@article {pmid41090337,
year = {2025},
author = {Cherdantsev, AI and Kulagin, KA and Polyakova, AN and Karpov, VL and Sosnovtseva, AO and Karpov, DS},
title = {[DNA Double-Strand Break Repair System by a Mechanism of Non-Homologous End Joining Provides Resistance to DNA-Damaging and Oxidizing Stresses in the Yeast Debaryomyces hansenii].},
journal = {Molekuliarnaia biologiia},
volume = {59},
number = {4},
pages = {616-628},
doi = {10.31857/S0026898425040083},
pmid = {41090337},
issn = {0026-8984},
mesh = {*DNA End-Joining Repair ; *DNA Breaks, Double-Stranded ; *Oxidative Stress/genetics ; CRISPR-Cas Systems ; *Debaryomyces/genetics/metabolism ; *Fungal Proteins/genetics/metabolism ; Osmotic Pressure ; },
abstract = {The unconventional halotolerant yeast Debaryomyces hansenii is of great importance in biotechnology and the food industry, and in basic research it serves as a model for studying the molecular mechanisms of resistance to increased salinity and osmotic stress. We have previously established an efficient method for editing the D. hansenii genome using the CRISPR/Cas9 system. In turn, this has stimulated further investigation of the structure and physiological role of DNA double-strand break repair pathways in D. hansenii. The aim of the present work was to evaluate the involvement of key components of the DNA double-stranded break repair system by the non-homologous end joining (NHEJ) mechanism in the resistance of D. hansenii to DNA-damaging compounds and compounds that induce oxidative, high salinity, and osmotic stress. Using the CRISPR/Cas9 system, mutant strains with knockout of the DEHA2F10208g (DhKU70), DEHA2B01584g (DhKU80) , and DEHA2G04224g (DhLIG4) genes encoding key components of NHEJ were obtained. It was found that mutant strains, unlike the wild-type strain, are sensitive to chemical compounds that damage DNA, as well as to compounds that cause oxidative stress. Osmotic and high salinity stresses and vanillin do not cause significant changes in the rate of colony formation of mutant strains. Unexpectedly, mutant strains exhibit increased resistance to caffeine compared to the wild-type strain. The data indicate that the NHEJ systems of D. hansenii play a significant role in the response to DNA-damaging and oxidative types of stress. The importance of the NHEJ system in the processes of maintaining yeast cell homeostasis should be taken into account when creating strains producing valuable substances.},
}

*DNA End-Joining Repair
*DNA Breaks, Double-Stranded
*Oxidative Stress/genetics
CRISPR-Cas Systems
*Debaryomyces/genetics/metabolism
*Fungal Proteins/genetics/metabolism
Osmotic Pressure

@article {pmid41090155,
year = {2025},
author = {Li, JM and Huang, J and Liao, Y and Hu, T and Wang, CL and Zhang, WZ and Huang, CW},
title = {Gene and RNA Editing: Revolutionary Approaches to Treating Diseases.},
journal = {MedComm},
volume = {6},
number = {10},
pages = {e70389},
pmid = {41090155},
issn = {2688-2663},
abstract = {Gene editing and RNA editing technologies are advancing modern medicine by enabling precise manipulation of genetic information at the DNA and RNA levels, respectively. The third-generation gene editing tools, particularly Clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) system, have transformed genetic disease treatment with high efficiency, precision, and cost effectiveness, while RNA editing, via adenosine deaminase acting on RNA (ADAR) enzymes and CRISPR-Cas13, offers reversible regulation to avoid genomic integration risks. Despite advancements, challenges persist in delivery efficiency, tissue specificity, and long-term safety, limiting their clinical translation. This review systematically discusses the molecular mechanisms and technological evolution of these tools, focusing on their promising applications in treating nervous system disorders (e.g., Alzheimer's, Parkinson's), immune diseases (e.g., severe combined immunodeficiency, lupus), and cancers. It compares their technical attributes, analyzes ethical and regulatory issues, and highlights synergies between the two technologies. By bridging basic research and clinical translation, this review provides critical insights for advancing precision medicine, reshaping disease diagnosis, prevention, and treatment paradigms.},
}

@article {pmid41088816,
year = {2025},
author = {Mo, T and Ren, HY and Zhang, XX and Lu, YW and Teng, ZQ and Zhang, X and Dai, LP and Hou, L and Zhao, N and He, J and Qin, T},
title = {Phenotypic Function of Legionella pneumophila Type I-F CRISPR-Cas.},
journal = {Biomedical and environmental sciences : BES},
volume = {38},
number = {9},
pages = {1105-1119},
doi = {10.3967/bes2025.107},
pmid = {41088816},
issn = {2214-0190},
mesh = {*Legionella pneumophila/genetics/physiology/pathogenicity ; *CRISPR-Cas Systems ; Biofilms/growth & development ; Phenotype ; Bacterial Proteins/genetics/metabolism ; Gene Deletion ; },
abstract = {OBJECTIVE: CRISPR-Cas protects bacteria from exogenous DNA invasion and is associated with bacterial biofilm formation and pathogenicity.

METHODS: We analyzed the type I-F CRISPR-Cas system of Legionella pneumophila WX48, including Cas1, Cas2-Cas3, Csy1, Csy2, Csy3, and Cas6f, along with downstream CRISPR arrays. We explored the effects of the CRISPR-Cas system on the in vitro growth, biofilm-forming ability, and pathogenicity of L. pneumophila through constructing gene deletion mutants.

RESULTS: The type I-F CRISPR-Cas system did not affect the in vitro growth of wild-type or mutant strains. The biofilm formation and intracellular proliferation of the mutant strains were weaker than those of the wild type owing to the regulation of type IV pili and Dot/Icm type IV secretion systems. In particular, Cas6f deletion strongly inhibited these processes.

CONCLUSION: The type I-F CRISPR-Cas system may reduce biofilm formation and intracellular proliferation in L. pneumophila.},
}

*Legionella pneumophila/genetics/physiology/pathogenicity
*CRISPR-Cas Systems
Biofilms/growth & development
Phenotype
Bacterial Proteins/genetics/metabolism
Gene Deletion

@article {pmid41086252,
year = {2025},
author = {Libri, AB and Wang, J and Marton, T and Yu, W and Dossin, F and Balmus, G and Reina-San-Martin, B and Frock, R and Lescale, C and Deriano, L},
title = {Senataxin promotes recombination fidelity during antigen receptor gene diversification.},
journal = {Science signaling},
volume = {18},
number = {908},
pages = {eadv8801},
doi = {10.1126/scisignal.adv8801},
pmid = {41086252},
issn = {1937-9145},
mesh = {Animals ; *V(D)J Recombination/genetics ; Mice ; DNA End-Joining Repair ; *RNA Helicases/genetics/metabolism ; Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; DNA Breaks, Double-Stranded ; Multifunctional Enzymes ; CRISPR-Cas Systems ; Mice, Knockout ; *DNA Helicases/genetics/metabolism ; Humans ; *Receptors, Antigen, T-Cell/genetics ; Precursor Cells, B-Lymphoid/metabolism/immunology ; },
abstract = {Antigen receptor diversity depends on the assembly of variable (V), diverse (D), and joining (J) exons in genes encoding immunoglobulins (Igs) and T cell receptors (TCRs). During V(D)J recombination, DNA double-strand breaks (DSBs) introduced by the RAG1/2 nuclease complex are repaired by the process of nonhomologous end-joining (NHEJ). We hypothesized that functional redundancies between NHEJ and the chromatin DSB response, which depends on the kinase ATM, potentially masked the activity of additional factors that regulate V(D)J recombination. We performed targeted CRISPR-Cas9 knockout screens for genes implicated in V(D)J recombination in pro-B cells that were either untreated or treated with an ATM inhibitor. We found that loss of the RNA/DNA helicase senataxin (SETX) impaired V(D)J recombination and led to the formation of aberrant hybrid joints between coding ends and signal ends, both in vitro and in mice. The loss of SETX in a background deficient in the NHEJ factor XLF or in which ATM was inhibited led to substantial impairment of V(D)J recombination and to the presence of unsealed coding ends. SETX limited aberrant activation-induced cytidine deaminase (AID)-induced DNA end-joining between Igh-containing alleles during the process of class-switch recombination. Together, our findings reveal a previously uncharacterized role for SETX in promoting recombination fidelity during antigen receptor gene diversification.},
}

Animals
*V(D)J Recombination/genetics
Mice
DNA End-Joining Repair
*RNA Helicases/genetics/metabolism
Ataxia Telangiectasia Mutated Proteins/genetics/metabolism
DNA Breaks, Double-Stranded
Multifunctional Enzymes
CRISPR-Cas Systems
Mice, Knockout
*DNA Helicases/genetics/metabolism
Humans
*Receptors, Antigen, T-Cell/genetics
Precursor Cells, B-Lymphoid/metabolism/immunology

@article {pmid41085803,
year = {2025},
author = {de Souza, HCA and Panzenhagen, P and Portes, AB and Dos Santos, AMP and Fidelis, J and Junior, CAC},
title = {CRISPR-Cas systems in combating antimicrobial resistance: which system to choose? A systematic review.},
journal = {World journal of microbiology & biotechnology},
volume = {41},
number = {10},
pages = {381},
pmid = {41085803},
issn = {1573-0972},
support = {FinanceCode001//Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/ ; 313119/2020-1//Conselho Nacional de Desenvolvimento Científico e Tecnológico/ ; E26/202.227/2018//Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Bacteria/genetics/drug effects ; *Drug Resistance, Bacterial/genetics ; Humans ; Anti-Bacterial Agents/pharmacology ; },
abstract = {Antimicrobial resistance (AMR) poses a growing threat to global public health, progressively compromising the efficacy of available antimicrobials. Technologies based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) have emerged as promising tools for controlling resistant pathogens, offering high specificity and versatility. However, a comprehensive and systematic synthesis of CRISPR strategies applied to AMR remains limited. From February 12, 2025, we conducted a systematic review of the PubMed, Embase, and Scopus databases, using the following search strategy: Population (resistant bacteria or plasmid-mediated resistance), Intervention (CRISPR, including variants such as CRISPR-Cas9, Cas3, Cas12, Cas13, and CRISPR interference [CRISPRi]), and Outcomes (bacterial resensitization or plasmid curing). The CRISPR-Cas9 system was the most frequently employed (75.7%), with conjugation identified as the primary delivery method. We identified the advantages and limitations of each system, highlighting CRISPRi and CRISPR-Cas13a as alternatives to overcome the constraints of direct genome editing. Delivery efficiency remains a central challenge, although nanocarrier- and bacteriophage-based methodologies show promising potential. We also propose a decision map that guides the selection of the most appropriate CRISPR-Cas system and delivery strategy, considering factors such as therapeutic objective, gene location, methodology efficiency, application environment, and clinical feasibility. This review provides an updated and structured synthesis of CRISPR strategies applied to AMR, emphasizing their potential translational and clinical applications. The findings can inform the development of CRISPR-based therapeutics, guide the design of preclinical studies, and support future strategies for combating multidrug-resistant infections in clinical settings.},
}

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

@article {pmid41085033,
year = {2025},
author = {Levassor, L and Whitford, CM and Petersen, SD and Blin, K and Weber, T and Frandsen, RJN},
title = {StreptoCAD: An Open-Source Software Toolbox Automating Genome Engineering Workflows in Streptomycetes.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00261},
pmid = {41085033},
issn = {2161-5063},
abstract = {Streptomycetes hold immense potential for discovering novel bioactive molecules for applications in medicine or sustainable agriculture. However, high-throughput exploration is hampered by the current Streptomyces genetic engineering methods that involve the manual design of complex experimental molecular biological engineering strategies for each targeted gene. Here, we introduce StreptoCAD, an open-source software toolbox that automates and streamlines the design of genome engineering strategies in Streptomyces, supporting various CRISPR-based and gene overexpression methods. Once initiated, StreptoCAD designs all necessary DNA primers and CRISPR guide sequences, simulates plasmid assemblies (cloning) and the resulting modification of the genomic target(s), and further summarizes the information needed for laboratory implementation and documentation. StreptoCAD currently offers six design workflows, including the construction of overexpression libraries, base-editing, including multiplexed CRISPR-BEST plasmid generation, and genome engineering using CRISPR-Cas9, CRISPR-Cas3, and CRISPRi systems. In addition to automating the design process, StreptoCAD further secures compliance with the FAIR principles, ensuring reproducibility and ease of data management via standardized output files. To experimentally demonstrate the design process and output of StreptoCAD, we designed and constructed a series of gene overexpression strains, and performed CRISPRi knockdowns in Streptomyces Gö40/10, underscoring the tool's efficiency and user-friendliness.. This tool simplifies complex genetic engineering tasks and promotes collaboration through standardized workflows and design parameters. StreptoCAD is set to transform genome engineering in Streptomyces, making sophisticated genetic manipulations accessible for all and accelerating natural product discovery.},
}

@article {pmid41084992,
year = {2025},
author = {Han, J and Ganguly, R and Yi, JY and Yun, H and Jung, SY and Sung, C and Lee, CS},
title = {Osmotically Tunable Microdroplets Enable Amplification-Free CRISPR Detection of Gene Doping.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e15861},
doi = {10.1002/advs.202515861},
pmid = {41084992},
issn = {2198-3844},
abstract = {Gene doping is an increasing challenge in sports, demanding highly sensitive and specific detection tools beyond the limitations of the current amplification-dependent methods. Here, an innovative amplification-free clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) 12a assay integrated with osmotically tunable double emulsion (DE) droplets is reported for rapid and ultrasensitive gene doping detection. Target DNA and CRISPR/Cas12a complexes are encapsulated within DE droplets, where osmotic shrinkage rapidly concentrates the reaction components, thereby enhancing the fluorescent signal intensity without nucleic acid amplification. This platform enables the detection of the human erythropoietin (hEPO) gene at unprecedented attomolar levels within 30 min, achieving a 25-fold improvement in sensitivity compared with that of nonshrinkable formats. Notably, the assay demonstrated a robust and specific performance in complex serum samples with minimal matrix interference. This novel approach offers a rapid, reliable, and inherently contamination-free solution for gene doping surveillance with broad potential for versatile amplification-free nucleic acid diagnostics.},
}

@article {pmid41083939,
year = {2025},
author = {Menelih, A and Girma, A and Aemiro, A},
title = {Advancing nutritional quality in oilseed crops through genome editing: a comprehensive review.},
journal = {GM crops & food},
volume = {16},
number = {1},
pages = {709-732},
pmid = {41083939},
issn = {2164-5701},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics/metabolism ; *Nutritive Value ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Plant Oils/metabolism ; Seeds/genetics ; Fatty Acid Desaturases/genetics/metabolism ; Genome, Plant ; },
abstract = {Genome editing has emerged as a powerful approach to enhancing the nutritional quality of oilseed crops. Clustered regularly interspaced short palindromic repeats case9 (CRISPR/Cas9) is the predominant editing tool, while transcription activator-like effector nucleases (TALENs) and base editors are used less commonly. Key fatty acid desaturase genes such as FAD2 and FAD3 are prime targets because of their critical functions in fatty acid desaturation. This review summarizes recent progress in editing genes associated with oil composition and related traits across oilseed species. Visual data representations including, Sankey diagrams, heat maps, and crop-trait matrices illustrate shared editing priorities and emerging trait targets across crops. Despite its promise, genome editing still faces challenges in transformation efficiency, field-level validation, and regulatory acceptance. This review underscores the increasing impact of target gene editing on nutritional trait improvement and its potential to accelerate the development of healthier, more sustainable oilseed varieties.},
}

*Gene Editing/methods
*Crops, Agricultural/genetics/metabolism
*Nutritive Value
CRISPR-Cas Systems
Plants, Genetically Modified/genetics
*Plant Oils/metabolism
Seeds/genetics
Fatty Acid Desaturases/genetics/metabolism
Genome, Plant

@article {pmid41002041,
year = {2025},
author = {Clark, MB and Funk, AT and Paporakis, A and Brown, GP and Beach, SJ and Tay, A and Deering, S and Cooper, C and Tizard, M and Jolly, CJ and Ward-Fear, G and Waddle, AW and Shine, R and Maselko, M},
title = {Efficient CRISPR-Cas9-Mediated Genome Editing of the Cane Toad (Rhinella marina).},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {321-332},
doi = {10.1177/25731599251382427},
pmid = {41002041},
issn = {2573-1602},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Monophenol Monooxygenase/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Bufo marinus/genetics ; Albinism/genetics ; Introduced Species ; },
abstract = {Invasive species inflict major ecological, economic, and cultural harm worldwide, highlighting the urgent need for innovative control strategies. Genome editing offers exciting possibilities for targeted control methods for invasive species. Here, we demonstrate CRISPR-Cas9 genome editing in the cane toad (Rhinella marina), one of Australia's most notorious invasive species, by targeting the tyrosinase gene to produce albino phenotypes as visual markers for assessing editing efficiency. Microinjection of Cas9 protein and guide RNAs into one-cell zygotes resulted in 87.6% of mosaic larvae displaying nearly complete albinism, with 2.3% exhibiting complete albinism. For completely albino individuals, genomic analysis confirmed predominantly frameshift mutations or large deletions at the target site, with no wild-type alleles detected. Germline transmission rates reflected the extent of albinism in the mosaic adult, with maternal transmission approaching 100%. This first application of CRISPR-Cas9 in the Bufonidae family opens possibilities for exploring basic research questions and population control strategies.},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Monophenol Monooxygenase/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Bufo marinus/genetics
Albinism/genetics
Introduced Species

@article {pmid40875132,
year = {2025},
author = {Skipper, TS and Dickson, KA and Denes, CE and Waller, MA and Du, TY and Neely, GG and Bowden, NA and Faiz, A and Marsh, DJ},
title = {Revealing genetic drivers of ovarian cancer and chemoresistance: insights from whole-genome CRISPR-knockout library screens.},
journal = {Cellular oncology (Dordrecht, Netherlands)},
volume = {48},
number = {5},
pages = {1245-1265},
pmid = {40875132},
issn = {2211-3436},
mesh = {Humans ; *Ovarian Neoplasms/genetics/drug therapy ; Female ; *Drug Resistance, Neoplasm/genetics ; *Gene Knockout Techniques ; *CRISPR-Cas Systems/genetics ; *Gene Library ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Understanding genetic dependencies in cancer is key to identifying novel actionable drug targets to advance precision medicine. Whole-genome CRISPR-knockout library screening methods have facilitated this goal. Pooled libraries of single guide RNAs (sgRNAs) targeting over 90% of the annotated protein coding genome are used to induce gene knockouts in pre-clinical cancer models. Novel genes of interest are identified by evaluating sgRNA dropout or enrichment following selection pressure application. This method is particularly beneficial for researching cancers where effective treatment strategies are limited. One example of a commonly chemoresistant cancer, particularly at relapse, is the low survival malignancy epithelial ovarian cancer (EOC), made up of multiple histotypes with distinct molecular profiles. CRISPR-knockout library screens in pre-clinical EOC models have demonstrated the ability to predict biomarkers of treatment response, identify targets synergistic with standard-of-care chemotherapy, and determine novel actionable targets which are synthetic lethal with cancer-associated mutations. Robust experimental design of CRISPR-knockout library screens, including the selection of strong pre-clinical cell line models, allows for meaningful conclusions to be made. We discuss essential design criteria for the use of CRISPR-knockout library screens to discover genetic dependencies in cancer and draw attention to discoveries with translational potential for EOC.},
}

Humans
*Ovarian Neoplasms/genetics/drug therapy
Female
*Drug Resistance, Neoplasm/genetics
*Gene Knockout Techniques
*CRISPR-Cas Systems/genetics
*Gene Library
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid40789680,
year = {2025},
author = {Sun, X and Li, M and Wang, H and Yang, Y and Kang, Y and Sun, P and Dong, J and Jin, M and Jin, W},
title = {Possible Reversion of CRISPR-Cas9-Edited Sequences in Octoploid Strawberry.},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {375-389},
doi = {10.1177/25731599251361374},
pmid = {40789680},
issn = {2573-1602},
mesh = {*Fragaria/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plant Leaves/genetics ; Polyploidy ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics ; Seedlings/genetics ; Plant Proteins/genetics ; },
abstract = {Gene editing is more challenging in octoploids due to the presence of multiple copies of each gene. However, the ability to edit genes in these plants would allow editing in commercial varieties. Here, we delivered sequences targeting FaMYB9 into octoploid strawberry "Honeoye" and identified several gene-edited lines. Among them, the heterozygous gene-edited line FaMYB9[CR]-15 had curved and wrinkled leaves at 3 months, whereas leaves of 3-month-old wild-type (WT) strawberry seedlings were elliptical with a smooth surface. At that stage, FaMYB9[CR]-15 leaves also had large patches of wax. We identified 11,402 differentially expressed genes, divided into four clusters, between WT and FaMYB9[CR]-15 seedlings at 3 months. Notably, cluster 4 genes-related to nonhomologous end joining, microhomology-mediated end joining repairs, homologous recombination, nucleotide excision repair, and mismatch repair-were more highly expressed in the gene-edited line than in the WT. Surprisingly, by 6 months of age, FaMYB9[CR]-15 leaves had become smooth with small patches of wax, and expression levels of cluster 4 genes were significantly lower than at 3 months. Over the same period, the percentage of FaMYB9 loci harboring the mutant allele decreased from 70.2% to 43.7%. These findings lead us to conclude that there could be reversion of mutated sequences in octoploid strawberry, emphasizing the challenges of gene editing high-ploidy materials.},
}

*Fragaria/genetics
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Plant Leaves/genetics
Polyploidy
Gene Expression Regulation, Plant
Plants, Genetically Modified/genetics
Seedlings/genetics
Plant Proteins/genetics

@article {pmid40675779,
year = {2025},
author = {Sherkow, JS},
title = {A "Bare Hope of A Result": The Second CRISPR Patent Appeal.},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {317-320},
doi = {10.1177/25731599251361362},
pmid = {40675779},
issn = {2573-1602},
mesh = {*Patents as Topic/legislation & jurisprudence ; Humans ; *Gene Editing/legislation & jurisprudence ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; United States ; Dissent and Disputes/legislation & jurisprudence ; },
abstract = {On May 12, 2025, the US Court of Appeals for the Federal Circuit issued its second decision in the long-running CRISPR patent dispute between the Regents of the University of California and related institutions (CVC) and the Broad Institute. This Perspective recounts the principal dispute to date, reviews the Federal Circuit's recent opinion, and provides a critique of its analysis. In particular, this Perspective highlights how the decision is self-contradictory and in tension with patent law's conception doctrine-when an inventor has formed a "definite and permanent" idea of an invention in the mind or whether the invention was little more than a "bare hope" of a result. This Perspective briefly concludes with the implications of this recent decision and where the underlying dispute is likely headed.},
}

*Patents as Topic/legislation & jurisprudence
Humans
*Gene Editing/legislation & jurisprudence
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
United States
Dissent and Disputes/legislation & jurisprudence

@article {pmid40659333,
year = {2025},
author = {Manuvera, VA and Bobrovsky, PA and Kharlampieva, DD and Grafskaia, EN and Brovina, KA and Serebrennikova, MY and Lazarev, VN},
title = {Bacterial Expression System with Deep Repression and Activation via CRISPR-Cas9.},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {353-365},
doi = {10.1177/25731599251358852},
pmid = {40659333},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; Plasmids/genetics ; *Gene Editing/methods ; Promoter Regions, Genetic ; *Gene Expression Regulation, Bacterial ; Green Fluorescent Proteins/genetics ; 5' Untranslated Regions ; Terminator Regions, Genetic ; },
abstract = {Incomplete repression of recombinant genes encoding toxic polypeptides can suppress cell growth even in the absence of a transcription inducer. To address this issue, we developed a CRISPR-Cas9-based genome editing approach that directly modifies the plasmid encoding the toxic peptide during Escherichia coli cultivation. The constructed plasmids contained a transcription terminator between the promoter and coding region, preventing full gene expression through abortive transcription. Upon CRISPR-Cas9 activation, this region is excised, thus restoring the functional gene. To implement this approach, we modified widely used pET-series expression plasmids by adding extra terminators in the 5'-untranslated region of the recombinant gene. Four antimicrobial peptides with strong bactericidal properties served as toxic gene products, while green fluorescent protein was used to assess the efficiency of expression repression. As a result, we developed an expression system with strong repression, which is activated by CRISPR-Cas9-mediated excision of a DNA fragment from the plasmids.},
}

*CRISPR-Cas Systems/genetics
*Escherichia coli/genetics
Plasmids/genetics
*Gene Editing/methods
Promoter Regions, Genetic
*Gene Expression Regulation, Bacterial
Green Fluorescent Proteins/genetics
5' Untranslated Regions
Terminator Regions, Genetic

@article {pmid40637801,
year = {2025},
author = {Han, J and Yu, B and Jing, J and He, X and Hua, Y and Xu, G},
title = {EGFR blockade confers sensitivity to pan-RAS inhibitors in KRAS-mutated cancers.},
journal = {Cellular oncology (Dordrecht, Netherlands)},
volume = {48},
number = {5},
pages = {1317-1335},
pmid = {40637801},
issn = {2211-3436},
mesh = {Humans ; *ErbB Receptors/antagonists & inhibitors/metabolism ; Animals ; *Proto-Oncogene Proteins p21(ras)/genetics/antagonists & inhibitors ; Cell Line, Tumor ; *Mutation/genetics ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; Mice ; *Neoplasms/genetics/drug therapy/pathology ; CRISPR-Cas Systems/genetics ; Cell Proliferation/drug effects ; Drug Resistance, Neoplasm/drug effects ; *ras Proteins/antagonists & inhibitors ; },
abstract = {INTRODUCTION: KRAS is one of the most commonly occurring mutated oncogene in human cancers. Development of KRAS G12C or G12D inhibitors exhibit promising clinical activities, but patients harboring other hotspot KRAS mutations cannot benefit from those strategies. Recent development in pan-RAS inhibitors have broad therapeutic implications and merit clinical investigation. However, intrinsic and acquired drug resistance caused by tumor heterogeneity greatly limit the clinical application, posing a significant challenge in this field.

