal-Organic Frameworks/chemistry ; SELEX Aptamer Technique ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Kidney injury molecule-1 (KIM-1) has emerged as a pivotal prognostic biomarker for renal allograft function. However, its detection remains challenging due to sensitivity and accuracy limitations. An innovative biosensing platform synergizing aptamer recognition, CRISPR trans-cleavage and nanozyme amplification for dual-readout KIM-1 detection has been presented in this work. This platform employs a meticulously selected high-affinity aptamer with capillary electrophoresis SELEX for specific target recognition, leverages the CRISPR/Cas12a system for signal transduction and cascade amplification, and utilizes engineered FeNi MOF@AgNPs nanozyme for dual-signal output. With the presence of KIM-1, whose binding with aptamer, effectively inhibits the trans-cleavage activity of the CRISPR/Cas12a system, and uninhibited Cas12a subsequently cleaves nanozyme-conjugated magnetic probes, releasing FeNi MOF@AgNPs nanozymes that catalyze a TMB-based reaction to generate intense colorimetric and fluorescent dual-readout signals. The as developed aptasensor demonstrates satisfied sensitivity achieving detection limits of 58.7 pg/mL (colorimetric) and 34.4 pg/mL (fluorometric), and dependable accuracy achieving average relative deviation of -2.7% (colorimetric) and 3.2% (fluorometric) with commercial ELISA kit in urine samples from patients with acute kidney injury. Moreover, longitudinally track the dynamic changes in urinary KIM-1 concentrations over the first 8 days following renal transplantation was successfully realized. This work not only provides a robust analytical tool for KIM-1 detection but also establishes a generic research approach for extending CRISPR-based systems to the precise detection of proteins.},
}

*Biosensing Techniques/methods
Humans
*Hepatitis A Virus Cellular Receptor 1/isolation & purification
*Aptamers, Nucleotide/chemistry
CRISPR-Cas Systems
*Kidney Transplantation
Limit of Detection
Prognosis
Metal Nanoparticles/chemistry
Silver/chemistry
Colorimetry/methods
Metal-Organic Frameworks/chemistry
SELEX Aptamer Technique
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41707427,
year = {2026},
author = {Tu, Z and Wang, Y and Qian, X and Chen, J and Li, L and Wang, T and Chen, H and Wei, H and Yang, P and Pan, J and Wang, S and Rong, Z},
title = {Plasmonic magnetic nanoparticles-enabled universal enrichment, photothermal lysis, and duplex CRISPR detection of bacteria in urine samples.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118518},
doi = {10.1016/j.bios.2026.118518},
pmid = {41707427},
issn = {1873-4235},
mesh = {Humans ; *Escherichia coli/isolation & purification/genetics ; *Magnetite Nanoparticles/chemistry ; *Enterococcus faecalis/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Urinary Tract Infections/microbiology/urine/diagnosis ; CRISPR-Cas Systems ; Surface Plasmon Resonance/methods ; *Escherichia coli Infections/urine/microbiology/diagnosis ; Limit of Detection ; Boronic Acids/chemistry ; },
abstract = {Urinary tract infections (UTIs), exacerbated by antibiotic resistance and evolving pathogen diversity, demand rapid and sensitive diagnostics. This study introduces an integrated platform combining magnetic enrichment, photothermal lysis, and CRISPR-based detection (ME-CRISPR) for simultaneous identification of Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) in UTIs. We engineered plasmonic magnetic nanoparticles functionalized with 4-mercaptophenylboronic acid for broad-spectrum bacterial capture within 10 min. Subsequent near-infrared laser irradiation leveraged localized surface plasmon resonance to lyse captured bacteria in situ. The released nucleic acids were directly analyzed in a single-step duplex RPA-CRISPR/Cas12a-Cas13a assay. This 40-min workflow achieved a limit of detection of 10 CFU/mL for both pathogens. Validation with 90 clinical samples (39 E. coli, 26 E. faecalis, and 25 negative controls) demonstrated 100% sensitivity and specificity, matching qPCR performance while significantly reducing turnaround time. The platform overcomes limitations of centrifugation and Gram-class-dependent lysis, offering a rapid and ultrasensitive point-of-care testing tool to curb antibiotic misuse.},
}

Humans
*Escherichia coli/isolation & purification/genetics
*Magnetite Nanoparticles/chemistry
*Enterococcus faecalis/isolation & purification/genetics
*Biosensing Techniques/methods
*Urinary Tract Infections/microbiology/urine/diagnosis
CRISPR-Cas Systems
Surface Plasmon Resonance/methods
*Escherichia coli Infections/urine/microbiology/diagnosis
Limit of Detection
Boronic Acids/chemistry

@article {pmid41707429,
year = {2026},
author = {Nong, J and Pan, Z and Li, Y and Wei, J and Gong, Y and Li, J and Zhang, K and Liao, X},
title = {COF-confined CsPbBr3 nanocomposite with CRISPR/Cas12a-driven DNA walking for ultrasensitive electrochemiluminescent detection of circulating tumor DNA.},
journal = {Biosensors & bioelectronics},
volume = {302},
number = {},
pages = {118536},
doi = {10.1016/j.bios.2026.118536},
pmid = {41707429},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Nanocomposites/chemistry ; Luminescent Measurements/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Limit of Detection ; *Circulating Tumor DNA/blood/isolation & purification/genetics ; Endodeoxyribonucleases/chemistry ; DNA Probes/chemistry ; Metal-Organic Frameworks/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {A highly sensitive electrochemiluminescence biosensor was developed for circulating tumor DNA detection by integrating a covalent organic framework-confined CsPbBr3 nanocomposite with a CRISPR/Cas12a-driven amplification strategy. The covalent organic framework not only stabilizes CsPbBr3 nanocrystals in aqueous environments but also regulates interfacial charge transfer and enables programmable immobilization of DNA probes. Upon recognition of the target sequence, Cas12a is activated and progressively cleaves surface-tethered quencher-modified DNA strands, leading to stepwise restoration of electrochemiluminescence emission. This surface-confined signal amplification eliminates the need for polymerase chain reaction or isothermal preamplification. Under optimized conditions, the biosensor exhibited a linear response over a concentration range from 10 fM to 10 nM, with a detection limit of 5.4 fM. The method demonstrated good selectivity toward single-base mismatches and satisfactory performance in diluted serum and clinical plasma samples. These results highlight a synergistic material-enzyme strategy for sensitive and robust nucleic acid detection and provide a proof-of-concept platform for electrochemiluminescence-based circulating tumor DNA analysis.},
}

*Biosensing Techniques/methods
Humans
*Nanocomposites/chemistry
Luminescent Measurements/methods
CRISPR-Cas Systems/genetics
Electrochemical Techniques/methods
Limit of Detection
*Circulating Tumor DNA/blood/isolation & purification/genetics
Endodeoxyribonucleases/chemistry
DNA Probes/chemistry
Metal-Organic Frameworks/chemistry
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41708610,
year = {2026},
author = {Klann, M and Miura, S and Lee, SH and Vianello, SD and Ross, R and Watanabe, M and Gairin, E and Liang, Y and Hutto, HW and McCluskey, BM and Herrera, M and Solnica-Krezel, L and Besseau, L and Pigolotti, S and Parichy, DM and Kinoshita, M and Laudet, V},
title = {Cell-cell communication as underlying principle governing color pattern formation in teleost fishes.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41708610},
issn = {2041-1723},
mesh = {Animals ; *Cell Communication/genetics/physiology ; Zebrafish/genetics ; Gap Junctions/metabolism ; *Pigmentation/genetics/physiology ; Mutation, Missense ; Connexins/genetics/metabolism ; Fish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Genome-Wide Association Study ; Phenotype ; Gene Editing ; *Perciformes/genetics ; },
abstract = {The diverse pigmentation patterns of animals are crucial for predation avoidance and behavioral display. This diversity arises from interactions among distinct pigment cell types, yet mechanisms generating pattern variation across teleost fishes remain incompletely understood. In zebrafish, Turing models have been proposed to explain stripe patterns, but it is unclear if they apply to other fishes. Here, we investigate the Snowflake mutant of the anemonefish Amphiprion ocellaris, which displays enlarged white bars with irregular boundaries. Using genome-wide association mapping and targeted sequencing, we identify a missense mutation (E42K) in gja5b, encoding the gap junction protein Connexin 41.8. CRISPR/Cas9-mediated genome editing recapitulates the Snowflake phenotype, while pharmacological inhibition of gap junctions phenocopies the boundary defects, supporting a causal role for impaired intercellular communication. Expression analyses reveal that, unlike zebrafish, anemonefish gja5b is predominantly expressed in iridophores. With functional in vitro assays we demonstrate that the E42K mutation acts as a dominant negative, strongly reducing gap junctional coupling. Introducing the same mutation in zebrafish reveals context-dependent effects on pigment patterning. Taken together our findings highlighting gap junction-mediated communication as a conserved but flexible mechanism controlling pigment boundary positioning and pattern diversification.},
}

Animals
*Cell Communication/genetics/physiology
Zebrafish/genetics
Gap Junctions/metabolism
*Pigmentation/genetics/physiology
Mutation, Missense
Connexins/genetics/metabolism
Fish Proteins/genetics/metabolism
CRISPR-Cas Systems
Genome-Wide Association Study
Phenotype
Gene Editing
*Perciformes/genetics

@article {pmid41708664,
year = {2026},
author = {Huo, Y and Mei, J and Zhang, D and Yan, B and Zhang, D and Dong, C and Yin, S and Liu, M and Wang, X and Chen, D and Guan, Y and Song, G and Du, B and Wang, Y and Zheng, Z and Liu, H and Li, D and Yang, L and Wang, L},
title = {Engineered Un1Cas12f1 for multiplex genome editing with enhanced activity and targeting scope.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41708664},
issn = {2041-1723},
support = {U24A20677//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32025023//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32230064//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32311530111//National Natural Science Foundation of China (National Science Foundation of China)/ ; 24J22800400//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Humans ; Mice ; HEK293 Cells ; Genome, Human ; Mutation ; Genetic Therapy/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {The compact CRISPR-Cas12f system is promising for AAV-delivered gene therapy, but its application has been constrained by restrictive PAM recognition (e.g., TTTR) and suboptimal editing efficiency. Through bacterial library screening and mammalian cell validation, we engineer evoCas12f, an optimized variant incorporating five key mutations, that dramatically expands PAM recognition to NTNR/NYTR. This advancement reduces median distance between two neighbouring PAM sites to 2 nucleotides in the human genome. It also demonstrates 1.4-fold enhanced activity at TTTR sites compared to wild-type Un1Cas12f1, achieving up to 91% editing efficiency. Remarkably, evoCas12f enables efficient generation of homozygous mutations in F0 generation mice, even at non-canonical PAM sites. We further adapt this system for robust transcriptional activation and precise base editing with a well-defined editing window. As a compact yet highly efficient platform, evoCas12f represents a significant advance in CRISPR technology, enabling multiplexed editing for high-resolution targeting applications and expanding possibilities for therapeutic genome engineering.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Animals
Humans
Mice
HEK293 Cells
Genome, Human
Mutation
Genetic Therapy/methods
*CRISPR-Associated Proteins/genetics/metabolism

