@article {pmid41372121,
year = {2025},
author = {Cheng, KW and Bhave, M and Markhard, AL and Peng, D and Bhatt, KD and Travisano, KA and Medicielo, JV and Anaya, A and Lembirik, S and Njoya, L and Anantpadma, M and Kuhn, JH and Puschnik, AS and Kistler, AL},
title = {Replicon-based genome-wide CRISPR knockout screening for the identification of host factors involved in viral replication.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11028},
pmid = {41372121},
issn = {2041-1723},
mesh = {*Virus Replication/genetics ; Humans ; *Replicon/genetics ; *Dengue Virus/genetics/physiology ; Chikungunya virus/genetics/physiology ; *CRISPR-Cas Systems ; Gene Knockout Techniques/methods ; Ebolavirus/genetics/physiology ; Cell Line ; Animals ; *Host-Pathogen Interactions/genetics ; Membrane Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; HEK293 Cells ; Hexosyltransferases/genetics/metabolism ; },
abstract = {We describe a viral replicon-based CRISPR knockout (KO) screening approach to specifically identify host factors essential for viral replication which are often missed in live virus screens. We benchmark the replicon screening using a stable fluorescent dengue virus type 2 (DENV-2) replicon cell line and successfully identify host genes known to be required for viral DENV-2 replication (e.g., endoplasmic reticulum membrane complex and oligosaccharyltransferase complex components), along with additional genes that have not been reported in prior CRISPR KO screens with DENV-2. We extend this replicon screening approach to chikungunya virus (CHIKV), a positive-sense RNA virus, and Ebola virus (EBOV), a negative-sense RNA virus, and identify distinct sets of genes required for replication of each virus. Our findings indicate that viral replicon-based CRISPR screens are a useful approach to identify host factors essential for replication of diverse viruses and to elucidate potential novel targets for host-directed medical countermeasures.},
}

*Virus Replication/genetics
Humans
*Replicon/genetics
*Dengue Virus/genetics/physiology
Chikungunya virus/genetics/physiology
*CRISPR-Cas Systems
Gene Knockout Techniques/methods
Ebolavirus/genetics/physiology
Cell Line
Animals
*Host-Pathogen Interactions/genetics
Membrane Proteins/genetics/metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
HEK293 Cells
Hexosyltransferases/genetics/metabolism

@article {pmid41371329,
year = {2025},
author = {Chen, B and Gao, J and Sun, H and Zhao, Y and Liu, Y and Qiu, X and Li, Y},
title = {Integrating CRISPR with SERS: Toward intelligent point-of-care diagnostics of the future.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {581},
number = {},
pages = {120782},
doi = {10.1016/j.cca.2025.120782},
pmid = {41371329},
issn = {1873-3492},
abstract = {In recent years, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas) system has emerged as a transformative genome-editing platform. Beyond its editing applications, the CRISPR/Cas system has attracted growing interest in molecular diagnostics particularly for nucleic acid detection due to its exceptional sensitivity and target specificity. Meanwhile, surface-enhanced Raman spectroscopy (SERS), which relies on plasmonic nanoparticles or nanostructures, has become a powerful biosensing technology known for its high sensitivity and distinct spectral fingerprinting capability. The integration of CRISPR/Cas-mediated molecular recognition with the ultrasensitive detection of SERS offers a rapid, low-volume, and direct strategy for identifying diverse nucleic acid targets. This synergistic combination has inspired the development of innovative biosensing platforms designed for ultrasensitive and precise molecular diagnostics. In this review, we first outline the fundamental principles of CRISPR/Cas and SERS, then summarize their hybrid applications in nucleic acid detection. Finally, we discuss the current progress, challenges, and future perspectives of CRISPR/Cas-integrated SERS biosensing.},
}

@article {pmid41371153,
year = {2025},
author = {Longhi Cervantes, DS and Leal, GM and Fortirer, JDS and de Oliveira, LF and Navarro, BV and Buckeridge, MS},
title = {microRNAs and stress adaptation in grasses: A systematic review.},
journal = {Plant physiology and biochemistry : PPB},
volume = {230},
number = {},
pages = {110783},
doi = {10.1016/j.plaphy.2025.110783},
pmid = {41371153},
issn = {1873-2690},
abstract = {MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression after transcription, playing crucial roles in plant development and stress adaptation. In grasses, this regulation is vital under isolated biotic and abiotic stress conditions and combined stress scenarios, although many regulatory modules remain unexplored. This systematic review examined 60 studies out of 1823 publications indexed in Scopus, focusing on grass miRNAs with validated targets through Degradome-Seq and/or RACE approaches. Results indicate that miRNA-target modules were validated more often under abiotic stress than biotic or combined stress conditions. The most frequently studied miRNA families include miR156, miR159, miR164, miR169, and miR396, which are commonly linked to various types of stress, whether isolated or combined. Most research has concentrated on major crops such as rice and maize, with limited studies on other agriculturally important grasses. This review highlights advances in miRNA-phytohormone interactions, systemic signaling, and target validation strategies. It also underscores the potential of biotechnological tools such as RNAi, artificial miRNAs, target mimicry, and CRISPR/Cas for engineering more resilient grasses. Integrating multi-omics approaches and an increasing focus on combined stress responses suggest promising strategies for sustainable agriculture, food security, and bioenergy production amidst climate challenges. Together, these advances strengthen the potential of microRNA-based regulation as a key tool for enhancing crop resilience and adaptation.},
}

@article {pmid41370200,
year = {2025},
author = {Shi, X and Hu, C and Jia, L and Lei, Z and Guo, B and Zhou, J and Wang, F},
title = {An SpC editor targeting pre-mRNA splicing for precise CRISPR control and enhanced antitumor efficacy.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
doi = {10.1093/nar/gkaf1328},
pmid = {41370200},
issn = {1362-4962},
support = {32271512//National Natural Science Foundation of China/ ; 82572281//National Natural Science Foundation of China/ ; 2022JC-56//Natural Science Basic Research Program of Shaanxi/ ; 2023-JC-ZD-43//Natural Science Basic Research Program of Shaanxi/ ; 2024JC-YBQN-0168//Natural Science Basic Research Program of Shaanxi/ ; 2023A1515110886//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *RNA Precursors/genetics/metabolism ; *RNA Splicing/drug effects ; Animals ; Mice ; Cell Line, Tumor ; Spliceosomes/genetics/metabolism ; Macrolides/pharmacology ; Apoptosis/genetics ; Neoplasms/genetics/therapy ; RNA, Guide, CRISPR-Cas Systems/genetics ; Epoxy Compounds ; },
abstract = {The CRISPR/Cas9 system is a powerful genome editing tool that has the potential to be applied to a variety of biomedical applications. Despite the considerable potential of this gene editing technology, there are numerous safety concerns including the possibility of unpredictable off-target effects. The splicing process, which involves the removal of introns from pre-mRNA and the alignment of exons to produce mature transcripts, is a critical step in gene expression in most eukaryotes. In this study, we present a spliceosome-responsive CRISPR/Cas9 (SpC) editor that utilizes the splicing inhibitor pladienolide B (PB) to regulate pre-mRNA splicing and control the expression of the anti-CRISPR protein AcrIIA4, thereby modulating the activity of the Cas9 nuclease. This approach allows for precise regulation of the gene editing process, thereby effectively mitigating off-target effects. The reliability and robustness of the SpC editor were demonstrated through in vitro and in vivo bioluminescence imaging. Furthermore, a dual-target sgRNA was designed to target the diphtheria toxin A gene, resulting in apoptosis induction and growth inhibition of tumor cells across various types of cancer cells. Our results indicate that this SpC editor has the capacity to precisely regulate tumor cell growth, thus providing new insights and significant implications for cancer gene therapy.},
}

Humans
*CRISPR-Cas Systems
*Gene Editing/methods
*RNA Precursors/genetics/metabolism
*RNA Splicing/drug effects
Animals
Mice
Cell Line, Tumor
Spliceosomes/genetics/metabolism
Macrolides/pharmacology
Apoptosis/genetics
Neoplasms/genetics/therapy
RNA, Guide, CRISPR-Cas Systems/genetics
Epoxy Compounds

@article {pmid41369371,
year = {2025},
author = {Gardner-Kay, A and Le, L and Filla, M and Kibiryeva, N and O'Brien, JE and Bittel, DC},
title = {CRISPR Disruption of scaRNA1 Reduces Pseudouridylation in Spliceosomal RNA U2 at U89 and Perturbs the Transcriptome in HEK293T Cells.},
journal = {Cells},
volume = {14},
number = {23},
pages = {},
pmid = {41369371},
issn = {2073-4409},
support = {Intramural Faculty grant//Kansas City University/ ; },
mesh = {Humans ; HEK293 Cells ; *Spliceosomes/metabolism/genetics ; *Transcriptome/genetics ; *Pseudouridine/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Small Nuclear/metabolism/genetics ; RNA Splicing/genetics ; },
abstract = {Small Cajal body-associated RNAs (scaRNAs) are essential for biochemical modification of spliceosomal RNAs and spliceosome function. Changes in scaRNA expression level have been associated with developmental issues, including cancer and congenital heart defects (CHDs), although the mechanism remains unclear. Small Cajal body-associated RNA 1 (scaRNA1) guides pseudouridylation at uridine 89 (Ψ89) of the spliceosomal RNA U2, a highly conserved modification that may be critical for spliceosome function. To investigate the role of scaRNA1 in splicing regulation, CRISPR-Cas9 genome editing was used to introduce targeted deletions in the scaRNA1 locus in HEK293T cells. Edited clones were identified by T7 endonuclease I assay and confirmed by Sanger sequencing. Pseudouridylation at Ψ89 was quantified using CMC-based reverse transcription followed by quantitative PCR, and global mRNA splicing alterations were assessed by RNA sequencing. Clones harboring scaRNA1 disruptions exhibited a significant reduction in Ψ89 pseudouridylation, consistent with impaired scaRNA1 function. Transcriptome analysis (of mRNA from two clones) revealed >300 protein coding genes with significant changes in transcript isoform level, including >100 genes related to RNA-binding activity. These results indicate that scaRNA1 disruption alters spliceosomal function and leads to substantial changes in mRNA splicing. The dysregulated splicing of RNA-binding proteins may impair RNA processing and gene expression programs required for normal development, providing new insight into how noncoding RNA dysfunction may contribute to developmental pathogenesis.},
}

Humans
HEK293 Cells
*Spliceosomes/metabolism/genetics
*Transcriptome/genetics
*Pseudouridine/metabolism/genetics
*CRISPR-Cas Systems/genetics
*RNA, Small Nuclear/metabolism/genetics
RNA Splicing/genetics

@article {pmid41369349,
year = {2025},
author = {Cheng, X and Wang, D and Zhang, X and Li, L and Liu, Y and Cao, G and Zhang, Y},
title = {Regulation of the Homeostasis of Early Embryo Development in Dairy Cows by Targeted Editing of the PRLR Gene-Mediated Activation of the Anti-Heat Stress Pathway.},
journal = {Cells},
volume = {14},
number = {23},
pages = {},
pmid = {41369349},
issn = {2073-4409},
support = {No. 2023ZD04050//Guifang Cao/ ; },
mesh = {Animals ; Cattle ; *Gene Editing/methods ; *Heat-Shock Response/genetics ; *Homeostasis/genetics ; *Embryonic Development/genetics ; *Receptors, Prolactin/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Female ; Reactive Oxygen Species/metabolism ; Fibroblasts/metabolism ; Nuclear Transfer Techniques ; Oocytes/metabolism ; },
abstract = {The intensification of global climate warming exacerbates the issue of heat stress in dairy cows, making the SLICK mutation in the prolactin receptor (PRLR) gene a critical target for enhancing heat tolerance in these animals. This study aims to investigate the effects of CRISPR/Cas9-mediated editing of the PRLR gene on the biological characteristics of bovine fibroblasts and early embryonic development following somatic cell nuclear transfer (SCNT). Using the CRISPR/Cas9 system, we targeted and edited a 20 bp-150 bp region within exon nine of the PRLR gene. After conducting off-target predictions and activity screenings, we identified optimal guide RNA (sgRNA) sequences and established stable transgenic cell lines. Transcriptome sequencing was performed on edited cells to identify key genes and validate their expression profiles. Edited cells were utilized as donor cells for SCNT, during which we assessed oocyte levels of reactive oxygen species (ROS), glutathione (GSH), and mitochondrial function to analyze embryonic developmental performance. We constructed a cellular stress resistance network aimed at mitigating damage transmission while maintaining embryonic developmental homeostasis. This research provides technical support and theoretical reference for genetic editing breeding programs aimed at improving heat tolerance in dairy cattle.},
}

Animals
Cattle
*Gene Editing/methods
*Heat-Shock Response/genetics
*Homeostasis/genetics
*Embryonic Development/genetics
*Receptors, Prolactin/genetics/metabolism
CRISPR-Cas Systems/genetics
Female
Reactive Oxygen Species/metabolism
Fibroblasts/metabolism
Nuclear Transfer Techniques
Oocytes/metabolism

@article {pmid41367214,
year = {2025},
author = {Selokar, NL and Singh, P and Jose, B and Gautam, D and Patel, K and Verma, R and De, S and Singh, MK and Singh, D},
title = {A Myostatin (MSTN[-/-]) Knockout Buffalo Produced by CRISPR-Cas9 Mediated Genome Editing and Somatic Cell Nuclear Transfer.},
journal = {The CRISPR journal},
volume = {8},
number = {6},
pages = {436-442},
doi = {10.1177/25731599251401528},
pmid = {41367214},
issn = {2573-1602},
mesh = {Animals ; *Myostatin/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Buffaloes/genetics ; *Nuclear Transfer Techniques ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Fibroblasts/metabolism ; Female ; Animals, Genetically Modified/genetics ; },
abstract = {CRISPR-Cas9 genome editing offers significant opportunities to improve livestock traits; however, its application in buffalo has been very limited, with no prior reports of live gene-edited animals. Here, we report the successful birth of a buffalo edited in the myostatin (MSTN) gene. To achieve this, five single-guide RNAs (sgRNAs) targeting the buffalo MSTN gene were designed and tested in skin-derived fibroblasts. Among these, sgRNA5 exhibited the highest editing efficiency, approaching ∼50%, as confirmed by T7 Endonuclease I assay, Tracking of Indels by Decomposition, and Inference of CRISPR Edits analyses. Single-cell cloning identified six edited fibroblast clonal populations, including one with a bi-allelic frameshift mutation predicted to severely truncate the MSTN protein. These bi-allelic clonal cells were subsequently used as nuclear donors to produce somatic cell nuclear transfer (SCNT) embryos, which were transferred into recipient buffaloes (n = 15). This effort established three pregnancies and resulted in the birth of one live MSTN knockout buffalo calf. Phenotypically, the calf displayed accelerated growth and increased muscle fiber number and size while maintaining normal meat composition. In conclusion, this study reports the world's first gene-edited buffalo generated through CRISPR-Cas9-mediated genome editing combined with SCNT. These findings provide a proof-of-concept for genome editing in buffalo and demonstrate that MSTN disruption can effectively enhance muscle growth and meat production traits.},
}

Animals
*Myostatin/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Buffaloes/genetics
*Nuclear Transfer Techniques
*Gene Knockout Techniques/methods
RNA, Guide, CRISPR-Cas Systems/genetics
Fibroblasts/metabolism
Female
Animals, Genetically Modified/genetics

@article {pmid41367203,
year = {2025},
author = {Patel, J and Patel, D and Raval, A},
title = {Artificial Intelligence for Predictive Modeling in CRISPR/Cas9 Gene Editing: a Survey of Methods and Design Strategies.},
journal = {The journal of gene medicine},
volume = {27},
number = {12},
pages = {e70061},
doi = {10.1002/jgm.70061},
pmid = {41367203},
issn = {1521-2254},
mesh = {*CRISPR-Cas Systems ; *Artificial Intelligence ; *Gene Editing/methods ; Humans ; Machine Learning ; Algorithms ; },
abstract = {Ongoing developments in genome editing most notably the continued evolution of CRISPR-Cas systems and their orthogonal or modified counterparts have substantively altered both experimental and applied practices in biomedicine, agriculture, and therapeutic design. More recently, the systematic incorporation of artificial intelligence and machine learning methodologies has augmented the specificity, throughput, and explanatory capacity of genome-editing workflows, thereby refining the prediction of on-target efficiencies, the appraisal of off-target liabilities, and the tailoring of molecular therapeutic configurations. The present contribution offers an integrative survey of these computational developments, emphasizing (i) predictive algorithms, (ii) machine-learning and deep-learning frameworks, (iii) data-centric procedural strategies, and (iv) dedicated applications in oncology, neurology, rare-disease research, and precision-medicine contexts. Throughout, we evaluate architectural choices, sequence-encoding representations, and lingering dataset-related biases, while additionally addressing current constraints concerning model interpretability, ethical viability, and the procedural prerequisites for clinical translation. Moreover, we advance a structured taxonomy that organizes AI-mediated genome-editing approaches according to methodological lineage and functional scope, and we delineate extant research lacunae. By combining these elements, we supply a prospective assessment of the means by which artificial intelligence might be further leveraged to support secure, efficacious, and equitably accessible genome engineering outcomes.},
}

*CRISPR-Cas Systems
*Artificial Intelligence
*Gene Editing/methods
Humans
Machine Learning
Algorithms