RESULTS: In this study, through CRISPR/Cas9 sgRNA screening using a human kinome sgRNA library, EGFR was discovered to correlate with the sensitivity of KRAS-mutated tumors to pan-RAS inhibitor RMC-7977. Through multiple in vitro cell proliferation or viability assays, EGFR loss or pharmacological EGFR inhibition significantly enhances the effectiveness of pan-RAS inhibitors in multiple KRAS[G12C] or KRAS[G12D] cancer cell lines, disregarding their cellular origins. Mechanistically, co-inhibition of EGFR and pan-RAS may further dampen the RTK-RAS-RAF-MEK-ERK pathway activation than either alone, thereby enhancing the anti-tumor activity of pan-RAS inhibitors. Strikingly, with the LL/2 syngeneic mice tumor model, the combination of pan-RAS inhibitors and EGFR inhibitors demonstrated more significant in vivo therapeutic efficacy compared to either single agent.

CONCLUSION: In conclusion, this study employed high-throughput CRISPR/Cas9 sgRNA screening to identify the enhanced anti-cancer effects when combining EGFR inhibitors with pan-RAS inhibitors in multiple human KRAS-mutated cancer cell lines as well as a mouse syngeneic tumor model. This synergy underscores the potential for a combinational therapy strategy, leveraging EGFR and pan-RAS inhibitors to improve treatment outcomes for patients with KRAS-driven cancers.},
}

Humans
*ErbB Receptors/antagonists & inhibitors/metabolism
Animals
*Proto-Oncogene Proteins p21(ras)/genetics/antagonists & inhibitors
Cell Line, Tumor
*Mutation/genetics
*Protein Kinase Inhibitors/pharmacology/therapeutic use
Mice
*Neoplasms/genetics/drug therapy/pathology
CRISPR-Cas Systems/genetics
Cell Proliferation/drug effects
Drug Resistance, Neoplasm/drug effects
*ras Proteins/antagonists & inhibitors

@article {pmid40637619,
year = {2025},
author = {Guerra, I and Jensen, K and Perez-Pinera, P},
title = {Implementation of an Undergraduate Laboratory-Based Mammalian Genome Editing Course.},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {366-374},
doi = {10.1089/crispr.2025.0017},
pmid = {40637619},
issn = {2573-1602},
mesh = {*Gene Editing/methods ; Humans ; Curriculum ; Animals ; Genetic Engineering/methods ; Laboratories ; Students ; Mammals/genetics ; CRISPR-Cas Systems ; Universities ; },
abstract = {Genome engineering methods can be utilized to perform complex genetic manipulations in living cells with remarkable efficiency and precision. Given the transformative potential of these enabling technologies, their applications are steadily expanding into most biology and biomedical fields where they play a central role in many experimental frameworks. For these reasons, in order to effectively prepare future generations of biologists and bioengineers for successful careers, there is a high need to incorporate courses teaching genome editing fundamentals into existing curricula. To accomplish this objective, lecture-based courses are rapidly integrating genome editing concepts; however, there are few laboratory courses that teach the practical skills needed to successfully perform genome editing experiments. Here, we describe the development and implementation of a semester-long laboratory course that teaches students not only the techniques needed to perform gene knockout, gene activation, gene repression, and base editing in mammalian cells but also prepares them to design and troubleshoot experiments, write scientific manuscripts, as well as prepare and deliver scientific presentations. Course evaluations demonstrate that this class effectively equips students with the knowledge and hands-on experience needed to succeed in careers related to genome engineering, cell and tissue engineering, and, more broadly, biology.},
}

*Gene Editing/methods
Humans
Curriculum
Animals
Genetic Engineering/methods
Laboratories
Students
Mammals/genetics
CRISPR-Cas Systems
Universities

@article {pmid41083470,
year = {2025},
author = {McCallion, O and Du, W and Glaser, V and Milward, K and Short, S and Bilici, M and Cross, A and Stark, H and Franke, C and Kath, J and Valkov, M and Yang, M and Amini, L and Künkele, A and Polansky, JK and Schmueck-Henneresse, M and Volk, HD and Reinke, P and Wagner, DL and Hester, J and Issa, F},
title = {HLA matching or CRISPR editing of HLA class I/II enables engraftment and effective function of allogeneic human regulatory T cell therapy in a humanized mouse transplantation model.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9090},
pmid = {41083470},
issn = {2041-1723},
support = {825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 211122/Z/18//Wellcome Trust (Wellcome)/ ; FS/ 12/72/29754//British Heart Foundation (BHF)/ ; },
mesh = {Animals ; Humans ; *T-Lymphocytes, Regulatory/immunology/transplantation ; Mice ; Skin Transplantation ; Gene Editing/methods ; Graft Survival/immunology ; CRISPR-Cas Systems ; Transplantation, Homologous ; *Histocompatibility Antigens Class I/genetics/immunology ; Adoptive Transfer ; Forkhead Transcription Factors/metabolism/genetics ; CD8-Positive T-Lymphocytes/immunology ; },
abstract = {Regulatory T cells (Tregs) hold promise for treating autoimmune disease and transplant rejection, yet generation of autologous products for adoptive transfer can suffer donor variability and slow turnaround, limiting their use in urgent indications. We therefore examine whether allogeneic, pre-manufactured ('off-the-shelf') Tregs could overcome these barriers. In a human skin-xenograft model, HLA-mismatched Tregs are swiftly eliminated by recipient CD8[+] T cells and fail to protect grafts. Stringent matching of HLA class I and II restores efficacy but is clinically impractical. Using non-viral CRISPR editing we disrupt B2M and CIITA while inserting an HLA-E-B2M fusion, generating hypo-immunogenic Tregs that evade both T and NK cell attack. Engineered cells retain FOXP3 stability and potent in vitro suppression, and after a single low-dose infusion, prolong human skin graft survival in a humanized mouse model comparably to autologous Tregs. Histology and spatial transcriptomics reveal minimal cytotoxic infiltration and enrichment of immunoregulatory and tissue-repair programmes. Multiplex HLA engineering thus enables ready-to-use allogeneic Tregs that withstand host immune attack for adoptive transfer.},
}

Animals
Humans
*T-Lymphocytes, Regulatory/immunology/transplantation
Mice
Skin Transplantation
Gene Editing/methods
Graft Survival/immunology
CRISPR-Cas Systems
Transplantation, Homologous
*Histocompatibility Antigens Class I/genetics/immunology
Adoptive Transfer
Forkhead Transcription Factors/metabolism/genetics
CD8-Positive T-Lymphocytes/immunology

@article {pmid41082405,
year = {2025},
author = {Yang, L and Fang, Y and Lian, Y and Kong, Z and Miao, J and Chen, Y and Li, W and Chen, F and Zhang, B and Chen, Y and Bian, Y},
title = {Aloe-Emodin Targeting FOXC2 Disrupts NETs Formation and EMT-Driven Postoperative Peritoneal Adhesion Through TGF-β1-Smad2/3 Pathway.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e11013},
doi = {10.1002/advs.202511013},
pmid = {41082405},
issn = {2198-3844},
support = {81704084//National Natural Science Foundation of China/ ; 0121/2022/A3//Science and Technology Development Fund, Macau SAR/ ; 0127/2023/RIA2//Science and Technology Development Fund, Macau SAR/ ; SJCX24_1123//Postgraduate Research & Practice Innovation Program of Jiangsu Province/ ; SJCX25_0992//Postgraduate Research & Practice Innovation Program of Jiangsu Province/ ; //333 High-Level Talents Cultivation Project of Jiangsu Province/ ; },
abstract = {Postoperative peritoneal adhesion (PPA) develops through TGF-β1-driven fibrotic remodeling, characterized by neutrophil extracellular trap (NETs)-induced aberrant epithelial-to-mesenchymal transition (EMT) deposition. Although aloe-emodin (AE) exhibits anti-fibrosis potential, its molecular mechanisms remain elusive. Forkhead box protein C2 (FOXC2) is a critical regulator of fibrotic tissue formation, yet its role in PPA is unknown. Here, it is demonstrated that FOXC2 expression is elevated in human ileostomy tissue, PPA rodent model, and TGF-β1-exposed peritoneal mesothelial cells (PMCs), where it orchestrates NETs formation and extracellular matrix (ECM) remodeling. Mechanically, CRISPR/Cas-based knockdown and overexpression of FOXC2 alter EMT changes in PMCs, which is achieved via TGF-β1-Smad2/3 signaling. FOXC2 functions as a dual mediator and amplifier through the TGF-β1-Smad2/3 pathway feedback loop to drive EMT alterations. Its overexpression further induces neutrophil recruitment and NETs formation, exacerbating EMT in PMCs. Notably, AE ameliorates FOXC2-driven peritoneal fibrosis by impeding NETs formation and EMT changes through the TGF-β1-Smad2/3 pathway. Moreover, AE binds directly to FOXC2, and the Ser125 residue is critical for the binding of FOXC2 to AE. These findings identify FOXC2 as a pivotal effector in fibrotic responses during PPA formation and reveal that AE targeting the Ser125 residue of FOXC2 may be a promising therapeutic approach to attenuate PPA.},
}

@article {pmid41082121,
year = {2026},
author = {Kim, WD and Huber, RJ},
title = {Modeling Lysosomal Disease in Dictyostelium discoideum: Examining the Trafficking and Secretion of Lysosomal Enzymes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2976},
number = {},
pages = {189-207},
pmid = {41082121},
issn = {1940-6029},
mesh = {*Dictyostelium/genetics/metabolism/enzymology ; *Lysosomes/enzymology/metabolism ; Protein Transport ; *Lysosomal Storage Diseases/genetics/metabolism/enzymology ; Gene Editing/methods ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Protozoan Proteins/metabolism/genetics ; },
abstract = {Non-mammalian models are powerful systems for enhancing our understanding of lysosomal function and lysosomal storage diseases. The social amoeba Dictyostelium discoideum is an excellent model organism for studying lysosomal function, as its genome encodes many proteins associated with lysosomal disease. Methods for gene knockout are straightforward in D. discoideum and include restriction enzyme-mediated integration (REMI) mutagenesis, homologous recombination via the Cre-loxP system, and CRISPR/Cas9-mediated gene editing, which collectively allow researchers to study protein function (e.g., lysosomal enzymes) in a genetically tractable biomedical model system. Additionally, activity assays for conserved lysosomal enzymes are well-established in D. discoideum. In this chapter, we outline methods for studying the intracellular localization and secretion of conserved lysosomal proteins in D. discoideum.},
}

*Dictyostelium/genetics/metabolism/enzymology
*Lysosomes/enzymology/metabolism
Protein Transport
*Lysosomal Storage Diseases/genetics/metabolism/enzymology
Gene Editing/methods
CRISPR-Cas Systems
Gene Knockout Techniques
Protozoan Proteins/metabolism/genetics

@article {pmid41082119,
year = {2026},
author = {Chear, S and Chiam, A and Talbot, J and Thorne, BN and Wilkinson, EJ and Hewitt, AW and Cook, AL},
title = {Generation of Donor-Specific iPSC for Modelling Lysosomal Storage Disorders.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2976},
number = {},
pages = {151-173},
pmid = {41082119},
issn = {1940-6029},
mesh = {Humans ; *Lysosomal Storage Diseases/pathology/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; Fibroblasts/metabolism/cytology ; *Cell Culture Techniques/methods ; CRISPR-Cas Systems ; Cells, Cultured ; Tissue Donors ; },
abstract = {iPSC technology has enabled the generation of human cell-based models of lysosomal storage disorders and has provided disease-relevant systems to undertake drug discovery or pre-clinical testing of gene- or cell-based therapies. Here, we provide a protocol to generate iPSCs derived from people with lysosomal storage disorders and illustrate expected results using a CLN2 disease donor-specific skin fibroblast culture. Protocol steps include lipofection of episomal plasmids, picking of putative iPSC colonies following live cell TRA-1-60 immunofluorescence, and quality control steps such as immunofluorescence for expression of undifferentiated cell markers, germ layer differentiation, and confirmation of pathological variant genotype. The iPSC generated by this protocol can be differentiated to several cell lineages and can be used with CRISPR/Cas technology to generate isogenic disease models.},
}

Humans
*Lysosomal Storage Diseases/pathology/genetics/metabolism
*Induced Pluripotent Stem Cells/metabolism/cytology
Cell Differentiation
Fibroblasts/metabolism/cytology
*Cell Culture Techniques/methods
CRISPR-Cas Systems
Cells, Cultured
Tissue Donors

@article {pmid41082037,
year = {2025},
author = {Kim, HJ and Kwon, MY and Song, S and Cheon, SW and Kim, HJ},
title = {Engineered Lactiplantibacillus plantarum and Levilactobacillus brevis utilizing ribonucleoprotein-mediated editing for inactivation of hemolysin gene.},
journal = {World journal of microbiology & biotechnology},
volume = {41},
number = {10},
pages = {373},
pmid = {41082037},
issn = {1573-0972},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/metabolism ; *Hemolysin Proteins/genetics ; Probiotics ; Bacterial Proteins/genetics ; Plasmids/genetics ; *Lactobacillus plantarum/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Lactobacillus/genetics ; },
abstract = {Lactiplantibacillus plantarum and Levilactobacillus brevis are widely used probiotics with significant potential as chassis organisms for probiotic engineering. However, their bioengineering remains underdeveloped compared to that of other probiotic bacteria due to the limited availability of genetic tools. Although CRISPR-Cas systems have shown promise for genome editing in Lactobacillus species, strain- or site-specific targeting challenges must be overcome to enhance their broader applicability. This study aimed to develop a novel editing system with reduced dependency on plasmids and antibiotics in L. plantarum WCFS1, L. plantarum SPC 72 - 1 and L. brevis SPC-SNU 70 - 2 using a Cas9-gRNA ribonucleoprotein (RNP) complex. Although the hlyIII gene has been annotated as a hemolysin-related gene in several Lactobacillus genomes, no functional hemolytic activity has been definitively demonstrated to date. In this study, hlyIII was selected as a target to evaluate genome editing efficiency and to assess its potential relevance to strain safety. To construct ΔhlyIII strains, the RNP complex targeting hlyIII was separately transformed with recombinase RecE/T and double-stranded donor DNA. As a result, ΔhlyIII mutants were obtained under optimized electroporation conditions. Sequencing analysis revealed a 50 bp deletion and the introduction of a stop codon in hlyIII across all mutant strains. The hemolytic activity test showed a reduction in free hemoglobin levels in the ΔhlyIII strains compared to the wild type: 27.0%, 74.3%, and 5.0% in L. plantarum WCFS1, L. plantarum SPC 72 - 1, and L. brevis SPC-SNU 70 - 2, respectively. These results suggest strain-dependent differences in hemolytic activity and indicate that inactivation of hlyIII may contribute to reduced hemolysis, although further validation is needed to clarify its functional role. In conclusion, the hlyIII gene was successfully edited in L. plantarum and L. brevis using Cas9-gRNA ribonucleoprotein-mediated editing, demonstrating the feasibility of this genome editing platform for application in probiotic strains.},
}

*Gene Editing/methods
CRISPR-Cas Systems
*Ribonucleoproteins/genetics/metabolism
*Hemolysin Proteins/genetics
Probiotics
Bacterial Proteins/genetics
Plasmids/genetics
*Lactobacillus plantarum/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
*Lactobacillus/genetics

@article {pmid41039923,
year = {2025},
author = {Xiao, J and Hu, X and Chen, H and Diao, B and Huang, X and Liu, L},
title = {CRISPR-Programmed CuO Nanocatalyst Release for Ultrasensitive Detection of Pathogens in Sterile Body Fluids.},
journal = {Analytical chemistry},
volume = {97},
number = {40},
pages = {22427-22435},
doi = {10.1021/acs.analchem.5c05043},
pmid = {41039923},
issn = {1520-6882},
mesh = {*Copper/chemistry ; Humans ; Catalysis ; *CRISPR-Associated Proteins/metabolism/chemistry ; Benzidines/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; Limit of Detection ; RNA, Ribosomal, 16S/genetics/analysis ; *Body Fluids/microbiology ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; Bacterial Proteins/metabolism/chemistry ; Colorimetry ; },
abstract = {Treatment of sterile body fluids (SBFs) infections is delayed by conventional methods that require up to 72 h to detect pathogens. Here, we present a CRISPR-associated protein 12a (Cas12a)-programmed nanocatalyst release (CNR) method for culture-free diagnostics. To enhance both sensitivity and coverage, three starter DNA (sDNA)-complementary DNA (cDNA) probe pairs were designed for conserved regions and additional three pairs for variable regions of bacterial 16S or fungal 18S rRNA. Upon target recognition, cDNA undergoes strand displacement, releasing sDNA to activate Cas12a. The activated Cas12a cleaves copper oxide nanoparticle (CuONPs)-loaded magnetic probes, releasing tandem CuONPs. Upon acid dissolution, each CuONP generates Cu[2+] ions that catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), producing a visible colorimetric signal. This quadruple signal amplification strategy integrates high-copy rRNA targets, multi-cDNA recognition, Cas12a-mediated continuous release of tandem CuONPs, and Cu[2+]-driven chromogenic amplification. This nucleic acid amplification-free assay detects pathogens at 0.69 CFU mL[-1] in original SBFs samples (after 10-fold centrifugation) within 70 min. In 64 clinical samples, it achieved 100% sensitivity and 100% specificity versus culture. Notably, one culture-negative but clinically confirmed case was correctly identified. Overall, the CNR method offers a rapid, ultrasensitive, and accessible diagnostic solution for resource-limited settings.},
}

*Copper/chemistry
Humans
Catalysis
*CRISPR-Associated Proteins/metabolism/chemistry
Benzidines/chemistry
*Endodeoxyribonucleases/metabolism/chemistry
Limit of Detection
RNA, Ribosomal, 16S/genetics/analysis
*Body Fluids/microbiology
*Metal Nanoparticles/chemistry
*CRISPR-Cas Systems
Bacterial Proteins/metabolism/chemistry
Colorimetry

@article {pmid40980924,
year = {2025},
author = {Deng, L and He, X and Zhou, S and Gu, T and Dong, J and Zhu, S and Luo, X and Huo, D and Hou, C},
title = {A sticky end-driven PAM-free RPA-CRISPR/Cas12a dual amplification system for ultrasensitive detection of KRAS G12C.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {83},
pages = {16282-16285},
doi = {10.1039/d5cc04401d},
pmid = {40980924},
issn = {1364-548X},
mesh = {*Proto-Oncogene Proteins p21(ras)/genetics ; *CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; Limit of Detection ; DNA/chemistry/genetics ; *Nucleic Acid Amplification Techniques ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Herein, a fluorescent biosensing platform was constructed for KRAS G12C single base mutation detection by CRISPR/Cas12a-coupled RPA without the PAM site. The KRAS G12C gene sequence was cleaved into double-stranded DNA containing a sticky end using HindIII enzyme cleavage site specificity. Sticky end dsDNA activated the trans-cleavage activity of Cas12a and generates an intense fluorescent signal. This strategy detected KRAS G12C targets in a linear range of 10 aM-10 pM with a detection limit of 1.5 aM. What's more, the method was able to distinguish 0.1% KRAS G12C mutation in a total of 10 pM gene concentration and demonstrated excellent detection performance in real samples.},
}

*Proto-Oncogene Proteins p21(ras)/genetics
*CRISPR-Cas Systems
Humans
*Biosensing Techniques/methods
Limit of Detection
DNA/chemistry/genetics
*Nucleic Acid Amplification Techniques
*Endodeoxyribonucleases/metabolism
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid40721863,
year = {2025},
author = {Rybarikova, M and Rey, M and Hasanovic, E and Sipion, M and Rambousek, L and Déglon, N},
title = {Gene editing for Spinocerebellar ataxia type 3 taking advantage of the human ATXN3L paralog as replacement gene.},
journal = {Gene therapy},
volume = {32},
number = {5},
pages = {462-474},
pmid = {40721863},
issn = {1476-5462},
support = {FN 310030_184761/1//Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; No. 31ER30_179594//EC | EU Framework Programme for Research and Innovation H2020 | H2020 European Institute of Innovation and Technology (H2020 The European Institute of Innovation and Technology)/ ; },
mesh = {*Machado-Joseph Disease/therapy/genetics ; Animals ; *Ataxin-3/genetics ; Humans ; *Gene Editing/methods ; Mice, Transgenic ; Mice ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Exons ; Cerebellum/metabolism ; Disease Models, Animal ; Repressor Proteins ; },
abstract = {Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by a CAG expansion of the ataxin-3 gene (ATXN3). SCA3 patients suffer from ataxia, spasticity and dystonia in mid-adulthood, with spinocerebellar dysfunction and degeneration. As a monogenic disease for which only symptomatic treatment is available, ATXN3 is an attractive target for gene editing. We used the KamiCas9, a self-inactivating gene editing system, to explore gene editing strategies suitable for all SCA3 patients. We first tested the deletion of exon 10 or the introduction of a premature stop codon into exon 9. High editing events were observed in vitro, but efficiency was very low in SCA3 transgenic mice. We then evaluated an ablate-and-replace strategy. The ablate experiments resulted in 55 ± 18% cerebellar editing of the ATXN3 gene. A human ATXN3L paralog, expressed in the brains of SCA3 patients, may act as a natural, CRISPR-resistant replacement gene. In a proof-of-principle study, ablate and ablate-and-replace strategies were evaluated in SCA3 transgenic mice. Two months after injection, similar editing efficiencies were obtained in the ablate and ablate-and-replace groups. Immunofluorescence and RT-qPCR analyses of cerebellar markers support the development of this strategy for SCA3 treatment.},
}

*Machado-Joseph Disease/therapy/genetics
Animals
*Ataxin-3/genetics
Humans
*Gene Editing/methods
Mice, Transgenic
Mice
*Genetic Therapy/methods
CRISPR-Cas Systems
Exons
Cerebellum/metabolism
Disease Models, Animal
Repressor Proteins

@article {pmid40581821,
year = {2025},
author = {Luo, Y and Zhan, X and Zhang, Y and Wang, B and Wang, G and Zhang, Y and Li, G and Liu, Q and Shen, X and Chen, D and Hong, Y and Wu, W and Ye, G and Cheng, S and Pan, G and Cao, L},
title = {CRISPR-Cas9-mediated knockup of OsDREB1C enhances rice yield without compromising grain quality.},
journal = {Plant communications},
volume = {6},
number = {10},
pages = {101433},
doi = {10.1016/j.xplc.2025.101433},
pmid = {40581821},
issn = {2590-3462},
mesh = {*Oryza/genetics/growth & development ; *CRISPR-Cas Systems/genetics ; *Edible Grain/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/genetics ; Gene Editing ; Gene Knock-In Techniques ; },
abstract = {This study presents a CRISPR-Cas9-based strategy for engineering structural variations in the OsDREB1C gene in rice, leading to a yield increase of over 20% without compromising grain quality. The resulting homozygous plants are transgene-free, highlighting the potential of this approach for precise and effective crop improvement.},
}

*Oryza/genetics/growth & development
*CRISPR-Cas Systems/genetics
*Edible Grain/genetics
*Plant Proteins/genetics/metabolism
Plants, Genetically Modified/genetics
Gene Editing
Gene Knock-In Techniques

@article {pmid39572737,
year = {2025},
author = {Yan, RE and Corman, A and Katgara, L and Wang, X and Xue, X and Gajic, ZZ and Sam, R and Farid, M and Friedman, SM and Choo, J and Raimondi, I and Ganesan, S and Katsevich, E and Greenfield, JP and Dahmane, N and Sanjana, NE},
title = {Pooled CRISPR screens with joint single-nucleus chromatin accessibility and transcriptome profiling.},
journal = {Nature biotechnology},
volume = {43},
number = {10},
pages = {1628-1634},
pmid = {39572737},
issn = {1546-1696},
support = {DP2HG010099//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; GM136573//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R03OD034499//U.S. Department of Health & Human Services | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Humans ; *Chromatin/genetics/metabolism ; *Gene Expression Profiling/methods ; Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; Transcriptome/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Cell Nucleus/genetics ; },
abstract = {Pooled single-cell CRISPR screens have profiled either gene expression or chromatin accessibility but not both modalities. Here we develop MultiPerturb-seq, a high-throughput CRISPR screening platform with joint single-nucleus chromatin accessibility, transcriptome and guide RNA capture using combinatorial indexing combined with droplet microfluidics to scale throughput and integrate all three modalities. We identify key differentiation genes in a rare pediatric cancer and establish ZNHIT1 as a potential target for cancer reprogramming therapy.},
}

Humans
*Chromatin/genetics/metabolism
*Gene Expression Profiling/methods
Single-Cell Analysis/methods
*CRISPR-Cas Systems/genetics
Transcriptome/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
*Cell Nucleus/genetics

@article {pmid39482449,
year = {2025},
author = {Jabalera, Y and Tascón, I and Samperio, S and López-Alonso, JP and Gonzalez-Lopez, M and Aransay, AM and Abascal-Palacios, G and Beisel, CL and Ubarretxena-Belandia, I and Perez-Jimenez, R},
title = {A resurrected ancestor of Cas12a expands target access and substrate recognition for nucleic acid editing and detection.},
journal = {Nature biotechnology},
volume = {43},
number = {10},
pages = {1663-1672},
pmid = {39482449},
issn = {1546-1696},
mesh = {*Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Humans ; *CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; DNA/metabolism/genetics ; *Bacterial Proteins/genetics/metabolism ; Substrate Specificity ; },
abstract = {The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs from hydrobacteria to reconstruct a common ancestor, ReChb, characterized by near-PAMless targeting and the recognition of diverse nucleic acid activators and collateral substrates. ReChb shares 53% sequence identity with the closest Cas12a ortholog but no longer requires a T-rich PAM and can achieve genome editing in human cells at sites inaccessible to the natural FnCas12a or the engineered and PAM-flexible enAsCas12a. Furthermore, ReChb can be triggered not only by double-stranded DNA but also by single-stranded RNA and DNA targets, leading to non-specific collateral cleavage of all three nucleic acid substrates with similar efficiencies. Finally, tertiary and quaternary structures of ReChb obtained by cryogenic electron microscopy reveal the molecular details underlying its expanded biophysical activities. Overall, ReChb expands the application space of Cas12a nucleases and underscores the potential of ASR for enhancing CRISPR technologies.},
}

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

@article {pmid38472508,
year = {2025},
author = {Gould, SI and Wuest, AN and Dong, K and Johnson, GA and Hsu, A and Narendra, VK and Atwa, O and Levine, SS and Liu, DR and Sánchez Rivera, FJ},
title = {High-throughput evaluation of genetic variants with prime editing sensor libraries.},
journal = {Nature biotechnology},
volume = {43},
number = {10},
pages = {1648-1662},
pmid = {38472508},
issn = {1546-1696},
support = {V2022-028//V Foundation for Cancer Research (V Foundation)/ ; P30-CA14051//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; U01AI142756//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; RM1HG009490//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; *Genetic Variation/genetics ; *Gene Editing/methods ; *Tumor Suppressor Protein p53/genetics ; *High-Throughput Screening Assays/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; Neoplasms/genetics ; },
abstract = {Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53-the most frequently mutated gene in cancer-to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein-protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.},
}