@article {pmid41709648,
year = {2026},
author = {Doghish, AS and Ghaiad, HR and Elfar, N and El Said, NH and Radwan, AF and Abd-Elmawla, MA and Mohamed, HH and Mohammed, OA and Rizk, HA},
title = {Unraveling the Function of lncRNAs in Gliomas: Interaction With Signaling Pathways and Therapeutic Opportunities.},
journal = {Journal of biochemical and molecular toxicology},
volume = {40},
number = {3},
pages = {e70756},
doi = {10.1002/jbt.70756},
pmid = {41709648},
issn = {1099-0461},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Glioma/genetics/metabolism/therapy/pathology ; *Signal Transduction ; *Brain Neoplasms/metabolism/genetics/therapy/pathology ; Animals ; *Gene Expression Regulation, Neoplastic ; *RNA, Neoplasm/metabolism/genetics ; },
abstract = {Brain tumors represent some of the most formidable challenges in neuro-oncology due to their aggressive clinical course, resistance to therapy, and profound molecular heterogeneity. Among the emerging regulatory elements reshaping our understanding of tumor biology are long non-coding RNAs (lncRNAs), a diverse class of RNA transcripts that modulate gene expression and cellular behavior without encoding proteins. This review provides an in-depth and integrative examination of the biogenesis, regulatory mechanisms, and functional roles of lncRNAs in brain tumor development and progression. We systematically explore both canonical and non-canonical pathways of lncRNA biogenesis, detailing how these influence structural specificity and molecular interactions. This review synthesized evidence retrieved from PubMed/MEDLINE, Scopus, and Web of Science, covering publications from January 2010 to June 2025. This analysis highlights key gaps, such as context-dependent therapeutic effects that limit translational applicability. A major focus is placed on the interplay between lncRNAs and core oncogenic signaling pathways, including Phosphoinositide 3-kinase (PI3K)/serine/threonine kinase (AKT), Signal Transducer and Activator of Transcription 3 (STAT3), Wingless/Int-1 (Wnt)/β-catenin, and Transforming Growth Factor-Beta (TGF-β), which drive malignant transformation, invasion, stemness, and therapeutic resistance in gliomas. Furthermore, we dissect the molecular functions of lncRNAs as epigenetic regulators, competitive endogenous RNAs (ceRNAs), and structural scaffolds, and discuss their contribution to the dynamic tumor microenvironment. By synthesizing the latest findings, this review underscores the academic and translational importance of targeting lncRNA-associated networks. It also highlights emerging therapeutic approaches, such as antisense oligonucleotides, RNA interference, CRISPR-Cas systems, and natural lncRNA-modulating compounds, which collectively represent a promising frontier in precision medicine for brain tumors. This work offers a critical framework for future research and therapeutic innovation in the lncRNA landscape of neuro-oncology.},
}

Humans
*RNA, Long Noncoding/genetics/metabolism
*Glioma/genetics/metabolism/therapy/pathology
*Signal Transduction
*Brain Neoplasms/metabolism/genetics/therapy/pathology
Animals
*Gene Expression Regulation, Neoplastic
*RNA, Neoplasm/metabolism/genetics

@article {pmid41709870,
year = {2026},
author = {Feng, L and Yu, P and He, N and Zhang, Q and Tang, Y and Geng, J and Lu, Q and Song, C and Chen, F},
title = {Label-Free MicroRNA Diagnostics: From CRISPR Nucleases to Nanomaterial-Enhanced Transducers.},
journal = {ACS synthetic biology},
volume = {15},
number = {3},
pages = {915-954},
doi = {10.1021/acssynbio.5c00868},
pmid = {41709870},
issn = {2161-5063},
mesh = {*MicroRNAs/genetics/analysis ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Nanostructures/chemistry ; },
abstract = {Label-free detection of microRNAs (miRNAs) has emerged as a pivotal approach in molecular diagnostics, integrating the programmability of CRISPR systems with the high sensitivity of nanomaterial-based transduction. miRNAs are short, noncoding RNAs that play central roles in gene regulation and disease pathogenesis, serving as valuable biomarkers for early diagnosis and prognosis. Conventional miRNA detection methods rely on labeling and multistep amplification, which hinder their adaptability for rapid and point-of-care applications. In contrast, label-free biosensing translates molecular recognition into intrinsic optical, electrochemical, or mechanical signals, enabling real-time, amplification-free analysis. This review summarizes recent advances in label-free miRNA biosensing, with emphasis on CRISPR/Cas12a, Cas13a, and Cas14a systems that couple target recognition with signal transduction, and nanomaterial-assisted platforms including gold and silver nanoparticles, carbon nanotubes, quantum dots, silica nanostructures, and magnetic composites. Particular attention is given to innovations that achieve attomolar-level sensitivity, single-nucleotide discrimination, and multiplex detection. We also discuss integration into microfluidic and wearable platforms, addressing persistent challenges in repeatability and stability, antifouling performance, and clinical translation. Emerging trends in artificial intelligence-assisted data processing, molecular logic circuits, and digital single-molecule biosensing are highlighted. These advances collectively outline the pathway toward intelligent, amplification-free, and portable miRNA diagnostics, bridging molecular biology and synthetic bioengineering for next-generation healthcare applications.},
}

*MicroRNAs/genetics/analysis
*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems/genetics
*Nanostructures/chemistry

@article {pmid41710881,
year = {2026},
author = {Karaoglu, IC and Odabas, A and Önder, T and Kizilel, S},
title = {Single-gene knockout of RNLS or HIVEP2 are insufficient to protect β-cell spheroids from allo- and xeno-rejection.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1759835},
pmid = {41710881},
issn = {1664-3224},
mesh = {Animals ; Mice ; *Insulin-Secreting Cells/transplantation/immunology/metabolism ; Humans ; *Graft Rejection/genetics/immunology/prevention & control ; Gene Knockout Techniques ; *Spheroids, Cellular/immunology/transplantation/metabolism ; CRISPR-Cas Systems ; *Islets of Langerhans Transplantation ; Cell Line ; Gene Editing ; Transplantation, Heterologous ; },
abstract = {INTRODUCTION: β-Cell replacement therapy offers a potential cure for type 1 diabetes, but its success is limited by rapid graft rejection. While genome-wide CRISPR screens have recently identified RNLS and HIVEP2 as candidate genes capable of protecting β-cells from autoimmune destruction, their efficacy against the distinct mechanisms of allogeneic and xenogeneic rejection remains unknown. This study aimed to test the hypothesis that single-gene ablation of RNLS or HIVEP2 protects β-cell spheroids from allo- and xenorejection in immunocompetent hosts.

METHODS: Murine β-TC-6 and human EndoC-βH1 β-cell lines were genetically edited using CRISPR-Cas9 to knockout RNLS or HIVEP2. Editing efficiencies were confirmed via T7 endonuclease I assay and Tracking of Indels by Decomposition (TIDE) analysis. Cells were aggregated into uniform, size-controlled spheroids using an optimized agarose suspension culture. Functional integrity was assessed via glucose-stimulated insulin secretion (GSIS). To evaluate immune evasion in vivo, luciferase-labeled spheroids were transplanted subcutaneously into immunocompetent CD-1 mice, modelling allogeneic (murine-to-murine) and xenogeneic (human-to-murine) rejection, with graft survival monitored longitudinally by bioluminescence imaging.

RESULTS: Robust editing efficiencies were achieved for both targets. Functional characterization indicated that Rnls deletion modestly impaired GSIS in murine cells, whereas HIVEP2 deletion showed no functional alterations in either cell line. In vivo assessment revealed no protective effects of RNLS or HIVEP2 deletion; grafts from both knockout groups displayed rejection kinetics indistinguishable from non-targeting controls. While allogeneic grafts survived longer than xenogeneic grafts, both were ultimately cleared by the host immune system regardless of genotype.

DISCUSSION: These data indicate that single-gene deletions of RNLS or HIVEP2 are insufficient to protect β-cell grafts from the barriers of allo- or xenorejection. By defining the limitations of these targets in isolation, our findings highlight the necessity for combinatorial genome editing strategies or complementary integration with immunomodulatory biomaterials to achieve effective and sustained β-cell graft survival.},
}

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

@article {pmid41710969,
year = {2026},
author = {Xu, Z and Wei, M and Jiang, M and Wang, Y and He, K},
title = {Single-step duplex CRISPR coupled with lateral flow assay for point-of-care detection of human immunodeficiency virus and Treponema pallidum.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {10},
pages = {1996-2004},
doi = {10.1039/d5ay02168e},
pmid = {41710969},
issn = {1759-9679},
mesh = {Humans ; *Treponema pallidum/genetics/isolation & purification ; *Point-of-Care Systems ; *HIV Infections/diagnosis/virology ; *Syphilis/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; *HIV/genetics/isolation & purification ; Point-of-Care Testing ; Sensitivity and Specificity ; },
abstract = {Continued acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) and syphilis caused by Treponema pallidum (TP) pose great challenges for global health, highlighting the need for rapid and sensitive diagnostics. Here, we introduced DIAL (Duplex Integrated All-in-one CRISPR Lateral flow assay), a simplified CRISPR-based diagnostic system for simultaneous detection of HIV and TP. We developed a single-step duplex CRISPR assay for dual-target recognition, which incorporated reverse transcription, multiplex recombinase polymerase amplification, transcription, CRISPR/Cas12a detection, and CRISPR/Cas13a detection in one pot. Then, we combined CRISPR detection assay with a "line-elimination" dual-line lateral flow assay for low-cost and equipment-free visual readout. We further developed reagent lyophilization and extraction-free sample lysis techniques to meet the point-of-care testing. The optimized DIAL system detected as low as 100 copies per µL of both HIV RNA and TP DNA within 45 minutes. In a clinical evaluation of 46 samples, it demonstrated 100% sensitivity and 97.5% specificity for HIV and 90% sensitivity and 100% specificity for TP, relative to quantitative PCR. The DIAL provided a scalable, accessible approach for decentralized screening of sexually transmitted infections or other pathogens in resource-limited settings.},
}