@article {pmid41366257,
year = {2025},
author = {Launspach, M and Macos, J and Afzal, S and Hohmann, J and Appis, ML and Pilgram, M and Beez, S and Ohlendorf, E and van der Ven, CFT and Lachiheb, C and Töws, K and Andersch, L and Jens, M and Zirngibl, F and Kath, J and Stecklum, M and Rodriguez-Fos, E and Anders, K and Wagner, DL and Henssen, AG and Kühn, R and Eggert, A and Künkele, A},
title = {Personalized CRISPR knock-in cytokine gene therapy to remodel the tumor microenvironment and enhance CAR T cell therapy in solid tumors.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10987},
pmid = {41366257},
issn = {2041-1723},
mesh = {*Tumor Microenvironment/genetics/immunology ; Humans ; Animals ; Gene Knock-In Techniques ; Mice ; *CRISPR-Cas Systems ; *Immunotherapy, Adoptive/methods ; *Genetic Therapy/methods ; Chemokine CXCL10/genetics ; *Cytokines/genetics ; Cell Line, Tumor ; *Neuroblastoma/therapy/immunology/genetics ; Receptors, Chimeric Antigen/genetics ; T-Lymphocytes/immunology ; Chemokine CXCL11/genetics ; *Neoplasms/therapy/immunology/genetics ; Interferon-gamma/genetics ; Precision Medicine ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; },
abstract = {The immunosuppressive tumour microenvironment (TME) remains a central barrier to effective immunotherapy in solid tumours. We present a gene-therapeutic strategy that enables localized remodelling of the TME via tumour-intrinsic cytokine expression. Central to this approach is CancerPAM, a multi-omics bioinformatics pipeline that identifies and ranks patient-specific, tumour-exclusive CRISPR-Cas9 knock-in sites with high specificity and integration efficiency. Using neuroblastoma as a model, CancerPAM analysis of tumour sequencing data identifies optimal knock-in sites for pro-inflammatory cytokines (CXCL10, CXCL11, IFNG), and CancerPAM rankings correlate strongly with target-site specificity and knock-in efficiency, validating its predictive performance. CRISPR-mediated CXCL10 knock-in enhances CAR T cell infiltration and antitumour efficacy in vitro and in vivo, including humanized CD34[+] HuNOG mice, where CXCL10-expressing tumours show stronger immune infiltration and prolonged tumour control within a reconstituted human immune microenvironment. Our findings establish a framework for safe and effective CRISPR-based cytokine delivery, integrating localized TME remodelling with cellular immunotherapies to enhance CAR T cells and other treatments in immune-refractory solid tumours.},
}

*Tumor Microenvironment/genetics/immunology
Humans
Animals
Gene Knock-In Techniques
Mice
*CRISPR-Cas Systems
*Immunotherapy, Adoptive/methods
*Genetic Therapy/methods
Chemokine CXCL10/genetics
*Cytokines/genetics
Cell Line, Tumor
*Neuroblastoma/therapy/immunology/genetics
Receptors, Chimeric Antigen/genetics
T-Lymphocytes/immunology
Chemokine CXCL11/genetics
*Neoplasms/therapy/immunology/genetics
Interferon-gamma/genetics
Precision Medicine
Clustered Regularly Interspaced Short Palindromic Repeats
Female

@article {pmid41366133,
year = {2025},
author = {Bian, W and Mcquarrie, DWJ and Haussmann, IU and Arnold, R and Soller, M},
title = {Genetic evaluation of CRISPR-Cas9 off-target effects from deleterious mutations on Drosophila male single X chromosome.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {270},
pmid = {41366133},
issn = {1438-7948},
mesh = {Animals ; Male ; *CRISPR-Cas Systems ; *X Chromosome/genetics ; *Mutation ; Gene Editing/methods ; *Drosophila/genetics ; *Drosophila melanogaster/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease protein 9 (Cas9) is a powerful tool used for genome engineering, but concerns remain about off-target effects. Here we evaluate potential deleterious effects of CRISPR-Cas9 by combining sequence analysis and the genetics of the male X chromosome in a Drosophila model. Since males have only one X chromosome deleterious mutations on the X chromosome will manifest in reducing viability or result in visible phenotypes and thus provide sensitive readouts of off-target activity. Our data do not support large scale off-target effects in Drosophila. To optimize sgRNA selection, we incorporated off-target evaluation into the PlatinumCRISPr sgRNA selection tool for a broad range of organisms.},
}

Animals
Male
*CRISPR-Cas Systems
*X Chromosome/genetics
*Mutation
Gene Editing/methods
*Drosophila/genetics
*Drosophila melanogaster/genetics
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41365538,
year = {2025},
author = {Jung, SC and Oh, H and Eom, W and Jin, YS and Park, SH and Park, K and Koh, HG},
title = {Scarless Genetic Engineering of Saccharomyces cerevisiae for Enhanced Guanosine Monophosphate Production as a Natural Flavor Enhancer.},
journal = {Journal of microbiology and biotechnology},
volume = {35},
number = {},
pages = {e2508034},
doi = {10.4014/jmb.2508.08034},
pmid = {41365538},
issn = {1738-8872},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Metabolic Engineering/methods ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Fermentation ; *Guanosine Monophosphate/biosynthesis/metabolism ; *Flavoring Agents/metabolism ; *Genetic Engineering/methods ; Promoter Regions, Genetic ; Inosine Monophosphate/metabolism ; Pentose Phosphate Pathway/genetics ; },
abstract = {Saccharomyces cerevisiae and Cyberlindnera jadinii are widely utilized in the natural food seasoning industry as sources of flavor enhancing nucleotides such as inosine monophosphate (IMP) and guanosine monophosphate (GMP), which contribute to umami taste and support sodium reduction in food. However, wild type yeast strains produce GMP at levels that are inadequate for industrial scale applications, necessitating metabolic engineering strategies to increase production efficiency. This study employed a CRISPR-Cas9-based scarless genome engineering approach to enhance GMP biosynthesis in S. cerevisiae via promoter replacement. The key genes IMD3 and GUA1, responsible for converting IMP to GMP, were overexpressed to redirect purine flux toward GMP production. To address precursor limitations, ZWF1 and RKI1, involved in the pentose phosphate pathway, were also overexpressed. In parallel, the expression of STB5 and RAP1 was increased to enhance NADPH regeneration and relieve transcriptional bottlenecks. As a result, the final engineered strain SCJ-7 demonstrated a 1.77-fold increase in GMP titer and a 1.40-fold increase in GMP content during flask fermentation compared to the wild-type. In fed-batch fermentation, GMP titer was further improved by 27.6%. These findings demonstrate that combining metabolic flux enhancement with transcriptional regulation provides an effective and scalable strategy for boosting GMP production in S. cerevisiae, offering strong potential for industrial application in the food industry.},
}

*Saccharomyces cerevisiae/genetics/metabolism
*Metabolic Engineering/methods
Saccharomyces cerevisiae Proteins/genetics/metabolism
CRISPR-Cas Systems
Fermentation
*Guanosine Monophosphate/biosynthesis/metabolism
*Flavoring Agents/metabolism
*Genetic Engineering/methods
Promoter Regions, Genetic
Inosine Monophosphate/metabolism
Pentose Phosphate Pathway/genetics

@article {pmid41365534,
year = {2025},
author = {Skeate, JG and Lee, CJ and Stewart, C and Fischbach, MJ and Kar, B and Tsai, AK and Kenderian, SS and Stromnes, IM and Largaespada, DA and Moriarity, BS and Rogers, LM},
title = {Functional genomics for improving adoptive T-cell transfer therapies.},
journal = {Journal for immunotherapy of cancer},
volume = {13},
number = {12},
pages = {},
doi = {10.1136/jitc-2025-013286},
pmid = {41365534},
issn = {2051-1426},
mesh = {Humans ; *Genomics/methods ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/immunology/transplantation ; Gene Editing/methods ; Animals ; *Neoplasms/therapy/immunology/genetics ; CRISPR-Cas Systems ; },
abstract = {Adoptive cell therapy (ACT) has shown remarkable success in the treatment of some malignancies, particularly leukemia. However, there are multiple factors that limit the durability of ACT in solid tumors, including dose-limiting toxicities, the immunosuppressive tumor microenvironment, and T-cell exhaustion. As the manufacture and preparation of adoptive T-cell therapies allows time and adequate conditions for ex vivo T-cell engineering, forward genetic screens can identify novel genetic targets that could improve their effectiveness. CRISPR is a commonly used functional genomics tool that has been successfully used to both enhance our understanding of mechanisms of resistance and to discover potential genetic edits to improve ACT. A complementary approach, Sleeping Beauty transposon mutagenesis provides additional opportunities to identify novel genetic edits without being constrained by the annotated human genome. Here, we summarize forward genetic screens and their tools to uncover strategies to enhance ACT. Complementary approaches can be combined and improved on to identify translatable genetic editing strategies through studies that accurately recapitulate disease-specific challenges.},
}

Humans
*Genomics/methods
*Immunotherapy, Adoptive/methods
*T-Lymphocytes/immunology/transplantation
Gene Editing/methods
Animals
*Neoplasms/therapy/immunology/genetics
CRISPR-Cas Systems

@article {pmid41242662,
year = {2026},
author = {Zhang, N and Zhou, X and Jiao, X and Liang, Z and Jiang, W and Liu, S and Wu, P},
title = {Field-deployable CRISPR-Dx for BmNPV and Nosema bombycis: DNA-extraction-free one-pot RPA-Cas12a and Cas12a/Cas13a dual-gene assays with handheld devices.},
journal = {Insect biochemistry and molecular biology},
volume = {186},
number = {},
pages = {104449},
doi = {10.1016/j.ibmb.2025.104449},
pmid = {41242662},
issn = {1879-0240},
mesh = {Animals ; *Nosema/isolation & purification/genetics ; *Nucleopolyhedroviruses/isolation & purification/genetics ; *Bombyx/virology/microbiology ; *CRISPR-Cas Systems ; },
abstract = {Simple, accurate, sensitive, and rapid pathogen diagnosis is crucial for effective control of silkworm diseases. Although CRISPR-based nucleic acid detection systems show great potential for on-site detection of silkworm pathogens, their practicality is hindered by complex workflows and reagent-storage constraints. To address these limitations and enhance field suitability, we developed a DNA extraction-free one-pot RPA-CRISPR/Cas12a (DEORC) system and a dual-gene assay for detecting Bombyx mori nucleopolyhedrovirus (BmNPV) and Nosema bombycis using a handheld device. The DEORC assay detects BmNPV in hemolymph samples as early as 6 h post-infection (hpi) and N. bombycis at 10[3] spores/mL in spore suspensions. The entire process from sampling to visual readout is completed in approximately 70 min without requiring sophisticated equipment. To further enable off-grid deployment, we lyophilized the Cas12a detection reagents using 1 M betaine as a lyoprotectant, which retained measurable activity for at least one month at 4 °C under our test conditions, facilitating short-term refrigerated transport and field storage. Additionally, the dual-gene assay detects 10[3] copies/μL of a double-reference plasmid and simultaneously detects both BmNPV and N. bombycis in a single tube from midgut samples at 48 hpi; when combined with extraction-free techniques, it enables simultaneous detection of both pathogens in hemolymph samples at 72 hpi. Collectively, these advancements provide sensitive and portable tools for on-site sericulture disease management, offering faster and more practical workflows than two-step single-gene and traditional approaches.},
}

Animals
*Nosema/isolation & purification/genetics
*Nucleopolyhedroviruses/isolation & purification/genetics
*Bombyx/virology/microbiology
*CRISPR-Cas Systems

@article {pmid41241162,
year = {2026},
author = {Huang, G and Tang, Y and Zhang, S and Ning, H and Xu, J and Li, F and Zhang, X},
title = {Multifunctional nano-polymer-based targeted delivery system for CRISPR/Cas9-Mediated hepatocellular carcinoma therapy.},
journal = {International journal of pharmaceutics},
volume = {687},
number = {},
pages = {126392},
doi = {10.1016/j.ijpharm.2025.126392},
pmid = {41241162},
issn = {1873-3476},
mesh = {*Carcinoma, Hepatocellular/therapy/genetics ; *Liver Neoplasms/therapy/genetics ; Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; Animals ; *Methyltransferases/genetics ; Cell Line, Tumor ; Plasmids/administration & dosage ; Polyethylene Glycols/chemistry ; *Polymers/chemistry ; Mice, Inbred BALB C ; Deoxycholic Acid/chemistry/administration & dosage ; *Nanoparticles/chemistry/administration & dosage ; Mice, Nude ; Hep G2 Cells ; Apoptosis ; Gene Transfer Techniques ; Mice ; Genetic Therapy/methods ; Polyethyleneimine/chemistry ; Disaccharides ; },
abstract = {CRISPR/Cas9 gene-editing technology exhibits substantial therapeutic potential for hepatocellular carcinoma (HCC); however, the targeted delivery of the CRISPR/Cas9 system into tumor cells remains a critical challenge requiring urgent exploration. Methyltransferase-Like 3 (METTL3), a key methyltransferase, drives HCC proliferation via multiple mechanisms. To address this challenge, a multifunctional delivery system was developed to efficiently deliver CRISPR/Cas9 plasmids targeting METTL3 (pMETTL3) into HCC cells. The cationic PEI, which facilitated the adsorption of pMETTL3 and protected it from lysosomal degradation, served as the polymeric backbone and was modified with deoxycholic acid (DOCA) to enhance its hydrophobicity. Meanwhile, lactobionic acid (LA) was grafted onto the structure to actively target HCC cells. The resulting pMETTL3/LPD was further functionalized with pH-sensitive and cleavable polyethylene glycol (PEG), aiming to reduce toxicity and enhance prolonged circulation. Results demonstrated that the delivery system maintains stability in physiological pH environments while achieving significantly enhanced accumulation in tumor tissues. Furthermore, the efficient cellular uptake of CRISPR/Cas9 plasmids enables precise gene editing, thereby effectively disrupting METTL3 expression, inducing apoptosis, and ultimately inhibiting HCC growth. This study presents a promising therapeutic strategy targeting METTL3 for HCC treatment and further expands the application of CRISPR/Cas9 gene-editing technology in cancer therapy.},
}

*Carcinoma, Hepatocellular/therapy/genetics
*Liver Neoplasms/therapy/genetics
Humans
*CRISPR-Cas Systems
Gene Editing/methods
Animals
*Methyltransferases/genetics
Cell Line, Tumor
Plasmids/administration & dosage
Polyethylene Glycols/chemistry
*Polymers/chemistry
Mice, Inbred BALB C
Deoxycholic Acid/chemistry/administration & dosage
*Nanoparticles/chemistry/administration & dosage
Mice, Nude
Hep G2 Cells
Apoptosis
Gene Transfer Techniques
Mice
Genetic Therapy/methods
Polyethyleneimine/chemistry
Disaccharides

@article {pmid41193657,
year = {2025},
author = {Li, H and Melnyk, JE and Fu, BXH and Shrestha, R and Zhang, M and Sjöström, M and Feng, S and Anderson, JA and Han, W and Chesner, LN and Shin, HJ and Farsh, T and Suarez, HJ and Nath, S and Chou, J and Das, R and Egusa, EA and Calvert, M and Kishishita, A and Barpanda, A and Zhu, J and Maheshwari, A and Chen, WS and Alshalalfa, M and Winters, A and Hua, JT and Liu, T and Davicioni, E and Wiita, AP and Stohr, BA and Siddiqui, J and Huang, B and Small, EJ and Shokat, KM and Nelson, PS and Quigley, DA and Wasmuth, EV and Gilbert, LA and Feng, FY},
title = {Genome-scale CRISPR screens identify PTGES3 as a direct modulator of androgen receptor function in advanced prostate cancer.},
journal = {Nature genetics},
volume = {57},
number = {12},
pages = {3027-3038},
pmid = {41193657},
issn = {1546-1718},
support = {21YOUN12//Prostate Cancer Foundation (PCF)/ ; Young Investigator Award//Prostate Cancer Foundation (PCF)/ ; YI//Prostate Cancer Foundation (PCF)/ ; PC230420//Prostate Cancer Foundation (PCF)/ ; young investigator//Prostate Cancer Foundation (PCF)/ ; 17CHAL06//Prostate Cancer Foundation (PCF)/ ; CA204602//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; P50CA097186 PNW Prostate Cancer SPORE Career Enhancement Program//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 1F32CA236347-01//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; CA230516-02S1//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 1R01CA221969-01//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 1R01CA244550//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; P30 CA015704/CA/NCI NIH HHS/United States ; R01 CA234715/CA/NCI NIH HHS/United States ; R01 CA266452/CA/NCI NIH HHS/United States ; P50 CA097186/CA/NCI NIH HHS/United States ; 1R01CA230516-01//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; 1R01CA227025//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; P50CA186786//U.S. Department of Health & Human Services | NIH | National Cancer Institute (NCI)/ ; DP2 CA239597/CA/NCI NIH HHS/United States ; Prostate Cancer Program 2021 Pilot Research Awards//UC | UC San Francisco | Department of Medicine, University of California, San Francisco (UCSF Department of Medicine)/ ; 2018-00382//Vetenskapsrådet (Swedish Research Council)/ ; HT9425-23-1-0462//U.S. Department of Defense (United States Department of Defense)/ ; W81XWH-20-1-0136//U.S. Department of Defense (United States Department of Defense)/ ; R01GM124334//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01GM131641//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R00 GM140264/GM/NIGMS NIH HHS/United States ; V2024-016//V Foundation for Cancer Research (V Foundation)/ ; P30 CA015704/CA/NCI NIH HHS/United States ; R01 CA234715/CA/NCI NIH HHS/United States ; R01 CA266452/CA/NCI NIH HHS/United States ; P50 CA097186/CA/NCI NIH HHS/United States ; DP2 CA239597/CA/NCI NIH HHS/United States ; R00 GM140264/GM/NIGMS NIH HHS/United States ; },
mesh = {Male ; *Receptors, Androgen/genetics/metabolism ; Humans ; *Prostatic Neoplasms/genetics/pathology/metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems/genetics ; Homeodomain Proteins/genetics/metabolism ; GATA2 Transcription Factor/genetics/metabolism ; Drug Resistance, Neoplasm/genetics ; },
abstract = {The androgen receptor (AR) is a critical driver of prostate cancer (PCa). Here, to study regulators of AR protein levels and oncogenic activity, we developed a live-cell quantitative endogenous AR fluorescent reporter. Leveraging this AR reporter, we performed genome-scale CRISPRi flow cytometry sorting screens to systematically identify genes that modulate AR protein levels. We identified and validated known AR protein regulators, including HOXB13 and GATA2, and also unexpected top hits including PTGES3-a poorly characterized gene in PCa. PTGES3 repression resulted in loss of AR protein, cell-cycle arrest and cell death in AR-driven PCa models. Clinically, analysis of PCa data demonstrates that PTGES3 expression is associated with AR-directed therapy resistance. Mechanistically, we show PTGES3 binds directly to AR, regulates AR protein stability and is necessary for AR function in the nucleus at AR target genes. PTGES3 represents a potential therapeutic target for overcoming known mechanisms of resistance to existing AR-directed therapies in PCa.},
}