Humans
*Genetic Variation/genetics
*Gene Editing/methods
*Tumor Suppressor Protein p53/genetics
*High-Throughput Screening Assays/methods
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems/genetics
Neoplasms/genetics

@article {pmid41081710,
year = {2025},
author = {Serreze, DV and Tousey-Pfarrer, M and Racine, JJ},
title = {Humanized Mouse Models for Type 1 Diabetes.},
journal = {Current protocols},
volume = {5},
number = {10},
pages = {e70224},
doi = {10.1002/cpz1.70224},
pmid = {41081710},
issn = {2691-1299},
mesh = {*Diabetes Mellitus, Type 1/immunology/genetics ; Animals ; *Disease Models, Animal ; Mice, Inbred NOD ; Humans ; Mice ; CRISPR-Cas Systems ; },
abstract = {T cell-mediated autoimmune type 1 diabetes (T1D) is under complex polygenic control in both humans and the NOD mouse model. However, in both species, particular major histocompatibility complex (MHC; designated HLA in humans) haplotypes provide the primary T1D risk factor. Both MHC/HLA class I and II variants interactively contribute to T1D by respectively driving autoreactive CD8 and CD4 T cell responses that cooperatively destroy insulin-producing pancreatic β cells. While NOD mice have provided important insights to the pathogenic basis of T1D, the model has so far provided only a limited means to identify possible clinically translatable disease intervention approaches. This highlights a need to humanize NOD mice in ways that their pathogenic basis of T1D development becomes more similar to that characterizing the disease course in patients. In this review, we discuss the use of CRISPR/Cas9-generated murine-MHC-deficient NOD mice as a platform for introduction of patient-relevant HLA and T cell receptor molecules. These mice provide ever-improving models for development of clinically applicable interventions for T1D and other autoimmune diseases. © 2025 The Author(s) Current Protocols published by Wiley Periodicals LLC.},
}

*Diabetes Mellitus, Type 1/immunology/genetics
Animals
*Disease Models, Animal
Mice, Inbred NOD
Humans
Mice
CRISPR-Cas Systems

@article {pmid41081480,
year = {2025},
author = {Ma, F and Zheng, Q and Sun, Y and Zhang, N and Xu, W},
title = {Toward Tissue-Free Plant Engineering: Emerging Platforms for Sustainable Horticultural Transformation.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c06803},
pmid = {41081480},
issn = {1520-5118},
abstract = {Genetic transformation in horticultural crops is being reshaped by the emergence of nontissue culture technologies that bypass entrenched barriers of genotype dependence, regeneration inefficiency, and sterile culture requirements. This review surveys recent in planta methods, including regenerative activity-dependent in Planta injection delivery (RAPID), cut-dip-budding (CDB), virus-based delivery, nanoparticle-mediated transformation, and Agrobacterium rhizogenes-induced regeneration, and evaluates their operational versatility across species. We further examine their integration with developmental regulators (BABY BOOM [BBM], WUSCHEL [WUS]), visual markers (RUBY), and CRISPR/Cas systems to enhance transformation efficiency and precision. Case studies across fruit, vegetable, and ornamental crops illustrate broad applicability and growing technical maturity. Despite these advances, unresolved challenges in biosafety, reproducibility, and regulatory alignment remain. We advocate a new transformation paradigm that is rapid, genotype-independent, and environmentally compatible, enabling scalable and more accessible broadly applicable crop improvement in horticultural biotechnology.},
}

@article {pmid41080519,
year = {2025},
author = {Chaturvedi, A and Ranjan, R},
title = {Strategies for plant-virus disease management from gene editing to nanotechnology.},
journal = {Physiology and molecular biology of plants : an international journal of functional plant biology},
volume = {31},
number = {8},
pages = {1293-1308},
pmid = {41080519},
issn = {0971-5894},
abstract = {Plant viruses are a global agricultural threat and can result in large financial losses. The globalization of agriculture and its international trading are the major causes of viruses and their vectors expanding to new environmental niches. Conventional methods are not effective in managing virus infection. To mitigate the virus spread, one of the cutting-edge biotechnological approaches, CRISPR/Cas is a robust tool. CRISPR/Cas is a powerful genome editing technology, and provides a highly specific viral genome targeting. Additionally, nanotechnology is a cutting-edge method for mitigating plant viruses. Nanoparticles in biosensors aid in the early identification of plant viruses, hence preventing the spread of disease in the future. Moreover, nanoparticles can also be used as a flexible delivery system. Nanoparticle-mediated delivery of dsRNA ensures minimal off-target while maintaining biosafety. This review explores the genome editing approach and nanotechnological strategies for ensuring sustainable agriculture practices for virus disease management, focusing on biosafety, efficacy, and practical applicability. It also aims to provide a clear insight into the limitations and strengths of each approach.},
}

@article {pmid41076478,
year = {2025},
author = {Wu, M and Wang, F and Wang, Y and Wu, YX and Tian, BY and Ou, XY and Xu, QC and Wu, XY and Han, C and Liu, WL and Xing, S},
title = {Mismatch-introduced crRNA guided PCR-CRISPR/Cas12a platform improves EGFR point mutation detection in single tumor cell.},
journal = {Mikrochimica acta},
volume = {192},
number = {11},
pages = {727},
pmid = {41076478},
issn = {1436-5073},
support = {2021B1515230006//the Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {Humans ; ErbB Receptors/genetics ; *Point Mutation ; *CRISPR-Cas Systems ; *Polymerase Chain Reaction/methods ; *Single-Cell Analysis/methods ; Cell Line, Tumor ; Neoplastic Cells, Circulating ; DNA Mutational Analysis/methods ; Base Pair Mismatch ; },
abstract = {Dynamic monitoring of epidermal growth factor receptor (EGFR) mutations is essential for the early identification of resistance and treatment adaptation. Single-cell heterogeneity analysis is crucial for precision cancer medicine, yet sensitive and specific detection methods for individual tumor cells remain challenging. Here, we develop a PCR-CRISPR/Cas12a platform enhanced by the incorporation of mismatched base in crRNA at specific site for single-cell point mutation detection. This platform demonstrated high specificity and sensitivity, detecting point mutation at a frequency of 0.1% and in as low as 1.02 ng of genomic DNA, which represents an improvement over the amplification-refractory mutation system PCR (ARMS-PCR). Notably, the accuracy of the platform is highly consistent with next-generation sequencing (NGS), as evidenced by Kappa test values surpassing 0.9. By utilizing a conical-pore membrane with optimized porosity for single circulating tumor cell (CTC) enrichment, our platform enables point mutations detection in individual tumor cells, offering potential enhancements in precision and reliability for EGFR mutation analysis. This novel methodology holds potential for more accurate and personalized cancer treatment strategies.},
}

Humans
ErbB Receptors/genetics
*Point Mutation
*CRISPR-Cas Systems
*Polymerase Chain Reaction/methods
*Single-Cell Analysis/methods
Cell Line, Tumor
Neoplastic Cells, Circulating
DNA Mutational Analysis/methods
Base Pair Mismatch

@article {pmid41075940,
year = {2025},
author = {Zhang, J and Dai, P and Weng, Z and Xu, R and Li, Y and Liu, X and Lei, J},
title = {Efficient CRISPR/Cas-based gene editing in cotton induced by cotton leaf crumple virus.},
journal = {Journal of biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jbiotec.2025.10.001},
pmid = {41075940},
issn = {1873-4863},
abstract = {Plant viral vectors can replicate autonomously and spread within host cells, making them an ideal tool for the delivery of CRISPR/Cas gene-editing elements. Here, we constructed a cotton CRISPR/Cas system mediated by cotton leaf crumple virus (CLCrV) as a delivery vector. We first inoculated Pro35s::Cas9 and ProUbi::Cas9 cotton with sgRNAs designed to knock out GhAGL16, GhPDS, and GhCLA1 target genes via the CLCrV vector and then compared the effects of these two transformation receptors on the editing efficiency of the same target genes. We next explored the feasibility of simultaneous multi-target editing in cotton via pooled virus inoculation. Finally, we used a cotton line overexpressing nCas9-TadA7.10 as the transformation receptor to explore the feasibility of CLCrV-mediated adenine base editing and verify the specificity of gene editing in this system. Mutation detection and deep sequencing revealed that the Pro35s::Cas9 and ProUbi::Cas9 cotton lines did not differ significantly in editing efficiency, and both could be used as successful receptors for the CLCrV-mediated Cas9 system. Pooled inoculation with CLCrV-sgRNAs enabled the simultaneous editing of multiple target genes in Pro35s::Cas9 and ProUbi::Cas9 cotton, although this approach had somewhat lower editing efficiency than inoculation with single sgRNAs. The CLCrV-mediated adenine base-editing system enabled A-to-G conversion at target sites in cotton GhPEBP and showed high gene-editing specificity. In summary, this study establishes an efficient CLCrV-mediated CRISPR system in cotton, providing a powerful technical tool for editing of multiple target genes and base editing.},
}

@article {pmid41074985,
year = {2025},
author = {Gupta, I and Sharma, JG and Kaul, T},
title = {Nanoparticle-driven CRISPR-Cas9 genome editing: a new frontier in crop improvement.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {1015},
pmid = {41074985},
issn = {1573-4978},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; *Nanoparticles/chemistry ; Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; Nanotechnology/methods ; Genetic Engineering/methods ; Agriculture/methods ; },
abstract = {The agricultural sector has experienced unpredictable and extreme climatic aberrations, which have severely hampered food production. However, applying advanced nanotechnological approaches in agriculture will be crucial for ensuring more secure and sustainable food production. The revolutionizing phyto-nanotechnology enables the precise delivery of biomolecules i.e., nucleotides and proteins, and the modulated release of agrochemicals, including fungicides and pesticides. In addition, CRISPR-Cas-based genetic engineering holds great promise for food security, agriculture, and environmental sustainability. However, its application in plants faces challenges, including cargo delivery, germline transformation, species independence, HDR efficiency, and overall editing effectiveness. Nanomaterials offer innovative and effective solutions to overcome these challenges by enhancing genome-editing tools precision, efficiency, and delivery mechanisms. This review examines the key limitations of CRISPR-mediated plant genome editing and how nanoparticle technologies can overcome them. We highlight essential nanotech innovations that enhance genome modification, paving the way for a faster, more versatile genomic toolbox in plant biotechnology.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Crops, Agricultural/genetics
*Nanoparticles/chemistry
Genome, Plant/genetics
Plants, Genetically Modified/genetics
Nanotechnology/methods
Genetic Engineering/methods
Agriculture/methods

@article {pmid41074026,
year = {2025},
author = {Frey, T and Kandolf-Zumpf, C and Kaempf, A and Schaffer, K and Hollenstein, M and Lampl, A and Kovarik, JJ and Strobl, J and Stary, G and Eckl-Dorna, J and Schmidt, R and Schmetterer, KG},
title = {T cell receptor associated transmembrane adaptor 1 (TRAT1) modulates human Th17 and Treg responses via PI3-kinase and STAT dependent mechanisms.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {431},
pmid = {41074026},
issn = {1478-811X},
support = {P34728-B//Austrian Science Funds/ ; P34728-B//Austrian Science Funds/ ; P34728-B//Austrian Science Funds/ ; },
mesh = {Humans ; *T-Lymphocytes, Regulatory/metabolism/immunology/cytology ; *Th17 Cells/metabolism/immunology/cytology ; Signal Transduction ; *Phosphatidylinositol 3-Kinases/metabolism ; *STAT Transcription Factors/metabolism ; *Adaptor Proteins, Signal Transducing/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Adaptor proteins associated with the T cell receptor (TCR) play critical roles in regulating immune responses by Translating receptor engagement into intracellular signals. T cell Receptor Associated Transmembrane Adaptor 1 (TRAT1) has been implicated in modulating TCR complex stability, but its functional role in human effector and regulatory CD4[+] T cell subsets remains poorly understood. This study aimed to elucidate the role of TRAT1 in regulating T cell activation and differentiation, particularly in helper T cells function and regulatory T cells.

METHODS: Primary human CD4⁺ T cells, including thymus-derived and induced regulatory T cells (Treg), were genetically modified by CRISPR/Cas9-mediated gene deletion or retro-/lentiviral overexpression of TRAT1. Functional assays, flow cytometry, cytokine quantification, and RNA sequencing were performed to evaluate modulation of T cell functions. Mechanistic studies included pathway inhibition using small molecules and phospho-protein analysis. The influence of TRAT1 on Treg function was further assessed in a CAR Treg context in an immune organoid model of allo-rejection.

RESULTS: Thymus-derived, TGFb-induced and FOXP3-transgenic Treg displayed reduced expression of TRAT1 compared to effector T cells, which showed pronounced up-regulation of TRAT1 following activation. In effector T cells, deletion of TRAT1 led to increased signaling through the phosphoinositide 3-kinase pathway resulting in enhanced proliferation and increased expression of activation markers. However, this was accompanied by reduced production of interleukin-17, which was linked to elevated activity of STAT6 as shown by inhibition experiments using small molecule inhibitors. Overexpression and CRISPR/Cas9-mediated knockout of TRAT1 in Treg enhanced suppression of CD4⁺ target cells via up-regulation of LAP/GARP but reduced suppression of CD8⁺ target cells, an effect confirmed in HLA-A2-specific CAR Treg in a human organoid model of allo-rejection.

CONCLUSIONS: TRAT1 acts as a dual regulator of human CD4⁺ T cell function, limiting effector activation through modulation of intracellular signaling and supporting regulatory T cell-mediated suppression. These findings reveal a novel mechanism of immune regulation with potential implications for the development of cell-based immunotherapies.},
}

Humans
*T-Lymphocytes, Regulatory/metabolism/immunology/cytology
*Th17 Cells/metabolism/immunology/cytology
Signal Transduction
*Phosphatidylinositol 3-Kinases/metabolism
*STAT Transcription Factors/metabolism
*Adaptor Proteins, Signal Transducing/metabolism
CRISPR-Cas Systems

@article {pmid41073589,
year = {2025},
author = {Das, A and Debnath, S and Pramanik, S and Monshi, FI and Rahimi, M},
title = {Bio-digital feedback loop systems: a synergistic integration of predictive genomics, genome editing, and AI-driven phenomic synthesis for next-generation edible and medicinal mushroom breeding.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {11},
pages = {168},
pmid = {41073589},
issn = {1572-9699},
mesh = {*Gene Editing/methods ; *Genomics/methods ; *Agaricales/genetics ; *Artificial Intelligence ; CRISPR-Cas Systems ; *Phenomics/methods ; *Breeding/methods ; },
abstract = {Edible mushrooms face persistent challenges in yield optimization, bioactive compound production, and climate resilience that conventional breeding methods struggle to address. Traditional approaches such as cross-breeding, protoplast fusion, and mutagenesis are limited by genetic noise, laborious screening, and unstable trait inheritance. This review proposes a transformative paradigm built upon converging advances in molecular biology and data science: the bio-digital feedback loop (BDFL) framework, integrating multi-omics, CRISPR-engineered chassis strains, and predictive phenomics for precision mushroom breeding. Our framework employs multi-omics to decipher gene networks governing critical traits, such as substrate degradation enzymes, developmental synchrony regulators, and secondary metabolite pathways. CRISPR-Cas9 and synthetic biology tools then deploy these insights to verify and design modular gene circuits in pre-engineered "plug-and-play" chassis strains, enabling conflict-free stacking of desirable traits. Artificial intelligence serves as the linchpin, not only automating high-throughput phenotyping through advanced imaging but also accelerating the entire breeding cycle by predicting trait heritability from omics data and optimizing the design of CRISPR guide RNAs and genetic constructs for efficient editing. The BDFL we describe iteratively refines strains by feeding phenomics data back into AI algorithms, enabling rapid trait optimization cycles. This transcends the trial-and-error limitations of classical methods, accelerating development of climate-smart mushrooms for circular bioeconomies including strains engineered to thrive on agricultural waste, overproduce immunomodulatory compounds, or resist emerging pathogens. The integration of predictive genomics, AI-driven phenomics, and CRISPR-edited chassis strains heralds a new era of precision mycology, where mushrooms are computationally designed as sustainable solutions for global food security, pharmaceutical innovation, and ecological resilience, ultimately transforming fungi into programmable biological factories tailored to address pressing agricultural and ecological challenges.},
}

*Gene Editing/methods
*Genomics/methods
*Agaricales/genetics
*Artificial Intelligence
CRISPR-Cas Systems
*Phenomics/methods
*Breeding/methods

@article {pmid40959957,
year = {2025},
author = {Chen, T and Hu, G and Fu, J and Tu, J},
title = {Structural Basis of PAM-Induced Conformational Changes in SpCas9: A Molecular Dynamics Study.},
journal = {Journal of chemical information and modeling},
volume = {65},
number = {19},
pages = {10624-10633},
doi = {10.1021/acs.jcim.5c01626},
pmid = {40959957},
issn = {1549-960X},
mesh = {*Molecular Dynamics Simulation ; *CRISPR-Associated Protein 9/chemistry/metabolism ; Protein Conformation ; DNA/metabolism/chemistry ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry ; CRISPR-Cas Systems ; },
abstract = {As the most widely utilized CRISPR gene-editing enzyme, SpCas9 has been extensively studied and applied. However, its strict dependence on the canonical NGG PAM sequence significantly restricts its targeting scope. Although recent research has successfully engineered SpCas9 variants capable of recognizing noncanonical (non-NGG) PAMs, these variants still exhibit limitations when binding noncanonical PAMs, including substantially reduced cleavage efficiency. To elucidate the molecular mechanisms underlying noncanonical PAM recognition by SpCas9, we employed molecular dynamics simulations to compare the structural differences within the Cas9-gRNA-DNA ternary complex when bound to various PAM sequences. Our analysis revealed significant conformational changes within SpCas9 upon engagement with noncanonical PAMs and uncovered the regulatory mechanisms underpinning these changes. We further identified key dynamic determinants governing the extensive conformational transitions occurring during the noncanonical PAM binding process. These findings provide insights into the dynamic landscape of noncanonical PAM recognition, offering crucial mechanistic guidance for designing efficient, PAM-compatible Cas9 variants.},
}

*Molecular Dynamics Simulation
*CRISPR-Associated Protein 9/chemistry/metabolism
Protein Conformation
DNA/metabolism/chemistry
RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry
CRISPR-Cas Systems

@article {pmid40925496,
year = {2025},
author = {Yamazaki, M and Ueta, A and Nakanishi, T and Tachikawa, K and Kawai, M and Ozono, K and Michigami, T},
title = {Involvement of impaired phosphate production and aberrant extracellular ATP signaling in the pathogenesis of hypophosphatasia: Analysis of ALPL-Knockout human iPS cell models.},
journal = {Bone},
volume = {201},
number = {},
pages = {117629},
doi = {10.1016/j.bone.2025.117629},
pmid = {40925496},
issn = {1873-2763},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *Hypophosphatasia/metabolism/pathology/genetics ; *Alkaline Phosphatase/metabolism/genetics/deficiency ; *Phosphates/metabolism ; *Adenosine Triphosphate/metabolism ; *Signal Transduction ; Gene Knockout Techniques ; Osteogenesis ; Cell Differentiation ; Osteoblasts/metabolism/pathology ; CRISPR-Cas Systems/genetics ; *Models, Biological ; *Extracellular Space/metabolism ; },
abstract = {Hypophosphatasia (HPP) is caused by inactivating variants of ALPL, the gene encoding tissue non-specific alkaline phosphatase (TNSALP). In order to deepen our understanding of the pathogenic mechanisms of HPP, we herein generated ALPL-knockout (KO) human induced pluripotent stem (iPS) cells by applying CRISPR/Cas9-mediated gene deletion to an iPS clone derived from a healthy subject. We analyzed two ALPL-KO clones, one ALPL-hetero KO clone, and a control clone isogenic except for ALPL. In an osteogenic culture using β-glycerophosphate, which generates inorganic phosphate (Pi) by TNSALP-mediated degradation, ALPL-KO clones showed impaired mineralization, elevated levels of extracellular pyrophosphate (PPi), and reduced levels of extracellular Pi. Osteogenic induction using 3 mM Pi instead of β-glycerophosphate rescued the decreased content of hydroxyapatite in ALPL-KO cells despite the still high levels of extracellular PPi; however, abnormal distribution of hydroxyapatite was noted. Osteoblast lineage cells differentiated from ALPL-KO iPS clones showed the up-regulation of SPP1 and the down-regulation of ANKH and the genes for type III sodium/phosphate co-transporters in the culture using β-glycerophosphate, but not when 3 mM Pi was used. Extracellular ATP levels were elevated in osteoblast lineage cells derived from ALPL-KO iPS clones in both culture conditions, which was associated with the down-regulation of P2X7 encoding a purinergic receptor. Moreover, osteoblast lineage cells differentiated from ALPL-KO iPS clones in the culture using β-glycerophosphate showed a change in cellular response to extracellular Pi. These results suggest that the reduced local production of extracellular Pi and aberrant ATP signaling play substantial roles in the pathogenesis of HPP.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/pathology
*Hypophosphatasia/metabolism/pathology/genetics
*Alkaline Phosphatase/metabolism/genetics/deficiency
*Phosphates/metabolism
*Adenosine Triphosphate/metabolism
*Signal Transduction
Gene Knockout Techniques
Osteogenesis
Cell Differentiation
Osteoblasts/metabolism/pathology
CRISPR-Cas Systems/genetics
*Models, Biological
*Extracellular Space/metabolism

@article {pmid40884048,
year = {2025},
author = {Mikdar, M and Shabani, E and Grüring, C and Chaand, M and Kanjee, U and Goldberg, JM and Azouzi, S and Tennessen, JA and Elsworth, B and Keutcha, C and Barteneva, NS and Doench, JG and Duraisingh, MT},
title = {Sialyl-T Antigen: A Novel Red Blood Cell Determinant for Plasmodium falciparum Invasion.},
journal = {American journal of hematology},
volume = {100},
number = {11},
pages = {1952-1962},
doi = {10.1002/ajh.70037},
pmid = {40884048},
issn = {1096-8652},
support = {P300P3_151146//Schweizerischer Nationalfonds zur Frderung der Wissenschaftlichen Forschung/ ; PBSKP3_140144//Schweizerischer Nationalfonds zur Frderung der Wissenschaftlichen Forschung/ ; 5R01AI140751//National Institute of Allergy and Infectious Diseases/ ; 5R01HL139337//National Heart Lung and Blood Institute/ ; 23POST1017743//American Heart Association/ ; },
mesh = {*Plasmodium falciparum/pathogenicity/physiology ; Humans ; *Erythrocytes/parasitology/metabolism ; *Malaria, Falciparum/parasitology/blood/genetics ; *Galactosyltransferases/genetics/metabolism ; Animals ; N-Acetylneuraminic Acid/metabolism ; CRISPR-Cas Systems ; },
abstract = {Malaria continues to pose significant health challenges globally despite advances in control measures. Plasmodium falciparum, the parasite responsible for most severe malaria cases, uses multiple redundant invasion pathways to enter the red blood cell (RBC) during the blood stage of infection. Through a combination of RNA interference screening in erythroid cells and validation by CRISPR/Cas9-mediated knockout in primary human hematopoietic stem cells, we identified the glycosyltransferase Core 1 Synthase Glycoprotein-N-Acetylgalactosamine 3-Beta-Galactosyltransferase 1 (C1GALT1) as a novel host determinant for P. falciparum invasion. Analyses of C1GALT1-deficient cultured reticulocytes and RBCs with the glycophorin A/B-null MkMk blood group phenotype demonstrated that the C1GALT1-dependent α(2-3) sialic acid structures within mucin-type O-glycans are crucial for efficient invasion of both sialic acid-dependent and sialic acid-independent P. falciparum strains, but not the primate malaria parasite Plasmodium knowlesi. However, different P. falciparum parasite strains exhibit variable dependencies on distinct sialic acid configurations on the RBC surface. Overall, our findings highlight a key role for RBC glycans in malaria infection.},
}

*Plasmodium falciparum/pathogenicity/physiology
Humans
*Erythrocytes/parasitology/metabolism
*Malaria, Falciparum/parasitology/blood/genetics
*Galactosyltransferases/genetics/metabolism
Animals
N-Acetylneuraminic Acid/metabolism
CRISPR-Cas Systems

@article {pmid40992249,
year = {2025},
author = {Greisle, T and Kunze, I and Wang, X and Malinowski, AR and Böttcher, A and Lickert, H and Burtscher, I},
title = {Generation of a Flattop-T2A-H2B-Venus x C-peptide-mCherry double reporter human iPSC line to monitor WNT/Planar cell polarity pathway activity.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103838},
doi = {10.1016/j.scr.2025.103838},
pmid = {40992249},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cell Polarity ; *Wnt Signaling Pathway ; Cell Line ; CRISPR-Cas Systems ; Insulin-Secreting Cells/metabolism/cytology ; Genes, Reporter ; C-Peptide/metabolism/genetics ; Cell Differentiation ; },
abstract = {Deriving functional β-cells from human induced pluripotent stem cells (hiPSCs) holds potential for cell replacement therapy, disease modeling, and drug testing in diabetes research. Wnt/Planar cell polarity (PCP) signaling is crucial for endocrine cell development and β-cell maturation in murine models and can be tracked by the expression of the tissue-specific effector gene Flattop. Here, we report the generation of a human fluorescent FLTP/CFAP126 (Flattop-T2A-H2B-Venus) and FLTP-Insulin (Flattop-T2A-H2B-Venus x C-peptide-mCherry) double reporter by CRISPR/Cas9 gene editing. These hiPSC reporter lines allow monitoring of WNT/PCP signaling during endocrine cell formation and studying its role in β-cells in a human model system.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
*Cell Polarity
*Wnt Signaling Pathway
Cell Line
CRISPR-Cas Systems
Insulin-Secreting Cells/metabolism/cytology
Genes, Reporter
C-Peptide/metabolism/genetics
Cell Differentiation