Humans
*Treponema pallidum/genetics/isolation & purification
*Point-of-Care Systems
*HIV Infections/diagnosis/virology
*Syphilis/diagnosis/microbiology
*CRISPR-Cas Systems/genetics
*HIV/genetics/isolation & purification
Point-of-Care Testing
Sensitivity and Specificity

@article {pmid41711690,
year = {2026},
author = {Shi, H and Chi, H},
title = {Next-generation CRISPR screens enable causal systems immunology.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {3},
pages = {},
doi = {10.1084/jem.20241266},
pmid = {41711690},
issn = {1540-9538},
support = {//American Lebanese Syrian Associated Charities/ ; CA253188/NH/NIH HHS/United States ; CA281868/NH/NIH HHS/United States ; AI105887/NH/NIH HHS/United States ; AI131703/NH/NIH HHS/United States ; AI140761/NH/NIH HHS/United States ; AI150241/NH/NIH HHS/United States ; AI150514/NH/NIH HHS/United States ; //Lupus Research Alliance/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Systems Biology/methods ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Analysis ; Immunoinformatics ; },
abstract = {Mapping the causal circuits that shape the phenotypic and functional landscape of immune cells remains a formidable challenge. Recent advances in pooled CRISPR-based screens, coupled with multiplexed single-cell profiling and imaging-based spatial readouts, make this goal increasingly attainable. In this Perspective, we discuss how CRISPR-based genetic screens will fundamentally transform our understanding of immunobiology. We highlight the applications of state-of-the-art, high-throughput pooled perturbation approaches, including emerging methodologies for bulk, single-cell, and spatial CRISPR screens, to advance our understanding of immunity and in vivo biology. Additionally, we summarize new strategies to address the complexity of combinatorial perturbations to uncover genetic interactions and mechanistic drivers of immunity at unprecedented scale and resolution. By integrating CRISPR screening data with experimental insights, we advocate a new framework in immunology research that leverages perturbation-driven regulatory effects and networks to discover new therapeutic targets and establish causal systems biology and immunology for advancing immunological knowledge and therapeutic application.},
}

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

@article {pmid41711853,
year = {2026},
author = {Guo, Y and Zhao, J and Li, X and Zheng, X and Yuan, Y and Deng, J and Ren, Y and Xu, K},
title = {Detection of alkaline phosphatase activity based on ATP hydrolysis and CRISPR/Cas12a.},
journal = {Analytical and bioanalytical chemistry},
volume = {418},
number = {8},
pages = {2337-2344},
pmid = {41711853},
issn = {1618-2650},
support = {2023JJ30430//Natural Science Foundation of Hunan Province/ ; 23A0075//Education Department of Hunan Province/ ; 82173572//National Natural Science Foundation of China/ ; 82574159//National Natural Science Foundation of China/ ; },
mesh = {*Alkaline Phosphatase/metabolism/analysis ; *Adenosine Triphosphate/metabolism/chemistry ; *CRISPR-Cas Systems ; Hydrolysis ; Animals ; Limit of Detection ; Milk/enzymology ; Aptamers, Nucleotide/chemistry ; *CRISPR-Associated Proteins/metabolism ; Biosensing Techniques/methods ; Cattle ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; },
abstract = {Alkaline phosphatase (ALP) activity serves as a crucial biomarker for livestock disease diagnosis, nutritional management, and the evaluation of milk pasteurization efficacy, holding significant implications for food safety and food science. In this study, we developed a novel method for detecting ALP activity in food samples, based on ATP hydrolysis coupled with a CRISPR/Cas12a system. The assay employs a DNA molecular recognition lock probe, comprising an ATP-specific aptamer and an activator strand designed to trigger the CRISPR/Cas12a trans-cleavage activity. In the absence of ALP, ATP acts as a trans-hydrolysis substrate to open the lock structure, releasing the activator strand. This subsequently activates the Cas12a protein, leading to the generation of a fluorescent signal. Conversely, when the target ALP is present, it catalyzes the hydrolysis of ATP via a dephosphorylation reaction. This prevents the opening of the "Aptamer-Activator" molecular lock, thereby inhibiting Cas12a activation and resulting in a corresponding decrease in fluorescence intensity. Under optimized conditions, the assay demonstrated a detection limit of 2.52 mU/mL for ALP activity, with a linear range of 0-18.75 mU/mL. The total detection time was 70 min. The method was successfully applied to detect ALP activity in samples from various livestock (chickens, pigs, sheep, cattle) and in milk, achieving recovery rates between 92 and 99%. In conclusion, we have developed a sensitive, cost-effective, and rapid method for ALP detection. This work provides a promising strategy for the development of point-of-care testing (POCT) devices in food safety monitoring.},
}

*Alkaline Phosphatase/metabolism/analysis
*Adenosine Triphosphate/metabolism/chemistry
*CRISPR-Cas Systems
Hydrolysis
Animals
Limit of Detection
Milk/enzymology
Aptamers, Nucleotide/chemistry
*CRISPR-Associated Proteins/metabolism
Biosensing Techniques/methods
Cattle
*Endodeoxyribonucleases/metabolism
Bacterial Proteins

@article {pmid41712102,
year = {2026},
author = {Lin, J and Hazaisi, H and Guan, Y and Bai, M},
title = {Multiplex gene editing drives revolution in crop breeding: overlaid editing of multiple genes and customization of complex traits.},
journal = {Advanced biotechnology},
volume = {4},
number = {1},
pages = {5},
pmid = {41712102},
issn = {2948-2801},
support = {2023ZD040360104//Chinese Academy of Agricultural Sciences from Institute of Crop Science/ ; },
abstract = {Modern agriculture currently demands higher standards for the simultaneous improvement of crop yield, quality and stress resistance. However, traditional crop breeding methods can no longer meet the needs of modern agricultural development. Improving a single trait is no longer sufficient to meet the multifaceted demands of modern agricultural production and consumer expectations. Multiple traits breeding has increasingly become a key objective in current crop breeding. Over the past decade, CRISPR/Cas9-based multiplex genome editing (MGE) has enabled efficient pyramiding and precise regulation of multiple traits via targeted editing of multiple gene loci, revolutionizing crop breeding. In this review, we briefly describe the core CRISPR/Cas-based MGE strategies and technical workflows, and thoroughly discuss the practical outcomes of MGE applications in various fields, such as enhancing crop stress resistance, increasing yield and improving quality. This review aims to provide a summary and theoretical reference for crop breeding, as well as open up new ideas for achieving different breeding goals.},
}

@article {pmid41712409,
year = {2026},
author = {Nenad, WC and Kuhlers, PC and Sturgill, IR and Biju, I and Bucklan, M and Hernandez, L and Zhu, LC and Hoadley, KA and Raab, JR},
title = {Hepatocyte-targeted Bap1 reduction in the liver primes an inflammatory transcriptional response.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {5},
pages = {},
pmid = {41712409},
issn = {2160-1836},
support = {/GM/NIGMS NIH HHS/United States ; },
mesh = {*Ubiquitin Thiolesterase/genetics/metabolism ; Animals ; *Hepatocytes/metabolism ; *Tumor Suppressor Proteins/genetics/metabolism ; Mice ; *Liver/metabolism/pathology ; *Inflammation/genetics/metabolism/pathology ; Mice, Knockout ; Transcriptome ; CRISPR-Cas Systems ; *Transcription, Genetic ; Gene Expression Profiling ; },
abstract = {BRCA1-associated protein 1 (BAP1) is a deubiquitinase, frequently altered in cancers including hepatocellular carcinoma and cholangiocarcinoma. While Bap1 has been shown to play key roles in metabolism, maintenance of tissue homeostasis, and immune cell development, little is known about its normal functions in the liver in vivo. Using AAV8-mediated CRISPR/CAS9 genome editing, we generated a mouse hepatocyte-specific model of Bap1 knockout to define the changes that occur in liver biology in an in vivo system and characterize how loss of Bap1 alters the liver's response to injury. Single-cell resolution spatial transcriptomics were performed in conjunction with immunohistochemistry to analyze cell-type composition and immune cell recruitment changes. Bulk RNA-sequencing was performed to further assess the impact of Bap1 loss on transcription. Hepatocyte-specific depletion of Bap1-induced transcriptional changes shared with acute injury. We observed a strong dysregulation of inflammatory pathways associated with Bap1 loss. Moreover, the transcriptional response of Bap1 depletion in hepatocytes to damage was markedly different than in control liver, with Bap1-depleted livers showing a decreased hepatocyte identity based on gene expression. Spatial transcriptomics and quantitative texture analysis of immunohistochemistry revealed an altered immune environment prior to damage and an impaired recruitment of immune cells in Bap1-depleted livers after damage. Our data suggest Bap1 is a critical modulator in the liver's immune cell response and its loss leads to an inflammatory environment prior to damage and disrupts the recruitment immune cells. Our quantitative spatial analysis highlights the power of such approaches to characterize the spatial distribution of different cell types in a tissue.},
}

*Ubiquitin Thiolesterase/genetics/metabolism
Animals
*Hepatocytes/metabolism
*Tumor Suppressor Proteins/genetics/metabolism
Mice
*Liver/metabolism/pathology
*Inflammation/genetics/metabolism/pathology
Mice, Knockout
Transcriptome
CRISPR-Cas Systems
*Transcription, Genetic
Gene Expression Profiling

@article {pmid41712528,
year = {2026},
author = {Magdy, M and Tinker-Kulberg, R and Josephs, EA},
title = {Polyvalent Guide RNAs Enhance the CRISPR-Mediated Suppression of a Human Coronavirus.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1738-1750},
doi = {10.1021/acssynbio.5c00574},
pmid = {41712528},
issn = {2161-5063},
support = {R35GM133483/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Coronavirus 229E, Human/genetics ; Gene Editing/methods ; RNA, Viral/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; },
abstract = {While CRISPR enzymes have become important tools for targeted gene editing in mammalian cells, they can also be used to specifically target and deplete viral nucleic acids to treat infections; this can be accomplished by delivering an RNA-targeting CRISPR effector like Cas13 along with a guide RNA (gRNA) that recognizes sequences from the genomes of single-stranded RNA (ssRNA) viruses. Previously, we hypothesized that by designing individual gRNAs able to target multiple, similar-but-not-identical viral sequences simultaneously ("polyvalent" guide RNAs or pgRNAs), gRNA's polyvalency would overcome any deficits caused by mispairing between the gRNA and the viral targets and, hence, still increase Cas13's antiviral potency and prevent mutagenic escape. We subsequently demonstrated this was the case using a model of viral infection in plants; however, it was not determined whether this strategy would also work against a human virus. Here, pgRNAs were designed to target multiple RNA sequences within human coronavirus 229E (hCoV-229E) and delivered along with Cas13 into a human lung epithelial cell line infected by hCoV-229E. CRISPR antiviral treatments using pgRNAs exhibited significant viral suppression in a CRISPR-dependent manner─more so than their single-target gRNA counterparts, even when multiple single-target gRNAs were used simultaneously. This improvement was also observed even as Cas13 with those same pgRNAs exhibited less "collateral" or nonspecific RNase activity relative to their single-target counterparts, which could imply that they may have greater specificity and safety profiles as therapeutic agents. Our findings demonstrate a computational and experimental pipeline by which pgRNAs, created using an unconventional gRNA design strategy, can be generated and validated to target human viruses using CRISPR antiviral biotechnologies more effectively.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Coronavirus 229E, Human/genetics
Gene Editing/methods
RNA, Viral/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
HEK293 Cells