Male
*Receptors, Androgen/genetics/metabolism
Humans
*Prostatic Neoplasms/genetics/pathology/metabolism
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems/genetics
Homeodomain Proteins/genetics/metabolism
GATA2 Transcription Factor/genetics/metabolism
Drug Resistance, Neoplasm/genetics

@article {pmid41170598,
year = {2025},
author = {Yoo, D and Park, H and Lim, H and Kim, G and Kim, D and Seo, Y and An, J and Kang, S and Park, C and Lee, MH and Lee, T},
title = {Versatile Biosensing Tool: CRISPR-Cas12a System-Integrated Electrochemical Biosensor for Severe Fever with Thrombocytopenia Syndrome Virus Detection in Clinical and Environmental Conditions.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {49},
pages = {e06860},
doi = {10.1002/smll.202506860},
pmid = {41170598},
issn = {1613-6829},
support = {2021R1C1C1005583//National Research Foundation of Korea (NRF)/ ; RS-2024-00416117//National Research Foundation of Korea (NRF)/ ; No.2020R1A5A1018052//National Research Foundation of Korea (NRF)/ ; RS-2024-00507931//Materials & Components Technology Development Program/ ; },
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Phlebovirus/isolation & purification/genetics ; *Severe Fever with Thrombocytopenia Syndrome/virology/diagnosis ; *Electrochemical Techniques/methods ; },
abstract = {Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly lethal zoonotic virus with a maximum mortality rate of 30%. Despite its risks and potential for human-to-human transmission, the standard diagnostic method has been absent for SFTSV detection. Therefore, this study introduces a versatile SFTSV biosensing technology using the electrochemical-clustered regularly interspaced short palindromic repeat (EC-CRISPR) system. The sensing membrane is functionalized with the 2WJ DNA@Au-MoS2 signal probe as a strategy to amplify the EC response resulting from target detection events of the CRISPR system. The sensor exhibits selective, sensitive, and reproducible detection capabilities in phosphate-buffered saline, human serum, and Haemaphysalis longicornis genomic DNA diluted conditions with detection limits of 210.7, 189.0, and 285.1 fM, respectively. This verifies the versatility of the fabricated system, which significantly contributes to the early SFTSV detection in various fields. In the meantime, the sufficient sensing performance is demonstrated in identifying of SFTSV from infectious agent DNA. Furthermore, the proposed EC-CRISPR biosensing platform can be considered as a flexible foundational technique for the diagnosis of zoonotic infectious diseases, as it demonstrates practical applicability for the detection of Orientia tsutsugamushi by utilizing a customized CRISPR system programming strategy.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*Phlebovirus/isolation & purification/genetics
*Severe Fever with Thrombocytopenia Syndrome/virology/diagnosis
*Electrochemical Techniques/methods

@article {pmid41123840,
year = {2025},
author = {Remiszewski, P and Siedlecki, E and Wełniak-Kamińska, M and Mikula, M and Czarnecka, AM},
title = {Genetically Modified Mouse Models for Sarcoma Research: A Comprehensive Review.},
journal = {Current oncology reports},
volume = {27},
number = {11},
pages = {1221-1248},
pmid = {41123840},
issn = {1534-6269},
support = {2019/35/O/NZ2/03761 (AMC)//Narodowe Centrum Nauki/ ; },
mesh = {*Sarcoma/genetics/pathology ; Animals ; Humans ; *Disease Models, Animal ; Mice ; CRISPR-Cas Systems ; Animals, Genetically Modified ; Gene Editing ; },
abstract = {PURPOSE OF REVIEW: Sarcomas are a heterogeneous group of over 170 malignant tumours of mesenchymal origin. The poor prognosis highlights the need for novel therapeutic strategies. Preclinical modelling is essential, yet challenging, given that sarcomas differ substantially from carcinomas and resources are very limited.

RECENT FINDINGS: GEMMs allow for the precise modelling of recurrent sarcoma genetics. The Cre-loxP system offer spatial and temporal control over the activation of oncogenes or the loss of tumour suppressors, while the CRISPR-Cas9 system enables the rapid, simultaneous editing of key drivers such as Trp53, Nf1, Kras and Pten. These models reproduce key features of human sarcomas, including their histopathology, the initiation of tumours in specific lineages and sites, and tumour-immune interactions within immune-competent hosts. GEMMs have been used to investigate hypotheses about the cells of origin, to test radiotherapy and immunotherapy, and to compare fusion-driven sarcomas with those with a complex karyotype. Despite variability, GEMMs remain essential tools for investigating the mechanisms of initiation, progression, and response to therapy. GEMMs offer mechanistic fidelity, but their use is limited by factors such as breeding burden, variability in recombination, off-target effects of CRISPR, underrepresentation of genomic complexity and inconsistent metastasis. These weaknesses reduce their predictive value, particularly with regard to advanced disease and immunotherapy. Progress will require the integration of Cre-loxP with CRISPR-Cas9, the standardisation of induction and reporting, and a closer alignment with distinct sarcoma subtypes, in order to enhance translational relevance.},
}

*Sarcoma/genetics/pathology
Animals
Humans
*Disease Models, Animal
Mice
CRISPR-Cas Systems
Animals, Genetically Modified
Gene Editing

@article {pmid41110509,
year = {2026},
author = {Faber, NR and Ashok, K and Venkatesan, T and Wertheim, B and Bulgarella, M},
title = {Leveraging advances in RNAi and CRISPR for improved biological pest control.},
journal = {Current opinion in insect science},
volume = {73},
number = {},
pages = {101453},
doi = {10.1016/j.cois.2025.101453},
pmid = {41110509},
issn = {2214-5753},
mesh = {*RNA Interference ; *Pest Control, Biological/methods ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; *Insecta/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The limitations of chemical pesticides and their associated risks highlight the need for more sustainable pest management strategies. Biological control using natural enemies offers an eco-friendly alternative but is sometimes constrained by efficiency and scalability. Emerging molecular tools-RNA interference (RNAi) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based gene editing-present novel opportunities to enhance existing biological control or to control pests directly. RNAi induces targeted gene knockdown via a non-heritable, transient response. CRISPR enables precise genetic modifications and could improve traits in beneficial insects or disrupt essential genes in pests, optionally including a gene drive for increased power. Although limitations remain for several species, these technologies could be valuable tools for integrated pest management. Their future implementation raises biosafety and regulatory considerations, particularly for self-propagating systems like gene drives. This review showcases developments in RNAi and CRISPR-based pest control, and calls for risk-based, adaptive governance to enable their responsible use in sustainable agriculture.},
}

*RNA Interference
*Pest Control, Biological/methods
Animals
*CRISPR-Cas Systems
*Gene Editing
*Insecta/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid40997504,
year = {2025},
author = {Berger, P and Wilming, L and Jürgens, R and Minchuk, Y and Leußink, S and Gandhi, S and Walter, C and Wind, SM and Heider, D and Lamparter, L and Galic, M and Stoll, M and Jorch, SK and Roth, J and Austermann, J and Fehler, O},
title = {PSTPIP1 and pyrin, two key regulators of macrophage differentiation.},
journal = {European journal of cell biology},
volume = {104},
number = {4},
pages = {151518},
doi = {10.1016/j.ejcb.2025.151518},
pmid = {40997504},
issn = {1618-1298},
mesh = {*Macrophages/metabolism/cytology ; *Cell Differentiation ; Humans ; *Pyrin/metabolism/genetics ; *Adaptor Proteins, Signal Transducing/metabolism/genetics ; CRISPR-Cas Systems ; *Cytoskeletal Proteins/metabolism/genetics ; Animals ; },
abstract = {BACKGROUND: Monocytes develop from hematopoietic stem cells; migrate into the tissue, where they undergo a stimulation-dependent and tissue specific differentiation into macrophages imprinting specific inflammatory functions. The development of inflammatory functions during differentiation of progenitor cells into macrophages remained incompletely understood.

OBJECTIVE: We intended to identify regulatory factors driving monocyte/macrophage differentiation.

METHODS: A Genome-wide CRISPR/Cas9 knockout screen (GeCKO) in ER-HoxB8 macrophages was used to identify key drivers of macrophage differentiation which were verified in independent knock-out and knock-in cells. Immunophenotyping was studied by FACS, morphology and migration by fluorescence microscopy, the inflammatory response by ELISA. Transcriptomic data were obtained by next generation mRNA sequencing and validated by quantitative polymerase chain reaction and immunoblotting.

RESULTS: Genome-wide CRISPR/Cas9 knockout screen identified the cytosolic cytoskeleton-associated adaptor molecule PSTPIP1 (proline-serine-threonine phosphatase interacting protein 1) as a regulatory factor of macrophage differentiation. Interestingly, mutations in PSTPIP1 cause autoinflammatory disorders (PAPA syndrome). Deletion of PSTPIP1 resulted in hampered differentiation, decreased inflammatory response, changed morphology, altered cell adhesion and migration properties. PSTPIP1 is a regulator of Pyrin inflammasome activity which drives autoinflammation in familial Mediterranean fever (FMF). Deletion of Pyrin also resulted in a strong alteration of cellular dynamics in macrophages.

CONCLUSION: PSTPIP1 and Pyrin are crucial factors in macrophage differentiation. Their deletion or mutation resulted in a hampered differentiation of macrophages resulting in strong morphological alterations and impacting phagocyte key functions as adhesion and migration. Impaired differentiation of macrophages may represent a significant factor in the pathophysiology of autoinflammatory diseases like FMF and PAPA.},
}

*Macrophages/metabolism/cytology
*Cell Differentiation
Humans
*Pyrin/metabolism/genetics
*Adaptor Proteins, Signal Transducing/metabolism/genetics
CRISPR-Cas Systems
*Cytoskeletal Proteins/metabolism/genetics
Animals

@article {pmid40997217,
year = {2025},
author = {Cosgrove, BD and Bounds, LR and Taylor, CK and Su, AL and Rizzo, AJ and Barrera, A and Sun, T and Safi, A and Song, L and Whitlow, T and Tata, A and Iglesias, N and Diao, Y and Tata, PR and Hoffman, BD and Crawford, GE and Gersbach, CA},
title = {Mechanosensitive genomic enhancers potentiate the cellular response to matrix stiffness.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6778},
pages = {eadl1988},
doi = {10.1126/science.adl1988},
pmid = {40997217},
issn = {1095-9203},
mesh = {*Extracellular Matrix/physiology ; Humans ; Fibroblasts/physiology/metabolism ; *Enhancer Elements, Genetic ; *Epigenesis, Genetic ; *Mechanotransduction, Cellular/genetics ; Lung/cytology ; Single-Cell Analysis ; Cell Movement/genetics ; Cell Proliferation/genetics ; Apoptosis/genetics ; Cell Adhesion/genetics ; Gene Editing ; Epigenome ; CRISPR-Cas Systems ; },
abstract = {Epigenetic control of gene expression and cellular phenotype is influenced by changes in the local microenvironment, yet how mechanical cues precisely influence epigenetic state to regulate transcription remains largely unmapped. In this study, we combined genome-wide epigenome profiling, epigenome editing, and phenotypic and single-cell RNA sequencing CRISPR screening to identify a class of genomic enhancers that responds to the mechanical microenvironment. These "mechanoenhancers" can be preferentially activated on either soft or stiff extracellular matrix contexts and regulate transcription to influence critical cell functions including apoptosis, adhesion, proliferation, and migration. Epigenetic editing of mechanoenhancers reprograms the cellular response to the mechanical microenvironment and modulates the activation of disease-related genes in lung fibroblasts from healthy and fibrotic donors. Epigenetic editing of mechanoenhancers holds potential for precise targeting of mechanically driven diseases.},
}

*Extracellular Matrix/physiology
Humans
Fibroblasts/physiology/metabolism
*Enhancer Elements, Genetic
*Epigenesis, Genetic
*Mechanotransduction, Cellular/genetics
Lung/cytology
Single-Cell Analysis
Cell Movement/genetics
Cell Proliferation/genetics
Apoptosis/genetics
Cell Adhesion/genetics
Gene Editing
Epigenome
CRISPR-Cas Systems

@article {pmid41364162,
year = {2025},
author = {Murtaza, M and Gupta, P and Choudhary, P and Manzoor, M and Sharma, S and Jaglan, S},
title = {Strategies to decipher silent biosynthetic gene clusters in actinomycetes.},
journal = {Archives of microbiology},
volume = {208},
number = {1},
pages = {53},
pmid = {41364162},
issn = {1432-072X},
mesh = {*Actinobacteria/genetics/metabolism ; *Multigene Family/genetics ; *Biosynthetic Pathways/genetics ; Anti-Bacterial Agents/biosynthesis ; CRISPR-Cas Systems ; },
abstract = {Actinobacteria have a huge, mainly untapped potential for the production of secondary metabolites. These metabolites are an important source of bioactive compounds. However, a majority of biosynthetic gene clusters (BGCs) are either under-expressed or fully silent under standard laboratory conditions, limiting their potential. The present review article aims to explore the biosynthetic gene clusters (BGCs) of actinobacteria using strategies that aid in unlocking these silent BGCs. The strategies discussed are PCR-Targeted Gene Replacement (PCR-TR); Cre-LoxP recombination system; Transcription factor decoys, Ribosome engineering, and CRISPR/Cas technologies. Besides, elicitors also helped with the identification of these cryptic or silent BGCs and advanced our ability to explore these natural products. Combining experimental and computational platforms provides an opportunity to unlock the hidden chemical diversity in nature, thereby accelerating the identification of new bioactive substances. The new antibiotics explored by all the strategies could help in the fight against antimicrobial resistance (AMR).},
}

*Actinobacteria/genetics/metabolism
*Multigene Family/genetics
*Biosynthetic Pathways/genetics
Anti-Bacterial Agents/biosynthesis
CRISPR-Cas Systems

@article {pmid41362674,
year = {2026},
author = {Toofan, P and Singh, M and Brooks, A and McLuckie, K},
title = {Non-clinical safety considerations on genome editing using the CRISPR/Cas system.},
journal = {Genes & diseases},
volume = {13},
number = {2},
pages = {101785},
pmid = {41362674},
issn = {2352-3042},
abstract = {Recent advances in gene editing using the CRISPR/Cas system have revolutionized genome editing, opening new horizons for human cellular and gene therapy products. Genome editing technologies are rapidly being adopted in clinical trials. However, critical non-clinical safety considerations are required to address challenges in translating research to the clinic. Here, we review current ex vivo and in vivo genome editing approaches using the CRISPR/Cas system and discuss the practical use of these methods in pre-clinical studies and in the clinic. We also discuss known limitations of genome editing in humans and the mitigation of risk factors associated with it from a non-clinical safety perspective. This review aims to aid researchers in acquiring a perspective that is essential for the safe translation of genome editing to the clinic.},
}

@article {pmid41361988,
year = {2025},
author = {Min, YH and Lee, DG and Lee, HY and Yoo, JH and Lee, KH and Shin, YB and Byun, JY},
title = {CRISPR/Cas12a with Antisense Oligonucleotide-Regulated Translational Amplification for Ultrasensitive Nucleic Acid Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c03081},
pmid = {41361988},
issn = {2379-3694},
abstract = {Highly sensitive nucleic acid testing-assisted early disease detection is crucial for effective disease prevention and management, particularly when targeting low-abundance genetic materials in molecular diagnostics. This study describes CRATE (CRISPR/Cas controlled antisense oligonucleotide (ASO)-mediated translational signal enhancement), a novel ultrasensitive approach for nucleic acid detection by integrating Cas12a trans-cleavage, ASO-controlled gene expression, and cell-free signal protein amplification. This assay leverages the target-induced trans-cleavage of ASO-controlled gene expression for the amplification of signal proteins, with luminescent signal allowing for attomolar-level target DNA detection, as well as antigenic protein application enabling visual detection by lateral flow assay. The CRATE assay improves sensitivity using ASO-modified locked nucleic acid, achieving a 10-aM-level DNA detection. The proof of concept demonstrates 0.1 copies/μL detection of HPV genomic DNA from HPV-positive cancer cells as well as colorimetric lateral flow tests with ∼10 copies/μL sensitivity. The CRATE assay can detect the HBV target in plasma from HBV-positive patients with 100% sensitivity and specificity. With high specificity and accuracy, the CRATE assay retains the potential for detecting any nucleic acid of interest. By integration of precise CRISPR-based cleavage, ASO regulation, and efficient protein signal amplification, this approach provides a promising solution for highly selective and sensitive nucleic acid detection and potential applications in clinical diagnostics and point-of-care testing.},
}