@article {pmid40983220,
year = {2025},
author = {Madhusudhan, K and Padmanaban, A and Parvathi, VD},
title = {Early detection of Parkinson's disease via aptamer-CRISPR platform.},
journal = {Neuroscience},
volume = {586},
number = {},
pages = {163-195},
doi = {10.1016/j.neuroscience.2025.09.027},
pmid = {40983220},
issn = {1873-7544},
mesh = {Humans ; *Parkinson Disease/diagnosis/genetics ; *Aptamers, Nucleotide ; Early Diagnosis ; *CRISPR-Cas Systems ; Biomarkers ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Parkinson's disease (PD) is a neurodegenerative disorder with a worldwide prevalence of around 9.4 million that is expected to double by 2040. It's extended prodromal phase allows irreversible neuronal loss to occur before manifestation of symptoms. Current diagnostic approaches, primarily based on clinical assessment and neuroimaging, are often delayed and lack sensitivity in the early stages, highlighting the need for an early, conclusive, and minimally invasive test. This review focuses on the integration of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) diagnostics with aptamers to detect PD-associated biomarkers. CRISPR systems utilising Cas12 and Cas13 enzymes offer high specificity and collateral cleavage activity that can be harnessed for signal amplification. Aptamers are short, single-stranded oligonucleotides that can be designed to identify nucleic and non-nucleic acid targets. Their fusion with CRISPR may enable the sensitive detection of key PD biomarkers such as α-Syn, dopa decarboxylase, glial fibrillary acidic protein, and neurofilament light chain in biological fluids like blood, CSF, urine, saliva, and sweat. We explore various strategies for aptamer-CRISPR integration, detection, and multiplexing techniques for parallel biomarker detection. We also examine existing diagnostic platforms and discuss barriers to clinical translation. Ultimately, aptamer-CRISPR diagnostics could represent a powerful, next-generation approach for early PD detection.},
}

Humans
*Parkinson Disease/diagnosis/genetics
*Aptamers, Nucleotide
Early Diagnosis
*CRISPR-Cas Systems
Biomarkers
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40953792,
year = {2025},
author = {Watts, JL and Willeke, L and Stottmann, RW},
title = {Mouse variants in Taf1c result in reduced survival to birth.},
journal = {Developmental biology},
volume = {528},
number = {},
pages = {143-151},
doi = {10.1016/j.ydbio.2025.09.011},
pmid = {40953792},
issn = {1095-564X},
mesh = {Animals ; Mice ; *TATA-Binding Protein Associated Factors/genetics/metabolism ; Humans ; *Transcription Factor TFIID/genetics ; Mutation, Missense ; Female ; Craniofacial Abnormalities/genetics ; Phenotype ; Ribosomes/metabolism/genetics ; Male ; CRISPR-Cas Systems ; },
abstract = {Ribosome biogenesis is a key cellular function and disruptions in this process can lead to congenital anomalies or "ribosomopathies" with varying phenotypes including craniofacial malformations and neurodevelopment symptoms. Classically, the mouse is a robust model to understand the molecular mechanisms underlying ribosomopathies to further elucidate human pathogenesis. We identified novel compound heterozygous missense variants in the TATA-box binding protein associated factor, RNA polymerase I subunit C (TAF1C) locus in a patient with some phenotypes consistent with ribosomopathies. TAF1C encodes a subunit of the SL1 complex which is critical for the RNA PolI complex to initiate ribosomal RNA transcription. We hypothesized that functional TAF1C is required at developmental stages critical for craniofacial and neurodevelopment. To test this hypothesis, we created mouse Taf1c variants orthologous to the human variants using CRISPR-CAS9 technology (Taf1c[R202Q] and Taf1c[S428A]). We also created an 11bp deletion to complement the missense variants (Taf1c[11bpdel]). We created multiple allelic combinations to determine the roles for Taf1c in survival and craniofacial development. Homozygous mice for any of these novel variants were underrepresented at organogenesis stages. We did not observe craniofacial anomalies in any surviving mice. Our results suggest that these specific TAF1C variants are not the cause of any human phenotype present in the patient motivating the study. However, we showed that Taf1c is required for embryonic survival and our studies contribute to knowledge about the role of ribosome biogenesis machinery throughout organogenesis.},
}

Animals
Mice
*TATA-Binding Protein Associated Factors/genetics/metabolism
Humans
*Transcription Factor TFIID/genetics
Mutation, Missense
Female
Craniofacial Abnormalities/genetics
Phenotype
Ribosomes/metabolism/genetics
Male
CRISPR-Cas Systems

@article {pmid40929753,
year = {2025},
author = {Herbrich, S and Ramachandran, H and Seibt, A and Tolle, I and Zink, A and Prigione, A and Rossi, A and Distelmaier, F},
title = {CRISPR/Cas9-mediated editing of COQ4 in induced pluripotent stem cells: A model for investigating COQ4-associated human coenzyme Q10 deficiency.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103825},
doi = {10.1016/j.scr.2025.103825},
pmid = {40929753},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *CRISPR-Cas Systems/genetics ; *Ubiquinone/deficiency/analogs & derivatives/genetics/metabolism ; *Gene Editing/methods ; *Mitochondrial Diseases/genetics/pathology/metabolism ; *Ataxia/genetics/pathology/metabolism ; *Mitochondrial Proteins/genetics/metabolism ; *Muscle Weakness/genetics/pathology/metabolism ; Cell Line ; },
abstract = {Pathogenic variants in the gene COQ4 cause primary coenzyme Q10 deficiency, which is associated with symptoms ranging from early epileptic encephalopathy up to adult-onset ataxia-spasticity spectrum disease. We genetically modified commercially available wild-type iPS cells by using a CRISPR/Cas9 approach to create heterozygous and homozygous isogenic cell lines carrying the disease-causing COQ4 variants c.458C > T, p.Ala153Val and c.437T > G, p.Phe146Cys, respectively. All iPSCs lines exhibited a normal cell morphology, expression of pluripotency markers, and the ability to differentiate into the three primary germ layers. The COQ4-deficient cell lines will provide a helpful tool to investigate the disease mechanism and to develop therapeutic strategies.},
}

*Induced Pluripotent Stem Cells/metabolism/cytology
Humans
*CRISPR-Cas Systems/genetics
*Ubiquinone/deficiency/analogs & derivatives/genetics/metabolism
*Gene Editing/methods
*Mitochondrial Diseases/genetics/pathology/metabolism
*Ataxia/genetics/pathology/metabolism
*Mitochondrial Proteins/genetics/metabolism
*Muscle Weakness/genetics/pathology/metabolism
Cell Line

@article {pmid40925290,
year = {2025},
author = {Raabe, J and Lewandowski, V and Fuchs, S and Hammerschmidt, A and Piasecki, A and Orthey, E and Krämer, E and Ehler, E and Cuello, F},
title = {Generation of a biallelic NRAP-knockout mutant from a human iPSC line.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103829},
doi = {10.1016/j.scr.2025.103829},
pmid = {40925290},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; Cell Line ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Alleles ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; Myocytes, Cardiac/metabolism/cytology ; Mutation ; },
abstract = {Cardiomyopathies, a leading cause of mortality, are associated with dysfunctional intercalated discs, which connect neighbouring cardiomyocytes and ensure proper contractility. In human cardiac diseases, loss-of-function mutations of the intercalated disc-associated protein Nebulin-Related Anchoring Protein (NRAP) have been reported. NRAP plays a crucial role in myofibril assembly and mechanotransduction, however, its regulatory functions remain unclear. To investigate the effects of NRAP loss-of-function in cardiac disease, a human induced pluripotent stem cell (hiPSC) line was generated carrying a biallelic NRAP-knockout (KO) using the CRISPR-Cas9 technology. Control and mutant cell lines were assessed for karyotype integrity, pluripotency, off-target effects, mycoplasma contamination, and differentiation into ectoderm, mesoderm, and endoderm. This hiPSC line provides a valuable tool to study how NRAP modulates cardiac function and contributes to disease progression.},
}

Humans
*Induced Pluripotent Stem Cells/metabolism/cytology
Cell Differentiation
Cell Line
CRISPR-Cas Systems
Gene Knockout Techniques
Alleles
*Adaptor Proteins, Signal Transducing/genetics/metabolism
Myocytes, Cardiac/metabolism/cytology
Mutation

@article {pmid40902326,
year = {2025},
author = {Ran, Y and Ruan, J and Wang, Y and Feng, X and Tan, P and Guan, Y and Guo, X},
title = {Generation of a PHF19 knockout human embryonic stem cell line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103824},
doi = {10.1016/j.scr.2025.103824},
pmid = {40902326},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Transcription Factors/genetics/metabolism/deficiency ; Cell Line ; *Gene Knockout Techniques ; *DNA-Binding Proteins/genetics/metabolism/deficiency ; },
abstract = {PHD finger protein 19 (PHF19) is a polycomb protein that promoted cardiac hypertrophy via epigenetic targeting SIRT2. To determine the role of PHF19 in myocardial hypertrophy, we established a large fragment knockout model of PHF19 gene in human embryonic stem cells (hESCs-H7) using the CRISPR/Cas9 system based on a vector. This PHF19-KO cell line has a normal karyotype, classical human pluripotent stem cell morphology, strong pluripotency, and significantly reduced PHF19 gene expression, which will become a useful tool for further in-depth research on the pathogenesis of PHF19 gene deficiency induced myocardial hypertrophy.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
*Transcription Factors/genetics/metabolism/deficiency
Cell Line
*Gene Knockout Techniques
*DNA-Binding Proteins/genetics/metabolism/deficiency

@article {pmid40850232,
year = {2025},
author = {Kim, JW and Jo, S and Kang, EH and Ryu, JH and Noh, H and Park, HJ and Kim, H},
title = {Generation of human embryonic stem cell line expressing dCas9-TET1 fusion protein for epigenetic editing.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103811},
doi = {10.1016/j.scr.2025.103811},
pmid = {40850232},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *Proto-Oncogene Proteins/genetics/metabolism ; *Mixed Function Oxygenases/genetics/metabolism ; *Epigenesis, Genetic ; CRISPR-Cas Systems ; Cell Line ; *CRISPR-Associated Protein 9/metabolism/genetics ; Epigenome Editing ; },
abstract = {CRISPR-based epigenome editing systems can induce site-specific transcriptional activation or repression of target genes. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) is a transcriptional activation effector involved in the cytosine demethylation of CpG dinucleotides in gene regulatory regions. In this study, we generated a human embryonic stem cell line that stably expresses catalytically dead Cas9 (dCas9) fused to the catalytic domain of TET1 via lentiviral transduction. This cell line can be used for locus-specific transcriptional activation in combination with single guide RNAs and serves as a valuable tool for epigenetic regulation in stem cell and organoid models.},
}

Humans
*Human Embryonic Stem Cells/metabolism/cytology
*Gene Editing/methods
*Proto-Oncogene Proteins/genetics/metabolism
*Mixed Function Oxygenases/genetics/metabolism
*Epigenesis, Genetic
CRISPR-Cas Systems
Cell Line
*CRISPR-Associated Protein 9/metabolism/genetics
Epigenome Editing

@article {pmid40848400,
year = {2025},
author = {Cota-Coronado, A and Manning, M and Kim, DH and Lee, J and Gibbons, A and Rosenbluh, J and Hill, RA and Sundram, S},
title = {Generation of two Betacellulin CRISPR-Cas9 knockout hiPSC lines to study the affected EGF system paradigm in schizophrenia.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103808},
doi = {10.1016/j.scr.2025.103808},
pmid = {40848400},
issn = {1876-7753},
mesh = {*Schizophrenia/genetics/metabolism/pathology ; Humans ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Betacellulin/genetics/metabolism ; *Epidermal Growth Factor/metabolism/genetics ; Gene Knockout Techniques ; Cell Differentiation ; Cell Line ; },
abstract = {Several members of the epidermal growth factor (EGF) family have been implicated in the biology of schizophrenia (Ketharanathan et al., 2024). The EGF-related ligand, Betacellulin (BTC), plays an important role in the proliferation and differentiation of neural stem cells and our group found markedly reduced BTC levels in patients with schizophrenia. Nevertheless, the interplay of affected BTC and its participation in neural specification and neurodevelopment remains elusive. We generated Knockout (KO) - BTC clones from an existing hiPSC line through CRISPR/Cas9-mediated modification. Furthermore, we validated BTC-KO through genotyping/sequencing, FACS and Western Blot. Finally, we demonstrated trilineage differentiation potential in vitro.},
}

*Schizophrenia/genetics/metabolism/pathology
Humans
*CRISPR-Cas Systems/genetics
*Induced Pluripotent Stem Cells/metabolism/cytology
*Betacellulin/genetics/metabolism
*Epidermal Growth Factor/metabolism/genetics
Gene Knockout Techniques
Cell Differentiation
Cell Line

@article {pmid41073543,
year = {2025},
author = {Shi, Z and Cheng, TL},
title = {UGI relocation inside Cas9 reduces Cas9 dependent off target effects in cytosine base editors.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {35518},
pmid = {41073543},
issn = {2045-2322},
support = {20ZR1403100//Shanghai Natural Science Foundation/ ; },
mesh = {*Gene Editing/methods ; *Cytosine/metabolism ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; *Uracil-DNA Glycosidase/antagonists & inhibitors/metabolism ; Humans ; HEK293 Cells ; },
abstract = {Cytosine base editors (CBEs) achieve precise C-to-T conversions by addition of uracil DNA glycosylase inhibitor (UGI) with Cas9 nickase (nCas9) and cytidine deaminase, and the conventional fusion at the nCas9 carboxyl terminus effectively inhibits uracil excision repair to enhance editing efficiency. However, despite potent on-target activity, classical CBEs exhibit significant Cas9-dependent DNA off-target effects that necessitate optimization for future applications. Here we present a strategic UGI relocation through internal fusion within the nCas9 architecture. This spatial reorganization maintains comparable on-target editing efficiency while substantially reducing Cas9-dependent DNA off-target activity. Our findings establish an alternative engineering paradigm to develop high-fidelity CBEs, offering an improved platform for widespread genome editing applications.},
}

*Gene Editing/methods
*Cytosine/metabolism
*CRISPR-Associated Protein 9/metabolism/genetics/chemistry
*CRISPR-Cas Systems
*Uracil-DNA Glycosidase/antagonists & inhibitors/metabolism
Humans
HEK293 Cells

@article {pmid41073413,
year = {2025},
author = {Zhang, H and Li, M and Wang, G and Zhu, K and Guo, G and Fu, H and Hu, C and Chu, Z and Hu, J and Wu, Q and Chen, Y and Qiu, D and Xie, J and Li, D and Li, B and Li, W and Dong, L and Hou, Y and Cui, X and Huang, B and Liu, Y and Li, Y and Li, H and Yuan, C and Dong, L and Liu, Z and Lu, P},
title = {Paired NLRs originated from Triticum dicoccoides coordinately confer resistance to powdery mildew in wheat.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9040},
pmid = {41073413},
issn = {2041-1723},
mesh = {*Triticum/genetics/microbiology/immunology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; *Ascomycota/physiology/pathogenicity ; *Plant Proteins/genetics/metabolism ; *NLR Proteins/genetics/metabolism ; Haplotypes ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Wheat has evolved diverse resistance genes against powdery mildew, typically controlled by single-gene-encoded proteins. Here, we report the map-based cloning of PmWR183, a resistance locus encoding two adjacent NLR proteins (PmWR183-NLR1 and PmWR183-NLR2) from wild emmer wheat. Stable transformation and CRISPR/Cas9 knockout experiments demonstrate that the two NLRs function cooperatively: neither gene alone confers resistance, but their co-expression restores immunity, while disruption of either gene abolishes resistance. PmWR183 mediates a developmental stage-dependent response, with susceptibility at the seedling stage and strong resistance at the adult stage. Protein interaction assays reveal constitutive association of PmWR183-NLR1 and PmWR183-NLR2, supporting their cooperative role. Geographical and haplotype analyses show the locus originates from wild emmer and is rare in cultivated wheat, exhibiting at least nine haplotypes. Together, our findings uncover a rare NLR gene pair conferring effective resistance to powdery mildew, providing valuable resources for wheat breeding.},
}

*Triticum/genetics/microbiology/immunology
*Disease Resistance/genetics
*Plant Diseases/microbiology/genetics/immunology
*Ascomycota/physiology/pathogenicity
*Plant Proteins/genetics/metabolism
*NLR Proteins/genetics/metabolism
Haplotypes
Gene Expression Regulation, Plant
CRISPR-Cas Systems

@article {pmid41073016,
year = {2025},
author = {Nie, Y and Wang, W and Wang, N and Yuan, M and Huang, L and Sun, Y and Li, K and Liu, Z and Mu, Y},
title = {PCR-CRISPR/Cas12a-based fluorescence and lateral flow dipstick platforms for efficient screening of CD71 biallelic mutants.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344622},
doi = {10.1016/j.aca.2025.344622},
pmid = {41073016},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Receptors, Transferrin/genetics ; *Polymerase Chain Reaction/methods ; *Mutation ; *Antigens, CD/genetics ; *Alleles ; Fluorescence ; *CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR/Cas9 technology plays a pivotal role in gene editing and has been extensively utilized in gene function studies, disease modeling, and gene therapy. However, efficient and accurate detection of CRISPR/Cas9-induced mutants remains a challenge due to the complexity, time-consuming nature, and high cost of existing detection methods. Meanwhile, CRISPR/Cas12a systems have been widely applied in molecular diagnostics due to the non-specific trans-cleavage activity of Cas12a, yet their application in detecting CRISPR/Cas9-induced mutations remains limited. In this study, we developed a PCR-CRISPR/Cas12a-based method to enable the rapid and accurate screening of CD71 biallelic mutants. The detection system was mainly composed of CRISPR RNA specific to the CD71 gene-editing site, Cas12a protein, target DNA, and ssDNA probes for fluorescence or lateral flow dipstick assays. The system demonstrated high specificity in distinguishing CD71 biallelic mutants, with validation through TA cloning confirming its accuracy. Additionally, the method exhibited high sensitivity, establishing it as an efficient tool for biallelic mutated cell clone screening. These findings underscore the potential of PCR-CRISPR/Cas12a as a rapid, sensitive, and cost-effective approach for the precise identification of biallelic mutants, contributing to advancements in gene-editing research and molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
Humans
*Receptors, Transferrin/genetics
*Polymerase Chain Reaction/methods
*Mutation
*Antigens, CD/genetics
*Alleles
Fluorescence
*CRISPR-Associated Proteins/genetics/metabolism
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41073014,
year = {2025},
author = {Cao, R and Wang, S and Guo, Q and Xie, H and Wang, Y and Li, T and Chang, F and Shi, H and Ding, S and Min, X and Duan, X},
title = {DNAzyme-driven SDA reaction regulates CRISPR/Cas12a for highly sensitive and selective analysis of underexpressed miRNA.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344620},
doi = {10.1016/j.aca.2025.344620},
pmid = {41073014},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics/metabolism ; *DNA, Catalytic/metabolism/chemistry ; *CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Male ; Prostatic Neoplasms/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Underexpressed microRNA (miRNA) exerts a pivotal influence across a spectrum of physiological and pathological processes, with their role being particularly pronounced in the incipient stages of tumorigenesis. However, there are several challenges to analyzing these underexpressed miRNAs for their lower abundance and relative decreases in some cancers. Here, we developed a novel fluorescence biosensor based on the DNAzyme-driven strand displacement amplification (SDA) regulating CRISPR/Cas12a for the sensitive and selective detection of underexpressed miRNA, using prostate cancer-associated miR-222 as a proof-of-concept. This study innovatively expanded the application of DNAzyme substrates, designed as templates to trigger SDA and CRISPR/Cas12a reaction, which could effectively generate a positive signal output for detecting underexpressed miRNA. In the absence of miR-222, DNAzyme formation was blocked, allowing the complete substrate to activate SDA, which generated ssDNA that triggered CRISPR/Cas12a trans-cleavage activity to produce a strong fluorescent signal. In contrast, intact DNAzymes (in the presence of miR-222) cleaved the substrates into short DNA fragments, preventing SDA and CRISPR/Cas12a activation, thereby maintaining the sensor in a low fluorescent state. The biosensor demonstrated a linear detection range spanning from 0.1 pmol/L to 1 nmol/L, accompanied by a detection limit of 33.5 fmol/L. Moreover, it exhibited excellent specificity and anti-interference capacity, enabling the successful detection of miR-222 in blood samples. This "DNAzyme-SDA-CRISPR" fluorescence strategy offers a effective, programmability and scalable solution for detecting underexpressed miRNAs in early cancer screening, which is expected to become a powerful tool in early tumor diagnostics and precision therapy.},
}

*MicroRNAs/analysis/genetics/metabolism
*DNA, Catalytic/metabolism/chemistry
*CRISPR-Cas Systems
Humans
*Biosensing Techniques/methods
*Nucleic Acid Amplification Techniques/methods
Limit of Detection
Male
Prostatic Neoplasms/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41043311,
year = {2026},
author = {Hu, M and Zhou, C and Li, M and Zhao, J},
title = {From 3D culture to clinical decision-making: Systematic innovations in breast cancer organoids.},
journal = {Biomaterials advances},
volume = {179},
number = {},
pages = {214528},
doi = {10.1016/j.bioadv.2025.214528},
pmid = {41043311},
issn = {2772-9508},
mesh = {Humans ; *Organoids/pathology ; *Breast Neoplasms/pathology/therapy/genetics ; Female ; *Clinical Decision-Making ; Animals ; CRISPR-Cas Systems ; *Cell Culture Techniques, Three Dimensional/methods ; Tumor Microenvironment ; Gene Editing ; Precision Medicine ; },
abstract = {Breast cancer is a malignant tumour with high heterogeneity. Traditional research models rely mainly on 2D cell culture and patient-derived tumour xenografts (PDXs). However, these models have limited use in clinical trials because of their shortcomings in mimicking the tumour microenvironment and preserving the genetic background. In recent years, organoids, emerging models capable of self-organizing to form 3D structures in vitro, have become key tools for overcoming the traditional dilemma and are promising alternatives for breast cancer research. This review integrates cutting-edge technologies such as organ-on-a-chip and CRISPR/Cas9 gene editing to summarize the multidimensional generation strategy of breast cancer organoids and discusses the clinical value of translation from diagnosis to therapy. Compared with existing studies, this review provides a systematic solution from "model generation" to "precision medicine" for breast cancer research, and the hope is that this review will pave the way for the further development of organoids.},
}

Humans
*Organoids/pathology
*Breast Neoplasms/pathology/therapy/genetics
Female
*Clinical Decision-Making
Animals
CRISPR-Cas Systems
*Cell Culture Techniques, Three Dimensional/methods
Tumor Microenvironment
Gene Editing
Precision Medicine

@article {pmid41015100,
year = {2025},
author = {Deng, D and Yi, X and Wen, W and He, L and Peng, W},
title = {The role of the transformer gene in sex determination and its employment in CRISPR/Cas9-based homing gene drive in the global fruit pest Drosophila suzukii.},
journal = {Insect biochemistry and molecular biology},
volume = {184},
number = {},
pages = {104406},
doi = {10.1016/j.ibmb.2025.104406},
pmid = {41015100},
issn = {1879-0240},
mesh = {Animals ; *Sex Determination Processes/genetics ; CRISPR-Cas Systems ; Female ; Male ; *Drosophila Proteins/genetics/metabolism ; *Drosophila/genetics/growth & development ; *Gene Drive Technology ; Nuclear Proteins ; },
abstract = {Sex determination of Diptera is established by the cascade genes such as transformer (tra), though the primary signals for sex determination differ among different insects. Here, we report the isolation, expression and function of tra gene in an invasive pest, Drosophila suzukii, and study the potential use of the D. suzukii tra (Dstra) gene in CRISPR/Cas9-based homing gene drive for genetic-based pest management. The Dstra gene is highly conserved in structure and has a sex-specific transcript. To test the function of this gene in sex determination, Dstra dsRNA was injected into embryos. Almost all XX embryos developed into masculinized phenotypic male adults with intersex morphology. Abnormal ovaries were revealed in XX pseudomales upon dissection. Based on the necessary role of Dstra for female development, we developed and evaluated a homing gene drive that targets Dstra in D. suzukii. The drive component consisting of multiplex Dstra single guide RNAs and Cas9 with Dsvasa promoter was introduced into the Dstra locus. Abnormal development of both the external genitalia and gonads was observed in G0 and G1 chromosomal female adults that expressed the male-specific doublesex (dsx) transcript. Interestingly, knocking out Dstra led to significantly reduced fertility in adults of corresponding sex and moderate transmission rates of the DsRed gene (63.54 %) were observed. Our results not only confirm the conserved function of the Dstra gene in sex determination, but also highlight the potential of sex conversion-based suppression gene-drive strategy targeting the Dstra gene in controlling of D. suzukii populations.},
}

Animals
*Sex Determination Processes/genetics
CRISPR-Cas Systems
Female
Male
*Drosophila Proteins/genetics/metabolism
*Drosophila/genetics/growth & development
*Gene Drive Technology
Nuclear Proteins

@article {pmid41072992,
year = {2025},
author = {Shi, S and Qin, F and Wu, J and Yang, J and Zhang, X and Wang, S and Wen, W and Wu, Z},
title = {Ultrasensitive single-particle collision electrochemical platform employing CRISPR/Cas12a for ctDNA biosensing.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344590},
doi = {10.1016/j.aca.2025.344590},
pmid = {41072992},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *Circulating Tumor DNA/blood/analysis/genetics ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; Silver/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; },
abstract = {Circulating tumor DNA (ctDNA) is a characteristic tumor biomarker used for cancer diagnosis, treatment, and prognosis. However, the low concentration of ctDNA in peripheral blood and the interference of complex matrices with signals make the detection of ctDNA extremely challenging. Single-particle collision electrochemistry (SPCE) has been widely used in bioanalysis due to its advantages such as high throughput, simple operation, high sensitivity, and low detection limit. In this work, a novel SPCE biosensor for the rapid detection of ctDNA was developed by combining CRISPR/Cas12a with excellent cleavage activity and magnetic beads (MBs) with good separation and enrichment capabilities. The trans-cleavage ability of CRISPR/Cas12a can only be triggered in the presence of target ctDNA to effectively cleave the ssDNA2 on the surface of Ag NPs-ssDNA2 within 1 h, thereby activating the collision activity of silver nanoparticles (Ag NPs). ctDNA was quantified by the collision frequency of Ag NPs. The detection limit of the developed SPCE biosensor for ctDNA was as low as 4.2 fM, and the linear range was 10 fM-1 nM. In addition, MBs allow the biosensor to detect ctDNA in complex samples by directly sampling from complex matrices, with excellent sensitivity and specificity, demonstrating the great potential of the developed SPCE biosensor in the detection of patient samples.},
}