@article {pmid41712626,
year = {2026},
author = {Sünderhauf, D and Ringger, JR and Payne, LJ and Pinilla-Redondo, R and Gaze, WH and Brown, SP and van Houte, S},
title = {CRISPR-Cas is beneficial in plasmid competition, but limited by competitor toxin-antitoxin activity when horizontally transferred.},
journal = {PLoS biology},
volume = {24},
number = {2},
pages = {e3003658},
pmid = {41712626},
issn = {1545-7885},
mesh = {*Plasmids/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Transfer, Horizontal ; Escherichia coli/genetics ; *Toxin-Antitoxin Systems/genetics ; },
abstract = {Bacteria can encode dozens of different immune systems that protect them from infection by mobile genetic elements (MGEs). MGEs themselves may also carry immune systems, such as CRISPR-Cas, to target competitor MGEs. It is unclear when this is favored by natural selection, and whether toxin-antitoxin (TA) systems-common competitive mechanisms carried by plasmids-can alter their efficacy. Here, we develop and test novel theory to analyze the outcome of competition between plasmids when one carries a CRISPR-Cas system that targets the other plasmid. Our mathematical model and experiments using Escherichia coli and competing IncP plasmids reveal that plasmid-borne CRISPR-Cas is beneficial to the plasmid carrying it when the plasmid has not recently transferred to a new host. However, CRISPR-Cas is selected against when the plasmid carrying it transfers horizontally, if a resident competitor plasmid encodes a TA system that elicits post-segregational killing. Consistent with a TA barrier to plasmid-borne CRISPR-Cas, a bioinformatic analysis reveals that naturally occurring CRISPR-Cas-bearing plasmids avoid targeting other plasmids with TA systems across bacterial genera. Our work shows how the benefit of plasmid-borne CRISPR-Cas is severely reduced against TA-encoding competitor plasmids, but only when plasmid-borne CRISPR-Cas is horizontally transferred. These findings have key implications for the distribution of prokaryotic defenses and our understanding of their role in competition between MGEs, and the utility of CRISPR-Cas as a tool to remove plasmids from pathogenic bacteria.},
}

*Plasmids/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Transfer, Horizontal
Escherichia coli/genetics
*Toxin-Antitoxin Systems/genetics

@article {pmid41713039,
year = {2026},
author = {Zhang, H and Cui, C and Wang, X and Liu, S and Wang, X and Wang, Y and Ge, S and Cai, Y and Bao, J and Wang, Z},
title = {Development of a one-pot RT-RAA/CRISPR-Cas13a assay for rapid genotyping of Nipah virus in pigs.},
journal = {Diagnostic microbiology and infectious disease},
volume = {115},
number = {2},
pages = {117316},
doi = {10.1016/j.diagmicrobio.2026.117316},
pmid = {41713039},
issn = {1879-0070},
mesh = {Animals ; Swine ; *Nipah Virus/genetics/isolation & purification/classification ; Sensitivity and Specificity ; *Swine Diseases/virology/diagnosis ; Genotype ; *CRISPR-Cas Systems ; *Genotyping Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *Henipavirus Infections/veterinary/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; Polymorphism, Single Nucleotide ; },
abstract = {INTRODUCTION: Nipah virus (NiV) is a highly pathogenic zoonotic virus transmitted from bats to humans through pigs as a key intermediate host. Given the existence of two distinct NiV genotypes, which differ in clinical manifestations and transmission patterns in both humans and pigs, rapid and sensitive method for detection and genotyping is crucial for effective disease control. Isothermal amplification combined with CRISPR/Cas-based assay provides a promising approach to meet this need.

METHODS: Conserved regions were identified by aligning the N gene sequences from 67 NiV strains. Specific primers and probes were designed for reverse transcription recombinase-aided amplification (RT-RAA) to detect NiV. Subsequently, single nucleotide polymorphisms within the conserved region were analyzed, and corresponding crRNAs were designed to establish a one-pot RT-RAA/CRISPR-Cas13a assay for NiV genotyping. The assays were evaluated using simulated pig serums spiked with NiV pseudovirus.

RESULTS: The RT-RAA assay exhibited a detection sensitivity of 10[-2] Infection Unit/mL (IU/mL) for NiV pseudovirus, outperforming conventional qRT-PCR in simulated pig serum samples. No cross-reactivity was observed with viral RNA or DNA of PCV2, PEDV, PRRSV, PRV and SVA, confirming high specificity. The entire one-pot RT-RAA/CRISPR-Cas13a assay could be completed within 1 hour and clearly discriminated between the two NiV genotypes without requiring sophisticated instruments. Evaluation with simulated samples showed a sensitivity of 100% (95% CI, 92.87-100%) and a specificity of 94% (95% CI, 83.78-98.36%), with a detection limit of 10[-1] IU/mL for NiV pseudovirus.

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

Animals
Swine
*Nipah Virus/genetics/isolation & purification/classification
Sensitivity and Specificity
*Swine Diseases/virology/diagnosis
Genotype
*CRISPR-Cas Systems
*Genotyping Techniques/methods
*Nucleic Acid Amplification Techniques/methods
*Henipavirus Infections/veterinary/diagnosis/virology
*Molecular Diagnostic Techniques/methods
Polymorphism, Single Nucleotide

@article {pmid41713565,
year = {2026},
author = {Shashikala, T and Yogi, D and Akshay, K and Nagesh, SN and Manamohan, M and Venkataravanappa, V and Jha, GK and Ashok, K and Asokan, R},
title = {First report of CRISPR/Cas13a-based rapid detection of groundnut bud necrosis virus without amplification.},
journal = {Methods (San Diego, Calif.)},
volume = {249},
number = {},
pages = {9-22},
doi = {10.1016/j.ymeth.2026.02.010},
pmid = {41713565},
issn = {1095-9130},
mesh = {*CRISPR-Cas Systems/genetics ; *Plant Diseases/virology ; RNA, Viral/genetics/isolation & purification ; *Tospovirus/genetics/isolation & purification ; Vigna/virology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Globally, the groundnut bud necrosis virus (GBNV) (Bunyaviridae), pose a serious threat to solanaceous and leguminous crops causing serious crop loss. This requires a rapid and sensitive diagnostics for initially identifying at the earliest stage and further to initiate disease management. This study presents the first report of a CRISPR/Cas13a-based diagnostic assay for GBNV detection without amplification. GBNV was maintained in cowpea cv. C152 through mechanical inoculation, further viral RNA was isolated to clone the two target genes viz. nucleocapsid (NP) and movement protein (MP) genes. These genes were ligated to pTZ57R/T vector and sequenced. Similarly, the LshCas13a gene was cloned from pUC19 into pET28a, expressed in E. coli BL21, and purified using Ni-NTA affinity chromatography. Guide RNAs targeting conserved regions of NP and MP genes were synthesized by in vitro transcription and mixed with Cas13a protein to form ribonucleoprotein (RNP) complex. Target RNA, obtained either by in vitro-transcription or crude extract of infected tomato was used to detect GBNV, using a fluorescence-based reporter assay. This method found to be highly sensitive that could detect GBNV at as low as 0.01 ng. From the field perspective, GBNV could be detected from the crude extract of the GBNV infected tomato leaves using an alkaline PEG buffer. Thus CRISPR/Cas13a-based assay provides a rapid, amplification-free, and field-deployable diagnostic platform for GBNV. This lays the groundwork for a field adoptable CRISPR diagnostics for other plant RNA viruses also.},
}

*CRISPR-Cas Systems/genetics
*Plant Diseases/virology
RNA, Viral/genetics/isolation & purification
*Tospovirus/genetics/isolation & purification
Vigna/virology
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41713574,
year = {2026},
author = {Hu, T and Hou, Z and Zhang, Y and Jing, P and Dai, X and Wang, H},
title = {Development of a one-pot integrated rapid detection method for white spot syndrome virus based on RAA-CRISPR/Cas12a technology.},
journal = {Journal of invertebrate pathology},
volume = {216},
number = {},
pages = {108569},
doi = {10.1016/j.jip.2026.108569},
pmid = {41713574},
issn = {1096-0805},
mesh = {*White spot syndrome virus 1/isolation & purification ; Animals ; *Penaeidae/virology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Aquaculture ; Sensitivity and Specificity ; },
abstract = {Pathogenic microorganisms, particularly white spot syndrome virus (WSSV), pose a major threat to global shrimp aquaculture, causing mass mortalities and substantial economic losses. To address the urgent need for rapid, accurate, and field-deployable detection methods, this study developed an innovative one-pot RAA-CRISPR/Cas12a assay. The platform integrates recombinase-aided amplification (RAA) with CRISPR/Cas12a technology using sucrose-mediated density gradient phase separation in a closed-tube format: sucrose acts as a density modifier to form distinct layers, spatially isolating RAA amplification reagents from CRISPR/Cas12a detection components to avoid cross-interference and enable sequential reactions without manual intervention. Under isothermal conditions at 37℃ for 60 min, the optimized assay achieves a limit of detection as low as 1 copy/μL, validated by both fluorescence and lateral flow dipstick (LFD) readouts. High specificity was confirmed by the absence of cross-reactivity with four other prevalent shrimp pathogens: infectious hypodermal and hematopoietic necrosis virus (IHHNV), Decapod iridescent virus 1 (DIV1), Enterocytozoon hepatopenaei (EHP), and Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (VpAHPND). Clinical validation with 30 field samples showed concordant results with the chinese national detection standard (GB/T 28630.2-2012). This novel nucleic acid detection platform combines highly sensitive, excellent specificity, and user-friendly visual interpretation, making it highly suitable for point-of-care testing and large-scale disease surveillance in shrimp aquaculture.},
}

*White spot syndrome virus 1/isolation & purification
Animals
*Penaeidae/virology
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Aquaculture
Sensitivity and Specificity