@article {pmid41361700,
year = {2025},
author = {Ain, QU and McCarthy, A and Nadeem, A and Javed, M and Niakan, K and Nashta, AF},
title = {CRISPR/Cas9-mediated generation of GATA3 knockout in Bovine Fibroblast and MDBK cell lines to assess sgRNAs targeting efficiency.},
journal = {Functional & integrative genomics},
volume = {25},
number = {1},
pages = {269},
pmid = {41361700},
issn = {1438-7948},
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems ; *GATA3 Transcription Factor/genetics/metabolism ; Fibroblasts/metabolism/cytology ; Cell Line ; *Gene Knockout Techniques ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {GATA3 is expressed in the outer cells of the morula stage during embryonic development and is considered a key driver of the regulation of early lineage development in bovines. This research presents an optimised somatic cell validation resource, successfully generating GATA3 knockout (KO) Bovine Fetal Fibroblasts and MDBK cells using CRISPR/Cas9-mediated genome editing for their future implications in vivo studies designed to definitively understand the role of GATA3 in cell lineage specification and bovine embryo development. This involved designing single-guide RNAs (sgRNAs) targeting different regions of the GATA3 gene, cloning them into the px459 plasmid, delivering the CRISPR clone into bovine fibroblast cells and the MDBK cell line, screening for successful targeting and knockouts, and MiSeq analysis to verify successful disruption of the GATA3 gene. A total of eleven guides were designed targeting the functional domains in Exons 4 and 5 and the transcription initiation site in Exon 2. Designed guides were first optimized and screened using an in vitro cleavage assay. The guides with the best cutting efficiencies were then tested in vivo by targeting bovine fetal fibroblast (BFFs) and MDBK cell line followed by MiSeq analysis to verify the successful knockouts. A total of two effective guides were identified targeting the zinc-finger (ZnF) functional domains of the GATA3 gene (sgRNA#5 and sgRNA#8 in Exon 4 and Exon 5, respectively) and one in Exon 2 (sgRNA#1) targeting the transcription initiation site of the GATA3 gene. MiSeq data from targeted bovine cells showed indel frequency of 47.40%, 55.5%, and 42.4% in bovine fetal fibroblasts, 11.03%, 28.9% and 7.3% for MDBK cells for top three sgRNAs. Overall, MiSeq data for 3 selected sgRNAs showed successful disruption of the GATA3 gene, inserting a base pair 2-3 bp upstream of the PAM site, ultimately resulting in a premature stop codon TAA in the downstream region. This study established and validated highly efficient sgRNAs targeting the GATA3 gene, forming a molecular basis for forthcoming functional investigations in bovine embryos to explore gene function and protein-level effects.},
}

Animals
Cattle
*CRISPR-Cas Systems
*GATA3 Transcription Factor/genetics/metabolism
Fibroblasts/metabolism/cytology
Cell Line
*Gene Knockout Techniques
*RNA, Guide, CRISPR-Cas Systems/genetics
Gene Editing/methods

@article {pmid41361167,
year = {2025},
author = {Fast, L and Omar, M and Kanis, P and Schaffer, T and Chowdhury, D and Rakava, E and Pääbo, S and Riesenberg, S},
title = {Search-and-remove genome editing allows selection of cells by DNA sequence.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10985},
pmid = {41361167},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; Humans ; Animals ; CRISPR-Cas Systems/genetics ; Neanderthals/genetics ; Mutation, Missense ; CRISPR-Associated Protein 9/metabolism ; Base Sequence ; Cell Line, Tumor ; },
abstract = {The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However, such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here, we present SNIPE, a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells, which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations.},
}

*Gene Editing/methods
Humans
Animals
CRISPR-Cas Systems/genetics
Neanderthals/genetics
Mutation, Missense
CRISPR-Associated Protein 9/metabolism
Base Sequence
Cell Line, Tumor

@article {pmid41359835,
year = {2025},
author = {Roura-Martinez, D and Popa, N and Jaouen, F and Rombaut, C and Lepolard, C and Bachar, D and Borges, A and Cazorla, M and Villet, M and Moreno, S and Marie, H and Gascon, E},
title = {Combination of Cas9 and adeno-associated vectors enables efficient in vivo knockdown of precise miRNAs in the rodent and primate brain.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {50},
pages = {e2513076122},
doi = {10.1073/pnas.2513076122},
pmid = {41359835},
issn = {1091-6490},
support = {ANR-22-CE17-0034//Association Nationale de la Recherche et de la Technologie (ANRT)/ ; 6239//Fondation France Alzheimer/ ; 2022//Fondation Recherche Alzheimer/ ; },
mesh = {Animals ; *MicroRNAs/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Brain/metabolism ; Mice ; Genetic Vectors/genetics ; *Gene Knockdown Techniques/methods ; *Dependovirus/genetics ; Rats ; RNA, Guide, CRISPR-Cas Systems/genetics ; Neural Stem Cells/metabolism ; Neurons/metabolism ; Primates ; Receptors, AMPA/metabolism ; Olfactory Bulb/metabolism ; CRISPR-Associated Protein 9/metabolism ; },
abstract = {microRNAs (miRNAs) are key regulators of multiple biological functions. Although intensively studied, inactivating miRNAs in vivo is particularly challenging, especially in the brain. Here, we designed cell-specific tools aiming at downregulating defined miRNA species in vivo and investigating their function in discrete neuronal networks. Focusing on miR-124, a miRNA highly expressed in the mammalian brain and transcribed from three independent chromosomal loci, we designed and validated different guide RNAs. In vivo, our CRISPR-Cas9 designs strongly downregulate miR-124 levels without affecting the expression of other miRNAs. As a result, levels of endogenous miR-124 targets exhibit a significant increase supporting the release of its silencing activity. We provide evidence that specific deletion of miR-124 in neural stem cells of the subventricular zone altered migration of newly generated neurons into the olfactory bulb. We also showed that our vectors modified the Ca[2+] permeability of AMPA receptors, a robust functional output downstream of miR-124. We also extended our approach to other miRNAs, mammalian species, and Cas9 proteins, confirming the versatility of CRISPR-Cas9. These tool properties support their potential for elucidating miRNA functions in complex experimental in vivo settings such as brain networks.},
}

Animals
*MicroRNAs/genetics/metabolism
*CRISPR-Cas Systems/genetics
*Brain/metabolism
Mice
Genetic Vectors/genetics
*Gene Knockdown Techniques/methods
*Dependovirus/genetics
Rats
RNA, Guide, CRISPR-Cas Systems/genetics
Neural Stem Cells/metabolism
Neurons/metabolism
Primates
Receptors, AMPA/metabolism
Olfactory Bulb/metabolism
CRISPR-Associated Protein 9/metabolism

@article {pmid41359384,
year = {2025},
author = {Ichinose, M and Ohta, M and Shimajiri, Y and Akaiwa, Y and Nakamura, I and Shimamoto, M and Makinoda, R and Ozaki, S and Tamai, T and Maekawa, N and Tonomoto, M and Nakamura, T and Yagi, Y and Gutmann, B},
title = {RECODE: a programmable guide-free C-to-U RNA editing tool.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41359384},
issn = {1362-4962},
support = {//EditForce, Inc/ ; },
mesh = {*RNA Editing ; Humans ; Animals ; Mice ; *Cytidine Deaminase/genetics/metabolism/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; *Uridine/genetics/metabolism ; HEK293 Cells ; *Cytidine/metabolism/genetics ; RNA-Binding Proteins/genetics/metabolism ; Gene Editing/methods ; },
abstract = {Programmable RNA cytidine deaminase tools have been developed to convert cytidine-to-uridine (C-to-U) using CRISPR systems with guide RNAs. These tools, however, have limitations such as low editing efficiency, limited targetable sequence flexibility, and off-target RNA editing. Here, we present a novel guide-free C-to-U editing tool, named RECODE (RNA Editor for C-to-U with an Optimized DYW Enzyme), based on the RNA-binding pentatricopeptide repeat proteins, naturally fused to a C-terminal DYW cytidine deaminase domain. The RECODE specificity domain was engineered to enable retargeting, while its length and sequence were optimized to reduce off-target effects. Further optimization of the C-terminal catalytic region increased both the editing activity and the translation of the edited RNA. We showed that RECODE efficiently edits a wide range of targets in human cells, without affecting adjacent cytidines. It achieved over 50% editing efficiency for most sites, except those with an upstream guanine. Furthermore, we showed that RECODE is functional in mice, with high editing efficiency observed in specific tissues such as skeletal muscles using an AAV delivery system, suggesting its therapeutic potential for various diseases.},
}

*RNA Editing
Humans
Animals
Mice
*Cytidine Deaminase/genetics/metabolism/chemistry
RNA, Guide, CRISPR-Cas Systems/genetics
CRISPR-Cas Systems
*Uridine/genetics/metabolism
HEK293 Cells
*Cytidine/metabolism/genetics
RNA-Binding Proteins/genetics/metabolism
Gene Editing/methods

@article {pmid41308487,
year = {2026},
author = {Rodríguez-Estévez, D and Gil-Durán, C and Silva, R and Palma, D and Vaca, I and Chávez, R},
title = {CRISPR/Cas9-mediated development of Penicillium roqueforti strains deficient in roquefortine C and mycophenolic acid enables toxin-free blue cheese production.},
journal = {International journal of food microbiology},
volume = {446},
number = {},
pages = {111535},
doi = {10.1016/j.ijfoodmicro.2025.111535},
pmid = {41308487},
issn = {1879-3460},
mesh = {*Penicillium/genetics/metabolism ; *Cheese/microbiology/analysis ; *Mycophenolic Acid/metabolism ; *CRISPR-Cas Systems ; *Mycotoxins/biosynthesis ; Food Microbiology ; *Indoles/metabolism ; Heterocyclic Compounds, 4 or More Rings ; Piperazines ; },
abstract = {Penicillium roqueforti, a key fungus in the manufacture of blue-veined cheeses, can produce mycotoxins such as roquefortine C and mycophenolic acid. The production of these metabolites is highly strain- and condition-dependent. In industrial manufacture, hypotoxigenic P. roqueforti strains are typically used as controlled adjunct starters under standardized conditions, resulting in minimal mycotoxin accumulation, whereas naturally matured or artisan cheeses display more variable strain composition and ripening environments, which can elevate risk. In this context, the development of strains incapable of mycotoxin biosynthesis represents an important step toward safer cheese products. Here, we report the generation of P. roqueforti strains lacking the ability to synthesize roquefortine C and mycophenolic acid using CRISPR/Cas9. Single and double mutants deficient in one or both mycotoxins were obtained. Laboratory-scale cheeses produced under artisan-like conditions with these engineered strains contained no detectable levels of the target mycotoxins, in contrast to cheeses made with the wild-type strain. All mutants retained the ability to colonize cheese but displayed altered fungal biomass production compared to the native strain. These differences were consistent in curd and laboratory media and were not associated with changes in lipolytic or proteolytic activities. Further analyses revealed that while the absence of mycophenolic acid did not affect NaCl sensitivity, the lack of roquefortine C increased sensitivity to salt. Collectively, these results demonstrate the feasibility of producing mycotoxin-free blue cheeses using strains deficient in roquefortine C and mycophenolic acid biosynthesis, thereby laying the foundation for developing mycotoxin-free cheeses with engineered atoxigenic P. roqueforti strains.},
}

*Penicillium/genetics/metabolism
*Cheese/microbiology/analysis
*Mycophenolic Acid/metabolism
*CRISPR-Cas Systems
*Mycotoxins/biosynthesis
Food Microbiology
*Indoles/metabolism
Heterocyclic Compounds, 4 or More Rings
Piperazines

@article {pmid41271253,
year = {2025},
author = {Li, L and Xiong, Y and Guo, Y and Duan, H and Leng, Y and Huang, X and Chen, G and Xiong, Y},
title = {G-Quadruplex-Enhanced DNA Silver Nanoclusters Enable CRISPR/Cas12a System for Ultrasensitive Detection of Salmonella typhimurium.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {49},
pages = {31603-31610},
doi = {10.1021/acs.jafc.5c13828},
pmid = {41271253},
issn = {1520-5118},
mesh = {G-Quadruplexes ; *CRISPR-Cas Systems ; *Silver/chemistry ; *Salmonella typhimurium/genetics/isolation & purification ; Metal Nanoparticles/chemistry ; *Biosensing Techniques/methods/instrumentation ; Limit of Detection ; Milk/microbiology ; Animals ; *DNA/chemistry/genetics ; Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases ; },
abstract = {DNA-templated silver nanoclusters (tDNA-AgNCs) show considerable promise as fluorescence reporters for the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system because of their ease of synthesis, strong resistance to photobleaching, and large Stokes shift. However, the weak luminous intensity and low cleavage efficiency of tDNA-AgNCs limit the sensitivity of CRISPR assays. In this study, we developed a novel approach by introducing activator DNA (aDNA) with a G-quadruplex structure to enhance the luminous intensity of the tDNA-AgNCs. As a result of the high cleavage efficiency of free aDNA by activated CRISPR/Cas12a, the G-quadruplex-enhanced tDNA-AgNCs (GED-AgNCs) were integrated into recombinase polymerase amplification and CRISPR/Cas12a system for the ultrasensitive detection of Salmonella typhimurium. By optimizing the synthesis of tDNA-AgNCs and GED-AgNCs, our developed G-quadruplex-enhanced DNA-AgNC CRISPR assay (G-DACA) platform enabled the sensitive determination of S. typhimurium with a detection limit as low as 1 CFU/mL and a wide dynamic range of 10-10[8] CFU/mL. Moreover, our proposed method demonstrated good accuracy and reliability for the quantitative analysis of S. typhimurium in real pasteurized milk samples, with recovery rates ranging from 81.06% to 102.33% and relative standard deviations between 7.17% and 14.84%. Overall, our innovative G-DACA platform offers an economical and versatile solution for food safety and clinical diagnostics.},
}

G-Quadruplexes
*CRISPR-Cas Systems
*Silver/chemistry
*Salmonella typhimurium/genetics/isolation & purification
Metal Nanoparticles/chemistry
*Biosensing Techniques/methods/instrumentation
Limit of Detection
Milk/microbiology
Animals
*DNA/chemistry/genetics
Bacterial Proteins/genetics/metabolism
CRISPR-Associated Proteins/genetics/metabolism
Endodeoxyribonucleases

@article {pmid41270744,
year = {2025},
author = {Pei, Y and Li, S and Garipler, G and Kamimoto, K and Mazzoni, EO},
title = {Stem cell-based approach to identify regulatory TFs during mammalian cell differentiation.},
journal = {Stem cell reports},
volume = {20},
number = {12},
pages = {102716},
doi = {10.1016/j.stemcr.2025.102716},
pmid = {41270744},
issn = {2213-6711},
mesh = {*Cell Differentiation/genetics ; Animals ; Humans ; Mice ; *Transcription Factors/metabolism/genetics ; Motor Neurons/cytology/metabolism ; *Pluripotent Stem Cells/metabolism/cytology ; Transcriptome ; Single-Cell Analysis ; CRISPR-Cas Systems ; },
abstract = {Cell differentiation is regulated by transcription factors (TFs), but specific TFs needed for mammalian differentiation pathways are not fully understood. For example, during spinal motor neuron (MN) differentiation, 1,370 TFs are transcribed, yet only 55 have reported functional relevance. We developed a method combining pluripotent stem cell differentiation, single-cell transcriptomics, and a CRISPR-based TF loss-of-function screen and applied it to MN differentiation. The CRISPR screen identified 245 genes important for mouse MN differentiation, including 116 TFs. This screen uncovered important genes not showing differential transcription and identified a regulatory hub at the MN progenitor (pMN) stage. A secondary human screen of 69 selected candidates revealed a conservation between mouse pMN and human pMN and ventral pMN (vpMN) regulations. The validation of three hits required for efficient human MN differentiation supported the effectiveness of our approach. Collectively, our strategy offers a framework for identifying important TFs in various differentiation pathways.},
}

*Cell Differentiation/genetics
Animals
Humans
Mice
*Transcription Factors/metabolism/genetics
Motor Neurons/cytology/metabolism
*Pluripotent Stem Cells/metabolism/cytology
Transcriptome
Single-Cell Analysis
CRISPR-Cas Systems

@article {pmid41270297,
year = {2025},
author = {Kraiczy, J and Yu, B},
title = {Human fallopian tube epithelial organoids with TP53 mutation recapitulate features of serous tubal intraepithelial carcinoma (STIC).},
journal = {Gynecologic oncology},
volume = {203},
number = {},
pages = {198-208},
doi = {10.1016/j.ygyno.2025.10.038},
pmid = {41270297},
issn = {1095-6859},
mesh = {Humans ; Female ; *Tumor Suppressor Protein p53/genetics ; *Organoids/pathology ; *Fallopian Tubes/pathology ; *Fallopian Tube Neoplasms/genetics/pathology ; Mutation ; *Carcinoma in Situ/genetics/pathology ; *Ovarian Neoplasms/genetics/pathology ; DNA Copy Number Variations ; *Cystadenocarcinoma, Serous/genetics/pathology ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {OBJECTIVE: Serous tubal intraepithelial carcinoma (STIC) is the immediate precursor lesion for high-grade serous ovarian carcinoma (HGSOC) and harbors universal TP53 mutations. The lack of an appropriate in vitro model for STIC presents a major challenge in studying its pathogenesis. We aimed to develop a human in vitro model that mimics STIC lesions.