*Biosensing Techniques/methods
*Circulating Tumor DNA/blood/analysis/genetics
*Electrochemical Techniques/methods
Humans
*CRISPR-Cas Systems
Silver/chemistry
Metal Nanoparticles/chemistry
Limit of Detection

@article {pmid41072860,
year = {2025},
author = {Patel, RR and Arun, PP and Singh, SK and Singh, M},
title = {Overcoming Antimicrobial Resistance: Phage Therapy as a Promising Solution to Combat ESKAPE Pathogens.},
journal = {International journal of antimicrobial agents},
volume = {},
number = {},
pages = {107640},
doi = {10.1016/j.ijantimicag.2025.107640},
pmid = {41072860},
issn = {1872-7913},
abstract = {The global escalation of antimicrobial resistance (AMR) has intensified the search for alternative therapies, with bacteriophage (phage) therapy re-emerging as a promising solution. This review critically examines the therapeutic potential of phage therapy against multidrug-resistant (MDR) ESKAPE pathogens which are among the leading causes of hospital-acquired infections. The review discusses the distinct antibacterial strategies of phage namely, targeted lysis, enzymatic biofilm disruption, and synergy with antibiotics. It also explores the molecular regulation of phage life cycles, highlighting the therapeutic importance of the lytic-lysogenic switch. A central focus is the interplay between advanced delivery systems such as liposomes, hydrogels, nanofibers, and nanoemulsions, and specific administration routes including oral, topical, intravenous, intranasal, and intravesical approaches. These delivery strategies are essential for overcoming key physiological barriers such as gastric acidity, enzymatic degradation, and immune clearance, thereby enhancing phage stability, retention, and therapeutic efficacy. Recent innovations in phage engineering are also explored, particularly the use of CRISPR-Cas systems, synthetic biology, and continuous evolution platforms to broaden host range and optimize lytic function. The review further evaluates emerging clinical evidence, including outcomes from compassionate use cases and early-phase trials, which emphasize both the safety and therapeutic potential of phage therapy in real-world settings. Despite these advances, significant challenges persist, including bacterial resistance to phages, the need for regulatory clarity, and scalability of personalized treatments. With the integration of microbiology, nanotechnology, and clinical practice, phage therapy bridges the gap between ecological solutions and modern medicine, positioning itself as a versatile, sustainable pillar in the post-antibiotic era.},
}

@article {pmid41072406,
year = {2025},
author = {Henriques, WS and Bowman, J and Hall, LN and Gauvin, CC and Wei, H and Kuang, H and Zimanyi, CM and Eng, ET and Santiago-Frangos, A and Wiedenheft, B},
title = {Structures reveal how the Cas1-2/3 integrase captures, delivers, and integrates foreign DNA into CRISPR loci.},
journal = {Structure (London, England : 1993)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.str.2025.09.007},
pmid = {41072406},
issn = {1878-4186},
abstract = {Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify and determine structures of Cas1 and the Cas2/3 fusion proteins from Pseudomonas aeruginosa at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome.},
}

@article {pmid41071884,
year = {2025},
author = {Guo, Y and Xu, M and Xue, H and Ding, X and Wong, AM and Lin, N and Pu, D and Wong, AM and Wang, X and Zhao, H and Wong, N},
title = {Genome-wide CRISPR screen identifies splicing factor SF3B4 in driving hepatocellular carcinoma.},
journal = {Science advances},
volume = {11},
number = {41},
pages = {eadw7181},
doi = {10.1126/sciadv.adw7181},
pmid = {41071884},
issn = {2375-2548},
mesh = {*Carcinoma, Hepatocellular/genetics/pathology/metabolism/drug therapy ; Humans ; *Liver Neoplasms/genetics/pathology/metabolism/drug therapy ; Animals ; Mice ; *RNA Splicing Factors/genetics/metabolism ; Ferroptosis/genetics ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Phenylurea Compounds/pharmacology ; Drug Resistance, Neoplasm/genetics ; T-Box Domain Proteins/genetics/metabolism ; Quinolines ; },
abstract = {Although genome sequencings have recognized many cancer-associated genes in hepatocellular carcinoma (HCC), distinguishing their functional effect remains challenging. Leveraging on a genome-wide CRISPR knockout (KO) screening, we uncovered spliceosome factors as major survival essential genes in HCC and up-regulations of ferroptosis suppressors [particularly glutamate-cysteine ligase catalytic subunit (GCLC)] in lenvatinib resistance. Our KO screen in patient-derived HCC organoid showed splicing factor 3b subunit 4 (SF3B4) to be top-ranked, conferring prosurvival signal in HCC organoid and driving tumorigenic potentials in both hepatic progenitor organoids and hydrodynamic tail vein injection HCC murine model. The combined RNA immunoprecipitation sequencing, long-read isoform sequencing, and transcriptome revealed characteristic splicing landscape regulated by SF3B4 and identified T-box transcription factor 3 (TBX3) variant TBX3+2a as a potent downstream effector. Our findings highlighted vital roles of SF3B4 in HCC cell survival and tumor progression, and the phenomenon of ferroptosis resistance in patients unresponsive to first-line agent lenvatinib.},
}

*Carcinoma, Hepatocellular/genetics/pathology/metabolism/drug therapy
Humans
*Liver Neoplasms/genetics/pathology/metabolism/drug therapy
Animals
Mice
*RNA Splicing Factors/genetics/metabolism
Ferroptosis/genetics
Gene Expression Regulation, Neoplastic
*CRISPR-Cas Systems
Cell Line, Tumor
*Clustered Regularly Interspaced Short Palindromic Repeats
Phenylurea Compounds/pharmacology
Drug Resistance, Neoplasm/genetics
T-Box Domain Proteins/genetics/metabolism
Quinolines

@article {pmid41070887,
year = {2025},
author = {Zhang, Y and Wu, Y and Guo, A and Liu, Y and Sun, Q and Zou, X and Sun, Z},
title = {Fluorescent biosensors for the detection of foodborne pathogenic bacteria in food: a comprehensive review.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay01025j},
pmid = {41070887},
issn = {1759-9679},
abstract = {Foodborne pathogenic bacterial contamination poses a major challenge to global food safety and public health, making the development of rapid, sensitive, and specific detection technologies critically important. Conventional methods are limited by their long turnaround time, complex operations, and reliance on large-scale instruments, making them unsuitable for on-site rapid detection. Fluorescent biosensors, which combine highly specific biological recognition elements with highly sensitive fluorescent signal output, demonstrate significant advantages in detecting foodborne pathogens. This review systematically summarizes recent advances in fluorescent biosensors for the detection of common foodborne pathogenic bacteria, with a focus on the application of signal amplification strategies such as functional nanomaterials, amplification techniques, CRISPR/Cas systems, and Argonaute proteins. Furthermore, it analyzes performance metrics including multiplex pathogen detection, real-time quantification, anti-interference capability, and on-site applicability. Finally, future development trends and challenges are discussed, aiming to provide insights for the innovation of food safety monitoring technologies.},
}

@article {pmid41069986,
year = {2025},
author = {Pandey, V and Sharma, S and Pokharel, YR},
title = {Exploring CRISPR-Cas: The transformative impact of gene editing in molecular biology.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102717},
pmid = {41069986},
issn = {2162-2531},
abstract = {This review traces the evolution of clustered regularly interspaced short palindromic repeats (CRISPR) technology from a prokaryotic immune mechanism to a versatile tool for precise genome engineering. We compare CRISPR with traditional gene-editing methods like RNA interference (RNAi), zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), emphasizing its advantages in target specificity, multiplexing, and ease of design. We examine various Cas enzyme classes, engineered variants, and their applications in dissecting genetic alterations at the cellular level. The review further explores CRISPR's expanding role in developing disease models using tissues, organoids, and animal systems, enhancing our understanding of disease mechanisms. Finally, we discuss CRISPR's emerging applications in diagnostics and its transformative impact on immunotherapy and cell-based cancer treatments.},
}

@article {pmid41069170,
year = {2025},
author = {Zhu, Z and Xue, J and Cao, J and Zhang, Z and Gu, T and Sun, Y and Wang, H},
title = {One-pot assay for rapid detection of heterozygous herbicide resistance in Digitaria ciliaris var. chrysoblephara by combining CRISPR/Cas and LAMP.},
journal = {Pest management science},
volume = {},
number = {},
pages = {},
doi = {10.1002/ps.70279},
pmid = {41069170},
issn = {1526-4998},
support = {CX(22)5003//Jiangsu Agricultural Science and Technology Innovation Fund/ ; 2023YFD1401100//National Key R&D Program of China/ ; },
abstract = {BACKGROUND: Resistance to the acetyl-CoA carboxylase (ACCase) inhibitor herbicide cyhalofop-butyl in Digitaria ciliaris var. chrysoblephara is mainly caused by a mutation at the W2027C or W2027S site; however, the existing methods for this mutation site have insufficient detection performance and are difficult to achieve integrated detection in the field.

RESULTS: In this work, we have developed and optimized a One-Pot single-nucleotide polymorphism (SNP) detection for herbicide resistance based on CRISPR/Cas recognition coupled with the loop-mediated isothermal amplification (LAMP), named OpCas-LAMP. By designing specific CRISPR/Cas guide RNAs and LAMP primers, the OpCas-LAMP can accurately identify with 1% heterozygous mutants of the W2027S or W2027C mutations ACCase gene in D.ciliaris var. chrysoblephara. The optimized reaction system exhibits optimal amplification efficiency at 65°C, effectively distinguishing within 60 min (30-min LAMP detection after 30 min CRISPR/Cas pre-cleavage) between homozygous mutant (HM), heterozygous mutant (HT) and wild-type (WT).

CONCLUSION: This method enables real-time one-pot field detection by integrated with miniaturized detection devices, significantly enhancing its practicality and potential for widespread application. This work provides a novel technical approach for detecting herbicide resistance for global weed resistance monitoring and management. © 2025 Society of Chemical Industry.},
}

@article {pmid41068986,
year = {2025},
author = {Wu, J and Jang, H and Kwak, H and Son, M and Jiang, W and Hwang, HY and Jo, DH and Kim, D and Kim, HH and Kim, JH},
title = {Engineered virus-like particle-assembled Vegfa-targeting Cas9 ribonucleoprotein treatment alleviates neovascularization in wet age-related macular degeneration.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {346},
pmid = {41068986},
issn = {1474-760X},
support = {2022M3A9F3017506//National Research Foundation of Korea/ ; 2022M3A9E4017127//National Research Foundation of Korea/ ; 202200004004//Kun-hee Lee Child Cancer & Rare Disease Project/ ; 18-2023-0010//Seoul National University Hospital/ ; GTL24021-000//National Research Council of Science and Technology/ ; },
mesh = {Animals ; *Vascular Endothelial Growth Factor A/genetics/metabolism ; Mice ; Gene Editing/methods ; *Ribonucleoproteins/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Choroidal Neovascularization/therapy/genetics ; CRISPR-Cas Systems ; NIH 3T3 Cells ; Disease Models, Animal ; Humans ; *Wet Macular Degeneration/therapy/genetics ; Genetic Therapy ; *Macular Degeneration/therapy/genetics ; },
abstract = {BACKGROUND: Age-related macular degeneration, particularly the wet form, is a leading cause of vision loss, characterized by vascular endothelial growth factor A (VEGFA) overproduction. Engineered virus-like particles (eVLPs) combine the efficiency of viral systems with the transient nature of non-viral platforms to offer a potential solution for delivering VEGFA-targeting genome editing enzymes in a safe and efficient manner. Here, we investigate the therapeutic efficacy of eVLPs for transient delivery of Vegfa-targeting Cas9 ribonucleoprotein in a laser-induced choroidal neovascularization mouse model of wet age-related macular degeneration.

RESULTS: We find that Cas9-eVLPs enables efficient intracellular delivery in vitro, achieving up to 99% insertion and deletion frequency at Vegfa target locus and significant VEGFA protein downregulation in NIH/3T3 cells. A single subretinal injection of Cas9-eVLPs into the mouse retinal pigment epithelium effectively disrupts Vegfa expression, achieving an average indel efficiency of 16.7%. Compared to control groups, the laser-induced choroidal neovascularization mouse model exhibits significantly reduced choroidal neovascularization formation following Cas9-eVLPs intervention, and decreased VEGFA protein levels are detected in the retinal pigment epithelium. Furthermore, the retinal anatomical and functional toxicity are not affected after treatment.

CONCLUSIONS: eVLPs exhibit the potential as a safe and efficient delivery platform for Cas9 ribonucleoproteins, achieving precise Vegfa downregulation and significant reduction in choroidal neovascularization in a mouse model of wet age-related macular degeneration. With transient delivery of gene editing enzymes, high editing efficiency, and minimal risk of genomic integration, eVLPs present a promising alternative to conventional delivery systems for advancing genome editing therapies in retinal diseases.},
}

Animals
*Vascular Endothelial Growth Factor A/genetics/metabolism
Mice
Gene Editing/methods
*Ribonucleoproteins/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
*Choroidal Neovascularization/therapy/genetics
CRISPR-Cas Systems
NIH 3T3 Cells
Disease Models, Animal
Humans
*Wet Macular Degeneration/therapy/genetics
Genetic Therapy
*Macular Degeneration/therapy/genetics

@article {pmid41066575,
year = {2025},
author = {Groessl, S and Kalis, R and Snaebjornsson, MT and Wambach, L and Haider, J and Andersch, F and Schulze, A and Palm, W and Zuber, J},
title = {Acidosis orchestrates adaptations of energy metabolism in tumors.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6769},
pages = {eadp7603},
doi = {10.1126/science.adp7603},
pmid = {41066575},
issn = {1095-9203},
mesh = {*Energy Metabolism/genetics ; *Acidosis/metabolism/genetics ; Humans ; *Pancreatic Neoplasms/metabolism/genetics ; Mitochondria/metabolism ; Cell Line, Tumor ; *Adaptation, Physiological/genetics ; Animals ; Mice ; Stress, Physiological ; CRISPR-Cas Systems ; MAP Kinase Signaling System ; Mitochondrial Dynamics ; },
abstract = {Malignant tumors are characterized by diverse metabolic stresses, including nutrient shortages, hypoxia, and buildup of metabolic by-products. To understand how cancer cells adapt to such challenges, we conducted sequential CRISPR screens to identify genes that affect cellular fitness under specific metabolic stress conditions in cell culture and to then probe their relevance in pancreatic tumors. Comparative analyses of hundreds of fitness genes revealed that cancer metabolism in vivo was shaped by bioenergetic adaptations to tumor acidosis. Mechanistically, acidosis suppressed cytoplasmic activity of extracellular signal-regulated kinase (ERK), thereby preventing oncogene-induced mitochondrial fragmentation and promoting fused mitochondria. The resulting boost in mitochondrial respiration supported cancer cell adaptations to various metabolic stresses. Thus, acidosis is an environmental factor that alters energy metabolism to promote stress resilience in cancer.},
}

*Energy Metabolism/genetics
*Acidosis/metabolism/genetics
Humans
*Pancreatic Neoplasms/metabolism/genetics
Mitochondria/metabolism
Cell Line, Tumor
*Adaptation, Physiological/genetics
Animals
Mice
Stress, Physiological
CRISPR-Cas Systems
MAP Kinase Signaling System
Mitochondrial Dynamics

@article {pmid41065057,
year = {2025},
author = {Anjali, and Punetha, M and Kumar, A and Tripathi, MK and Kishor Kumar, DG and Khanna, S and Nanda, R and Yadav, P and Sharma, S and Maurya, VP and Singh, G and Chouhan, VS},
title = {Application of CRISPR/Cas9 for GDF9 Gene Editing in Caprine Granulosa Cells: Effects on Receptor Signalling and FGF2 Response.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {60},
number = {10},
pages = {e70128},
doi = {10.1111/rda.70128},
pmid = {41065057},
issn = {1439-0531},
support = {NASF/GTR-8004/2019-20//ICAR - National Agricultural Science Fund/ ; },
mesh = {Animals ; Female ; *Granulosa Cells/metabolism ; *Growth Differentiation Factor 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/veterinary ; *Goats/genetics ; *Fibroblast Growth Factor 2/pharmacology/metabolism ; Signal Transduction ; Receptors, FSH/metabolism/genetics ; },
abstract = {Fecundity-related genes, such as GDF9, play a critical role in regulating ovulation, fertilisation and early embryonic development. This study aimed to elucidate the functional role of GDF9 in caprine granulosa cells by employing CRISPR/Cas9-mediated gene editing. The CRISPR/Cas9 system, incorporating single guide RNA (sgRNA) and Cas9 endonuclease, was used to specifically disrupt the GDF9 gene. Successful GDF9 knockout was confirmed via the T7 Endonuclease I (T7E1) cleavage assay. Subsequent analyses assessed the impact of GDF9 disruption on the expression of GDF9 and its associated receptors-BMPR-1A, BMPR-1B and BMPR-II. Additionally, the study examined the modulatory effects of fibroblast growth factor 2 (FGF2) on receptor expression. FGF2 treatment led to increased mRNA expression of BMPR-1A, BMPR-1B and BMPR-II in wild-type granulosa cells. Furthermore, follicle-stimulating hormone receptor (FSHR) levels were significantly upregulated, whereas luteinising hormone receptor (LHR) expression decreased following FGF2 stimulation in wild-type cells. In contrast, GDF9-knockout cells showed elevated expression of both FSHR and LHR. The study also investigated the impact of GDF9 deletion on the expression of key steroidogenic genes, particularly StAR. The combined presence of GDF9 and FGF2 synergistically enhanced StAR expression. Cellular responses to FGF2 included a downregulation of CASPASE 3, indicating reduced apoptosis and an upregulation of PCNA, suggesting increased cell proliferation. In conclusion, this study provides novel insights into the regulatory role of GDF9 in ovarian granulosa cell function and highlights the utility of CRISPR/Cas9 technology for functional genomics in caprine species. The findings have significant implications for enhancing reproductive performance through targeted gene modulation.},
}

Animals
Female
*Granulosa Cells/metabolism
*Growth Differentiation Factor 9/genetics/metabolism
*CRISPR-Cas Systems
*Gene Editing/veterinary
*Goats/genetics
*Fibroblast Growth Factor 2/pharmacology/metabolism
Signal Transduction
Receptors, FSH/metabolism/genetics

@article {pmid41064997,
year = {2025},
author = {Chen, X and Ye, Q and Liang, Q and Li, J and Huang, Y and Xia, Q and Xiao, J and Liao, C and Lau, CH and Zhu, H},
title = {CRISPR-based platforms for detecting tumor-associated genetic materials in clinical samples.},
journal = {Bioanalysis},
volume = {},
number = {},
pages = {1-15},
doi = {10.1080/17576180.2025.2571023},
pmid = {41064997},
issn = {1757-6199},
abstract = {Tumor-associated genetic markers are useful for early cancer screening, diagnosis, and treatment monitoring. However, traditional detection methods are complex in operation procedures, time-consuming, and the equipment costs are expensive. CRISPR/Cas systems are becoming emerging detection tools for tumor detection due to their programmability, rapid reaction, high targeting specificity, and the ability to amplify the signals. CRISPR/Cas has made breakthroughs in the detection of tumor-associated genetic materials including gene mutations, DNA methylation, miRNA, lncRNA, and circRNA detection. Herein, we critically discuss these advancements and describe the key concepts of each CRISPR/Cas system for detecting tumor-associated genetic materials. The significance of these tumor-associated genetic materials in cancer diagnosis and prognosis is highlighted.},
}

@article {pmid41064763,
year = {2025},
author = {Ragulakollu, S and Loganathan, A and Swaminatham, M and Chellappan, G and Veeraswamy, R and Jegadeesan, R},
title = {Molecular breeding approaches for sustainable rice blast management: recent advances and challenges.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1551018},
pmid = {41064763},
issn = {1664-462X},
abstract = {Rice (Oryza sativa. L) is a staple crop globally, but blast disease caused by fungal pathogens Magnaporthe oryzae is one of the most devastating and results in severe economic losses in rice production worldwide. Recent technological advancements have opened new possibilities for developing blast resistance. The dynamic and highly adaptable nature of M. oryzae allows it to overcome plant defense mechanisms rapidly, posing a major threat to global food security and agricultural sustainability. While foundational to early resistance development, traditional breeding approaches have been limited by their time-consuming nature and reliance on phenotypic selection. These methods often require several generations to establish stable resistance traits. However, with the emergence of molecular breeding technologies, resistance breeding has experienced significant acceleration and precision. Tools such as marker-assisted selection (MAS), marker-assisted backcross breeding (MABB), and quantitative trait locus (QTL) mapping allow for the identification and introgression of resistance genes (R genes) more efficiently and accurately. Recent advances in genome engineering techniques, particularly CRISPR-Cas 9, have transformed the capability to manipulate resistance genes directly, enabling targeted editing and stacking of multiple genes (gene pyramiding) for durable resistance. Moreover, omics technologies-including genomics, transcriptomics, proteomics, and metabolomics-offer a comprehensive understanding of the molecular interactions between host and pathogen, facilitating the discovery of novel resistance mechanisms and regulatory pathways. The integration of allele mining with advanced biotechnological tools has further promoted the development of cisgenic and intragenic plants, where resistance genes from related cultivars or wild species are introduced without foreign DNA, thus addressing public concerns over transgenic crops. These strategies enhance resistance and help retain the desirable agronomic traits of elite rice varieties. Despite these advancements, the high mutation rate and genetic plasticity of M. oryzae enable it to evolve and overcome resistance provided by single R genes. Therefore, understanding host-pathogen interactions at the molecular and cellular levels remains essential. Emerging technologies such as nanotechnology show promise in developing targeted fungicide delivery systems and innovative diagnostic tools. Synthetic biology opens avenues for constructing synthetic resistance pathways or deploying plant biosensors. Additionally, machine learning and artificial intelligence (AI) algorithms are increasingly used to predict disease outbreaks, model gene interactions, and optimize breeding strategies based on large datasets. Thus, managing rice blast disease necessitates a holistic approach combining conventional breeding wisdom with modern molecular tools and emerging technologies. The synergy among these approaches holds promise to enhance resistance durability and protect global rice production against evolving fungal threats. This review emphasizes recent advancements in managing rice blast disease, offering valuable insights to sustain resilient breeding programs against this pathogen.},
}

@article {pmid41064460,
year = {2025},
author = {Rentz, L and Hellwig, L and Schneider, S and Schmitz, RA},
title = {Functional insights into Solo-Cas4 in Methanosarcina mazei Gö1.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf024},
pmid = {41064460},
issn = {2633-6693},
abstract = {Solo-Cas4 homologs are Cas4-family proteins found outside of canonical CRISPR-Cas operons. Here, we present the biochemical characterization of Solo-Cas4 from Methanosarcina mazei Gö1. We found significantly upregulated solo-cas4 transcript levels during stationary phase, while remaining constant under oxygen exposure, temperature shifts, high salt conditions or virus challenge. Heterologously expressed as a SUMO-fusion, the purified tag-free protein displays an absorption peak at 420 nm, indicative of a [4Fe-4S]-cluster . Size-exclusion-chromatography revealed that Solo-Cas4 forms a higher oligomeric complex, with an apparent molecular mass of 318 kDa. In vitro nuclease activity assays demonstrated that Solo-Cas4 cleaves metal-dependent linear dsDNA, with highest cleavage activity in the presence of Mn[2+], followed by Mg[2+], while Ca[2+] and Cu[2+] result in negligible cleavage. Isoleucine169 was identified to be crucial for catalysis, mutating it to alanine completely abolished nuclease activity . Mutating any of the four conserved cysteines-proposed to coordinate the [4Fe-4S]-cluster did not affect nuclease activity; however, it abolishes metal cluster binding. Supercoiled circular dsDNA was preferentially nicked by Solo-Cas4 in the presence of Mg[2+], whereas Mn[2+] also led to linearization followed by complete degradation. Besides, ssDNA was cleaved by Solo-Cas4 but with lower activity. In agreement, Microscale thermophoresis analysis revealed strong dsDNA binding with highest affinity to supercoiled circular DNA, and weak ssDNA binding. Overall, these findings indicate that M. mazei Solo-Cas4 is a high oligomeric Cas4-family nuclease that preferentially targets supercoiled dsDNA and is upregulated during stationary growth.},
}

@article {pmid40988554,
year = {2025},
author = {Li, H and Gui, P and Li, X and Lin, Y and Ma, Z and Yu, H and Ma, F},
title = {CRISPR/Cas9-Mediated Construction of a YPS Gene-Deficient Komagataella phaffii Strain for Enhanced Expression of BIAP Ⅱ.},
journal = {Yeast (Chichester, England)},
volume = {42},
number = {8-10},
pages = {195-205},
doi = {10.1002/yea.70002},
pmid = {40988554},
issn = {1097-0061},
support = {//This work was supported by Youth Innovation Promotion Association Fellowship Program, CAS (2022327), Shandong Provincial Laboratory Project (SYS202209), Key Technologies R&D Program of Guangdong Province (2022B1111050001), Natural Science Foundation of Shandong Province, China (ZR2021QB155)./ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Alkaline Phosphatase/genetics/metabolism ; *Saccharomycetales/genetics/metabolism/enzymology ; Cattle ; *Aspartic Acid Endopeptidases/genetics/metabolism ; Recombinant Proteins/genetics/metabolism ; Gene Editing ; Proteolysis ; Gene Expression ; },
abstract = {Multiple isoforms of bovine intestinal alkaline phosphatase (BIAP) have been identified, among which type Ⅱ (BIAP Ⅱ) exhibits the highest specific activity. While Komagataella phaffii has been successfully employed for the secretory expression of recombinant BIAP Ⅱ, substantial proteolytic degradation during the secretion and expression processes has been observed, leading to reduced protein yield and challenging purification procedures. Our investigation demonstrates that the proteolytic cleavage of BIAP Ⅱ is predominantly mediated by secretory pathway proteases, particularly the aspartic protease yapsin (Yps), with Yps1 playing a crucial role. Genetic disruption of the YPS1 gene resulted in a remarkable 2.5-fold increase in BIAP Ⅱ production yield compared to the parental strain, accompanied by significantly reduced proteolytic degradation. Through detailed analysis, we have identified the Yps1 cleavage site within the BIAP Ⅱ peptide chain, located between Lys137 and Lys138. To further minimize BIAP Ⅱ proteolysis, we developed a YPS multigene-deficient engineered strain using CRISPR/Cas9-mediated triple gene editing technology. Additionally, we have established a novel dual-color quantitative PCR (DC-qPCR) method that enables rapid and precise determination of target gene dosage, thereby enhancing screening efficiency while reducing experimental errors associated with repeated sample processing. The strategies and methodologies developed in this study may serve as a valuable reference for optimizing the expression of various secretory heterologous proteins in Komagataella phaffii.},
}

Animals
*CRISPR-Cas Systems
*Alkaline Phosphatase/genetics/metabolism
*Saccharomycetales/genetics/metabolism/enzymology
Cattle
*Aspartic Acid Endopeptidases/genetics/metabolism
Recombinant Proteins/genetics/metabolism
Gene Editing
Proteolysis
Gene Expression

@article {pmid40962170,
year = {2025},
author = {He, H and Huang, Z and Wen, F and Ge, N and Lin, X and Pan, J},
title = {PSPC1 knockout promotes radiosensitivity, inhibits EMT, and metastasis of nasopharyngeal carcinoma cells.},
journal = {Experimental cell research},
volume = {452},
number = {2},
pages = {114755},
doi = {10.1016/j.yexcr.2025.114755},
pmid = {40962170},
issn = {1090-2422},
mesh = {Humans ; *Radiation Tolerance/genetics ; *Nasopharyngeal Carcinoma/genetics/pathology/radiotherapy/metabolism ; Animals ; *Nasopharyngeal Neoplasms/pathology/genetics/radiotherapy ; Cell Line, Tumor ; *Epithelial-Mesenchymal Transition/genetics/radiation effects ; Cell Movement/genetics/radiation effects ; Apoptosis/genetics/radiation effects ; Mice ; Mice, Nude ; *RNA-Binding Proteins/genetics/metabolism ; Cell Proliferation/genetics ; *Nuclear Proteins/genetics/metabolism ; Gene Expression Regulation, Neoplastic/genetics ; Neoplasm Metastasis ; Mice, Inbred BALB C ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {PURPOSE: Paraspeckle component 1 (PSPC1) is upregulated in numerous cancers and is associated with reduced patient survival rates. Our previous research indicated that elevated PSPC1 levels in nasopharyngeal carcinoma (NPC) are positively linked to radiation resistance and tumor metastasis, two primary clinical challenges in NPC treatment. However, the precise role of PSPC1 in radiation resistance and metastasis of NPC cells remains unclear. This study aimed to explore the molecular mechanisms by which PSPC1 influences radiation resistance and metastasis in NPC.