@article {pmid41714766,
year = {2026},
author = {Taha, BA and Addie, AJ and Haider, AJ and Jasim, AJ and Ahmed, NM and Arsad, N},
title = {Perspective of smart nanocapsule swallowable laser-guided for integrated sensing and crispr-mediated cancer gene editing.},
journal = {Cancer gene therapy},
volume = {33},
number = {3},
pages = {351-365},
pmid = {41714766},
issn = {1476-5500},
mesh = {Humans ; *Gene Editing/methods ; *Nanocapsules/chemistry/administration & dosage ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; Lasers ; Biosensing Techniques/methods ; Animals ; *Genetic Therapy/methods ; },
abstract = {Current therapeutic techniques for cancer often lack specificity. They also cause systemic toxicity and lack genetic control. Thus, cancer ranks among the most complex and crucial global health issues. The novel concept of smart nanocapsules is discussed in this Perspective. These oral medications modify genes using CRISPR technology and integrate biosensing and laser-guided activation to enable more personalized cancer therapies. The creation of these versatile nanocapsules is driven by three objectives. First, they aim to enable controlled gene editing in the gastrointestinal tract. Second, they deliver treatments to specific target areas. Third, they detect tumors in real time. Nanocapsules equipped with biosensing components provide microenvironmental input. An external laser can trigger the release of light-absorbing agents. Moreover, these features reduce off-target effects and allow spatiotemporal precision, thhe enteric-coated architecture ensures oral stability. Surface functionalization enhances selective tumor accumulation. AI-guided control algorithms can manage diagnostic interpretation and activation. The CRISPR-based cancer medicines offer the potential for improved safety, specificity, and translational use in the future. Combining advanced nanotechnology, gene editing, and AI-guided control could create innovative solutions.},
}

Humans
*Gene Editing/methods
*Nanocapsules/chemistry/administration & dosage
*Neoplasms/genetics/therapy
*CRISPR-Cas Systems
Lasers
Biosensing Techniques/methods
Animals
*Genetic Therapy/methods

@article {pmid41715004,
year = {2026},
author = {Metz, P and Alves-Vasconcelos, S and Wallbank, R and Riepsaame, J and Brown, S and Hassan, AB},
title = {Variation in guide RNA library representation results in gene effect score bias in genome-wide CRISPR screens.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41715004},
issn = {1471-2164},
support = {OxPOS//F. Hoffmann-La Roche/ ; GEO//John Fell Fund, University of Oxford/ ; MPNST//Grenfell Shaw Charity/ ; Oxford Clinical Fellowships/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Gene Library ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Human ; },
abstract = {Genome wide CRISPR-based perturbation screens are powerful discovery tools enabling the identification of novel gene dependencies through either gain or loss of function. While genome wide guide RNA (gRNA) libraries have advantages when using enAsCas12a, such as multiplex single gRNAs per gene, they may be subject to similar confounding factors that can affect the interpretation of large genome-wide datasets. Here, we examine the impact of these variables in over twenty enAsCas12a multiple gRNA based perturbation screens performed using Humagne C, Humagne D and Inzolia libraries in human cells, as well as external datasets containing Cas9-based CRISPR library screens, including from DepMap. We demonstrate that the choice of CRISPR library is often the most significant factor that influences genetic perturbation results, outweighing other variables such as either target cell lines or culture media conditions. A potential contributor to this effect is gRNA representation within a given CRISPR library, where lower gRNA representation can lead to variable and more pronounced gene effect scores using either log fold change or Chronos analysis. These effects may be mitigated by using either multiple gRNA constructs per gene, by optimisation of CRISPR library production processes or by targeting with multiple independent gRNA libraries. Importantly, we also propose strategies for addressing gRNA representation bias during CRISPR screen hit prioritisation. CRISPR library gRNA representation dependent bias remains a major challenge in the interpretation of gene essentiality in perturbation screens.},
}

Humans
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
*Gene Library
*Clustered Regularly Interspaced Short Palindromic Repeats
Genome, Human

@article {pmid41715150,
year = {2026},
author = {Zhao, Y and Li, X and Du, Y},
title = {AI-driven CRISPR screening: optimizing gene editing through automation and intelligent decision support.},
journal = {Journal of translational medicine},
volume = {24},
number = {1},
pages = {},
pmid = {41715150},
issn = {1479-5876},
support = {No. Z04J2024E107-B-12//Bethune Charitable Foundation/ ; },
mesh = {Humans ; *Artificial Intelligence ; *Gene Editing ; *Automation ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Decision Support Techniques ; *CRISPR-Cas Systems/genetics ; Deep Learning ; *Genetic Testing/methods ; },
abstract = {BACKGROUND: CRISPR-based genetic screening has become a central methodology in functional genomics, enabling systematic interrogation of gene function, genetic interactions and context-dependent vulnerabilities at scale. However, the rapid expansion of screening modalities-including multi-condition designs, combinatorial perturbations, in vivo applications and single-cell readouts-has exposed fundamental limitations of heuristic-driven experimental design and post hoc statistical analysis.

MAIN BODY: This Review synthesizes how artificial intelligence is reshaping CRISPR screening by introducing predictive, adaptive and system-level intelligence across the experimental lifecycle. We organize recent advances into two tightly coupled modules. First, machine learning and deep learning (ML/DL) methods optimize experimental design by learning context-dependent perturbation behavior, anticipating confounding effects and enabling iterative, information-efficient screening strategies. Second, large language model-agent (LLM-agent) systems complement these advances by externalizing scientific reasoning, integrating biological knowledge at scale and coordinating analysis and decision-making in human-in-the-loop workflows.

CONCLUSIONS: Together, ML/DL and LLM-agent approaches reframe CRISPR screening from a static analytical pipeline into an intelligent experimental system, with important implications for robustness, scalability and biological discovery.},
}

Humans
*Artificial Intelligence
*Gene Editing
*Automation
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Decision Support Techniques
*CRISPR-Cas Systems/genetics
Deep Learning
*Genetic Testing/methods

@article {pmid41715936,
year = {2026},
author = {Shimizu, Y and Kataoka, K},
title = {DIPA-CRISPR Mediated Knockout of Vermilion Generates a Visible Eye Color Marker for The Band-Legged Ground Cricket Dianemobius nigrofasciatus.},
journal = {Archives of insect biochemistry and physiology},
volume = {121},
number = {2},
pages = {e70135},
doi = {10.1002/arch.70135},
pmid = {41715936},
issn = {1520-6327},
support = {JPJ009237//Bio-oriented Technology Research Advancement Institution, BRAIN/ ; 21K05614//JSPS KAKENHI Grant-in-Aid for Scientific Research (C)/ ; 21J23478/22KJ2609//Grant-in-Aid for JSPS Fellows/ ; },
mesh = {Animals ; Female ; *Gryllidae/genetics/growth & development/physiology ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; *Eye Color/genetics ; Photoperiod ; Gene Knockout Techniques ; Gene Editing ; },
abstract = {The molecular basis of photoperiodism, by which insects use photoperiodic cues to anticipate seasonal changes and regulate key life-history events such as development, diapause, and reproduction, remains poorly understood. Studies on the molecular mechanisms of photoperiodism in hemimetabolous insects are limited compared with those in holometabolous insects, largely due to the lack of appropriate model organisms. The band-legged ground cricket Dianemobius nigrofasciatus represents a valuable model system because it exhibits clear photoperiodic responses in the maternal induction of embryonic diapause, the wing morph, and the rate of nymphal development. With the recent availability of the D. nigrofasciatus genome sequence, the establishment of effective genome-editing methods and reliable marker genes is expected to promote functional genomic analyses. In this study, we aimed to establish a direct parental (DIPA)-CRISPR genome-editing approach and evaluate the utility of vermilion (Dn-v), a gene involved in ommochrome synthesis, as a visible eye color marker for mutant screening. Cas9 ribonucleoprotein complexes were injected into females 3-5 days after adult emergence, during the vitellogenic stage, successfully yielding Dn-v knockout mutants. These mutants had white compound eyes throughout development, with pigmentation reaching a vermilion color about 20 days after adult emergence. We further examined the photoperiodic response associated with maternal diapause induction in knockout mutants. Similar to the wild-type, knockout mutants exhibited low and high diapause incidence under long-day and short-day conditions, respectively. Our results demonstrate that DIPA-CRISPR is an effective genome-editing method in D. nigrofasciatus and that Dn-v serves as a practical and reliable marker gene. The establishment of these genomic tools provides a foundation for future functional analyses aimed at elucidating the molecular basis of photoperiodism in hemimetabolous insects.},
}

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

@article {pmid41717108,
year = {2026},
author = {Balasubramani, S and Li, Z and Gayathri, E and Gurusamy, D and Singh, A},
title = {Editorial: Advancing plant defense: genome editing, RNAi, and synthetic biology for sustainable pest control.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1785705},
pmid = {41717108},
issn = {1664-462X},
}

@article {pmid41717201,
year = {2026},
author = {Mittal, A and Manna, S and Nelson, V and Ladha, N},
title = {In Silico Design of gRNA for CRISPR System for Detection of Multidrug Resistant Tuberculosis Using Indian Mycobacterium tuberculosis Genomes: A Computational Study.},
journal = {Cureus},
volume = {18},
number = {1},
pages = {e101851},
pmid = {41717201},
issn = {2168-8184},
abstract = {Background Multidrug-resistant tuberculosis (MDR-TB) continues to pose a major challenge to TB elimination in India, where drug resistance and delayed diagnosis contribute significantly to ongoing transmission. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based diagnostics have emerged as versatile tools, compared to GeneXpert, capable of detecting resistance-associated mutations with rapid turnaround and high accuracy. This study aimed to design and in silico validate Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein (CRISPR-Cas)-based guide RNAs (gRNAs) targeting major drug-resistance mutations in Indian Mycobacterium tuberculosis (M. tuberculosis) isolates. Methods Whole-genome mutation profiles were analyzed using TBProfiler, and gRNAs were designed using CHOPCHOP. Off-target evaluation was performed using Cas-OFFinder and Basic Local Alignment Search Tool (BLAST). High-confidence mutations in gyrA, rpoB, katG, rpsL, embB, and ethA were selected based on prevalence in Indian isolates and WHO-defined resistance markers. Results Numerous drug resistance-associated mutations were identified in the drug-resistant tuberculosis genome isolates. The study identified six key genetic mutations identified in MTB isolates that are associated with phenotypic drug resistance, including gyrA (Asp94Gly), rpoB (Ser450Leu), and katG (Ser315Thr). For each of the six genes, the chromosome position, locus ID, mutation type, and affected amino acids were identified, and tailored guide RNAs were designed in silico. Top-ranked gRNAs demonstrated optimal GC content, high predicted cleavage efficiency, and zero off-target activity. Each resistance locus yielded multiple candidate gRNAs suitable for CRISPR-based assays. Conclusions This computational in silico analysis provides a robust panel of mutation-targeted gRNAs tailored to Indian MDR-TB genomic profiles. These findings lay a strong foundation for developing rapid, affordable CRISPR diagnostics for point-of-care detection of drug resistance. Future laboratory validation and clinical testing are essential for translation into diagnostic practice.},
}