METHODS: Using CRISPR-Cas9 gene editing, we generated human fallopian tube epithelial organoids with TP53 loss-of-function mutations (TP53[-/-] FTOs). We characterized TP53[-/-] FTOs on a cellular and molecular level using immunofluorescence confocal imaging, copy number variation (CNV) analysis, and RNA sequencing.

RESULTS: TP53[-/-] FTOs recapitulated key features of STIC lesions. They exhibited increased proliferation and nuclear abnormalities, including nuclear enlargement and atypical mitotic figures. Copy number variation analysis revealed aneuploidy in some TP53[-/-] FTOs. Compared to unedited controls, TP53[-/-] FTOs demonstrated significant transcriptomic changes, including the downregulation of DNA repair genes and upregulation of epithelial-mesenchymal transition (EMT) pathways. Similar to STIC lesions, TP53[-/-] FTOs showed a marked reduction in ciliated cells and ciliogenesis-associated gene expression.

CONCLUSIONS: These findings suggest that p53 loss in FTOs promotes a proliferative and genomically unstable state that is conducive to carcinogenesis. The TP53[-/-] FTO model we have generated provides a valuable tool for studying early events in ovarian carcinogenesis and for developing new strategies for the early detection and prevention of ovarian cancer.},
}

Humans
Female
*Tumor Suppressor Protein p53/genetics
*Organoids/pathology
*Fallopian Tubes/pathology
*Fallopian Tube Neoplasms/genetics/pathology
Mutation
*Carcinoma in Situ/genetics/pathology
*Ovarian Neoplasms/genetics/pathology
DNA Copy Number Variations
*Cystadenocarcinoma, Serous/genetics/pathology
CRISPR-Cas Systems
Gene Editing

@article {pmid41251373,
year = {2025},
author = {Pérez Antón, E and Dujeancourt-Henry, A and Rotureau, B and Glover, L},
title = {A CRISPR-based diagnostic tool to survey drug resistance in human African trypanosomiasis.},
journal = {Antimicrobial agents and chemotherapy},
volume = {69},
number = {12},
pages = {e0093325},
doi = {10.1128/aac.00933-25},
pmid = {41251373},
issn = {1098-6596},
support = {ANR-PRC 2021 SherPa//Agence Nationale de la Recherche/ ; LabEx IBEID//Labex/ ; },
mesh = {Humans ; *Trypanosomiasis, African/drug therapy/parasitology/diagnosis ; *Trypanocidal Agents/pharmacology ; *Drug Resistance/genetics ; *Trypanosoma brucei gambiense/drug effects/genetics ; Pentamidine/pharmacology/therapeutic use ; Melarsoprol/pharmacology/therapeutic use ; *CRISPR-Cas Systems/genetics ; Mutation ; Protozoan Proteins/genetics ; },
abstract = {The World Health Organization aims to eliminate human African trypanosomiasis caused by Trypanosoma brucei gambiense (gHAT) by 2030. With the decline of reported cases, maintaining active surveillance is essential, including for the potential emergence of drug-resistant parasites. We have developed new highly specific diagnostic tools, using the Cas13a-based Specific High-Sensitivity Reporter Enzymatic UnLOCKing (SHERLOCK) technology, for the detection of drug-resistant genotypes that (i) are already circulating, such as the AQP2/3(814) chimera providing resistance to pentamidine and melarsoprol or (ii) could emerge, such as the TbCPSF3 (N[232]H) mutation, associated with acoziborole resistance under laboratory conditions. The AQP2/3(814) SHERLOCK assay detected RNA from both cultured parasites and field strains isolated from gHAT patients who relapsed following melarsoprol or pentamidine treatment. The CPSF3(SNV) SHERLOCK assay discriminated between wild-type CPSF3 RNA and CPSF3 bearing a single A-C mutation that confers resistance to acoziborole in vitro. These SHERLOCK assays are amenable for use as a high-throughput screening method to monitor for drug-resistant-associated mutations in Trypanosoma brucei, providing a new molecular tool for epidemiological surveillance during the gHAT elimination phase.},
}

Humans
*Trypanosomiasis, African/drug therapy/parasitology/diagnosis
*Trypanocidal Agents/pharmacology
*Drug Resistance/genetics
*Trypanosoma brucei gambiense/drug effects/genetics
Pentamidine/pharmacology/therapeutic use
Melarsoprol/pharmacology/therapeutic use
*CRISPR-Cas Systems/genetics
Mutation
Protozoan Proteins/genetics

@article {pmid41237780,
year = {2025},
author = {Sivakumar, S and Wang, Y and Goetsch, SC and Pandit, V and Wang, L and Zhao, H and Sundarrajan, A and Armendariz, D and Takeuchi, C and Deng, M and Nzima, M and Chen, WC and Dederich, AE and El Hayek, L and Gao, T and Gogate, A and Kaur, K and Kim, HB and McCoy, MK and Niederstrasser, H and Oura, S and Pinzon-Arteaga, CA and Sanghvi, M and Schmitz, DA and Yu, L and Zhang, Y and Zhou, Q and Kraus, WL and Xu, L and Wu, J and Posner, BA and Chahrour, MH and Hon, GC and Munshi, NV},
title = {Benchmarking and optimizing Perturb-seq in differentiating human pluripotent stem cells.},
journal = {Stem cell reports},
volume = {20},
number = {12},
pages = {102713},
doi = {10.1016/j.stemcr.2025.102713},
pmid = {41237780},
issn = {2213-6711},
mesh = {Humans ; *Cell Differentiation/genetics ; *Pluripotent Stem Cells/cytology/metabolism ; Benchmarking ; Gene Regulatory Networks ; Myocytes, Cardiac/cytology/metabolism ; CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Perturb-seq is a powerful approach to systematically assess how genes and enhancers impact the molecular and cellular pathways of development and disease. However, technical challenges have limited its application in stem-cell-based systems. Here, we benchmarked Perturb-seq across multiple CRISPRi modalities, on diverse genomic targets, in multiple human pluripotent stem cells, during directed differentiation to multiple lineages, and across multiple single guide RNA (sgRNA) delivery systems. To ensure cost-effective production of large-scale Perturb-seq datasets as part of the Impact of Genomic Variants on Function (IGVF) consortium, our optimized protocol dynamically assesses experiment quality across the weeks-long procedure. Our analysis of 1,996,260 sequenced cells across benchmarking datasets reveals shared regulatory networks linking disease-associated enhancers and genes with downstream targets during cardiomyocyte differentiation. This study establishes open tools and resources for interrogating genome function during stem cell differentiation.},
}

Humans
*Cell Differentiation/genetics
*Pluripotent Stem Cells/cytology/metabolism
Benchmarking
Gene Regulatory Networks
Myocytes, Cardiac/cytology/metabolism
CRISPR-Cas Systems/genetics
High-Throughput Nucleotide Sequencing/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41218364,
year = {2025},
author = {Huang, X and Xiao, T and Zhao, X and Yang, Z and Sun, Z and Lu, K},
title = {Olfactory perception of trifluralin by GOBP2 decreases the susceptibility of Spodoptera litura to insecticides through modulation of 20E signaling pathway-mediated metabolic detoxification.},
journal = {Journal of hazardous materials},
volume = {500},
number = {},
pages = {140417},
doi = {10.1016/j.jhazmat.2025.140417},
pmid = {41218364},
issn = {1873-3336},
mesh = {Animals ; *Insecticides/toxicity/pharmacology ; *Spodoptera/drug effects/metabolism/genetics ; Signal Transduction/drug effects ; Inactivation, Metabolic ; *Trifluralin/pharmacology/toxicity ; Larva/drug effects/metabolism ; *Insect Proteins/genetics/metabolism ; Chlorpyrifos/toxicity ; CRISPR-Cas Systems ; *Herbicides/toxicity/pharmacology ; Oxazines ; },
abstract = {Herbicide contamination has emerged as a critical ecological concern due to its persistent environmental accumulation and unintended impacts on non-target species. This study reveals that olfactory exposure to the volatile herbicide trifluralin enhances metabolic detoxification in Spodoptera litura larvae, significantly reducing their susceptibility to indoxacarb and chlorpyrifos. Building on our previous discovery of GOBP2's trifluralin-binding capacity, CRISPR/Cas9-generated GOBP2 knockout (GOBP2[KO]) larvae exhibited compromised loss of cross-tolerance, accompanied by impaired induction of carboxylesterases (COEs) and glutathione S-transferases (GSTs). Mechanistically, trifluralin perception through GOBP2 activated a 20-hydroxyecdysone (20E)-dependent pathway, upregulating both 20E biosynthesis genes and ecdysone receptor (EcR)/ultraspiracle (USP) complexes in wild-type insects. Systemic disruption of this signaling axis via RNAi-mediated EcR/USP co-silencing abolished trifluralin-induced detoxification enzyme activation and restored insecticide vulnerability. Molecular validation through dual-luciferase assays and yeast-one hybrid systems confirmed EcR/USP-mediated transactivation of COE/GSTe genes via conserved response elements. Functional characterization identified GSTe1 and GSTe16 as key effectors, with GSTe16 exhibiting effective metabolic efficiency against indoxacarb (19.25 % degradation) and chlorpyrifos (13.04 % degradation), while GSTe1 showed significant activity toward indoxacarb metabolism (14.96 %) and chlorpyrifos degradation (10.17 %). Genetic evidence from CRISPR/Cas9-ablated S. litura and transgenic Drosophila models further established causal relationships between GSTe expression levels and insecticide tolerance. Our integrated analysis establishes that GOBP2-mediated endocrine signaling constitutes a central axis in trifluralin-induced insecticide tolerance, directly bridging herbicide perception to metabolic resistance through 20E-dependent regulatory cascades.},
}

Animals
*Insecticides/toxicity/pharmacology
*Spodoptera/drug effects/metabolism/genetics
Signal Transduction/drug effects
Inactivation, Metabolic
*Trifluralin/pharmacology/toxicity
Larva/drug effects/metabolism
*Insect Proteins/genetics/metabolism
Chlorpyrifos/toxicity
CRISPR-Cas Systems
*Herbicides/toxicity/pharmacology
Oxazines

@article {pmid41055127,
year = {2025},
author = {Lin, Y and Ye, X and Zeng, L and Luo, Y and Feng, Y and Zhang, Y},
title = {Heat shock-optimized CRISPR/Cas9 system for visible clonal analysis and mutant generation in Drosophila.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {12},
pages = {},
doi = {10.1093/g3journal/jkaf236},
pmid = {41055127},
issn = {2160-1836},
support = {LY21C070003//Natural Science Foundation of Zhejiang Province/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Heat-Shock Response/genetics ; *Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Drosophila/genetics ; Drosophila Proteins/genetics ; Clone Cells ; *Drosophila melanogaster/genetics ; Gene Editing ; },
abstract = {In Drosophila genetic studies, clonal analysis such as mosaic and Mosaic Analysis with a Repressible Cell Marker has been widely used to investigate gene function. Recently, the CRISPR/Cas9 system has been established as a powerful tool for efficient mutant generation; however, its application in clonal analysis has been rarely reported. Here, we present a suite of Gal4/UAS-Cas9 binary expression systems that integrate UAS-Cas9 and multiple-sgRNAs (single-guide RNAs) into a single plasmid. These systems facilitate versatile applications, enabling Gal4-driven direct phenotypic studies, approximate clonal analysis, in vitro cell transfection, and stable mutant generation, among which, the third-generation constructs: G3a/b incorporate visible labeling strategies for marking approximate clonal regions. In addition, compared to continuously active drivers, we found that the short-pulse-induced heat shock-Gal4 (hs-Gal4) was sufficient to induce high clonal efficiency and generate larger clones. In the germline, short-pulse heat shock is also effective. It reduces residual Cas9 activity in the germline stem cells, thereby minimizing the risk of affecting germline stem cell survival and improving mutant acquisition.},
}

Animals
*CRISPR-Cas Systems
*Heat-Shock Response/genetics
*Mutation
RNA, Guide, CRISPR-Cas Systems/genetics
*Drosophila/genetics
Drosophila Proteins/genetics
Clone Cells
*Drosophila melanogaster/genetics
Gene Editing

@article {pmid41052774,
year = {2025},
author = {Salama, R and Peet, E and Morrione, TL and Durant, S and Seager, M and Rennie, M and Scarlata, S and Nechipurenko, I},
title = {Functional classification of GNAI1 disorder variants in Caenorhabditis elegans uncovers conserved and cell-specific mechanisms of dysfunction.},
journal = {Genetics},
volume = {231},
number = {4},
pages = {},
doi = {10.1093/genetics/iyaf216},
pmid = {41052774},
issn = {1943-2631},
support = {//Charles H. Hood Foundation/ ; //Child Health Research Award/ ; R35 GM155316/NH/NIH HHS/United States ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics/metabolism ; Cilia/metabolism/genetics ; *GTP-Binding Protein alpha Subunits, Gi-Go/genetics/metabolism ; Humans ; *Caenorhabditis elegans Proteins/genetics/metabolism ; Neurons/metabolism ; CRISPR-Cas Systems ; Mutation ; Phenotype ; },
abstract = {Heterotrimeric G proteins transduce signals from G protein-coupled receptors, which mediate key aspects of neuronal development and function. Mutations in the GNAI1 gene, which encodes Gαi1, cause a disorder characterized by developmental delay, intellectual disability, hypotonia, and epilepsy. However, the mechanistic basis for this disorder remains unknown. Here, we show that GNAI1 is required for ciliogenesis in human cells and use Caenorhabditis elegans as a whole-organism model to determine the functional impact of 7 GNAI1-disorder patient variants. Using CRISPR-Cas9 editing in combination with robust cellular (cilia morphology) and behavioral (chemotaxis) assays, we find that T48I, K272R, A328P, and V334E orthologous variants impact both cilia assembly and function in AWC neurons, M88V and I321T have no impact on either phenotype, and D175V exerts neuron-specific effects on cilia-dependent sensory behaviors. Finally, we validate in human ciliated cell lines that D173V, K270R, and A326P GNAI1 variants disrupt ciliary localization of the encoded human Gαi1 proteins similarly to their corresponding orthologous substitutions in the C. elegans ODR-3 (D175V, K272R, and A328P). Overall, our findings determine the in vivo effects of orthologous GNAI1 variants and contribute to the mechanistic understanding of GNAI1-disorder pathogenesis as well as neuron-specific roles of ODR-3 in sensory biology.},
}

Animals
*Caenorhabditis elegans/genetics/metabolism
Cilia/metabolism/genetics
*GTP-Binding Protein alpha Subunits, Gi-Go/genetics/metabolism
Humans
*Caenorhabditis elegans Proteins/genetics/metabolism
Neurons/metabolism
CRISPR-Cas Systems
Mutation
Phenotype

@article {pmid40991383,
year = {2025},
author = {Suter, A and Graham, A and Kuah, JY and Crisologo, J and Gunatilake, C and Sourris, K and See, M and Rossello, FJ and Ramialison, M and Vlahos, K and Howden, SE},
title = {Efficient Installation of Heterozygous Mutations in Human Pluripotent Stem Cells Using Prime Editing.},
journal = {The CRISPR journal},
volume = {8},
number = {6},
pages = {401-411},
doi = {10.1177/25731599251380122},
pmid = {40991383},
issn = {2573-1602},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Mutation ; *Pluripotent Stem Cells/metabolism/cytology ; Heterozygote ; Induced Pluripotent Stem Cells/metabolism/cytology ; Fibroblasts/metabolism/cytology ; Leukocytes, Mononuclear/cytology ; Gene Knock-In Techniques ; Cellular Reprogramming ; },
abstract = {The utility of human pluripotent stem cells (hPSCs) is greatly enhanced by the ability to introduce precise, site-specific genetic modifications with minimal off-target effects. Although Cas9 endonuclease is an exceptionally efficient gene-editing tool, its propensity for generating biallelic modifications often limits its capacity for introducing heterozygous variants. Here, we use prime editing (PE) to install heterozygous edits in over 10 distinct genetic loci, achieving knock-in efficiencies of up to 40% without the need for subsequent purification or drug selection steps. Moreover, PE enables the precise introduction of heterozygous edits in paralogous genes that are otherwise extremely challenging to achieve using endonuclease-based editing approaches. We also show that PE can be successfully combined with reprogramming to derive heterozygous induced pluripotent stem cell clones directly from human fibroblasts and peripheral blood mononuclear cells. Our findings highlight the utility of PE for generating hPSCs with complex edits and represent a powerful platform for modeling disease-associated dominant mutations and gene-dosage effects in an entirely isogenic context.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Mutation
*Pluripotent Stem Cells/metabolism/cytology
Heterozygote
Induced Pluripotent Stem Cells/metabolism/cytology
Fibroblasts/metabolism/cytology
Leukocytes, Mononuclear/cytology
Gene Knock-In Techniques
Cellular Reprogramming