METHODS: Using the radiation-resistant R743 and radiosensitive CNE2 cell lines of NPC, we examined the impact of PSPC1 expression on post-radiation survival, cell cycle progression, apoptosis, migration, invasion, and tumor growth. CRISPR/Cas9 genome editing was employed to generate PSPC1 knockout (KO) lines in R743 cells, while PSPC1 overexpression (pcD-PSPC1) was achieved in CNE2 cells via pcDNA3.1(+)-PSPC1 plasmid transfection.

RESULTS: PSPC1 knockout converted R743 cells from radioresistant to radiosensitive, whereas PSPC1 overexpression decreased radiosensitivity in CNE2 cells. Cell cycle analysis revealed that PSPC1 KO arrested R743 cells in the G2/M phase post-irradiation, while PSPC1 overexpression prevented G2/M phase arrest in CNE2 cells. PSPC1 KO increased irradiation-induced apoptosis in R743 cells, whereas PSPC1 overexpression decreased it in CNE2 cells. Post-radiation, PSPC1 KO cells showed significantly reduced migration and invasion abilities. Bioinformatics analysis identified SFPQ as a PSPC1-interacting protein, with PSPC1 KO resulting in SFPQ downregulation. Additionally, PSPC1 KO enhanced the radiosensitivity of xenografted tumors in nude mice.

CONCLUSION: Our findings suggest that PSPC1 is a pivotal factor in enhancing the survival and spread of NPC cells post-radiation. Targeting PSPC1 or its downstream pathways could offer novel strategies to overcome radiation resistance and metastasis in NPC cells.},
}

Humans
*Radiation Tolerance/genetics
*Nasopharyngeal Carcinoma/genetics/pathology/radiotherapy/metabolism
Animals
*Nasopharyngeal Neoplasms/pathology/genetics/radiotherapy
Cell Line, Tumor
*Epithelial-Mesenchymal Transition/genetics/radiation effects
Cell Movement/genetics/radiation effects
Apoptosis/genetics/radiation effects
Mice
Mice, Nude
*RNA-Binding Proteins/genetics/metabolism
Cell Proliferation/genetics
*Nuclear Proteins/genetics/metabolism
Gene Expression Regulation, Neoplastic/genetics
Neoplasm Metastasis
Mice, Inbred BALB C
Gene Knockout Techniques
CRISPR-Cas Systems

@article {pmid40952014,
year = {2025},
author = {Llanga, T and Bush, K and Sun, Y and Yan, A and Zhou, J and Gorodkin, J and Sullenger, BA},
title = {Binding and Ligand Activation Driven Enrichment-Directed Evolution of SaCas9 gRNAs Improves Gene Editing Efficiency.},
journal = {Nucleic acid therapeutics},
volume = {35},
number = {5},
pages = {209-219},
doi = {10.1177/21593337251370553},
pmid = {40952014},
issn = {2159-3345},
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Staphylococcus aureus/genetics/enzymology ; *Directed Molecular Evolution/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Ligands ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats-based editing is inefficient at over two-thirds of genetic targets. A primary cause is ribonucleic acid (RNA) misfolding that can occur between the spacer and scaffold regions of the gRNA, which hinders the formation of functional Cas9 ribonucleoprotein (RNP) complexes. Here, we uncover hundreds of highly efficient gRNA variant scaffolds for Staphylococcus aureus (Sa)Cas9 utilizing an innovative binding and ligand activation driven enrichment (BLADE) methodology, which leverages asymmetrical product dissociation over rounds of evolution. SaBLADE-derived gRNA scaffolds contain 7%-42% of nucleotide variation relative to wild type. gRNA variants are able to improve gene editing efficiency at all targets tested, and they achieve their highest levels of editing improvement (>400%) at the most challenging DNA target sites for the wild-type SaCas9 gRNA. This arsenal of SaBLADE-derived gRNA variants showcases the power and flexibility of combinatorial chemistry and directed evolution to enable efficient gene editing at challenging, or previously intractable, genomic sites.},
}

*Gene Editing/methods
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
Staphylococcus aureus/genetics/enzymology
*Directed Molecular Evolution/methods
*CRISPR-Associated Protein 9/genetics/metabolism
*CRISPR-Cas Systems/genetics
Ligands
Humans

@article {pmid40935921,
year = {2025},
author = {Roh, H and Shen, SP and Hu, Y and Kwok, HS and Siegenfeld, AP and Lee, C and Zepeda, MU and Guo, CJ and Roseman, SA and Comenho, C and Sankaran, VG and Buenrostro, JD and Liau, BB},
title = {Coupling CRISPR scanning with targeted chromatin accessibility profiling using a double-stranded DNA deaminase.},
journal = {Nature methods},
volume = {22},
number = {10},
pages = {2083-2093},
pmid = {40935921},
issn = {1548-7105},
support = {R35GM153476//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; F31HL174076//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; R01DK103794//U.S. Department of Health & Human Services | National Institutes of Health (NIH)/ ; DGE1745303//National Science Foundation (NSF)/ ; },
mesh = {Humans ; *Chromatin/genetics/metabolism ; Hematopoietic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Cytidine Deaminase/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Genome editing enables sequence-function profiling of endogenous cis-regulatory elements, driving understanding of their mechanisms. However, these approaches lack direct, scalable readouts of chromatin accessibility across long single-molecule chromatin fibers. Here we leverage double-stranded DNA cytidine deaminases to profile chromatin accessibility at endogenous loci of interest through targeted PCR and long-read sequencing, a method we term targeted deaminase-accessible chromatin sequencing (TDAC-seq). With high sequence coverage at targeted loci, TDAC-seq can be integrated with CRISPR perturbations to link genetic edits and their effects on chromatin accessibility on the same single chromatin fiber at single-nucleotide resolution. We employed TDAC-seq to parse CRISPR edits that activate fetal hemoglobin in human CD34[+] hematopoietic stem and progenitor cells (HSPCs) during erythroid differentiation as well as in pooled CRISPR and base-editing screens tiling an enhancer controlling the globin locus. We further scaled the method to interrogate 947 variants in a GFI1B-linked enhancer associated with myeloproliferative neoplasm risk in a single pooled CRISPR experiment in CD34[+] HSPCs. Together, TDAC-seq enables high-resolution sequence-function mapping of single-molecule chromatin fibers by genome editing.},
}

Humans
*Chromatin/genetics/metabolism
Hematopoietic Stem Cells/metabolism/cytology
*Gene Editing/methods
*CRISPR-Cas Systems
*Cytidine Deaminase/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40921108,
year = {2026},
author = {Zhang, Z and Wang, J and Li, C and Sun, H and Bu, S and Jia, Q and Wan, Y and Zhao, Y and Zhou, H and Hao, Z and Li, N and Yu, S and Wang, L and Wan, J},
title = {An ultrasensitive biosensor for H1N1 virus coupled with 3D spherical DNA nanostructure and CRISPR-Cas12a.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {346},
number = {},
pages = {126905},
doi = {10.1016/j.saa.2025.126905},
pmid = {40921108},
issn = {1873-3557},
mesh = {*Influenza A Virus, H1N1 Subtype/isolation & purification/genetics ; *Biosensing Techniques/methods ; *Nanostructures/chemistry ; *CRISPR-Cas Systems ; Limit of Detection ; *DNA/chemistry ; Animals ; *CRISPR-Associated Proteins/metabolism/chemistry ; Humans ; Aptamers, Nucleotide/chemistry ; Cattle ; Milk/virology ; Chickens ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {To achieve ultrasensitive and real-time detection of the H1N1 influenza virus, this study designed a nucleic acid-free fluorescent biosensor based on 3D spherical DNA nanostructure and CRISPR/Cas12a (3D-SDNC). The biosensor constructs a rigid 3D nano-framework via self-assembly of six oligonucleotide chains, with H1N1-specific nucleic acid aptamers and Cas12a activator strands strategically positioned at multi-spined vertices for precise spatial coupling between viral recognition and signal transduction. Upon aptamer-virus binding, the induced conformational change liberates the activator strand, thereby activating the trans-cleavage activity of the Cas12a/crRNA complex to efficiently cleave the HEX/BHQ1 double-labeled fluorescent probe and initiate cascade signal amplification. Experimental results demonstrate a detection limit of 0.17 copies/μL (S/N = 3), achieving qPCR-comparable sensitivity, with spike recovery rates of 91.89 % to 104.03 % (RSD < 5.12 %) in chicken serum, bovine serum, and milk matrices. The innovative nucleic acid-free extraction design reduces the total detection time to 40 min; its efficiency is three times higher than qPCR. Notably, we not only discovered the ultra-high sensitivity of the sensor to H1N1 but also unexpectedly found that the rigid structure of the 3D spherical DNA nanostructure conferred enhanced stability under storage conditions. This work establishes a groundbreaking molecular engineering paradigm for rapid pathogen diagnosis, combining ultrahigh sensitivity, fast response, and clinical utility.},
}

*Influenza A Virus, H1N1 Subtype/isolation & purification/genetics
*Biosensing Techniques/methods
*Nanostructures/chemistry
*CRISPR-Cas Systems
Limit of Detection
*DNA/chemistry
Animals
*CRISPR-Associated Proteins/metabolism/chemistry
Humans
Aptamers, Nucleotide/chemistry
Cattle
Milk/virology
Chickens
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid40886669,
year = {2026},
author = {Liu, W and Xu, L and Lyu, Y and Yang, C},
title = {MXene-integrated CRISPR/Cas12a biosensor with Split activators for direct and rapid fluorescent detection of MicroRNA.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {346},
number = {},
pages = {126850},
doi = {10.1016/j.saa.2025.126850},
pmid = {40886669},
issn = {1873-3557},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; Limit of Detection ; Spectrometry, Fluorescence/methods ; *Endodeoxyribonucleases/metabolism ; DNA, Single-Stranded/chemistry ; Bacterial Proteins ; Nitrites ; Transition Elements ; CRISPR-Associated Proteins ; },
abstract = {Early and accurate cancer diagnosis is essential for reducing cancer-related mortality, and miRNA-21 has emerged as a critical biomarker for the early detection of various malignancies In this study, we developed a novel fluorescence biosensor, termed the MXene-SNA-Cas12a, that enables direct and amplification-free detection of miRNA-21 by integrating the CRISPR/Cas12a system with a chimeric split nucleic acid (SNA) activator and MXene-assisted fluorescence modulation. Specifically, a split activator comprising S12 ssDNA hybridized with miRNA-21 was employed to activate the trans-cleavage activity of Cas12a, effectively overcoming the system's inherent limitation in RNA recognition. Simultaneously, MXene nanosheets served as efficient quenchers by adsorbing FAM-labeled ssDNA reporters through non-covalent interactions and facilitating target-induced strand release, enabling a robust fluorescence "on/off" mechanism. This biosensor demonstrated excellent linearity over a miRNA-21 concentration range of 50 pM-25 nM, with a detection limit as low as 16 pM. It exhibited high specificity and strong resistance to interference, making it well-suited for complex biological environments. Moreover, the programmable nature of the split activator allows for easy adaptation to detect other RNA targets through rational sequence redesign, offering a versatile platform for CRISPR/Cas12a-based RNA diagnostics.},
}

*MicroRNAs/analysis/genetics
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Humans
Limit of Detection
Spectrometry, Fluorescence/methods
*Endodeoxyribonucleases/metabolism
DNA, Single-Stranded/chemistry
Bacterial Proteins
Nitrites
Transition Elements
CRISPR-Associated Proteins

@article {pmid40851311,
year = {2025},
author = {Kittock, CM and Karia, K and Kc, P and Evans, C and Wollman, J and Meyerink, BL and Pilaz, LJ},
title = {Modeling MPPH syndrome in vivo using Breasi-CRISPR.},
journal = {HGG advances},
volume = {6},
number = {4},
pages = {100497},
pmid = {40851311},
issn = {2666-2477},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Gene Editing/methods ; Humans ; *Hydrocephalus/genetics/pathology ; *Megalencephaly/genetics/pathology ; *Polydactyly/genetics/pathology ; Neural Stem Cells/metabolism ; },
abstract = {The increasing availability and affordability of genetic testing has resulted in the identification of numerous novel variants associated with neurodevelopmental disorders. There remains a need for methods to analyze the functional impact of these variants. Some methods, like expressing these variants in cell culture, may be rapid, but they lack physiologic context. Other methods, like making a whole-mouse model, may provide physiologic accuracy, but these are costly and time-consuming. We recently developed a technique, Breasi-CRISPR (Brain Easi-CRISPR), which results in efficient genome editing of neural precursor cells via electroporation of CRISPR-Cas9 reagents into developing mouse brains. Since Breasi-CRISPR is extremely rapid and enables the analysis of targeted genes in vivo, we wondered whether this technique would accelerate the study of monogenic neurodevelopmental disorders. Here, we use Breasi-CRISPR to model megalencephaly postaxial polydactyly polymicrogyria hydrocephalus (MPPH) syndrome. We found that 2 days after Breasi-CRISPR, we were able to see neurodevelopmental phenotypes known to be associated with MPPH syndrome, including increased cyclin D2 protein abundance and an increase in neural progenitor proliferation. Thus, Breasi-CRISPR can efficiently model MPPH syndrome and may be a powerful method to add to the toolbox of those investigating the functional impact of patient variants in neurodevelopmental disorders.},
}

Animals
Mice
*CRISPR-Cas Systems
Disease Models, Animal
*Gene Editing/methods
Humans
*Hydrocephalus/genetics/pathology
*Megalencephaly/genetics/pathology
*Polydactyly/genetics/pathology
Neural Stem Cells/metabolism

@article {pmid40706707,
year = {2025},
author = {Davydova, S and Meccariello, A},
title = {Engineering new clustered regularly interspaced short palindromic repeats-mediated population control for tephritid pests.},
journal = {Current opinion in insect science},
volume = {72},
number = {},
pages = {101415},
doi = {10.1016/j.cois.2025.101415},
pmid = {40706707},
issn = {2214-5753},
mesh = {Animals ; *Tephritidae/genetics ; *Pest Control, Biological/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Male ; Population Control ; },
abstract = {Tephritid fruit flies threaten the agricultural industry with a rising intensity on a worldwide scale. The application of clustered regularly interspaced short palindromic repeats (CRISPR) in insects has resulted in a current boost of CRISPR studies in tephritid pests. One of the primary pathways toward more efficient population management lies in genetic improvements to the Sterile Insect Technique. Herein, we review the pivotal advances in CRISPR application in non-model tephritid fruit flies in recent years. This consists of proof-of-principle studies to optimise CRISPR tools, applications for female elimination and male sterility, and the existing CRISPR-based systems for population control.},
}

Animals
*Tephritidae/genetics
*Pest Control, Biological/methods
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats
Female
Male
Population Control

@article {pmid40658346,
year = {2025},
author = {Uchigashima, M and Mikuni, T},
title = {Single-cell endogenous protein labeling via CRISPR-Cas9-mediated genome editing in the mouse brain.},
journal = {Anatomical science international},
volume = {100},
number = {4},
pages = {579-590},
pmid = {40658346},
issn = {1447-073X},
support = {20H05914//Japan Society for the Promotion of Science/ ; 20H05918//Japan Society for the Promotion of Science/ ; 23K18160//Japan Society for the Promotion of Science/ ; 24K02130//Japan Society for the Promotion of Science/ ; 22K21353//Japan Society for the Promotion of Science/ ; 23H04672//Japan Society for the Promotion of Science/ ; 23H02574//Japan Society for the Promotion of Science/ ; 23K27265//Japan Society for the Promotion of Science/ ; 24H01229//Japan Society for the Promotion of Science/ ; 24K22000//Japan Society for the Promotion of Science/ ; 25H02490//Japan Society for the Promotion of Science/ ; JP19dm0207080//Japan Agency for Medical Research and Development/ ; JP24wm0625117//Japan Agency for Medical Research and Development/ ; JPMJFR231M//Fusion Oriented REsearch for disruptive Science and Technology/ ; JPMJPR16F9//Precursory Research for Embryonic Science and Technology/ ; CDA00043/2019-C//Human Frontier Science Program/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; *Brain/metabolism/cytology ; *Single-Cell Analysis/methods ; Electroporation ; Somatosensory Cortex/cytology/metabolism ; Pyramidal Cells/metabolism ; },
abstract = {High-precision mapping of endogenous proteins is essential for understanding the molecular mechanism underlying neuronal functions in the brain. The SLENDR (single-cell labeling of endogenous proteins by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair) technique provides single-cell endogenous protein labeling with genetically encoded tags within the mammalian brain through precise genome editing via homology-directed repair (HDR). This technique is based on the introduction of HDR-mediated genome editing into neuronal progenitors in embryonic brains by in utero electroporation. Subsequent histological analyses enable high-resolution interrogation of the subcellular distribution of endogenous proteins within a single neuron using conventional fluorescent microscopy. Here, we describe a step-by-step protocol for the SLENDR technique to label endogenous proteins with genetically encoded tags in single pyramidal cells of the mouse primary somatosensory cortex. This protocol would be helpful to visualize the molecular organization underlying biological processes at single-neuron levels in the brain, such as signal processing from synaptic inputs to neuronal outputs across different scales.},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Mice
*Brain/metabolism/cytology
*Single-Cell Analysis/methods
Electroporation
Somatosensory Cortex/cytology/metabolism
Pyramidal Cells/metabolism

@article {pmid41063348,
year = {2025},
author = {Newman, A and Saha, A and Starrs, L and Arantes, PR and Palermo, G and Burgio, G},
title = {CRISPR-Cas12a REC2-Nuc interactions drive target-strand cleavage and constrain trans cleavage.},
journal = {Nucleic acids research},
volume = {53},
number = {18},
pages = {},
doi = {10.1093/nar/gkaf988},
pmid = {41063348},
issn = {1362-4962},
support = {//National Health and Medical Research Council/ ; //The Gordon and Gretel Bootes/ ; //National Computing Infrastructure/ ; //NCRIS/ ; //Australian Government Research Training Program/ ; R01GM141329/NH/NIH HHS/United States ; CHE-2144823//National Science Foundation/ ; 2138259//National Science Foundation/ ; 2138286//National Science Foundation/ ; 2138307//National Science Foundation/ ; 2137603//National Science Foundation/ ; 2138296//National Science Foundation/ ; FG-2023-20431//Alfred P. Sloan Foundation/ ; TC-24-063//Camille and Henry Dreyfus Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA Cleavage ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Escherichia coli/genetics ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Gene Editing ; Molecular Dynamics Simulation ; DNA/metabolism/chemistry/genetics ; *Bacterial Proteins/genetics/metabolism/chemistry ; Kinetics ; DNA, Single-Stranded/metabolism/genetics ; Mutation ; },
abstract = {CRISPR-Cas12a mediates RNA-guided cleavage of double-stranded DNA in cis, after which it remains catalytically active and non-specifically cleaves single-stranded DNA in trans. Native host defence by Cas12a employs cis cleavage, which can be repurposed for the genome editing of other organisms, and trans cleavage can be used for in vitro DNA detection. Cas12a orthologues have high structural similarity and a conserved mechanism of DNA cleavage, yet highly different efficacies when applied for genome editing or DNA detection. By comparing three well-characterized Cas12a orthologues (FnCas12a, LbCas12a, and AsCas12a), we sought to determine what drives their different cis and trans cleavage and how this relates to their applied function. We integrated in vitro DNA cleavage kinetics with molecular dynamics simulations, plasmid interference in Escherichia coli, and genome editing in human cell lines. We report large differences in cis cleavage kinetics between orthologues, which may be driven by dynamic REC2-Nuc interactions. We generated and tested REC2 and Nuc mutants, including a hitherto unstudied 'Nuc-loop', integrity of which is critical for the function of Cas12. In total, our in vitro, in vivo, and in silico survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications.},
}

*CRISPR-Cas Systems
*DNA Cleavage
Humans
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
Escherichia coli/genetics
*Endodeoxyribonucleases/metabolism/genetics/chemistry
Gene Editing
Molecular Dynamics Simulation
DNA/metabolism/chemistry/genetics
*Bacterial Proteins/genetics/metabolism/chemistry
Kinetics
DNA, Single-Stranded/metabolism/genetics
Mutation

@article {pmid41062879,
year = {2025},
author = {Paul, K and Raman K, V and Baaniya, M and Jadhav, I and Bhattacharjee, S and Tilgam, J and Saakre, M and Kumari, P and Das, S and Vijayan, J and Sreevathsa, R and Pattanayak, D},
title = {A novel recombinant CRISPR/Cas9 vector system for genome editing in plants.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {45},
pmid = {41062879},
issn = {1573-9368},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genetic Vectors/genetics ; Solanum tuberosum/genetics ; *Plants, Genetically Modified/genetics/growth & development ; Genome, Plant ; Arabidopsis/genetics ; Nicotiana/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic ; },
abstract = {Genome editing employing CRISPR/Cas9 systems has found widespread applications for knocking out targeted genes. In spite of exponential applications in plants for trait improvement, low editing efficiency in plants is a major concern. We report construction of a pCAMBIA2300 based binary vector cassette (pCR) harbouring novel recombinant CRISPR/Cas9 system for efficient genome editing in plants. The Cas9 cDNA with sequence encoding nuclear localization signals at the N-terminal and C-terminal ends had been codon optimized for better expression in plants. Undesirable internal restriction sites were removed. Small stretch of 5' UTR sequence of Rubisco small subunit (rcbS) of potato, harbouring in between potato granule bound starch synthase (GBSS) intron, was added at the 5' end of the Cas9 cDNA to function as 5' UTR. The recombinant Cas9 gene (rdCas9) was placed under the transcriptional control of CaMV 35S promoter and NOS terminator. The single guide RNA cassette (sgRNA) was comprised of Arabidopsis U6 promoter, 20-21 nucleotide (nt) spacer sequence, sgRNA scaffold sequence and potato U6 RNA Pol-III termination sequence. The 20-21 nt sgRNA spacer sequence could be added to the sgRNA construct by AarI or PaqCI digestion. The sgRNA construct had been designed in such a way so that single or multiplexed sgRNA could be cloned into the pCR vector cassette in a single step. Moreover, modular nature of this vector system can help to derive different combination of promoter, terminator with Cas9 and sgRNA constructs. The efficacy of the pCR vector system had been validated in Nicotiana tabacum and Solanum tuberosum by knocking out phytoene desaturase gene (PDS), through Agrobacterium-mediated transformation. The pCR binary vector system can be utilized as a versatile tool box for efficient genome editing of plant to improve agriculturally important traits.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Genetic Vectors/genetics
Solanum tuberosum/genetics
*Plants, Genetically Modified/genetics/growth & development
Genome, Plant
Arabidopsis/genetics
Nicotiana/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Promoter Regions, Genetic

@article {pmid41062549,
year = {2025},
author = {Sofras, D and Carolus, H and Subotić, A and Lobo Romero, C and Ennis, CL and Cuypers, L and Lagrou, K and Hernday, AD and Nobile, CJ and Rybak, JM and Van Dijck, P},
title = {A systematic comparison of CRISPR-Cas9 allele editing in Candida auris demonstrates unreliable cassette integration and effective episomal plasmid-based editing.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {35105},
pmid = {41062549},
issn = {2045-2322},
support = {11J8122N//Fonds Wetenschappelijk Onderzoek/ ; 11D7620N//Fonds Wetenschappelijk Onderzoek/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; PDMT2/23/032//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; R35GM124594/GM/NIGMS NIH HHS/United States ; R35GM124594/GM/NIGMS NIH HHS/United States ; F31DE028488/DE/NIDCR NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Plasmids/genetics ; *Candida auris/genetics ; Alleles ; Genome, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Candidozyma (Candida) auris is an emergent fungal pathogen of significant interest for molecular research. A handful of CRISPR-Cas9 based allele editing tools have been optimized for C. auris. Nonetheless, allele editing in this species remains a significant challenge, and different systems have different advantages and disadvantages. In this work, we compare four systems to introduce the genetic elements necessary for the production of Cas9 and the guide RNA molecule in the genome of C. auris, replacing the ENO1, LEU2 and HIS1 loci respectively, while the fourth system makes use of an episomal plasmid. We observed that the editing efficiency of all four systems was significantly different and strain-dependent. However, we did not detect correct integration of linear CRISPR cassette constructs in integration-based systems, in over 4,900 screened transformants. Still, all transformants, whether correctly edited or not, grew on selective nourseothricin media, suggesting ectopic integration of the CRISPR cassette, which was confirmed by long-read whole genome sequencing. The plasmid-based system showed the highest editing efficiency with an average of 41.9% correct transformants, despite yielding fewer transformants compared to the other systems. Transformation of protoplasts or silencing the non-homologous end joining (NHEJ) DNA repair pathway, by deleting two main NHEJ factors, KU70 and LIG4, did not improve the editing efficiency. While our research highlights important challenges in precise genome editing of C. auris by quantitatively evaluating the editing and targeting efficiencies of different methods, it also clearly shows the safety and usefulness of plasmid-based systems like EPIC, which we recommend for molecular work in this enigmatic fungal pathogen.},
}