@article {pmid41717698,
year = {2026},
author = {Tadokoro, T and Li, H and Gan, P and Xu, Z and Tan, W and Alzhanov, D and Sánchez-Ortiz, E and McAnally, JR and Guo, L and Xu, L and Ruan, P and Liu, N and Olson, EN},
title = {Ablation of PKCα Phosphorylation by CRISPR-Cas9 Base Editing Rescues Heart Failure.},
journal = {Circulation research},
volume = {138},
number = {7},
pages = {e326738},
pmid = {41717698},
issn = {1524-4571},
support = {P01 HL160488/HL/NHLBI NIH HHS/United States ; P50 HD087351/HD/NICHD NIH HHS/United States ; R01 HL157281/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Protein Kinase C-alpha/genetics/metabolism ; *Heart Failure/genetics/therapy/enzymology/physiopathology/pathology/metabolism ; Phosphorylation ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; Humans ; Myocytes, Cardiac/metabolism/enzymology ; Mice, Inbred C57BL ; Male ; Disease Models, Animal ; Induced Pluripotent Stem Cells ; },
abstract = {BACKGROUND: The prevalence of heart failure is increasing globally, with poor prognosis, highlighting the need for novel therapeutic strategies. PKCα (protein kinase C alpha), encoded by PRKCA, plays a central role in heart failure pathogenesis. Phosphorylation of PKCα at threonine 497 (T497) triggers a series of intramolecular phosphorylation events, leading to its activation. Ablation of T497 phosphorylation leads to reduced stability and activity of PKCα.

METHODS: We generated mice harboring a phospho-resistant PKCα (T497A) mutation in the germline using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated homology-directed repair. To assess the clinical feasibility of postnatal genome editing, we used CRISPR-Cas9 adenine base editing delivered by adeno-associated virus 9 to introduce the T497A substitution into the Prkca gene (Prkca[T497A]) in wild-type mice. Mice underwent transverse aortic constriction to model heart failure. Cardiac function, hypertrophy, fibrosis, and transcriptional changes were evaluated by echocardiography, wheat germ agglutinin staining, Masson's trichrome staining, and RNA-sequencing. The editing efficiency of Prkca[T497A] was assessed using Sanger sequencing and deep amplicon sequencing. To further explore its clinical potential, we introduced the PRKCA[T497A] mutation into human induced pluripotent stem cells by nucleofection-mediated adenine base editing. Ca[2+] homeostasis was analyzed in Fura-2-loaded human induced pluripotent stem cell-derived cardiomyocytes with PRKCA[T497A] under chronic AngII (angiotensin II) stimulation.

RESULTS: The T497A mutation in PKCα prevented its subsequent phosphorylation and led to PKCα protein degradation. Four weeks after transverse aortic constriction surgery, wild-type mice showed impaired cardiac function, cardiac remodeling, and increased lung weight. In contrast, PKCα phospho-resistant mice showed protection against heart failure-related aberrant changes in cardiac hypertrophy, fibrosis, and cardiac gene expression. Mice administered with adeno-associated virus 9 base editors to prevent T497 phosphorylation exhibited similar cardioprotective effects. In vitro, PKCα-edited induced pluripotent stem cell-derived cardiomyocytes were protected from AngII-induced impairments in contractility and Ca[2+] transients.

CONCLUSIONS: The editing of PRKCA[T497A] through adenine base editing represents a potential therapeutic approach for human cardiac diseases.},
}

Animals
*Protein Kinase C-alpha/genetics/metabolism
*Heart Failure/genetics/therapy/enzymology/physiopathology/pathology/metabolism
Phosphorylation
*CRISPR-Cas Systems
*Gene Editing/methods
Mice
Humans
Myocytes, Cardiac/metabolism/enzymology
Mice, Inbred C57BL
Male
Disease Models, Animal
Induced Pluripotent Stem Cells

@article {pmid41718462,
year = {2026},
author = {Ren, Y and Wu, R and Yang, X and Yao, B and Wang, Y},
title = {Membrane-Confined CRISPR-Cas12a Activation via Split Activator for Wash-Free Detection of Tumor-Specific Extracellular Vesicles.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2356-2365},
doi = {10.1021/acssensors.5c04166},
pmid = {41718462},
issn = {2379-3694},
mesh = {*Extracellular Vesicles/metabolism/chemistry ; *CRISPR-Cas Systems ; Humans ; Aptamers, Nucleotide/chemistry/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism ; *Neoplasms/diagnosis ; },
abstract = {Current CRISPR-Cas12a biosensing systems for protein analysis rely on the release of a blocked activator from an aptamer through a competitive mechanism. However, the design of the complementary length between the activator and the aptamer involves a critical trade-off: excessively long sequences impede aptamer-target binding, whereas overly short sequences fail to activate Cas12a effectively. To overcome this limitation, we split the full-length Cas12a activator into two short single-stranded DNA fragments (S1 and S2; 10 nt each). S1 was specifically sequestered within the hairpin structure of an allosteric aptamer. The binding of the target protein to the aptamer triggered a conformational change, exposing S1 and thereby enabling its assembly with S2 to activate Cas12a. The strategy successfully preserved the binding affinity of the aptamer without compromising the efficiency of Cas12a activation. Furthermore, we anchored the Cas12a substrate to the membrane surface of extracellular vesicles (EVs) via hydrophobic interactions, localizing the cleavage reaction to the EVs interface and thereby significantly enhancing local catalytic efficiency. Finally, the strategy provided highly sensitive detection of tumor-specific EVs, not only accurately distinguishing cancer patients from healthy individuals (77.5% accuracy for early-stage and 100% for advanced-stage) but also holding potential for monitoring dynamic postoperative changes. Overall, our study provided a wash-free, one-pot detection platform for EVs analysis and established a new paradigm for CRISPR-based clinical diagnostics.},
}

*Extracellular Vesicles/metabolism/chemistry
*CRISPR-Cas Systems
Humans
Aptamers, Nucleotide/chemistry/metabolism
*Biosensing Techniques/methods
*CRISPR-Associated Proteins/metabolism
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism
*Neoplasms/diagnosis

@article {pmid41718886,
year = {2026},
author = {Cao, X and He, Y and Guo, H and Cao, X and Zhang, D and Lai, Y and Yang, W and Ma, Z and Yu, X and Wang, L and Li, D and Zeng, Z},
title = {Optimization of RNP-CRISPR for high-efficiency gene editing in mouse intestinal organoids.},
journal = {Molecular and cellular biochemistry},
volume = {481},
number = {4},
pages = {1817-1827},
pmid = {41718886},
issn = {1573-4919},
support = {2019YFA0110802//National Key Research and Development Program of China/ ; No.32025023//National Natural Science Foundation of China/ ; No.32200732//National Natural Science Foundation of China/ ; 20MC1920400//Shanghai Municipal Commission for Science and Technology/ ; 22YF1437700//Shanghai Municipal Commission for Science and Technology/ ; 2019-01-07-00-05-E00054//Innovation Program of Shanghai Municipal Education Commission/ ; },
mesh = {Animals ; *Organoids/metabolism/cytology ; *Gene Editing/methods ; Mice ; *CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/metabolism ; *Intestines/cytology ; Wnt Signaling Pathway ; Mice, Knockout ; beta Catenin/metabolism ; },
abstract = {Mouse intestinal organoids are ideal models for investigating intestinal development and diseases. The full potential of these models hinges on the ability to precisely engineer their genome, yet traditional methods for CRISPR-based editing in 3D cultures often surfer from low efficiency, high cytotoxicity, and inconsistent post-editing differentiation, which limits their applications. Here, we developed an electroporation approach mediated by ribonucleoprotein (RNP)-CRISPR that achieves over 90% gene editing efficiency in mouse intestinal organoids. Using this optimized method, we generated APC-knockout organoids that exhibit Wnt pathway hyperactivation, demonstrated by R-spondin1-independent growth, increased nuclear β-catenin, and enhanced proliferation. Our method addresses a critical technical gap in murine organoid research, offering a scalable platform for intestinal disease modeling.},
}

Animals
*Organoids/metabolism/cytology
*Gene Editing/methods
Mice
*CRISPR-Cas Systems
*Ribonucleoproteins/genetics/metabolism
*Intestines/cytology
Wnt Signaling Pathway
Mice, Knockout
beta Catenin/metabolism

@article {pmid41719905,
year = {2026},
author = {Yu, F and Yue, D and Wang, F and Fu, B and Qin, G and Wei, S and Zang, W and Zhang, Q and Cui, L and Wang, T},
title = {Structure-initiated CHA variant coordinating SDA for cascade amplification in CRISPR/Cas12a-based miRNA analysis.},
journal = {Talanta},
volume = {304},
number = {},
pages = {129558},
doi = {10.1016/j.talanta.2026.129558},
pmid = {41719905},
issn = {1873-3573},
mesh = {*MicroRNAs/genetics/analysis/blood ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {MicroRNAs (miRNAs) are well-established biomarkers for tumor diagnosis and monitoring. Herein, we report a novel biosensing platform by engineering a structure-initiated variant of catalytic hairpin assembly (VCHA) that coordinates with strand displacement amplification (SDA) to drive cascade amplification for CRISPR/Cas12a-based detection. This system employs three hairpin probes which, upon recognizing the target miRNA, self-assemble into a key 5'-end dangling three-way conjugate (5'-DTC) structure. This structure serves as the exclusive trigger, simultaneously propagating the VCHA cycle and priming the SDA process through the coordinated action of polymerase and nicking enzyme. Consequently, VCHA and SDA operate synergistically within a unified circuit, generating abundant single-stranded activator DNA (acDNA) products. These acDNA molecules then activate the trans-cleavage activity of CRISPR/Cas12a, yielding a significantly amplified fluorescence readout. The VCHA-SDA/Cas12a platform demonstrated excellent performance for miRNA-155 detection, achieving a broad dynamic range from 1 pmol/L to 10 nmol/L with an ultra-low detection limit of 0.166 pmol/L. Furthermore, the platform successfully quantified miRNA levels in clinical plasma specimens and various cell lines, confirming its considerable potential as a robust tool for molecular diagnostics and clinical translation.},
}