@article {pmid40971219,
year = {2025},
author = {Basharat, R and Rizzo, G and Zoodsma, JD and Wollmuth, LP and Sirotkin, HI},
title = {Optimizing Prime Editing in Zebrafish.},
journal = {The CRISPR journal},
volume = {8},
number = {6},
pages = {426-435},
doi = {10.1177/25731599251380500},
pmid = {40971219},
issn = {2573-1602},
mesh = {*Zebrafish/genetics ; Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mutation, Missense ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; RNA, Messenger/genetics ; },
abstract = {Prime editing is a clustered regularly interspaced short palindromic repeats-based approach that enables the introduction of precise genetic modifications, including missense mutations, making it valuable for generating disease models. The comparative performance of novel prime editor (PE) variants in zebrafish remains largely unexplored. Here, we systematically evaluated the efficiency of five PEs-PE2, PE6b, PE6c, PEmax, and PE7-in zebrafish. We tested mRNA encoding for each of these PEs with prime editing guide RNAs (pegRNAs) designed to install five missense mutations. Efficient editing was achieved at four of the five sites with multiple PEs. Among these, PEmax emerged as the most efficient editor for introducing pure prime edits, with rates reaching 15.34%. We found that strategies proposed to block 3' degradation of pegRNAs (epegRNAs and addition of a La RNA binding motif to the PE) did not improve performance in our assays. Together, these findings establish PEmax as a robust tool to introduce missense mutations into zebrafish.},
}

*Zebrafish/genetics
Animals
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Mutation, Missense
RNA, Guide, CRISPR-Cas Systems/genetics
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
RNA, Messenger/genetics

@article {pmid40810623,
year = {2025},
author = {Salemdawod, A and Cooper, B and Liang, Y and Walczak, P and Vatter, H and Maciaczyk, J and Janowski, M},
title = {CRISPR-Cas9 Single Nucleotide Editing of Tuberous Sclerosis Complex 2 Gene in Mesenchymal Stem Cells.},
journal = {The CRISPR journal},
volume = {8},
number = {6},
pages = {412-425},
doi = {10.1177/25731599251367059},
pmid = {40810623},
issn = {2573-1602},
mesh = {Humans ; *Mesenchymal Stem Cells/metabolism ; *Tuberous Sclerosis Complex 2 Protein/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Point Mutation ; Tuberous Sclerosis ; },
abstract = {The tuberous sclerosis complex (TSC)2 gene regulates the mammalian target of rapamycin (mTOR) pathway, impacting cell proliferation and growth. The loss-of-function mutations, especially in mesenchymal progenitors, drive the development multiple benign and malignant tumors. TSC2 mutations in certain cancer types, e.g., breast cancer, are also associated with poorer prognosis. The databases of TSC2-mutations report point mutations as the most prevalent. We aimed to test the feasibility of inducing point mutations in mesenchymal stem cells (MSCs), targeting the most frequent point mutations of the TSC2 gene, TSC2. c.1864 C>T (p.Arg622Trp), TSC2. c.1832 G>A (p.Arg611Glu), and TSC2. c.5024 C>T (p.Pro1675Leu) using two delivery methods for CRISPR-Cas9. We report a high editing efficiency of up to 85% inducing TSC2 point mutations in hMSCs using lipofectamine-based transfection. Overall, the high editing efficiency of some TSC2 mutations enables the induction and reversal of mutations in primary hMSCs without needing resource-consuming derivation of cell lines frequently distinct from their primary counterparts.},
}

Humans
*Mesenchymal Stem Cells/metabolism
*Tuberous Sclerosis Complex 2 Protein/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Point Mutation
Tuberous Sclerosis

@article {pmid41359128,
year = {2025},
author = {Li, Z and Cheng, Y and Li, C and Wu, Q and Xin, Y},
title = {Harnessing microalgae for bioproducts: innovations in synthetic biology.},
journal = {World journal of microbiology & biotechnology},
volume = {41},
number = {12},
pages = {500},
pmid = {41359128},
issn = {1573-0972},
support = {32560020 and 31600059//National Natural Science Foundation of China/ ; RZ2300002678//Start-Up Funds of Hainan University/ ; DC2300001799//Open Project of State Key Laboratory of Marine Resource Utilization in South China Sea/ ; 2018YFA0902500//National Key Research and Development Program of China/ ; },
mesh = {*Microalgae/metabolism/genetics ; *Synthetic Biology/methods ; Biofuels ; *Metabolic Engineering/methods ; Gene Editing ; Lipids/biosynthesis ; CRISPR-Cas Systems ; Metabolic Networks and Pathways ; Photobioreactors ; },
abstract = {Microalgae are increasingly recognized as versatile platforms for sustainable production of biofuels and high-value bioproducts such as lipids, carotenoids and polyunsaturated fatty acids. Rapid progress in synthetic biology is transforming microalgal engineering by enabling precise rewiring of metabolic pathways and overcoming long-standing technical bottlenecks, particularly those related to transformation efficiency, genetic stability and strain scalability. Recent innovations (including CRISPR/Cas genome editing, modular cloning systems, synthetic promoter libraries and dynamic, environment-responsive regulatory circuits) have greatly expanded the genetic toolset available for both model and recalcitrant species. These advances support targeted control of lipid and pigment biosynthesis, improved flux distribution and more robust performance under industrially relevant conditions. When integrated with progress in photobioreactor design, automated cultivation, and process intensification, synthetic biology unlocks new potential for scalable, economically viable microalgal biomanufacturing. This review summarizes these developments, highlights remaining challenges in strain robustness and bioprocess translation, and outlines future pathways toward high-performance microalgal biofactories that can contribute meaningfully to a low-carbon, bio-based economy.},
}

*Microalgae/metabolism/genetics
*Synthetic Biology/methods
Biofuels
*Metabolic Engineering/methods
Gene Editing
Lipids/biosynthesis
CRISPR-Cas Systems
Metabolic Networks and Pathways
Photobioreactors

@article {pmid41356798,
year = {2026},
author = {Birappa, G and Perumalsamy, H and Hong, SH and Gowda, DAA and Chandrasekaran, AP and Karapurkar, JK and Rajkumar, S and Balusamy, SR and Jayachandran, A and Baek, KH and Lee, J and Matam, V and Kim, WJ and Kim, KS and Ramakrishna, S and Suresh, B},
title = {Single-cell RNA sequence analysis reveals USP32 as a therapeutic target to mitigate PD-L1-driven colorectal tumorigenesis in vitro and in vivo.},
journal = {Theranostics},
volume = {16},
number = {2},
pages = {986-1005},
pmid = {41356798},
issn = {1838-7640},
mesh = {Humans ; Animals ; *B7-H1 Antigen/metabolism/genetics ; *Colorectal Neoplasms/genetics/pathology/metabolism ; Mice ; *Ubiquitin Thiolesterase/genetics/metabolism ; Single-Cell Analysis/methods ; Ubiquitination ; *Carcinogenesis/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Sequence Analysis, RNA ; },
abstract = {Background: The expression levels of the programmed death-ligand 1 (PD-L1) protein serves as a prognostic indicator for patients with colorectal cancer (CRC). Advancement of CRC is facilitated by deubiquitinating enzymes (DUBs), which regulate oncoprotein levels via the ubiquitin-proteasomal pathway. The post-translational regulatory mechanisms governing PD-L1 protein abundance on CRC, in relation to different tumor grades and their clinical relevance, remains unknown. Methods: We analyzed single-cell RNA sequencing (scRNA-seq) data to identify DUB genes associated with PD-L1 expression in CRC. We used a loss-of-function-based CRISPR/Cas9 library to identify putative DUB genes that regulate the PD-L1 protein level. Immunoprecipitation was used to confirm the interaction between the USP32 and PD-L1 along with its ubiquitination status. A series of in vitro and in vivo carcinogenesis-related experiments were conducted to determine the clinical relevance between USP32 and PD-L1 expression in CRC progression. Results: In this study, we analyzed scRNA-seq data from extensive cohorts of human and mice at the single-cell level to identify DUB genes associated with PD-L1 expression in CRC. Our analysis identified multiple putative DUBs, including USP32 and USP12, as prognostic markers associated with PD-L1 expression, which was found to be elevated in T cells, macrophages, and classical monocytes cell types in patients with CRC. A secondary screening using CRISPR/Cas9-mediated loss-of-function analysis for DUBs found that USP32 modulates PD-L1 protein levels in CRC. Furthermore, we demonstrated that USP32 interacts with, stabilizes, and extends the half-life of PD-L1 by preventing its K-48-linked polyubiquitination as an underlying mechanism that contributes for tumorigenesis. Conclusion: A combination of scRNA-seq analysis and wet-lab experimental validation confirmed that USP32 mediates PD-L1 protein stabilization in colon cancer, identifying it as a potential therapeutic target for CRC. CRISPR/Cas9-mediated targeted knockout of the USP32 gene reduced PD-L1 protein levels and significantly mitigated colorectal cell proliferation and tumorigenesis, both in vitro and in vivo, in a xenograft mouse model, underscoring a novel and alternative approach to the treatment of CRC.},
}

Humans
Animals
*B7-H1 Antigen/metabolism/genetics
*Colorectal Neoplasms/genetics/pathology/metabolism
Mice
*Ubiquitin Thiolesterase/genetics/metabolism
Single-Cell Analysis/methods
Ubiquitination
*Carcinogenesis/genetics
Cell Line, Tumor
Gene Expression Regulation, Neoplastic
CRISPR-Cas Systems
Sequence Analysis, RNA

@article {pmid41356473,
year = {2025},
author = {Wei, C and Chen, Z},
title = {Comprehensive analysis of phage genomes from diverse environments reveals their diversity, potential applications, and interactions with hosts and other phages.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1686402},
pmid = {41356473},
issn = {1664-302X},
abstract = {Phages are ubiquitous and diverse, playing a key role in maintaining microbial ecosystem balance. However, their diversity, potential applications, and their interactions with hosts and other phages remain largely unexplored. To address this, we collected 59,652,008 putative viral genomes from our laboratory, 45 public viral datasets, and an integrated public viral genome database (IGN), covering seven habitats. We obtained 741,692 phage genomes with completeness ≥50% (PGD50), and most (93.83%, 695,938/741,692) of these phage genomes were classified into the Caudoviricetes class. We found that 158,522 species-level viral clusters that contained 28.96% (214,814/741,692) phage genomes without any known phage genomes in the IGN, indicating substantial novelty. Global phylogenetic trees for five iterations based on complete phage genomes significantly expanded the known diversity of the virosphere. Genome analysis revealed phage potential divergence with habitat types and highlighted the utilization of alternative genetic codes. Furthermore, 3D structural similarity searches demonstrated significant potential for annotating previously uncharacterized viral proteins. Analysis of CRISPR spacer inferred potential hosts of phages and competitive networks among phages, highlighting virulent phages as promising candidates for phage therapy against pathogenic bacteria. Intriguingly, diverse CRISPR-Cas systems were detected within phage genomes themselves, suggesting their enormous potential as novel gene editing tools. Collectively, this study provides a comprehensive phage genome resource, foundational for future research into phage-host and phage-phage interactions, phage therapy development, and the mining of next-generation genetic tools.},
}

@article {pmid41356196,
year = {2026},
author = {Zhang, Y and Deng, Q and Xu, Y and Wu, W and Wu, T and Huang, J and Hu, Y and Lin, W and Xu, X and Wu, J},
title = {ROS-responsive cellular vesicles with ferroptosis-targeting siACMSD delivery for acute kidney injury therapy.},
journal = {Theranostics},
volume = {16},
number = {4},
pages = {1941-1958},
pmid = {41356196},
issn = {1838-7640},
mesh = {*Ferroptosis/drug effects ; Animals ; *Reactive Oxygen Species/metabolism ; *Acute Kidney Injury/therapy/metabolism/drug therapy/pathology ; Mice ; Humans ; Cisplatin/pharmacology ; *Carboxy-Lyases/genetics/metabolism ; Disease Models, Animal ; Cell Line ; Male ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Mitochondria/metabolism/drug effects ; },
abstract = {Background: Acute kidney injury (AKI) is a severe and prevalent nephrotic syndrome which lack of definitive therapies. Alpha-amino-β-carboxymuconic acid-ε-semialdehyde decarboxylase (ACMSD) is a metabolic enzyme mainly expressed in the kidney which exacerbated AKI injury by promoting TCA cycle and inhibiting nicotinamide adenine dinucleotide (NAD[+]) production, whereas lack of effective intervention strategies for ACMSD-targeted therapy. Methods: Herein, we knocked out ACMSD in vitro through CRISPR-Cas9 method, and developed a reactive oxygen species (ROS)-responsive neutrophil-derived cellular vesicles (CVs) drugs (RNAi@ROS-CVs), which efficiently mediated ACMSD knockdown in vivo, exploring the mechanism of ACMSD-induced ferroptosis process in AKI. Results: ACMSD knockout effectively alleviated cisplatin (CP)-induced mitochondrial damage, suppressed TCA cycle progression, promoted NAD[+] synthesis, and inhibited ferroptosis in HK2 cells. In mice AKI model, RNAi@ROS-CVs effectively targeted the injured kidneys, downregulated ACMSD expression in renal tubular epithelial cells, reduced ROS production and lipid peroxidation, and alleviated CP or ischemia/reperfusion (I/R)-induced ferroptosis. Conclusion: These findings highlight the therapeutic potential of ACMSD-targeted knockout in AKI intervention and introduce a versatile and efficient controlled-release drug delivery platform for AKI-targeted therapy, with potential applicability to other acute renal diseases.},
}

*Ferroptosis/drug effects
Animals
*Reactive Oxygen Species/metabolism
*Acute Kidney Injury/therapy/metabolism/drug therapy/pathology
Mice
Humans
Cisplatin/pharmacology
*Carboxy-Lyases/genetics/metabolism
Disease Models, Animal
Cell Line
Male
Mice, Inbred C57BL
CRISPR-Cas Systems
Mitochondria/metabolism/drug effects

@article {pmid41356190,
year = {2026},
author = {Zhong, XY and Yang, YX and Xiong, YF and Ye, GC and Gong, X and Zhong, ML and He, HD and Wang, SG and Xia, QD},
title = {Programmable molecular microscopy: CRISPR/Cas fluorescent probes revolutionizing spatiotemporal genomic imaging.},
journal = {Theranostics},
volume = {16},
number = {4},
pages = {1877-1904},
pmid = {41356190},
issn = {1838-7640},
mesh = {*CRISPR-Cas Systems/genetics ; *Fluorescent Dyes ; Humans ; Animals ; *Molecular Imaging/methods ; *Genomics/methods ; Gene Editing/methods ; },
abstract = {Bioimaging technologies visually resolve spatiotemporal dynamics of biomolecules, cells, and tissues, enabling essential insights into gene regulation, disease mechanisms, and drug metabolism. CRISPR/Cas-based fluorescent probes transform CRISPR from "genetic scissors" into "molecular microscopes," providing an indispensable tool for in situ decoding of molecular events in living systems. Their high nucleic acid specificity establishes CRISPR/Cas as a pivotal technology for dynamically monitoring genomic and transcriptomic events at live-cell and in vivo levels. This work systematically outlines design strategies and functional mechanisms of mainstream CRISPR/Cas fluorescent probes for bioimaging, encompassing five categories: fluorescent proteins, synthetic dyes, smart gated probes, nanomaterials, and multimodal integrated probes. Recent advances and persistent challenges in achieving high-sensitivity targeted imaging, effective signal amplification, and precise delivery control are comprehensively examined, including analysis of their advantages, limitations, and adaptability in complex biological environments. Building on breakthroughs in in vivo delivery systems, diverse carriers demonstrate significant potential for enhancing CRISPR/Cas transport efficiency, improving tissue penetration, and enabling spatiotemporal controlled release. Continued innovation drives CRISPR/Cas imaging platforms toward higher sensitivity, enhanced biocompatibility, and multifunctional integration, thereby fostering the convergence and broad application of gene editing and molecular diagnostics.},
}

*CRISPR-Cas Systems/genetics
*Fluorescent Dyes
Humans
Animals
*Molecular Imaging/methods
*Genomics/methods
Gene Editing/methods

@article {pmid41354981,
year = {2025},
author = {Das, T and Barman, T and Prasad, A},
title = {Precision editing to improve fruit traits: CRISPR/Cas into the picture.},
journal = {Protoplasma},
volume = {},
number = {},
pages = {},
pmid = {41354981},
issn = {1615-6102},
abstract = {Crop growth, quality, and yield can be adversely affected by various biotic and abiotic stresses. Crop characteristics can be improved with conventional breeding and other variation-based breeding strategies. However, these strategies are time as well as resource consuming and to overcome this, novel approaches are necessary. CRISPR/Cas technique allows to improve desired traits more efficiently and accurately by targeting specific genes. Genome editing has become more versatile with CRISPR/Cas systems and is a valuable tool to protect food security by developing commercial crops optimized for yield and nutritional quality. Researchers are able to target and edit stress response pathway genes to develop crops with increased tolerance to stress. A lack of regeneration protocols and sufficient genome sequencing data has restricted fruit editing to only a few fruits (tomatoes, citrus, apple, kiwi, banana, grapes, strawberries, watermelon, etc.). This review is focused on CRISPR/Cas applications on the nutritional aspects of fruit engineering along with the challenges and opportunities. Another aspect which will be covered is the use of CRISPR/Cas technology to improve fruit resilience to biotic and abiotic stress, but not at the cost of yield. We discuss the pros and cons of using this technology, such as unintended effects on fruit traits or public concerns about GMOs. We conclude that the application of CRISPR/Cas9 technology has the potential to be of great benefit to the agricultural industry not only to improve nutritional aspects but also to help reduce crop losses.},
}