*Gene Editing/methods
*CRISPR-Cas Systems
*Plasmids/genetics
*Candida auris/genetics
Alleles
Genome, Fungal
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41062528,
year = {2025},
author = {Smidler, AL and Marrogi, EA and Scott, S and Mameli, E and Abernathy, D and Akbari, OS and Church, GM and Catteruccia, F and Esvelt, K},
title = {Engineering gene drive docking sites in a haplolethal locus in Anopheles gambiae.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {35074},
pmid = {41062528},
issn = {2045-2322},
support = {AI120480-02//Foundation for the National Institutes of Health/ ; R01 AI104956/AI/NIAID NIH HHS/United States ; R01 AI104956/AI/NIAID NIH HHS/United States ; R01AI151004//Foundation for the National Institutes of Health/ ; R01 AI104956/AI/NIAID NIH HHS/United States ; R00-DK102669-04//Foundation for the National Institutes of Health/ ; IRSA1016432//Burroughs Wellcome Fund/ ; IRSA1016432//Burroughs Wellcome Fund/ ; IRSA1016432//Burroughs Wellcome Fund/ ; OPP1158190/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*Anopheles/genetics ; Animals ; *Gene Drive Technology/methods ; Proteasome Endopeptidase Complex/genetics ; *Genetic Engineering/methods ; Mosquito Vectors/genetics ; *Genetic Loci ; CRISPR-Cas Systems ; },
abstract = {Gene drives are selfish genetic elements which promise to be powerful tools in the fight against vector-borne diseases such as malaria. We previously proposed population replacement gene drives designed to better withstand the evolution of resistance by homing through haplolethal loci. Because most mutations in the wild-type allele that would otherwise confer resistance are lethal, only successful drive homing and functional r1 alleles permits the cell to survive. Here we outline the development and characterization of two ΦC31-Recombination mediated cassette exchange gene drive docking lines with these features in Anopheles gambiae, a first step towards construction of robust gene drives in this important malaria vector. We outline adaption of the technique HACK (Homology Assisted CRISPR knockin) to knock-in two docking site sequences into a paired putative haplolethal-haplosufficient (Ribosome-Proteasome) locus, and confirm that these docking lines permit insertion of drive-relevant transgenes. We report the first anopheline proteasome knockouts, and identify ribosome mutants in the process reveal a major lethality and infertility hurdle that such designs must overcome to develop robust drives in the future. Although we do not achieve drive, this work provides a new tool for constructing future evolution-robust drive systems and reveals critical challenges that must be overcome for development of future gene drives designed to target haplolethal loci in anophelines and, potentially, other metazoans.},
}

*Anopheles/genetics
Animals
*Gene Drive Technology/methods
Proteasome Endopeptidase Complex/genetics
*Genetic Engineering/methods
Mosquito Vectors/genetics
*Genetic Loci
CRISPR-Cas Systems

@article {pmid41062469,
year = {2025},
author = {Han, J and Min, Y and Hu, L and Chen, JJ and Zhang, SX and Li, XF and Cheng, ZH and Liu, DF and Yu, HQ},
title = {Tailoring Cas12a functionality with a user-friendly and versatile crRNA variant toolbox.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8939},
pmid = {41062469},
issn = {2041-1723},
support = {52322002//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Mutation ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Escherichia coli/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Cas12a, with its unique targeting and cleavage activity towards DNA, has been widely applied in gene editing and molecular diagnostics. However, there currently lacks an activity regulation strategy that combines flexibility and simplicity to adapt Cas12a to different demands across various application scenarios. In this study, we present a simple yet effective strategy, wherein we systematically mutate the crRNA direct repeat (DR) sequence to uncover a range of distinct crRNA mutants, which are then compiled into a crRNA toolbox to enable flexible regulation of Cas12a activity. By harnessing the complementarity and synergy between these mutants, we successfully enhance Cas12a performance across various application scenarios. Our crRNA toolbox enables fine-tuned control over expression levels, improves base editing accuracy, enhances transformation and editing efficiency in prokaryote homologous recombination-mediated gene editing, and facilitates rapid, accurate, one-pot, semi-quantitative nucleic acid diagnostics. In summary, the DR sequence mutation strategy provides simple, flexible, and diverse options for Cas12a activity regulation and functional optimization.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/genetics/metabolism
Mutation
*Endodeoxyribonucleases/genetics/metabolism
*Bacterial Proteins/genetics/metabolism
Escherichia coli/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41061057,
year = {2025},
author = {Oonuma, K and Kuroda, R and Uchida, T and Zhang, SM},
title = {CRISPR/Cas9-germline editing of Biomphalaria glabrata: A breakthrough in genetic modification of snails that transmit schistosomiasis.},
journal = {Science advances},
volume = {11},
number = {41},
pages = {eadx5889},
pmid = {41061057},
issn = {2375-2548},
mesh = {Animals ; *Biomphalaria/genetics/parasitology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Schistosoma mansoni ; *Schistosomiasis/transmission/parasitology ; Humans ; },
abstract = {Human schistosomiasis remains one of the most devastating parasitic diseases worldwide, and the development of genetically modified vector snails has long been a goal in the field. Here, we report the successful creation of genetically modified Biomphalaria glabrata, an important intermediate host, using CRISPR/Cas9 gene editing. We targeted the fibrinogen-related protein 3.1 (FREP3.1) gene, confirmed stable germline transmission of the mutated gene, and established two different homozygous FREP3.1-edited lines. Disruption of the FREP 3.1 gene did not alter snail susceptibility to Schistosoma mansoni infection, possibly due to a limited role of FREP3.1 in resistance or to functional redundancy and/or compensatory expression within the highly diverse FREP gene family. Our study demonstrates successful germline editing, effective ex ovo culture of decapsulated embryos, and the generation of viable, genetically modified B. glabrata snails, thereby establishing a foundation for future genetic strategies to control schistosomiasis.},
}

Animals
*Biomphalaria/genetics/parasitology
*CRISPR-Cas Systems
*Gene Editing/methods
Schistosoma mansoni
*Schistosomiasis/transmission/parasitology
Humans

@article {pmid41060691,
year = {2025},
author = {Gao, F and Colles, FM and Ko, S and Luo, J and Sheppard, SK and Chen, M},
title = {Genomic epidemiology and the evolution of erm(B)-mediated macrolide resistance in Campylobacter.},
journal = {Microbial genomics},
volume = {11},
number = {10},
pages = {},
doi = {10.1099/mgen.0.001528},
pmid = {41060691},
issn = {2057-5858},
mesh = {*Campylobacter/genetics/drug effects/isolation & purification/classification ; *Macrolides/pharmacology ; Humans ; *Anti-Bacterial Agents/pharmacology ; Phylogeny ; Animals ; *Campylobacter Infections/epidemiology/microbiology ; *Drug Resistance, Bacterial/genetics ; China/epidemiology ; *Methyltransferases/genetics ; Genome, Bacterial ; Microbial Sensitivity Tests ; *Bacterial Proteins/genetics ; Gene Transfer, Horizontal ; Poultry/microbiology ; Evolution, Molecular ; },
abstract = {Campylobacter is a major foodborne bacterial pathogen that has become increasingly resistant to clinically important antimicrobials. Of particular concern is the emergence of erm(B)-mediated macrolide resistance, which has been increasingly documented across Campylobacter isolates from diverse ecological reservoirs. In this study, we investigated the genomic characteristics and epidemiology of erm(B)-carrying clinical Campylobacter isolates from Shanghai, alongside a globally representative dataset of all publicly available strains. Among clinical isolates obtained from a diarrhoeal outpatient surveillance programme between 2020 and 2023 in Shanghai, China, 16% (80/500) were erythromycin-resistant, with 23.8% (19/80) testing positive for erm(B). The genomes of these isolates were sequenced to identify erm(B) gene alleles. Phylogenetic analyses, pairwise comparisons of core and accessory genomes and examination of shared alleles revealed horizontal gene transfer as the predominant mechanism driving the transmission of erm(B) between isolates from various sources. Poultry was identified as a key reservoir for human infections caused by erm(B)-positive Campylobacter isolates. Comparative pangenome analyses of erm(B)-positive and negative isolates identified multiple accessory elements associated with erm(B) acquisition, among which the IS607 family transposon-associated tnpB gene exhibited sequence and structural homology to functional progenitors of CRISPR-Cas nucleases. These findings expand our understanding of the epidemiology of erm(B)-mediated macrolide resistance in Campylobacter and underscore the urgent need for enhanced antimicrobial stewardship in poultry production and targeted surveillance programmes to curb the spread of resistance.},
}

*Campylobacter/genetics/drug effects/isolation & purification/classification
*Macrolides/pharmacology
Humans
*Anti-Bacterial Agents/pharmacology
Phylogeny
Animals
*Campylobacter Infections/epidemiology/microbiology
*Drug Resistance, Bacterial/genetics
China/epidemiology
*Methyltransferases/genetics
Genome, Bacterial
Microbial Sensitivity Tests
*Bacterial Proteins/genetics
Gene Transfer, Horizontal
Poultry/microbiology
Evolution, Molecular

@article {pmid41042795,
year = {2025},
author = {Shen, Y and Fan, K and Gökbağ, B and Sun, N and Yang, C and Cheng, L and Li, L},
title = {A multi-layer encoder prediction model for individual sample specific gene combination effect (MLEC-iGeneCombo).},
journal = {PLoS computational biology},
volume = {21},
number = {10},
pages = {e1013547},
doi = {10.1371/journal.pcbi.1013547},
pmid = {41042795},
issn = {1553-7358},
mesh = {Humans ; Gene Knockout Techniques ; CRISPR-Cas Systems/genetics ; Computational Biology/methods ; Deep Learning ; *Models, Genetic ; Systems Biology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Using data from gene combination double knockout (CDKO) experiments, top ranked synthetic lethal (SL) gene pairs were highly inconsistent among different SL scores. This leads to a significant concern that SL prediction models highly depend on SL scores. In this paper, we introduce a new gene combination effect (GCE) measurement, log-fold change of dual-gRNA expression before and after CRISPR-cas9 lentivirus transfection. We show it is a direct and highly consistent measurement of GCE in all CDKO experiments. We therefore develop a multi-layer encoder model for individual sample specific GCE prediction, MLEC-iGeneCombo. Under a deep learning framework, MLEC-iGeneCombo is a systems biology model that contains sample specific multi-omics encoder, network encoder and cell-line encoder. For the first time, MLEC-iGeneCombo predicts GCE for a new cell. Using data from 18 CDKO experiments, MLEC-iGeneCombo achieves an average GCE prediction performance, 71.9%. All three encoders significantly improve the model's prediction performance (p[Formula: see text]), and their combined use yields the best GCE prediction performance. Our source code is available at https://github.com/karenyun/MLEC-iGeneCombo.},
}

Humans
Gene Knockout Techniques
CRISPR-Cas Systems/genetics
Computational Biology/methods
Deep Learning
*Models, Genetic
Systems Biology/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid40930101,
year = {2025},
author = {Vučićević, D and Hsu, CW and Lopez Zepeda, LS and Burkert, M and Hirsekorn, A and Bilić, I and Kastelić, N and Landthaler, M and Lacadie, SA and Ohler, U},
title = {Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation.},
journal = {Cell genomics},
volume = {5},
number = {10},
pages = {100984},
doi = {10.1016/j.xgen.2025.100984},
pmid = {40930101},
issn = {2666-979X},
mesh = {Humans ; *Single-Cell Analysis/methods ; Transcription Factors/genetics/metabolism ; Neuroblastoma/genetics ; Homeodomain Proteins/genetics ; Gene Regulatory Networks/genetics ; Enhancer Elements, Genetic/genetics ; CRISPR-Cas Systems/genetics ; *Genomics/methods ; Cell Line, Tumor ; },
abstract = {Enhancers are known to spatiotemporally regulate gene transcription, yet the identification of enhancers and their target genes is often indirect, low resolution, and/or assumptive. To identify and functionally perturb enhancers at their endogenous sites, we performed a pooled tiling CRISPR activation (CRISPRa) screen surrounding PHOX2B, a master regulator of neuronal cell fate and a key player in neuroblastoma, and found many CRISPRa-responsive elements (CaREs) that alter cellular growth. To determine CaRE target genes, we developed TESLA-seq (targeted single-cell activation), which combines CRISPRa screening with targeted single-cell RNA sequencing and enables the parallel readout of the effect of hundreds of enhancers on all genes in the locus. While most TESLA-revealed CaRE-gene relationships involved neuroblastoma-related regulatory elements, we found many CaREs and target connections normally active only in other tissues. This highlights the power of TESLA-seq to reveal gene regulatory networks, including edges active outside of a given experimental system.},
}

Humans
*Single-Cell Analysis/methods
Transcription Factors/genetics/metabolism
Neuroblastoma/genetics
Homeodomain Proteins/genetics
Gene Regulatory Networks/genetics
Enhancer Elements, Genetic/genetics
CRISPR-Cas Systems/genetics
*Genomics/methods
Cell Line, Tumor

@article {pmid40888415,
year = {2025},
author = {Zhao, T and Yu, L and Yin, M and Huang, S and Tian, R and Zhong, C and Nan, F and Zhang, H and Tian, X and Hu, Z},
title = {Enhanced One-Pot Cas12a-Based Nucleic Acid Detection via Epitope Insertion and Recruitment of Rad51.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {40},
pages = {e02417},
doi = {10.1002/smll.202502417},
pmid = {40888415},
issn = {1613-6829},
support = {32171465//National Natural Science Foundation of China/ ; 32371541//National Natural Science Foundation of China/ ; 82102392//National Natural Science Foundation of China/ ; 82172584//National Natural Science Foundation of China/ ; 2023M734091//China Postdoctoral Science Foundation/ ; 2023M734090//China Postdoctoral Science Foundation/ ; 2023M744121//China Postdoctoral Science Foundation/ ; 2024BCB057//Key Technology R&D Program of Hubei/ ; 0820250//Guangdong Special Support Plan Young Top-notch Talent/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Epitopes/metabolism ; *Rad51 Recombinase/metabolism ; Humans ; *Nucleic Acids/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The CRISPR-Cas12a system has emerged as a promising tool for nucleic acid-based diagnostics. However, its multi-step workflow and limited sensitivity hinder its integration into point-of-care testing (POCT). Here, the ECOT system (Engineered Cas12a for One-pot Test), a novel approach that combines protein engineering with one-pot detection, offering high sensitivity, specificity, and rapid response is introduced. By introducing GCN4 epitope insertions into LtCas12a and LbCas12a variants, their cis-cleavage activity, promoting efficient accumulation of amplification products is reduced. Additionally, the inclusion of scFv-Rad51 (single-chain variable fragment-Rad51) enhances Cas12a's trans-cleavage activity, amplifying signal intensity. The ECOT-Lb system demonstrated superior sensitivity in detecting low-copy HPV DNA samples, outperforming traditional qPCR in clinical tests. Achieving detection limits as low as 3 copies in under 30 min, the ECOT-Lb system is well-suited for home-based self-testing and widespread clinical diagnostics. This work provides a versatile and scalable protein engineering strategy that enhances the performance of CRISPR-based diagnostic tools, offering a promising platform for rapid molecular detection in diverse applications.},
}

*CRISPR-Cas Systems/genetics
*Epitopes/metabolism
*Rad51 Recombinase/metabolism
Humans
*Nucleic Acids/analysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40811280,
year = {2025},
author = {Chen, H and Jiang, Z},
title = {CrisprDA: A Data Augmentation Method Enhancing the Efficiency of sgRNA on-Target Activity Prediction.},
journal = {IEEE transactions on computational biology and bioinformatics},
volume = {22},
number = {5},
pages = {2313-2319},
doi = {10.1109/TCBBIO.2025.3591871},
pmid = {40811280},
issn = {2998-4165},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Computational Biology/methods ; *Deep Learning ; Neural Networks, Computer ; },
abstract = {The CRISPR/Cas9 system has emerged as a revolutionary technology in genome editing, yet the efficiency of this system is often limited by the activity level of single-guide RNAs (sgRNAs). In recent years, deep learning models have been increasingly utilized to predict sgRNA targeting activity. Notably, data scarcity rather than model architecture has become the predominant bottleneck in accurately predicting sgRNA activity. To overcome this challenge and enhance the performance of deep learning models, we propose Automix, a straightforward yet effective data augmentation method grounded in autoencoder technology. This method is complemented by CNLC (Confidence-based Nearest Label Correction), a pseudo-label correction technique designed to improve both the quality and quantity of training data. Additionally, we develop CrisprDA, a novel parallel architecture that integrates convolutional neural networks (CNNs) with attention mechanisms, for the precise prediction of sgRNA activity. Comprehensive experiments conducted on nine high-throughput datasets and eight functional datasets demonstrate that CrisprDA outperforms five compared methods, showing its superior predictive ability. Moreover, the application of Automix and CNLC to other comparative methods in our experiments further validates the effectiveness and generalizability of the proposed data augmentation strategy.},
}

*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Humans
*Computational Biology/methods
*Deep Learning
Neural Networks, Computer

@article {pmid41058711,
year = {2025},
author = {Barraclough, A and Bär, I and van Duijl, T and Fijnvandraat, K and Eikenboom, JCJ and Leebeek, FWG and Bierings, R and Voorberg, J and Trasanidou, D},
title = {Rewriting the script: gene therapy and genome editing for von Willebrand Disease.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1620438},
pmid = {41058711},
issn = {2673-3439},
abstract = {In recent years gene therapy has emerged as a powerful technology for treatment of a large variety of inherited disorders. With the FDA approval of in vivo gene therapy of hemophilia A and B using AAV-mediated transgene delivery to hepatocytes, the path towards a new treatment era seemed paved. Also, CRISPR-Cas based approaches have reached the clinic, as in the ex vivo treatment of hematopoietic stem cells for sickle cell disease and thalassemia patients. The question arises whether these innovative strategies will also be suitable for patients with von Willebrand Disease (VWD). Whilst in and ex vivo delivery to endothelial cells (ECs) has been demonstrated, and CRISPR-Cas9 gene editing has been successful in ECs, there are currently no gene therapy options available for VWD. The wide variety of pathogenic VWF mutations makes development of broadly applicable, cost-effective gene therapies challenging. While delivery of von Willebrand factor (VWF) as a therapeutic transgene would be optimal, the size of VWF challenges efficient delivery. Therefore, treatment of VWD requires targeted, personalized gene therapy; for instance by using the newest CRISPR-Cas technologies which can be tailored to facilitate alteration and restoration of various pathogenic VWD variants. This review describes the inherited bleeding disorder VWD and potential gene therapy approaches for management of the disease. Thereby we are exploring different CRISPR-Cas technologies and recent developments in the field. Moreover, we will discuss the ongoing advances of in vivo delivery systems, all with the scope on ECs.},
}

@article {pmid41057395,
year = {2025},
author = {Osgood, JA and Brown, AC and Burnham, KL and Mielczarek, O and Migliorini, G and Tay, C and Zhang, P and Palmer, MH and Davies, B and Cowley, SA and Knight, JC},
title = {Evidence for enhancer activity in intron 1 of TNFRSF1A using CRISPR/Cas9 in human induced pluripotent stem cell-derived macrophages.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {34885},
pmid = {41057395},
issn = {2045-2322},
support = {20773/VAC_/Versus Arthritis/United Kingdom ; 204969/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; 2018-I2M-2-002//Chinese Academy of Medical Sciences Innovation 537 Fund for Medical Science/ ; LC0910-004//Oxford Martin School, University of Oxford/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Macrophages/metabolism/cytology ; *Introns/genetics ; *Enhancer Elements, Genetic ; *Receptors, Tumor Necrosis Factor, Type I/genetics ; Gene Editing ; Cell Differentiation ; Genome-Wide Association Study ; Gene Expression Regulation ; },
abstract = {TNFα is a common drug target in the treatment of autoimmune diseases, with pro-inflammatory functions that are primarily mediated through its receptor, TNFRSF1A. TNFRSF1A has been genetically associated with many immune-mediated diseases including ankylosing spondylitis, multiple sclerosis, and inflammatory bowel disease. Many of the genetic variants within or near TNFRSF1A that have been associated with disease through genome-wide association studies (GWAS) lie in non-coding regions of the genome. Understanding the functional consequences of these genetic variants is limited by incomplete understanding of TNFRSF1A gene regulation, including for specific cellular contexts relevant to inflammation and immunity such as macrophages. This work used CRISPR/Cas9 in human induced pluripotent stem cells followed by differentiation into macrophages to investigate putative regulatory elements in the TNFRSF1A gene locus. Through gene editing, with functional genomic readouts including the assay for transposase-accessible chromatin using sequencing (ATAC-Seq), chromatin immunoprecipitation with sequencing (ChIP-Seq), and RNA-Seq to assess the consequences of these edits, we present evidence for an enhancer of TNFRSF1A contained within an intron of the gene. Understanding gene regulation and the genomic context in which GWAS variants lie could bring us closer to deconvoluting the genetic basis of common disease aetiology and uncover effective drug targets.},
}

Humans
*CRISPR-Cas Systems
*Induced Pluripotent Stem Cells/cytology/metabolism
*Macrophages/metabolism/cytology
*Introns/genetics
*Enhancer Elements, Genetic
*Receptors, Tumor Necrosis Factor, Type I/genetics
Gene Editing
Cell Differentiation
Genome-Wide Association Study
Gene Expression Regulation

@article {pmid41057263,
year = {2025},
author = {Yang, B and Wang, K},
title = {CRISPR-Cas-Directed Genome Editing in Maize.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.top108448},
pmid = {41057263},
issn = {1559-6095},
abstract = {Genetic engineering techniques are essential for both plant science and agricultural biotechnology, enabling functional genomics studies, dissection of complex traits, and targeted crop improvement. Among the various genetic tools currently in use, clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based genome editing has emerged as a transformative technology due to its precision, versatility, and ease of use. In particular, CRISPR-Cas9 has become the most widely adopted platform for genome manipulation in plant systems, including maize, owing to its high editing efficiency, multiplexing capabilities, and scalability for diverse applications. This review highlights the biological significance and technical considerations necessary to implement CRISPR-Cas9 in maize. We discuss critical components for successful editing, including the selection of strong and tissue-appropriate promoters for Cas gene and guide RNA expression, codon optimization of Cas nuclease genes, effective guide RNA design, and multiplexing strategies using RNA polymerase III (Pol III)- or Pol II-dependent promoter-driven polycistronic expression systems. Additionally, we provide insights into vector construction methodologies and reliable genotyping techniques to detect and validate genome edits. Together, these elements constitute a practical framework for deploying genome editing in maize research and breeding. By optimizing these parameters, researchers can enhance the efficiency and accuracy of CRISPR-mediated genome modifications, accelerating functional genomic discovery and the development of improved maize varieties tailored to meet future agricultural demands.},
}

@article {pmid41056201,
year = {2025},
author = {Weber, VJ and Reschigna, A and Gerhardt, MJ and Heigl, T and Hinrichsmeyer, KS and van den Engel, S and Otify, DY and Gavrilov, Z and Blaser, F and Meneau, I and Betz, C and Bolz, HJ and Biel, M and Michalakis, S and Becirovic, E},
title = {CRISPR/Cas-mediated activation of genes associated with inherited retinal dystrophies in human cells for diagnostic purposes.},
journal = {JCI insight},
volume = {},
number = {},
pages = {},
doi = {10.1172/jci.insight.189615},
pmid = {41056201},
issn = {2379-3708},
abstract = {Many patients suffering from inherited diseases do not receive a genetic diagnosis and are therefore excluded as candidates for treatments, such as gene therapies. Analyzing disease-related gene transcripts from patient cells would improve detection of mutations that have been missed or misinterpreted in terms of pathogenicity during routine genome sequencing. However, the analysis of transcripts is complicated by the fact that a biopsy of the affected tissue is often not appropriate, and many disease-associated genes are not expressed in tissues or cells that can be easily obtained from patients. Here, using CRISPR/Cas-mediated transcriptional activation (CRISPRa) we developed a robust and efficient approach to activate genes in skin-derived fibroblasts and in freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals. This approach was successfully applied to blood samples from patients with inherited retinal dystrophies (IRD). We were able to efficiently activate several IRD-linked genes and detect the corresponding transcripts using different diagnostically relevant methods such as RT-qPCR, RT-PCR and long- and short-read RNA sequencing. The detection and analysis of known and unknown mRNA isoforms demonstrates the potential of CRISPRa-mediated transcriptional activation in PBMCs. These results will contribute to ceasing the critical gap in the genetic diagnosis of IRD patients and other inherited diseases.},
}

@article {pmid41055650,
year = {2025},
author = {Che, X and Wei, Y and Wang, X and Wang, X and Wu, Z and Deng, J and Ge, S and Liu, X and Cai, Z and Zhang, H and He, L and Xu, J},
title = {Lipoxygenase ZmLOX3 Enhances Salt Tolerance of Maize Under the Regulation of ZmNAC032.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c02349},
pmid = {41055650},
issn = {1520-5118},
abstract = {Lipoxygenase (LOX) plays a critical role in plant biotic and abiotic stress responses by mediating lipid peroxidation and the production of jasmonic acid (JA). In this study, maize ZmLOX3 was identified as a positive regulator in salt stress tolerance. Overexpression of ZmLOX3 enhanced the salt tolerance of Arabidopsis. When maize seedlings were subjected to salt stress, the ZmLOX3[OE] lines exhibited a better growth phenotype than the control (B104) and the zmlox3[CR] (CRISPR/Cas) knockout mutants. Overexpression of ZmLOX3 improved ROS scavenging, Na[+]/K[+] homeostasis, and cell membrane stability. Transcriptome analyses revealed that ZmLOX3[OE] triggered the expression of genes involved in both the JA synthesis and signaling pathways. A transcription factor ZmNAC032 was identified via Y1H screening and was able to bind to the C[A/G]CG[T/G] sequence in the ZmLOX3 promoter and activate its expression. These findings are helpful for deciphering the function and regulatory status of ZmLOX in improving salt tolerance.},
}