*MicroRNAs/genetics/analysis/blood
*CRISPR-Cas Systems/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
*Biosensing Techniques/methods
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41719925,
year = {2026},
author = {Park, YJ and Song, DY and Jeon, HB and Kim, DM},
title = {Nucleic acid detection via protein readout through Cas-controlled gating of cell-free protein synthesis.},
journal = {Biosensors & bioelectronics},
volume = {301},
number = {},
pages = {118514},
doi = {10.1016/j.bios.2026.118514},
pmid = {41719925},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Cell-Free System ; *Protein Biosynthesis/genetics ; *Escherichia coli O157/genetics ; Bacillus anthracis/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Ribosomal, 16S/genetics/analysis ; *Nucleic Acids/analysis/genetics ; Bacterial Proteins/genetics ; },
abstract = {We present a modular platform that converts CRISPR target recognition into programmable protein outputs for nucleic acid detection. This system couples Cas-mediated collateral cleavage with cell-free protein synthesis. In the presence of a target, Cas-mediated collateral cleavage releases an extension-competent trigger DNA that gates reporter expression. Although collateral cleavage is inherently indiscriminate, we achieve deterministic fragment generation by employing chemically programmed precursors bearing backbone modifications-exemplified here by phosphorothioate linkages-that constrain cleavage to predefined sites. Using Bacillus anthracis and E. coli O157:H7 as a model, the developed CRIVER assay enables concurrent readouts of 16S rRNA together with the species-specific capB or ecf1 loci by integrating Cas13a-mediated RNA recognition and Cas12a-mediated DNA recognition into a dual-channel workflow. Taken together, proposed method establishes addressable signaling at the protein layer, supports protein-based outputs in a modular workflow, and provides a general route to sensitive, dual-channel nucleic acid detection.},
}

*Biosensing Techniques/methods
Cell-Free System
*Protein Biosynthesis/genetics
*Escherichia coli O157/genetics
Bacillus anthracis/genetics
*CRISPR-Cas Systems/genetics
*RNA, Ribosomal, 16S/genetics/analysis
*Nucleic Acids/analysis/genetics
Bacterial Proteins/genetics

@article {pmid41720308,
year = {2026},
author = {Shi, Q and Huang, W and Hu, D and Zhang, P and Chen, X and Hu, H and Wang, Y and Zhou, J and Weng, R and Quan, J and Zhao, D and Du, X and Yu, Y and Jiang, Y},
title = {The nationwide genomic characteristics and phylogenetic evolution of ST23-K1 hypervirulent Klebsiella pneumoniae in relation to virulence and antimicrobial resistance acquisition.},
journal = {The Journal of infection},
volume = {92},
number = {3},
pages = {106709},
doi = {10.1016/j.jinf.2026.106709},
pmid = {41720308},
issn = {1532-2742},
mesh = {*Klebsiella pneumoniae/genetics/pathogenicity/drug effects/classification ; *Klebsiella Infections/microbiology/epidemiology ; Virulence/genetics ; Phylogeny ; Animals ; China/epidemiology ; Mice ; Humans ; Plasmids/genetics ; Whole Genome Sequencing ; Genome, Bacterial ; Evolution, Molecular ; Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Bacterial Proteins/genetics ; Virulence Factors/genetics ; beta-Lactamases/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; },
abstract = {OBJECTIVES: Hypervirulent Klebsiella pneumoniae (hvKp) ST23-K1 poses a global health threat due to its high virulence and increasing antimicrobial resistance. This study aimed to characterise the genomic feature and phylogenetic evolution of ST23-K1 in China.

METHODS: K1 isolates from a nationwide epidemiological surveillance project underwent whole-genome sequencing. Virulence was assessed using hypermucoviscosity phenotyping and a murine infection model. For ST23-K1 carrying acquired antimicrobial resistance genes (ARGs), the CRISPR/Cas system, protospacers, anti-CRISPR (Acr) genes, and plasmidome were characterised. Time-resolved phylogenetic analysis was performed using integrated locally generated and publicly available data.

RESULTS: Among 400 K1 isolates, ST23 was the most prevalent sequence type, and its effective population size increased following CG23-I divergence. The CG23-I sub-lineage was widely distributed nationwide with limited evidence of clonal transmission. Isolates with an incomplete cps locus exhibited significantly reduced virulence compared with those carrying an intact locus. The prevalence of extended-spectrum β-lactamase-positive ST23-K1 isolates increased over time, whereas carbapenemase-producing isolates remained stable. Among acquired ARGs-positive ST23-K1 isolates, a conserved protospacer corresponding to a prevalent spacer was identified. This protospacer, together with AcrIE genes, was frequently co-located on IncFII-type plasmids.

CONCLUSION: ST23-K1 remains a hypervirulent lineage undergoing ongoing evolutionary expansion. The presence of acquired ARGs in ST23-K1 may be associated with AcrIE-harbouring IncFII plasmids, and functional validation is required to clarify the underlying mechanisms. Continuous genomic surveillance is essential to monitor the evolution and antimicrobial resistance trends of ST23-K1.},
}

*Klebsiella pneumoniae/genetics/pathogenicity/drug effects/classification
*Klebsiella Infections/microbiology/epidemiology
Virulence/genetics
Phylogeny
Animals
China/epidemiology
Mice
Humans
Plasmids/genetics
Whole Genome Sequencing
Genome, Bacterial
Evolution, Molecular
Anti-Bacterial Agents/pharmacology
*Drug Resistance, Bacterial/genetics
Bacterial Proteins/genetics
Virulence Factors/genetics
beta-Lactamases/genetics
Drug Resistance, Multiple, Bacterial/genetics

@article {pmid41720778,
year = {2026},
author = {Lee, YJ and Zhang, D and Stolze, SC and Saridis, G and Ebert, MK and Nakagami, H and Doehlemann, G},
title = {Ustilago maydis disrupts carbohydrate signaling networks to induce hypertrophy in host cells.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41720778},
issn = {2041-1723},
support = {DO1421/3-3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; //European Union’s Horizon 2020 research and innovation program/ ; },
mesh = {*Zea mays/microbiology/metabolism/genetics ; Signal Transduction ; Plant Proteins/metabolism/genetics ; *Plant Diseases/microbiology ; Starch/metabolism/biosynthesis ; Gene Expression Regulation, Plant ; Host-Pathogen Interactions ; Fungal Proteins/metabolism/genetics ; Plant Leaves/microbiology/metabolism ; Mesophyll Cells/metabolism/microbiology ; *Carbohydrate Metabolism ; Endoreduplication ; Virulence Factors/metabolism/genetics ; Cell Cycle/genetics ; CRISPR-Cas Systems ; Basidiomycota ; },
abstract = {Ustilago maydis infection in maize causes hypertrophic leaf tumors; however, the underlying mechanisms driving this excessive cell growth are unknown. In this study, we identify Hap1 (hypertrophy-associated protein 1) as an effector and virulence factor that regulates mesophyll cell hypertrophy. Using CRISPR-Cas9 mutagenesis, we demonstrate that Hap1 contributes to endoreduplication and starch accumulation in infected tissues. Transcriptomics revealed Hap1-dependent upregulation of starch biosynthesis and cell cycle genes, as well as suppression of plant defense. This links Hap1 to metabolic and cell cycle reprogramming, and immune suppression. To identify the target of Hap1 that drives metabolic reprogramming, we investigated its interaction with ZmSnRK1α in maize. We found that Hap1 interferes with the phosphorylation of SnRK1 substrates and that two Hap1-interacting effectors, Hip1 and Hip2, enhance its protein stability. We conclude that Hap1 contributes to the reprogramming of maize metabolism and cell cycle, as well as mesophyll cell hypertrophy, by modulating the SnRK1 signaling pathway to regulate starch biosynthesis and host defense responses.},
}

*Zea mays/microbiology/metabolism/genetics
Signal Transduction
Plant Proteins/metabolism/genetics
*Plant Diseases/microbiology
Starch/metabolism/biosynthesis
Gene Expression Regulation, Plant
Host-Pathogen Interactions
Fungal Proteins/metabolism/genetics
Plant Leaves/microbiology/metabolism
Mesophyll Cells/metabolism/microbiology
*Carbohydrate Metabolism
Endoreduplication
Virulence Factors/metabolism/genetics
Cell Cycle/genetics
CRISPR-Cas Systems
Basidiomycota

@article {pmid41720881,
year = {2026},
author = {Ong, JK and Bhunia, S and Hilbert, B and Kirschner, V and Duglosz, S and Zimmermann, F and Freichel, M and Cornean, A},
title = {ABE9 fused to SpRY Cas9 nickase enables precise generation of bystander free mouse models.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41720881},
issn = {2045-2322},
mesh = {Animals ; *Gene Editing/methods ; Mice ; *CRISPR-Cas Systems ; *Deoxyribonuclease I/genetics/metabolism ; Humans ; Disease Models, Animal ; *CRISPR-Associated Protein 9/genetics/metabolism ; Point Mutation ; },
abstract = {Point mutations cause many genetic disorders, but modelling them in organisms is technically challenging. Creating mouse models that mimic these mutations is crucial for establishing a causal relationship between mutations and disease phenotype, thereby supporting the development of therapeutic strategies. Adenine base editors (ABEs) can correct single-nucleotide variants (SNVs) in disease modelling without double-stranded breaks (DSBs) or donor DNA, achieving higher product purity than traditional Cas9 methods. Earlier ABE techniques faced issues like limited targetability, bystander editing, and off-target effects. By combining two editor advancements, we introduced and tested ABE9-SpRY, an improved ABE variant fused with a PAM-flexible SpRY-Cas9 nickase. Our results show that ABE9-SpRY effectively generates three out of four targeted A-to-G mutations in mouse embryos, achieving desired editing efficiencies of up to 96% in individual adult founder mice. Furthermore, we observe fewer off-target events at predicted DNA sites in mouse embryos and in an orthogonal R-loop assay compared with ABE8e-SpRY. ABE9-SpRY also enhances product purity in mouse embryos under pooled sgRNA injections and, as a proof-of-concept, at a single endogenous locus in human induced pluripotent stem cells (hiPSCs), relative to ABE8e-SpRY. Our findings support ABE9-SpRY's precision at the loci tested and PAM-flexible versatility. Although performance remains sequence-dependent, these data support ABE9-SpRY as a PAM-flexible tool for generating precise point-mutation models where bystander editing is a concern.},
}