@article {pmid41354953,
year = {2025},
author = {Nguyen, VT and Van, BTT and Uyen, TN and Tong, NX and Pham, TL and Vy, NHT and Thuy, DT and Thuy, NP and Kobayashi, M},
title = {Functional divergence of zebrafish keap1 paralogs revealed by CRISPR/Cas9-mediated gene editing: a specialized role for keap1b in inflammation.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {53},
pmid = {41354953},
issn = {1573-9368},
support = {108.06-2020.19//National Foundation for Science and Technology Development/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Oxidative Stress/genetics ; *Inflammation/genetics ; Kelch-Like ECH-Associated Protein 1/genetics ; NF-E2-Related Factor 2/genetics ; Signal Transduction/genetics ; Gene Editing ; Carrier Proteins ; },
abstract = {The Keap1/Nrf2 signaling pathway is a master regulator of cellular defense against oxidative and electrophilic stress. In teleosts like zebrafish (Danio rerio), whole-genome duplication resulted in two keap1 paralogs, keap1a and keap1b, whose functional specificities remain incompletely understood. This study investigates the divergent roles of these paralogs by comparing the responses of established keap1a and novel keap1b knockout larvae to distinct chemical stressors. By comparing the responses of keap1b[dl40], keap1a[dl07], and nfe2l2a[dl703] (Nrf2a) larvae to these stressors, we uncovered a striking functional dichotomy. While loss of either paralog conferred resistance to H2O2-induced oxidative stress, keap1b[dl40] larvae, unlike their keap1a[dl07] counterparts, exhibited extreme sensitivity to the lethal effects of CuSO4 exposure, with survival rates plummeting to ~ 25%. This heightened sensitivity to copper sulfate was associated with a blunted transcriptional response of inflammatory markers tnf-a and c3a, suggesting that Keap1b is critical for modulating the Nrf2a-mediated response to inflammatory stress in orchestrating a viable inflammatory response. This work clarifies the non-redundant, vital function of Keap1b in the response to heavy metal-induced stress and provides a valuable genetic resource (keap1b[dl40] null allele) for future studies.},
}

Animals
*Zebrafish/genetics
*Zebrafish Proteins/genetics/metabolism
*CRISPR-Cas Systems/genetics
Oxidative Stress/genetics
*Inflammation/genetics
Kelch-Like ECH-Associated Protein 1/genetics
NF-E2-Related Factor 2/genetics
Signal Transduction/genetics
Gene Editing
Carrier Proteins

@article {pmid41353974,
year = {2025},
author = {Madny, MA and Yadav, KS},
title = {Biomimetic oral drug delivery: Translating nature's design into therapeutic innovation.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {259},
number = {},
pages = {115348},
doi = {10.1016/j.colsurfb.2025.115348},
pmid = {41353974},
issn = {1873-4367},
abstract = {Oral drug delivery, the most patient friendly administration route offers convenience and compliance but faces formidable biological barriers. Enzymatic degradation, mucosal entrapment, efflux transport and extensive first-pass metabolism drastically reduce the effectiveness of sensitive therapeutics including peptides, proteins, nucleic acids and vaccines. Conventional formulations often fail to overcome these challenges highlighting the need for innovative approaches. Biomimetic drug delivery has emerged as a transformative strategy. By emulating structures and functions from cells, membranes, exosomes, viruses and gut microbiota these systems achieve immune evasion, mucus penetration, site-specific targeting and stimulus-responsive release. Such approaches improve formulation stability and in vivo absorption but also promise precise and patient centric therapies. This review provides a comprehensive overview of biomimetic oral systems highlighting their mechanisms, design principles and translational potential. Recent advances include cell membrane-coated nanoparticles for tumor targeting and immune modulation, exosome-inspired carriers for protein and RNA transport, virus-like particles (VLPs) for oral vaccines, and mucoadhesive or mucus-penetrating polymers modeled on pathogen strategies. Complementary pH, enzyme and redox-responsive platforms exploit gastrointestinal (GI) microenvironments to ensure controlled release. Emerging tools such as bioinspired computational modeling, 3D/4D printing, organoid-on-chip models and CRISPR/Cas-based platforms accelerate optimization and clinical translation. Although most technologies remain in preclinical development, early findings demonstrate superior pharmacokinetics, therapeutic efficacy, and safety over conventional systems. This article critically examines biomimetic oral drug delivery addressing advances and underlying mechanisms including regulatory considerations and future directions. They stand poised to form the foundation of next-generation precision therapeutics.},
}

@article {pmid41352919,
year = {2026},
author = {Guan, X and Wang, S and Wang, P and Zhang, J and Sun, S},
title = {Enhanced chemiluminescence aptasensing with triple cascade amplification for sensitive detection of tumor-derived exosomes.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344873},
doi = {10.1016/j.aca.2025.344873},
pmid = {41352919},
issn = {1873-4324},
mesh = {*Exosomes/chemistry/metabolism ; Humans ; *Aptamers, Nucleotide/chemistry/metabolism ; *Luminescent Measurements/methods ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; Mucin-1 ; Alkaline Phosphatase/chemistry/metabolism ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Biomarkers, Tumor ; Tetraspanin 30 ; },
abstract = {BACKGROUND: Tumor-associated exosomes hold significant clinical promise as liquid biopsy biomarkers. However, the accurate detection of these rare exosome subpopulations in clinical samples demands analytical platforms with exceptionally high sensitivity and specificity. While conventional nucleic acid amplification-based methods provide considerable detection sensitivity, they are often hampered by time-consuming procedures, operational complexity, and susceptibility to contamination. Therefore, it is imperative to develop practical exosome measurement platforms that combine high sensitivity, robustness, and rapid analysis capabilities to provide reliable evidence-based support for precision oncology.

RESULTS: In this work, a triple cascade-amplified aptasensor (TCAA) via functionalized gold nanoparticle (fAuNP), CRISPR/Cas12a, and alkaline phosphatase (ALP) was developed for enhanced chemiluminescence (CL) assay of tumor-derived exosomes without nucleic acid amplification. The target exosomes were initially recognized by CD63 and MUC1 aptamers. fAuNP-conjugated Trigger sequences then activated CRISPR/Cas12a to cleave single-stranded DNA and release ALP. Consequently, the ALP catalyzed substrate to produce CL signals correlating with the concentration of the analyte. By simultaneously integrating the signal amplification capabilities of multiple techniques, this TCAA achieved a limit of detection of 44 particles/μL for MUC1-positive exosomes within 60 min with excellent robustness. Compared with the single- and dual-amplification methods, the sensitivity was increased by 40-fold and 6-fold, respectively. Clinical trials showed that the area under the curve of this approach was 0.96, which was higher than that of the commercialized chemiluminescence immunoassay and effectively distinguished breast cancer-derived specimens.

SIGNIFICANCE: These findings indicate that the TCAA strategy provides a highly sensitive, rapid, and robust tool for the detection of low-abundance tumor exosome subpopulations without nucleic acid amplification. It effectively addresses the limitations of conventional methods and demonstrates high clinical utility. This work offers a reliable and practical platform for non-invasive liquid biopsy, holding great potential for trace-level detection of diverse biomarkers.},
}

*Exosomes/chemistry/metabolism
Humans
*Aptamers, Nucleotide/chemistry/metabolism
*Luminescent Measurements/methods
Gold/chemistry
Metal Nanoparticles/chemistry
Limit of Detection
Mucin-1
Alkaline Phosphatase/chemistry/metabolism
Nucleic Acid Amplification Techniques
CRISPR-Cas Systems
*Biosensing Techniques/methods
Biomarkers, Tumor
Tetraspanin 30

@article {pmid41330004,
year = {2025},
author = {Coşar, B and Kılıç, P and İşeri, ÖD},
title = {The intersection of CAR-T immunotherapy with emerging technologies.},
journal = {Cytokine & growth factor reviews},
volume = {86},
number = {},
pages = {238-259},
doi = {10.1016/j.cytogfr.2025.11.001},
pmid = {41330004},
issn = {1879-0305},
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/immunology/genetics ; *Neoplasms/therapy/immunology ; Animals ; Cytokines/immunology ; *T-Lymphocytes/immunology ; CRISPR-Cas Systems ; Gene Editing ; Tumor Microenvironment/immunology ; },
abstract = {Chimeric antigen receptor (CAR) T-cell (CAR-T) therapy is a transformative modality in cancer immunotherapy that employs genetically engineered T-cells to eliminate malignant cells selectively. Its efficacy and limitations are governed by cytokine- and growth factor-mediated signaling networks that shape T-cell activation, proliferation, differentiation, and persistence. This review traces the molecular evolution of CAR-T architecture across generations, highlighting how synthetic modulation of cytokine and co-stimulatory pathways enhances potency while reducing exhaustion and toxicity. We discuss strategies that incorporate cytokine engineering, metabolic reprogramming, and logic-gated activation to counteract the immunosuppressive tumor microenvironment. Recent technological advances-such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based cytokine pathway editing, induced pluripotent stem cell (iPSC)-derived "off-the-shelf" CAR-T platforms, and extracellular vesicle (EV)-mediated cytokine delivery-are reshaping adoptive immunotherapy. Framing CAR-T development through the lens of cytokine and growth factor biology, we outline how integrating these pathways enables safer, more durable, and scalable next-generation therapies for hematologic and solid tumors.},
}

Humans
*Immunotherapy, Adoptive/methods
*Receptors, Chimeric Antigen/immunology/genetics
*Neoplasms/therapy/immunology
Animals
Cytokines/immunology
*T-Lymphocytes/immunology
CRISPR-Cas Systems
Gene Editing
Tumor Microenvironment/immunology

@article {pmid41292433,
year = {2025},
author = {Lan, F and Chen, A and Ding, Y and Yang, C and Zhang, P and Fang, X},
title = {Sensitive and Specific Analysis of miRNAs in Single Tumor-Derived Extracellular Vesicles Using CRISPR-Based Nanoflow Cytometry.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26521-26531},
doi = {10.1021/acs.analchem.5c04700},
pmid = {41292433},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Extracellular Vesicles/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *Prostatic Neoplasms/genetics/diagnosis ; *Flow Cytometry/methods ; Male ; Limit of Detection ; Biomarkers, Tumor/genetics ; *Nanotechnology ; },
abstract = {Tumor-derived extracellular vesicle (TEV) microRNAs (miRNAs) are promising cancer biomarkers but pose detection challenges due to their low abundance and sequence homology. Here, we present a CRISPR/Cas13a-based nanoflow cytometry (nFCM) platform integrated with a DNA-guided orthogonal membrane fusion strategy for ultrasensitive miRNA detection of TEVs at the single particle level. TEVs were identified with aptamers against CD63 and EpCAM markers to create an orthogonal barcode-anchored TEV (Orth-TEV). Meanwhile, liposomes preloaded with CRISPR/Cas13a molecular sensing components were modified with cholesterol-tagged DNA probes to produce Tags-CRISPR/Cas13a@Lipo. The complementary DNA sequences on the Orth-TEV and Tags-CRISPR/Cas13a@Lipo vesicles facilitated zipper-like hybridization, thereby achieving specific membrane fusion to effectively eliminate the interference of nontarget vesicles or free molecules. The resulting TEV-CRISPR/Cas13a@Lipo vesicles allow in situ detection of three prostate cancer (PCa)-associated miRNAs in a single TEV via nFCM with a low detection limit (LOD) of 14.7 (miR-153), 16.0 (miR-183), and 23.7 (miR-940) particles/mL, respectively. The approach was further applied to plasma samples from PCa patients and healthy donors, showing significantly elevated miRNA signals in PCa-derived TEV. ROC analysis yielded AUC values of 0.931, 0.923, and 0.869 for the three target miRNAs, confirming excellent diagnostic performance. To enhance classification accuracy, we conducted a statistical multivariate analysis based on the PCA-LDA model, which achieved perfect group separation and a diagnostic accuracy of 91.3%. Overall, this CRISPR/Cas13a-based nFCM platform offers a robust, accurate, and clinically applicable platform for single-vesicle miRNA profiling with broad potential in liquid biopsy-based cancer diagnosis.},
}

*MicroRNAs/analysis/genetics
Humans
*Extracellular Vesicles/chemistry/genetics/metabolism
*CRISPR-Cas Systems
*Prostatic Neoplasms/genetics/diagnosis
*Flow Cytometry/methods
Male
Limit of Detection
Biomarkers, Tumor/genetics
*Nanotechnology

@article {pmid41292075,
year = {2025},
author = {Du, J and Hu, J and An, J and Li, H and Chen, B and Luo, J and Li, S and Teng, Y and Yuan, T and Zhu, X and Jiang, L and Xiong, E and Yang, R},
title = {Guanine-Quadruplex-Engineered crRNA Enables Light-Activated CRISPR/Cas12a System for Robust One-Pot Viral Assay.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26580-26589},
doi = {10.1021/acs.analchem.5c04848},
pmid = {41292075},
issn = {1520-6882},
mesh = {*G-Quadruplexes ; *CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; Humans ; *Guanine/chemistry ; *Light ; CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Conventional one-pot detection platforms integrating CRISPR/Cas12a with isothermal amplification significantly streamline the nucleic acid detection workflow, while minimizing the risk of aerosol contamination. However, the intrinsic cleavage activity of the CRISPR/Cas12a system can substantially interfere with the nucleic acid amplification efficiency, ultimately compromising detection sensitivity. Herein, we develop a light-activated CRISPR/Cas12a system by engineering the crRNA with a guanine-quadruplex (G4) motif at its 3'-terminal, achieving precise regulation of Cas12a activity via photoswitching G4 structure formation. Through coupling with a recombinase polymerase amplification (RPA) reaction, we establish a one-pot detection platform that demonstrates superior detection performance compared to traditional Cas12a-based one-pot systems. The detection sensitivity has been improved by 2 orders of magnitude, reaching a level of 1 copy/μL. Notably, the platform demonstrated comparable sensitivity and specificity to PCR, the gold standard method, in detecting clinical samples, such as Epstein-Barr virus (EBV) and Influenza A virus (IAV), making it a promising technology for clinical diagnostics.},
}

*G-Quadruplexes
*CRISPR-Cas Systems/genetics
Nucleic Acid Amplification Techniques/methods
Humans
*Guanine/chemistry
*Light
CRISPR-Associated Proteins/metabolism
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41289351,
year = {2025},
author = {Su, T and Wei, T and Wang, Z and Wu, H and Fan, Y and Su, S and Zhu, D and Wang, L},
title = {A Pre-Amplification-Free Modular Dual-CRISPR System for Enhanced Pathogen Detection Sensitivity.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26640-26648},
doi = {10.1021/acs.analchem.5c05145},
pmid = {41289351},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Nucleic Acid Hybridization ; *African Swine Fever Virus/genetics/isolation & purification ; Limit of Detection ; Humans ; *DNA, Viral/analysis/genetics ; Animals ; Nucleic Acid Amplification Techniques ; },
abstract = {CRISPR/Cas12a is extensively utilized for pathogen detection owing to its high specificity and efficiency. However, traditional single-CRISPR/Cas12a encounters challenges due to its limited sensitivity, requiring pre-amplification of nucleic acids. This increases the complexity of the procedure and the potential for cross-contamination and false positives. Herein, a modular dual-CRISPR approach was developed coupled with hybridization chain reaction (HCR) for the universal and sensitive detection of pathogen nucleic acids without the need for pre-amplification. The system comprises two core modules: the first CRISPR/Cas12a recognition module specifically identifies pathogen targets and releases the activating agent, while the second CRISPR/Cas12a signal module is activated by this agent to initiate the HCR reaction for generating a strong fluorescent signal through DNA nanostructure self-assembly. Through rational design, we demonstrate the ability of this dual-CRISPR system to achieve attomolar (aM) level sensitivity for pathogen nucleic acid detection without pre-amplification, showing over six-order-of-magnitude higher sensitivity than a traditional single-CRISPR/Cas12a system. Additionally, the flexibility and versatility of the modular dual-CRISPR system have been confirmed for diverse pathogen targets, such as African swine fever virus (ASFV), severe fever with thrombocytopenia syndrome virus (SFTSV), and human papillomavirus type 16 (HPV-16) DNA. The system's practicality was demonstrated by examining ASFV quality control samples in complex environments. The exploration of the pre-amplification-free dual-CRISPR system offers a new perspective on enhancing pathogen nucleic acid detection systems.},
}

*CRISPR-Cas Systems/genetics
Nucleic Acid Hybridization
*African Swine Fever Virus/genetics/isolation & purification
Limit of Detection
Humans
*DNA, Viral/analysis/genetics
Animals
Nucleic Acid Amplification Techniques

@article {pmid41265176,
year = {2026},
author = {Mao, S and Guo, Y and Dong, C and Wang, D and Wang, X and Weng, L and Yang, Y and Li, Y and Niu, T and Wu, Q and Zheng, Z and Shan, Z and Tan, X and Gao, Y and Jin, J and Wang, P and Ge, X and Shen, B and Yao, X and Fang, L},
title = {Loss of cyclin C drives resistance to anti-TIGIT therapy by upregulating CD155-mediated immune evasion.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {84},
number = {},
pages = {101318},
doi = {10.1016/j.drup.2025.101318},
pmid = {41265176},
issn = {1532-2084},
mesh = {Humans ; *Receptors, Immunologic/antagonists & inhibitors/immunology ; Killer Cells, Natural/immunology ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/immunology/genetics ; Up-Regulation ; *Neoplasms/immunology/drug therapy/genetics ; CRISPR-Cas Systems ; T-Lymphocytes/immunology ; Gene Expression Regulation, Neoplastic ; Immune Evasion ; Immune Checkpoint Inhibitors/pharmacology ; Tumor Escape ; Receptors, Virus ; },
abstract = {AIMS: CD155 is an immune checkpoint protein expressed in tumor cells that interacts with its ligand T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) on natural killer (NK) cells and T cells, mediating inhibitory regulation on immune cells. Blockade of the CD155-TIGIT interaction has demonstrated clinical benefits in patients with advanced cancers. The transcriptional and post-translational mechanisms governing CD155 expression remain largely unknown.