@article {pmid41054568,
year = {2025},
author = {Xia, B and Wang, Z and Fei, T and Ma, Y and Guo, Y and Fei, D and Shu, X and Zhao, G and Ma, M and Yuan, H},
title = {Development and application of a CRISPR/Cas12a-based reverse transcription-recombinase polymerase amplification assay with lateral flow dipstick and fluorescence detection for Getah virus.},
journal = {PeerJ},
volume = {13},
number = {},
pages = {e20119},
pmid = {41054568},
issn = {2167-8359},
mesh = {*CRISPR-Cas Systems ; *Alphavirus/isolation & purification/genetics ; Animals ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Fluorescence ; *Alphavirus Infections/diagnosis/virology/veterinary ; Swine ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Getah virus (GETV), a mosquito-borne alphavirus classified as a zoonotic disease, primarily infects livestock, particularly pigs and horses. In recent years, it has re-emerged in multiple Asian countries, posing a potential threat to animal husbandry and public health. In this study, we developed a rapid and sensitive GETV detection method based on reverse transcription-recombinase polymerase amplification (RT-RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system combined with a lateral flow dipstick (LFD) for visual readout. By leveraging sequence conservation in the GETV E2 envelope protein-coding regions, we engineered matched crRNA guides and amplification primers to develop a rapid CRISPR-Cas12a diagnostic workflow. The optimized platform combines RT-RPA (42 °C/20 min) with Cas12a's trans-nuclease activity, permitting multiplex detection via real-time fluorescence quantification or immunochromatographic strip visualization. Analytical evaluation demonstrated a detection capability of 10 copies/µL and exclusive specificity against four pathogen controls, including Japanese encephalitis virus and pseudorabies virus. Validation performed using simulated clinical samples revealed 100% concordance between the results of RT-RPA-CRISPR/Cas12a-LFD and quantitative polymerase chain reaction (PCR), while reducing the total detection time to 50 minutes. This approach eliminated the need for advanced instrumentation owing to its simplified operational design, enabling field-deployable rapid detection capabilities that establish essential technical infrastructure for initiating timely GETV containment measures. This approach has broad application potential in the fields of food safety, clinical diagnostics, and environmental science.},
}

*CRISPR-Cas Systems
*Alphavirus/isolation & purification/genetics
Animals
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Fluorescence
*Alphavirus Infections/diagnosis/virology/veterinary
Swine
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41026092,
year = {2025},
author = {Toyonishi, G and Nakazawa, T and Koshi, D and Horii, M and An, GH and Kawauchi, M and Honda, Y},
title = {CRISPR/Cas9-directed disruption of wc-2 leads to the absence of fruiting body development in Pleurotus ostreatus.},
journal = {FEMS microbiology letters},
volume = {372},
number = {},
pages = {},
doi = {10.1093/femsle/fnaf104},
pmid = {41026092},
issn = {1574-6968},
support = {18KK0178//Japan Society for the Promotion of Science/ ; 22H00380//Japan Society for the Promotion of Science/ ; 22KK0090//Japan Society for the Promotion of Science/ ; PJ0175072025//Rural Development Administration/ ; },
mesh = {*Pleurotus/genetics/growth & development/radiation effects ; *Fruiting Bodies, Fungal/growth & development/genetics/radiation effects ; *CRISPR-Cas Systems ; *Fungal Proteins/genetics/metabolism ; Light ; },
abstract = {Light, particularly blue light, is a key environmental factor that induces fruiting in certain agaricomycetes. In this study, we characterized mutant strains of Pleurotus ostreatus with disrupted wc-2, which encodes one of the white-collar proteins, Wc-2, to investigate the role of light in fruiting in P. ostreatus. We introduced two different plasmids containing expression cassettes for Cas9 and two different gRNAs targeting wc-2 separately into the dikaryotic P. ostreatus strain PC9×#64. Among the 11 dikaryotic hygromycin-resistant transformants, six strains did not form fruiting bodies. Genomic PCR followed by sequencing analysis suggested that all six fruitless strains were dikaryotic wc-2 disruptants. Small aggregate structures were not observed in the dikaryotic wc-2 disruptants grown under light conditions, as in PC9×#64 grown in a red box. These results suggest that Wc-2 is essential for the initiation of blue light-induced fruiting in P. ostreatus.},
}

*Pleurotus/genetics/growth & development/radiation effects
*Fruiting Bodies, Fungal/growth & development/genetics/radiation effects
*CRISPR-Cas Systems
*Fungal Proteins/genetics/metabolism
Light

@article {pmid41002250,
year = {2025},
author = {Gillmore, JD and Gane, E and Täubel, J and Pilebro, B and Echaniz-Laguna, A and Kao, J and Litchy, W and Shahda, S and Haagensen, A and Walsh, L and Smith, D and Kachadourian, J and Ward, JH and Lebwohl, D and Zhu, P and Xu, Y and Leung, A and Sonderfan, A and Gutstein, DE and Manvelian, G and Adams, D},
title = {Nexiguran Ziclumeran Gene Editing in Hereditary ATTR with Polyneuropathy.},
journal = {The New England journal of medicine},
volume = {393},
number = {14},
pages = {1375-1386},
doi = {10.1056/NEJMoa2510209},
pmid = {41002250},
issn = {1533-4406},
mesh = {Humans ; *Amyloid Neuropathies, Familial/therapy/genetics ; Male ; Female ; Middle Aged ; *Prealbumin/genetics/metabolism/antagonists & inhibitors ; Adult ; Aged ; *Gene Editing ; *Genetic Therapy/adverse effects/methods ; CRISPR-Cas Systems ; Neurofilament Proteins/blood ; Body Mass Index ; },
abstract = {BACKGROUND: Hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PN) is a rare, multisystem, progressive, debilitating, and fatal disease characterized by tissue deposition of misfolded transthyretin (TTR) in peripheral nerves. Nexiguran ziclumeran (nex-z) is an investigational in vivo therapy based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease) that is designed to reduce serum TTR levels through selective inactivation of TTR in the liver.

METHODS: In this phase 1, open-label study, we administered one infusion of nex-z to patients with ATTRv-PN. Primary objectives included assessment of the safety and pharmacodynamics of nex-z. Secondary end points included changes in the familial amyloid polyneuropathy stage, polyneuropathy disability score, serum neurofilament light chain (NfL) level, modified body-mass index (modified BMI, defined as the conventional BMI [weight in kilograms divided by square of height in meters] multiplied by the albumin level in grams per liter), and modified Neuropathy Impairment Score+7 (mNIS+7; range, 0 to 304, with higher scores indicating more impairment).

RESULTS: A total of 36 patients received nex-z; the mean follow-up was 27 months. The mean percent change from baseline in the serum TTR level was -90% at day 28, which was sustained through month 24 (-92%). Treatment-related adverse events included transient infusion-related reactions (in 21 patients), decreased thyroxine level without hypothyroidism or elevated thyrotropin level (in 8), and headache (in 4). One participant died from cardiac amyloidosis, and one withdrew owing to progressive decline in motor function. Serious adverse events were reported in 11 patients. At month 24, the familial amyloid polyneuropathy stage and polyneuropathy disability score remained stable in 29 and 27 patients, respectively; improved in 2 and 5, respectively; and worsened in 2 and 2, respectively. The mean change in the serum NfL level was -9.0 pg per milliliter, and the change in the modified BMI was 24.7. The mean change from baseline in the mNIS+7 was -8.5.

CONCLUSIONS: A single administration of nex-z in patients with ATTRv-PN was associated with rapid, deep, and durable reductions in serum TTR levels. The results support further investigation of nex-z to treat ATTRv-PN. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).},
}

Humans
*Amyloid Neuropathies, Familial/therapy/genetics
Male
Female
Middle Aged
*Prealbumin/genetics/metabolism/antagonists & inhibitors
Adult
Aged
*Gene Editing
*Genetic Therapy/adverse effects/methods
CRISPR-Cas Systems
Neurofilament Proteins/blood
Body Mass Index

@article {pmid40990297,
year = {2025},
author = {Sheng, J and Dong, Y and Sun, S and Zhang, Y and Li, C and Xu, X and Wang, H},
title = {Construction of a Sensing Platform Integrated with a CRISPR/Cas12a-Triggered Colorimetric Strategy for the Quantitative Detection of Meat Freshness.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {40},
pages = {25604-25614},
doi = {10.1021/acs.jafc.5c04851},
pmid = {40990297},
issn = {1520-5118},
mesh = {*Colorimetry/methods/instrumentation ; *Meat/analysis/microbiology ; CRISPR-Cas Systems ; Animals ; *Pseudomonas/genetics/isolation & purification/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Biosensing Techniques/instrumentation/methods ; *Endodeoxyribonucleases/genetics/metabolism ; CRISPR-Associated Proteins ; },
abstract = {Monitoring microbial determinants, such as Pseudomonas spp., is thus essential for assessing meat freshness. Here, a novel colorimetric sensing platform based on magnetic enzyme-labeled nanoparticles combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a without nucleic acid molecule preamplification was developed for detecting meat freshness. Under optimal conditions, a high-specificity crRNA was systematically verified, and the colorimetric sensor could accurately quantify Pseudomonas spp. loads with levels ranging from 1 × 10[3.7] to 1 × 10[8.7] CFU/mL, with a color change from colorless to yellow. A smart colorimetric platform, including a self-designed image acquisition device and self-programmed image analysis software, was developed and applied to the integrated determination of meat freshness by using the B-value in the RGB channel. The platform has been applied to both consumers and producers and has been validated by 48 actual samples of chilled meat. These findings provide new insights into the exploration of reliable tools for monitoring meat freshness.},
}

*Colorimetry/methods/instrumentation
*Meat/analysis/microbiology
CRISPR-Cas Systems
Animals
*Pseudomonas/genetics/isolation & purification/metabolism
*Bacterial Proteins/genetics/metabolism
*Biosensing Techniques/instrumentation/methods
*Endodeoxyribonucleases/genetics/metabolism
CRISPR-Associated Proteins

@article {pmid40985907,
year = {2025},
author = {Fei, S and Zhang, C and Zhang, X and Xie, Y and Fu, S and Wu, J},
title = {An Ultrasensitive Immunocapture (IC)-RPA-CRISPR/Cas12a Assay with Three Readout Modes for Detecting Xanthomonas oryzae pv. oryzicola of Rice Bacterial Leaf Streak.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {40},
pages = {25664-25675},
doi = {10.1021/acs.jafc.5c04360},
pmid = {40985907},
issn = {1520-5118},
mesh = {*Oryza/microbiology ; *Xanthomonas/genetics/isolation & purification ; *Plant Diseases/microbiology ; CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; Plant Leaves/microbiology ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Xanthomonas oryzae pv oryzicola (Xoc) is the causal agent of rice bacterial leaf streak (BLS) and causes enormous losses of rice yields in many countries every year. Development of sensitive diagnostic techniques is crucial for its prevention and control. Here, we developed an ultrasensitive IC-RPA-CRISPR/Cas12a assay with three readout modes [qPCR machine, UV lamp, and lateral flow strip (LFS)] for Xoc detection in rice, which combined advantages of immunocapture, recombinase polymerase amplification (RPA), and CRISPR/Cas12a-based cleavage. Especially, the immunocapture step allows to capture and enrich Xoc from samples, which minimizes the interference from rice debris to benefit nucleic acid release and amplification and enhances the specificity and sensitivity of this assay. The detection limits of its three readout modes for Xoc bacterial suspension is 2, 6, and 60 CFU/mL, respectively. Collectively, this study provides a specific, ultrasensitive, practical approach for quarantine and detection of Xoc that will benefit the prevention and control of BLS.},
}

*Oryza/microbiology
*Xanthomonas/genetics/isolation & purification
*Plant Diseases/microbiology
CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods/instrumentation
Plant Leaves/microbiology
Bacterial Proteins/genetics/metabolism

@article {pmid40970715,
year = {2025},
author = {Zhao, C and Li, G and Shen, C and Xie, Y and Chen, Y and Ying, X and Chen, Y and Zhang, C},
title = {An extraction-free and one-pot two-temperature CRISPR/Cas12b system for visual detection of Group B Streptococcus by integrating with RPA.},
journal = {Journal of clinical microbiology},
volume = {63},
number = {10},
pages = {e0081925},
doi = {10.1128/jcm.00819-25},
pmid = {40970715},
issn = {1098-660X},
support = {2024KY1444//Medical and Health Research Project of Zhejiang Province/ ; LTGC23H200004//Zhejiang Provincial Natural Science Foundation of China/ ; },
mesh = {*Streptococcus agalactiae/isolation & purification/genetics ; Humans ; *Streptococcal Infections/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Female ; Recombinases/metabolism ; Temperature ; Vagina/microbiology ; },
abstract = {UNLABELLED: Group B Streptococcus (GBS) is a major cause of neonatal infections, and rapid detection is essential for timely clinical intervention. In this study, we developed an extraction-free, one-pot CRISPR/Cas12b assay for visual detection of GBS by combining with isothermal amplification, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). The results showed that LAMP-CRISPR/Cas12b outperformed RPA-CRISPR/Cas12b system across all template concentrations, especially in low-copy template (30 and 10 copies/test) detection. To enhance the detection performance of RPA-CRISPR/Cas12b, we introduced a two-temperature protocol, with RPA reaction at 39°C followed by Cas12b activation at 62°C. Through the two-temperature approach, the detection rate of RPA-CRISPR/Cas12b system was significantly improved even in low-copy samples, achieving a sensitivity of 10 copies/test (1 copy/μL). Clinical validation using 60 vaginal-rectal swab samples showed 96.7% and 98.3% of concordance when compared to culture and qPCR methods, respectively. This assay offers a rapid (<1 h), highly sensitive, and user-friendly solution without requiring nucleic acid extraction or sophisticated instruments. Its compatibility with visual signal detection makes it ideal for point-of-care testing, especially in low-resource or time-sensitive settings. The platform can be adapted for broader pathogen detection in future field diagnostics.

IMPORTANCE: This study presents a rapid, convenient, and highly accurate method for Group B Streptococcus (GBS) detection by integrating the CRISPR/Cas12b system with recombinase polymerase amplification, an isothermal nucleic acid amplification technique. To streamline the workflow, we established a one-pot, extraction-free assay that significantly reduces the detection time. Through the systematic optimization of the dual-temperature conditions, we enhanced the amplification efficiency of target DNA, thereby improving the sensitivity of the CRISPR/Cas12b system. Additionally, the incorporation of a UV-visible detection system enables visual readout, facilitating instrument-free testing suitable for point-of-care (POC) applications.},
}

*Streptococcus agalactiae/isolation & purification/genetics
Humans
*Streptococcal Infections/diagnosis/microbiology
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Female
Recombinases/metabolism
Temperature
Vagina/microbiology

@article {pmid40899880,
year = {2025},
author = {Liu, P and Zhang, J and Gong, Y and Liu, W and Xiao, G and Liang, J and Wang, X and Bi, J and Zhang, G},
title = {Application of engineered CRISPR/Cas12a variants with altered protospacer adjacent motif specificities for the detection of isoniazid resistance mutations in Mycobacterium tuberculosis.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0016525},
pmid = {40899880},
issn = {2165-0497},
mesh = {*Isoniazid/pharmacology ; *Mycobacterium tuberculosis/genetics/drug effects/isolation & purification ; *CRISPR-Cas Systems/genetics ; *Antitubercular Agents/pharmacology ; Bacterial Proteins/genetics ; Humans ; *Drug Resistance, Bacterial/genetics ; Mutation ; Tuberculosis, Multidrug-Resistant/microbiology/diagnosis ; *Endodeoxyribonucleases/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Catalase/genetics ; Sensitivity and Specificity ; CRISPR-Associated Proteins/genetics ; Microbial Sensitivity Tests ; },
abstract = {UNLABELLED: Drug-resistant tuberculosis (TB) is a major global public health concern. Although isoniazid is currently considered one of the most effective first-line drugs for TB treatment, its efficacy is limited by the emergence of resistance. Therefore, it is imperative to develop new methods for detecting drug-resistant TB. In this study, we developed a nucleic acid detection system based on the clustered regularly interspaced short palindromic repeat (CRISPR) Cas12a_RR protein. The system combines recombinase polymerase amplification with an engineered CRISPR/Cas12a_RR protein to enable rapid and specific detection of the katG G944C mutation in isoniazid-resistant Mycobacterium tuberculosis (Mtb). It could detect the target DNA at concentrations as low as 1% in a mixed sample. Compared with TaqMan quantitative polymerase chain reaction and DNA sequencing, the CRISPR/Cas12a_RR system demonstrated superior detection performance in terms of sensitivity, specificity, and cost-effectiveness. Furthermore, it effectively differentiated between drug-resistant Mtb strains from wild-type Mtb strains in clinically isolated samples, with the entire detection process completed in 60 min. In conclusion, the CRISPR/Cas12a_RR detection system offers a novel, rapid, simple, sensitive, and specific approach for identifying isoniazid-resistant Mtb, with significant potential for clinical application, particularly in resource-limited settings.

IMPORTANCE: This study presents a novel method for detecting isoniazid-resistant Mycobacterium tuberculosis (Mtb) using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a mutants, offering rapid detection, cost-effectiveness, and high specificity, and thereby providing a promising new avenue for detecting isoniazid-resistant Mtb.},
}

*Isoniazid/pharmacology
*Mycobacterium tuberculosis/genetics/drug effects/isolation & purification
*CRISPR-Cas Systems/genetics
*Antitubercular Agents/pharmacology
Bacterial Proteins/genetics
Humans
*Drug Resistance, Bacterial/genetics
Mutation
Tuberculosis, Multidrug-Resistant/microbiology/diagnosis
*Endodeoxyribonucleases/genetics
Clustered Regularly Interspaced Short Palindromic Repeats
Catalase/genetics
Sensitivity and Specificity
CRISPR-Associated Proteins/genetics
Microbial Sensitivity Tests

@article {pmid40866699,
year = {2025},
author = {Pacesa, M and Nickel, L and Schellhaas, C and Schmidt, J and Pyatova, E and Kissling, L and Barendse, P and Choudhury, J and Kapoor, S and Alcaraz-Serna, A and Cho, Y and Ghamary, KH and Vinué, L and Yachnin, BJ and Wollacott, AM and Buckley, S and Westphal, AH and Lindhoud, S and Georgeon, S and Goverde, CA and Hatzopoulos, GN and Gönczy, P and Muller, YD and Schwank, G and Swarts, DC and Vecchio, AJ and Schneider, BL and Ovchinnikov, S and Correia, BE},
title = {One-shot design of functional protein binders with BindCraft.},
journal = {Nature},
volume = {646},
number = {8084},
pages = {483-492},
pmid = {40866699},
issn = {1476-4687},
support = {R35 GM138368/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; Gene Editing ; Protein Binding ; CRISPR-Cas Systems/genetics ; Allergens/immunology/metabolism/chemistry ; Immunoglobulin E/immunology/metabolism ; *Protein Engineering/methods ; *Proteins/metabolism/chemistry ; Receptors, Cell Surface/metabolism/chemistry ; Binding Sites ; Models, Molecular ; CRISPR-Associated Protein 9/metabolism ; Animals ; },
abstract = {Protein-protein interactions are at the core of all key biological processes. However, the complexity of the structural features that determine protein-protein interactions makes their design challenging. Here we present BindCraft, an open-source and automated pipeline for de novo protein binder design with experimental success rates of 10-100%. BindCraft leverages the weights of AlphaFold2 (ref. [1]) to generate binders with nanomolar affinity without the need for high-throughput screening or experimental optimization, even in the absence of known binding sites. We successfully designed binders against a diverse set of challenging targets, including cell-surface receptors, common allergens, de novo designed proteins and multi-domain nucleases, such as CRISPR-Cas9. We showcase the functional and therapeutic potential of designed binders by reducing IgE binding to birch allergen in patient-derived samples, modulating Cas9 gene editing activity and reducing the cytotoxicity of a foodborne bacterial enterotoxin. Last, we use cell-surface-receptor-specific binders to redirect adeno-associated virus capsids for targeted gene delivery. This work represents a significant advancement towards a 'one design-one binder' approach in computational design, with immense potential in therapeutics, diagnostics and biotechnology.},
}

Humans
Gene Editing
Protein Binding
CRISPR-Cas Systems/genetics
Allergens/immunology/metabolism/chemistry
Immunoglobulin E/immunology/metabolism
*Protein Engineering/methods
*Proteins/metabolism/chemistry
Receptors, Cell Surface/metabolism/chemistry
Binding Sites
Models, Molecular
CRISPR-Associated Protein 9/metabolism
Animals

@article {pmid40862592,
year = {2025},
author = {Shen, Y and Yi, C and Wang, H and Tang, Y and Li, J},
title = {Development of a rapid and sensitive RPA-CRISPR/Cas12a-based assay for the detection of Brucella melitensis.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0099825},
pmid = {40862592},
issn = {2165-0497},
mesh = {*Brucellosis/diagnosis/microbiology ; *Brucella melitensis/genetics/isolation & purification ; Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases/genetics/metabolism ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; Reproducibility of Results ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Brucellosis, a zoonotic disease caused by Brucella species, presents significant public health challenges due to its complex diagnosis and the limited availability of rapid detection methods. To address these challenges, we developed a novel detection method that integrates recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system, enabling dual readout through fluorescence (FL) and lateral flow strip (LFS) detection. The RPA-CRISPR/Cas12a-FL assay demonstrated an impressive detection limit of 1 copy/μL, which is 10 times more sensitive than quantitative polymerase chain reaction, while the RPA-CRISPR/Cas12a-LFS method achieved a detection limit of 10 copies/μL, comparable to nested PCR. Specificity testing confirmed the robustness of the assay, as it produced strong signals exclusively for Brucella without cross-reactivity with other bacterial species. Clinical validation using serum samples from 24 confirmed brucellosis patients and six healthy controls demonstrated a 100% concordance with serological results, underscoring the reliability of this method for clinical applications. This assay provides a rapid, sensitive, and specific tool for Brucella detection, suitable for both laboratory and field settings, and holds significant potential for enhancing the diagnosis and control of brucellosis.IMPORTANCEBrucellosis is a significant zoonotic disease, and rapid and accurate diagnosis is crucial for its treatment and control. To address this need, we developed a novel detection method that combines recombinant enzyme polymerase amplification with a CRISPR/Cas12a system, achieving dual readout through fluorescence and lateral flow strips. The test demonstrates excellent sensitivity and specificity, with clinical validation confirming complete concordance with serological results. This approach offers a fast, reliable, and field-deployable solution for brucellosis diagnosis, significantly enhancing disease management and public health outcomes.},
}

*Brucellosis/diagnosis/microbiology
*Brucella melitensis/genetics/isolation & purification
Humans
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Recombinases/genetics/metabolism
*Molecular Diagnostic Techniques/methods
Limit of Detection
Reproducibility of Results
Bacterial Proteins/genetics
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid40856920,
year = {2025},
author = {Thiel, J and Sürün, D and Brändle, DC and Teichert, M and Künzel, SR and Friedrich, U and Dahl, A and Schubert, K and Rzagalinski, I and Shevchenko, A and Traikov, S and Mirtschink, P and Wagenführ, L and Buchholz, F and Hölig, K and Tonn, T and Kronstein-Wiedemann, R},
title = {Knock Out of miRNA-30a-5p and Reconstitution of the Actin Network Dynamics Partly Restores the Impaired Terminal Erythroid Differentiation during Blood Pharming.},
journal = {Stem cell reviews and reports},
volume = {21},
number = {8},
pages = {2637-2653},
pmid = {40856920},
issn = {2629-3277},
support = {530364326//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Cell Differentiation/genetics ; *Actins/metabolism ; *Erythroid Cells/metabolism/cytology ; *Erythropoiesis/genetics ; Proto-Oncogene Mas ; Hematopoietic Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Erythrocytes/metabolism/cytology ; bcl-X Protein/metabolism/genetics ; Gene Knockout Techniques ; Cell Line ; },
abstract = {In vitro red blood cell (RBC) production offers a promising complement to conventional blood donation, particularly for patients with rare blood types. Previously, we developed imBMEP-A, the first erythroid cell line derived from reticulocyte progenitors, which maintains robust hemoglobin expression and erythroid differentiation in the presence of erythropoietin (EPO) despite its immortalized state. However, clinical translation remains hindered by the inability to scale up production due to impaired in vitro enucleation of RBC progenitor cell lines. Enhancing enucleation efficiency in imBMEP-A cells involved CRISPR/Cas9-mediated knockout (K.O.) of miR-30a-5p, a key enucleation inhibitor, moderately increasing rates to 3.3 ± 0.4%- 8.9 ± 1.7%. Further investigation of enucleation inefficiencies led to transcriptome and proteome comparisons between imBMEP-miR30a-K.O. cells and hematopoietic stem cells (HSCs). These analyses revealed altered gene expression and protein abundances linked to metabolic transitions, apoptosis promotion, and cytoskeletal regulation. Notably, forced expression of the proto-oncogene c-Myc, required for cell immortalization, emerged as a key driver of these physiological changes. Counteracting these effects required optimization of imBMEP-A cells by activating BCL-XL transcription and knocking out SCIN, which encodes the actin-severing protein scinderin. While BCL-XL is upregulated in normal erythropoiesis, it is downregulated in imBMEP-A. Conversely, SCIN, typically absent in erythroid cells, is highly expressed in imBMEP-A, disrupting actin organization. These interventions improved viability, restored actin network formation, and increased terminal erythropoiesis, yielding 22.1 ± 1.7% more orthochromatic erythroblasts. These findings establish a foundation for optimizing imBMEP-A cells for therapeutic use and advancing the understanding the pathophysiology of erythroleukemia.},
}

*MicroRNAs/genetics/metabolism
Humans
*Cell Differentiation/genetics
*Actins/metabolism
*Erythroid Cells/metabolism/cytology
*Erythropoiesis/genetics
Proto-Oncogene Mas
Hematopoietic Stem Cells/metabolism/cytology
CRISPR-Cas Systems
Erythrocytes/metabolism/cytology
bcl-X Protein/metabolism/genetics
Gene Knockout Techniques
Cell Line