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

@article {pmid41720886,
year = {2026},
author = {Nagalakshmi, U and Rodriguez, JE and Nguyen, T and Weissman, RF and Thornton, BW and Terrace, CI and Savage, DF and Dinesh-Kumar, SP},
title = {High-efficiency, transgene-free plant genome editing by viral delivery of an engineered TnpB.},
journal = {Nature plants},
volume = {12},
number = {3},
pages = {503-511},
pmid = {41720886},
issn = {2055-0278},
support = {IOS-2303522//National Science Foundation (NSF)/ ; DGE-2146752//National Science Foundation (NSF)/ ; },
mesh = {*Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Genome, Plant/genetics ; *Plant Viruses/genetics ; CRISPR-Cas Systems ; Transgenes ; *Nicotiana/genetics ; Genetic Vectors ; *Arabidopsis/genetics ; },
abstract = {Genome editing has revolutionized plant biology research[1], yet the efficient delivery of editing reagents remains a challenge. Current methods are labour intensive, involving lengthy tissue culture and complex transformation and regeneration steps. Viral delivery can mitigate these issues[2] but CRISPR-Cas nucleases exceed viral cargo limits, restricting guide RNA (gRNA) delivery into Cas9-expressing transgenic plants[2-11]. This requires generating an initial Cas9 transgenic line. Furthermore, gRNAs delivered by plant viral vectors can induce somatic edits, although only a few produce heritable edits[3-7,9-12]. Some engineered plant negative-strand rhabdoviruses can deliver both Cas9 and gRNA, but they face other challenges, including the need for tissue regeneration or pruning infected plants, and some rhabdoviruses can be delivered only through vector transmission[13-16]. Recently, smaller editors such as TnpBs were discovered, but they are significantly less active than Cas9[17-19]. Here we optimized a tobacco rattle virus-based system to deliver recently engineered, highly active ISDra2 TnpB variants. The eTnpBc variant enables effective somatic editing in systemic leaves and achieves up to 90% editing efficiency at target loci. In addition, up to 89% of offspring exhibit a mutant phenotype, with editing efficiencies reaching 100%. The design principles outlined here could promote wider use of eTnpBc for efficient, transformation- and transgene-free plant genome editing.},
}

*Gene Editing/methods
Plants, Genetically Modified/genetics
*Genome, Plant/genetics
*Plant Viruses/genetics
CRISPR-Cas Systems
Transgenes
*Nicotiana/genetics
Genetic Vectors
*Arabidopsis/genetics

@article {pmid41721525,
year = {2026},
author = {Chen, N and Gao, M and Bai, Y and Wang, M and Liu, M and Xiong, W and Mo, B},
title = {5'UTR Editing of the ribosomal protein UL3Z gene unveils its critical roles in pre-rRNA processing and global mRNA translation dynamics.},
journal = {Plant physiology},
volume = {200},
number = {3},
pages = {},
doi = {10.1093/plphys/kiag073},
pmid = {41721525},
issn = {1532-2548},
support = {32470592//National Natural Science Foundation of China/ ; 32270595//National Natural Science Foundation of China/ ; 2023B001//SZU 2035 Excellence Research Program/ ; },
mesh = {*5' Untranslated Regions/genetics ; *Ribosomal Proteins/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; *RNA Precursors/metabolism/genetics ; *Arabidopsis Proteins/genetics/metabolism ; *Protein Biosynthesis/genetics ; RNA, Messenger/metabolism/genetics ; Gene Expression Regulation, Plant ; *RNA Processing, Post-Transcriptional/genetics ; Gene Editing ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {The EMBRYO DEFECTIVE 2207 (EMB2207) gene, encoding ribosomal protein UL3Z, is critical for embryonic development in Arabidopsis, with loss of function resulting in embryo lethality. Despite its importance, the role of UL3Z in the complicated protein translation machinery in plants remains poorly understood due to the lack of viable hypomorphic alleles. In this study, we utilized CRISPR/Cas9 to edit the 5' untranslated region (5'UTR) of UL3Z, generating 5 ul3z mutants with varying degrees of reduced expression levels of UL3Z proteins. The mutant with the lowest expression exhibited the most severe developmental defects. In contrast, null mutants of its paralog UL3Y displayed no observable phenotypes. Interestingly, expression of UL3Y driven by the UL3Z/EMB2207 promoter successfully rescued the phenotypes of ul3z, demonstrating that these 2 paralogous ribosomal proteins actually possess functionally interchangeable roles. GUS staining results showed that UL3Z was constitutively expressed in all examined tissues, while UL3Y was only appreciably expressed in specific tissues. Molecular analysis further revealed the accumulation of ribosomal RNA (rRNA) maturation intermediates and increased polysome levels in ul3z mutants, indicating compromised pre-rRNA processing and disturbed global mRNA translation. Interestingly, 3' ends of many rRNA precursors in ul3z had higher frequency of non-encoded tails compared with Col-0. This study demonstrates that CRISPR/Cas9-mediated 5'UTR editing is an effective tool for generating viable hypomorphic alleles of lethal genes and uncovers the critical roles of UL3Z/EMB2207 in pre-rRNA processing and the maintenance of appropriate mRNA translation on ribosomes, underscoring its importance in plant development.},
}

*5' Untranslated Regions/genetics
*Ribosomal Proteins/genetics/metabolism
*Arabidopsis/genetics/metabolism
*RNA Precursors/metabolism/genetics
*Arabidopsis Proteins/genetics/metabolism
*Protein Biosynthesis/genetics
RNA, Messenger/metabolism/genetics
Gene Expression Regulation, Plant
*RNA Processing, Post-Transcriptional/genetics
Gene Editing
CRISPR-Cas Systems
Plants, Genetically Modified

@article {pmid41721881,
year = {2026},
author = {Jin, Z and Liu, M and Liu, M and Qi, X and Zhao, L and Yu, X and Guo, Y and Wen, Y},
title = {CRISPR/Cas9-mediated dual editing of VviGAI and VviFLC generates a novel early-flowering grapevine germplasm.},
journal = {Planta},
volume = {263},
number = {3},
pages = {},
pmid = {41721881},
issn = {1432-2048},
support = {No.32272670//Natural Science Foundation for Young Scientists of Shanxi Province/ ; No.31972986//the National Natural Science Foundation of China/ ; },
mesh = {*Vitis/genetics/growth & development/physiology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Flowers/genetics/growth & development/physiology ; *Plant Proteins/genetics/metabolism ; Phenotype ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; Mutation ; },
abstract = {CRISPR/Cas9-mediated dual knockout of VviGAI1 and VviFLC in grapevine promotes early flowering and induces distinctive morphological changes, offering novel genetic resources for breeding. CRISPR/Cas9-mediated genome editing offers a transformative approach for grapevine improvement. In this study, we achieved simultaneous knockout of two central flowering regulators VviGAI1, a DELLA protein ortholog, and VviFLC, a floral repressor in Vitis vinifera 'Cabernet Sauvignon' using a dual-sgRNA vector system. Remarkably, all 15 independent edited lines exhibited biallelic mutations in both genes, primarily consisting of frameshifts that led to premature termination. The dual-mutant plants displayed a range of distinctive phenotypic alterations, including dwarfism, shortened internodes, modified leaf morphology, and disrupted tendril development. Notably, one line (EL-43) showed precocious flowering under greenhouse conditions, underscoring the synergistic role of VviGAI1 and VviFLC in repressing floral transition. Comparative analysis with previously reported gai mutants revealed both conserved and novel traits, suggesting that structural variation within the DELLA domain contributes to phenotypic diversity. Collectively, our findings establish that dual editing of VviGAI1 and VviFLC not only accelerates flowering but also introduces unique vegetative and reproductive characteristics, providing a valuable genetic resource for future grapevine domestication and precision breeding efforts.},
}

*Vitis/genetics/growth & development/physiology
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Flowers/genetics/growth & development/physiology
*Plant Proteins/genetics/metabolism
Phenotype
Plants, Genetically Modified
Gene Expression Regulation, Plant
Mutation

@article {pmid41722369,
year = {2026},
author = {Boumpoureka, I and Gorgogietas, V and Petkovski, E and Massart, F and Mellick, GD and Krüger, R},
title = {Generation of two isogenic control iPSC lines (LCSBi001-A-2 and LCSBi001-A-3) from a Parkinson's disease patient line (LCSBi001-A) carrying the pathogenic VPS35 p.D620N mutation.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103944},
doi = {10.1016/j.scr.2026.103944},
pmid = {41722369},
issn = {1876-7753},
mesh = {Humans ; *Parkinson Disease/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology/pathology ; *Vesicular Transport Proteins/genetics ; *Mutation/genetics ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {The pathogenic mutation VPS35 p.D620N has been identified to cause autosomal dominant, late-onset Parkinson's disease (PD) in multiple individuals and families worldwide. Here, we describe the generation of two new isogenic control lines (LCSBi001-A-2 and LCSBi001-A-3) from an already established patient-derived line (LCSBi001-A) carrying the heterozygous VPS35 p.D620N mutation. The control lines were generated with CRISPR/Cas9 technology, and the correction of the mutation was verified with Sanger sequencing. Both lines express pluripotency markers, are capable of in vitro differentiation into the three germ layers, and have a normal karyotype. The mutant and control iPSC lines are available for research purposes.},
}

Humans
*Parkinson Disease/genetics/pathology/metabolism
*Induced Pluripotent Stem Cells/metabolism/cytology/pathology
*Vesicular Transport Proteins/genetics
*Mutation/genetics
Cell Line
CRISPR-Cas Systems
Cell Differentiation

@article {pmid41722572,
year = {2026},
author = {Abadie, FMC and Suiter, CC and Smith, NT and Daza, RM and Rominger, MC and Parrish, P and McDiarmid, TA and Lalanne, JB and Martin, B and Calderon, D and Ellison, A and Berger, AH and Shendure, J and Starita, LM},
title = {A multiplex, prime editing framework for identifying drug resistance variants at scale.},
journal = {Cell genomics},
volume = {6},
number = {5},
pages = {101167},
pmid = {41722572},
issn = {2666-979X},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Point Mutation/genetics ; *Drug Resistance, Neoplasm/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Protein Kinase Inhibitors/pharmacology ; Cell Line, Tumor ; },
abstract = {CRISPR-based genome editing has revolutionized functional genomics, enabling thousands of perturbations to be concurrently assayed in single experiments. However, for methods such as saturation genome editing (SGE), which aims to generate and assay libraries of point mutations, a challenge is that only one region (e.g., one exon) is studied per experiment. Here, we describe prime-SGE, a prime editing-based framework in which libraries of specific point mutations are installed into genes throughout the genome and then functionally assessed by sequencing of prime editing guide RNAs (pegRNAs) rather than the mutations themselves. We apply prime-SGE in two cell lines to assay thousands of point mutations in eight oncogenes for their ability to confer drug resistance to four tyrosine kinase inhibitors. Our prime-SGE strategy, combined with ongoing improvements in prime editing efficiency, opens the door to efficient positive selection screens of large numbers of point mutations at locations throughout the genome.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Point Mutation/genetics
*Drug Resistance, Neoplasm/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Protein Kinase Inhibitors/pharmacology
Cell Line, Tumor