METHODS: To identify regulators of CD155, we conducted a genome-wide CRISPR-Cas9 screen in cancer cells. Surface CD155 protein levels were analyzed via flow cytometry. The role of candidate regulators was validated through loss- and gain-of-function experiments with flow cytometry, Western blot, quantitative PCR, and chromatin immunoprecipitation (ChIP) assays. Additionally, ubiquitination assay was performed to examine post-translational modifications. Functional studies, including NK and T cell cytotoxicity assays, were conducted to assess the immune modulatory effects of CD155 regulation. Clinical relevance was evaluated by analyzing Cyclin C (CCNC) and CD155 expression in datasets of cancer patients who underwent immune checkpoint blockade therapy.

RESULTS: The CRISPR-Cas9 screen identified CCNC as a transcriptional suppressor of CD155. CCNC knockout led to increased surface CD155 expression in cancer cell lines. Mechanistically, CCNC inhibited CD155 transcription by suppressing the activity of the transcription factor FOSL2. Furthermore, CCNC was found to be ubiquitinated and degraded by the E3 ubiquitin ligase FBXO11, suggesting a post-translational regulatory mechanism. Functionally, loss of CCNC promoted CD155 upregulation, thereby enhancing tumor immune evasion from NK and T cell-mediated responses. Clinically, CCNC expression was negatively correlated with CD155 levels in cancer patients, particularly those receiving immune checkpoint blockade therapy.

CONCLUSION: This study identifies a previously unrecognized master regulator CCNC that functions as a suppressor of CD155-mediated cancer immune evasion. The findings of this study suggest that tumors with low CCNC expression may be resistant to monotherapy and highlight a combination immunotherapy (TIGIT/PD-1 co-blockade) as a promising anti-cancer therapeutic strategy to overcome immune evasion in CCNC-deficient tumors.},
}

Humans
*Receptors, Immunologic/antagonists & inhibitors/immunology
Killer Cells, Natural/immunology
Cell Line, Tumor
*Drug Resistance, Neoplasm/immunology/genetics
Up-Regulation
*Neoplasms/immunology/drug therapy/genetics
CRISPR-Cas Systems
T-Lymphocytes/immunology
Gene Expression Regulation, Neoplastic
Immune Evasion
Immune Checkpoint Inhibitors/pharmacology
Tumor Escape
Receptors, Virus

@article {pmid41259748,
year = {2025},
author = {Zheng, L and Zhou, X and Zhang, Y and Wang, W and Chen, C and Lin, X and Zheng, Y and Lou, Y},
title = {Rapid Bacterial Identification and Antimicrobial Susceptibility Testing Directly from Urine Samples via an Asymmetric Polymerase Chain Reaction-Cas12a Platform.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26466-26474},
doi = {10.1021/acs.analchem.5c04410},
pmid = {41259748},
issn = {1520-6882},
mesh = {Humans ; Microbial Sensitivity Tests ; *Bacteria/drug effects/isolation & purification/genetics ; *Anti-Bacterial Agents/pharmacology ; *Polymerase Chain Reaction/methods ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; },
abstract = {Antimicrobial resistance poses a critical global health challenge, largely due to the prolonged turnaround times of conventional pathogen identification (ID) and antimicrobial susceptibility testing (AST). Here, we present a clinically validated diagnostic platform integrating asymmetric polymerase chain reaction (aPCR) with CRISPR/Cas12a for direct bacterial ID and phenotypic AST from urine samples. Unlike traditional multiplex PCR requiring complex primer sets, our platform employs a singleplex aPCR targeting the V3-V4 region of 16S rDNA to generate single-stranded and double-stranded DNA. This design enables protospacer adjacent motif-free activation of Cas12a when required via the ssDNA fraction generated by aPCR, facilitating species-level multiplex detection of six common uropathogens at 10[3] CFU/mL via programmable CRISPR/Cas12a crRNAs. Phenotypic AST is accomplished within 60 min by quantifying nucleic acid changes following antibiotic exposure, allowing accurate discrimination between susceptible and resistant strains. When validated with 86 clinical urine samples, the aPCR-Cas12a platform achieved complete concordance with culture-based identification among the 45 samples carrying target pathogens and demonstrated high accuracy for AST, confirming its reliability for direct pathogen detection and susceptibility assessment from urine. The complete workflow requires only 5.5 h, significantly reducing the diagnostic time compared to standard methods (>48 h). This rapid, cost-effective, and scalable platform offers a promising solution for infection diagnosis and antimicrobial stewardship, with strong potential for integration into routine clinical microbiology and point-of-care settings.},
}

Humans
Microbial Sensitivity Tests
*Bacteria/drug effects/isolation & purification/genetics
*Anti-Bacterial Agents/pharmacology
*Polymerase Chain Reaction/methods
CRISPR-Cas Systems
*CRISPR-Associated Proteins/genetics
*Endodeoxyribonucleases/genetics/metabolism
Bacterial Proteins

@article {pmid41217936,
year = {2025},
author = {He, X and Deng, L and Zhou, S and Gu, T and Li, X and Zhu, S and Luo, X and Huo, D and Hou, C},
title = {Breaking the PAM Restriction: A Universal Double Stranded DNA Detection Method Based on the Sticky End-Mediated CRISPR/Cas12a Coupled RPA and Its Application to KRAS G12C Single Base Mutations.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26886-26896},
doi = {10.1021/acs.analchem.5c05908},
pmid = {41217936},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *Proto-Oncogene Proteins p21(ras)/genetics ; *DNA/analysis/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Point Mutation ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; *Recombinases/metabolism ; Bacterial Proteins ; },
abstract = {The CRISPR/Cas12a system facilitates efficient and specific nucleic acid detection, but its dependence on Protospacer Adjacent Motif (PAM) sequences and the complexity of existing sticky end-based methods pose challenges for stable and portable applications. To address these issues, this study developed a universal dsDNA detection method by integrating the sticky end-mediated CRISPR/Cas12a with recombinase polymerase amplification (RPA). By incorporating NlaIII recognition sites into RPA primers, precise cleavage of amplification products was achieved, generating uniform sticky ends and eliminating reliance on PAM sites. In comparison to flat end dsDNA containing PAM sites, the use of sticky end dsDNA significantly enhanced Cas12a activity. This strategy demonstrated sensitivity and specificity, achieving a detection limit of 40 aM and successfully identifying KRAS G12C mutations at a frequency of 0.1%, with genomic DNA results aligning with those obtained from FastNGS. Furthermore, we preliminarily explored a one-tube detection strategy, which effectively streamlined the operational process and reduced aerosol contamination. In summary, we established a simple, sensitive, and universal PAM-free CRISPR/Cas12a detection platform that integrates the advantages of isothermal amplification with a standardized sticky end design, thereby offering broad application prospects in molecular diagnostics and clinical translation.},
}

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

@article {pmid40974875,
year = {2026},
author = {Sun, D and Bo, L and Jiang, C and Lan, Y and Zhang, B and Zhang, C and Chen, ZS and Fan, Y},
title = {Beyond the boundary: The emerging roles of ATP-binding cassette transporters in multidrug resistance (MDR) and therapeutic targeting in cancer.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {84},
number = {},
pages = {101310},
doi = {10.1016/j.drup.2025.101310},
pmid = {40974875},
issn = {1532-2084},
mesh = {Humans ; *Drug Resistance, Neoplasm/drug effects/genetics ; *Neoplasms/drug therapy/genetics/pathology ; *Drug Resistance, Multiple/drug effects/genetics ; *ATP-Binding Cassette Transporters/genetics/metabolism/antagonists & inhibitors ; *Antineoplastic Agents/pharmacology/therapeutic use ; Animals ; Gene Editing/methods ; CRISPR-Cas Systems ; Molecular Targeted Therapy ; Immunoconjugates/pharmacology/therapeutic use ; Immunotherapy/methods ; },
abstract = {Multidrug resistance (MDR) remains a primary obstacle to successful cancer chemotherapy, with the overexpression of ATP-binding cassette (ABC) transporters being a principal cause. These transporters actively efflux a wide range of anticancer drugs, reducing their intracellular efficacy. Consequently, targeting ABC transporters represents a critical strategy for overcoming therapeutic resistance. This comprehensive review details the molecular architecture and functional mechanisms of all seven human ABC transporter subfamilies (ABCA-ABCG), elucidating their distinct roles in both cancer progression and the development of MDR. We trace the evolution of therapeutic interventions, from first, second, and third-generation small molecule inhibitors to the potential of natural products. Furthermore, this review explores advanced and emerging strategies designed to circumvent or neutralize ABC transporter activity. These include genetic approaches such as RNA interference and CRISPR-Cas9 gene editing, immunotherapy-based tactics like monoclonal antibodies and antibody-drug conjugates (ADCs), and the application of sophisticated nanoparticle delivery systems designed to bypass efflux mechanisms. By providing a holistic overview of the entire ABC transporter family and the broad array of strategies being developed to counteract their function, this article aims to equip researchers with a full-scope perspective on the field, identifying current challenges and illuminating future directions for combating MDR in cancer.},
}

Humans
*Drug Resistance, Neoplasm/drug effects/genetics
*Neoplasms/drug therapy/genetics/pathology
*Drug Resistance, Multiple/drug effects/genetics
*ATP-Binding Cassette Transporters/genetics/metabolism/antagonists & inhibitors
*Antineoplastic Agents/pharmacology/therapeutic use
Animals
Gene Editing/methods
CRISPR-Cas Systems
Molecular Targeted Therapy
Immunoconjugates/pharmacology/therapeutic use
Immunotherapy/methods

@article {pmid41352908,
year = {2026},
author = {Li, L and Tang, Z and Xu, H and Zhou, F and Ji, X and He, Z},
title = {Investigation on CRISPR-Cas12a-split crRNA system for successively detecting DNA and RNA in one tube.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344860},
doi = {10.1016/j.aca.2025.344860},
pmid = {41352908},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; *RNA, Viral/analysis/genetics ; Hepatitis B virus/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; HIV/genetics ; },
abstract = {Recently, CRISPR/Cas system has been proposed as a novel tool with simplicity and high accuracy. The CRISPR RNA (crRNA) can be divided into spacer crRNA and handle crRNA without losing its original function. In this work, we have investigated CRISPR Cas12a with split crRNA to detect HBV DNA and HIV RNA in a single tube. In the first step, Cas12a can recognize HBV DNA and initiate its trans-cleavage on FAM-BHQ1 reporter, after 1 h incubation, the fluorescence intensity was correlated with the concentration of HBV DNA. In the second step, the TAMRA BHQ2 ds DNA reporter was introduced in the same tube to bind with remained Cas12a proteins, HIV RNA and handle crRNA. The trans-cleavage from the first step would not interfere with HIV RNA and dsDNA reporter. With the incubation for another hour, HIV RNA can be quantified by the cis-cleavage of TAMRA BHQ2 reporter. we can successively identify the two nucleic acids with the limit of detection of 0.70 pM for HBV DNA, and 0.47 nM for HIV RNA, respectively. This special designed split crRNA can simplify detecting procedure and only need Cas12a protein in a single tube. Next, we expand this strategy in semi-quantifying two kinds of DNA in one tube. Overall, this study overcomes the limitation of conventional CRISPR-based methods and provides a new, inexpensive, and low-threshold approach based on Cas12a with split crRNA.},
}

*CRISPR-Cas Systems
*DNA, Viral/analysis/genetics
*RNA, Viral/analysis/genetics
Hepatitis B virus/genetics
*CRISPR-Associated Proteins/metabolism/genetics
Humans
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
HIV/genetics

@article {pmid41352906,
year = {2026},
author = {Fakhr, ZA and Xie, W and Zeng, S and Cai, S},
title = {Site accessibility-driven CRISPR/Cas13a activation for amplification-free RNA biosensing.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344858},
doi = {10.1016/j.aca.2025.344858},
pmid = {41352906},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *RNA/analysis ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; Kinetics ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {BACKGROUND: CRISPR-Cas13a biosensing enables rapid, amplification-free RNA diagnostics, yet assay sensitivity varies widely because guide RNAs (gRNAs) differ in their ability to activate the enzyme. Two factors, including the gRNA-target binding affinity and the structural accessibility of the target site, have been proposed to govern activation efficiency, but their relative importance remains unclear. In this study, we systematically disentangle these contributions by measuring binding affinities for gRNAs that span a spectrum of site accessibilities and by comparing their Michaelis-Menten kinetic parameters.

RESULTS: Three ciRS-7-specific gRNAs were designed with high, intermediate, and low spacer accessibility. Isothermal titration calorimetry (ITC) quantified site accessibility through entropy changes (ΔS = -862, -813, and -615 cal/mol/K), confirming greater structural exposure for less structured spacers, and also determined binding affinity for each gRNA-target pair. Michaelis-Menten analysis showed kcat values of 1.39, 1.31, and 1.16 s[-1] for the high, intermediate, and low-accessibility guides, respectively, establishing a clear relationship between structural accessibility and catalytic turnover. Importantly, the most structured gRNA exhibited lower activation efficiency compared with the gRNA that had higher site accessibility and lower binding affinity, demonstrating that site accessibility drives Cas13a activation. Detection-limit experiments also confirmed these results, showing that gRNAs with greater spacer accessibility yielded stronger signals and superior sensitivity.

SIGNIFICANCE: Our data establish site accessibility as a critical determinant of Cas13a activation for amplification-free RNA sensing. Prioritizing unstructured spacer regions enables improved enzyme activation efficiency, providing a clear design rule for next-generation CRISPR diagnostics. This accessibility-driven strategy will facilitate the development of faster, simpler, and more sensitive point-of-care assays for diverse RNA biomarkers.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems
*RNA/analysis
*RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry
Kinetics
*CRISPR-Associated Proteins/metabolism

@article {pmid41351274,
year = {2025},
author = {Hou, H and Li, Y and Su, N and Ding, Y and Shang, C and Li, X and Xiong, Z and Sun, Y and Zhan, W and Wang, Y and Zhang, X and Pan, Y and Wu, L and Li, J},
title = {Slmsh1-induced heritable enhancement of traits for tomato breeding improvement.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {5},
pages = {e70607},
doi = {10.1111/tpj.70607},
pmid = {41351274},
issn = {1365-313X},
support = {CSTB2023TIAD-KPX0026//Special Key Project of Technological Innovation and Application Development of Chongqing/ ; 31872123//National Natural Science Foundation of China/ ; 32172597//National Natural Science Foundation of China/ ; CARS-23-B08//China Agriculture Research System/ ; SWU-KF25027//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Solanum lycopersicum/genetics/physiology/growth & development ; *Plant Breeding/methods ; *Plant Proteins/genetics/metabolism ; Fruit/genetics/growth & development ; Droughts ; Quantitative Trait, Heritable ; Phenotype ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; },
abstract = {Vegetable grafting is a horticultural technique employed to develop specialized plant varieties by effectively enhancing resistance to both biotic and abiotic stresses, as well as improving fruit quality and yield. However, these advantageous traits are generally non-heritable. The MSH1 gene induced heritable enhancement-through-grafting (HEG) effect on growth vigor, demonstrating promising application potential. In this study, we employed the msh1 mutant tomato as a rootstock to induce heritable superior traits and combined this approach with hybridization techniques to enhance tomato cultivars. Three Slmsh1 mutants were generated using CRISPR/Cas9 which exhibited a dwarf phenotype with whitened spots. By grafting several distinct inbred lines onto Slmsh1, we observed significant HEG, drought stress tolerance, and fruit quality. Under drought conditions, Slmsh1-grafted tomato seedlings exhibited increased biomass and enhanced drought tolerance through the regulation of antioxidant enzyme activities. Differential expression and methylation analyses of the graft progeny revealed that these heritable enhanced traits (HETs) are likely attributable to epigenetic modifications in the expression of ROS-scavenging- and hormone-related genes. Furthermore, to explore practical applications, we crossed inbred lines with HETs and evaluated the growth, yield, and fruit quality of the resulting hybrid combinations. The results indicated that these hybrid combinations improved fruit yield and quality, enhancing the total soluble solids, soluble sugar, and soluble protein content. These findings suggest that Slmsh1-grafted progenies enhanced plant biomass and drought resistance, while their hybrid combinations positively influenced root growth, yield, and fruit quality, providing new insights into the synergistic integration of genome editing and conventional breeding.},
}

*Solanum lycopersicum/genetics/physiology/growth & development
*Plant Breeding/methods
*Plant Proteins/genetics/metabolism
Fruit/genetics/growth & development
Droughts
Quantitative Trait, Heritable
Phenotype
CRISPR-Cas Systems
Gene Expression Regulation, Plant

@article {pmid41350682,
year = {2025},
author = {Lyu, G and Li, P and Lang, W},
title = {A review of recent studies on CRISPR/Cas9-mediated genome editing in a variety of muscle-related genetic disorders.},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {1381},
pmid = {41350682},
issn = {1479-5876},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Animals ; *Muscular Diseases/genetics/therapy ; *Genetic Diseases, Inborn/genetics ; Genetic Therapy ; Muscular Dystrophies/genetics ; },
abstract = {The human body is capable of mutating a single gene to produce a wide range of debilitating disorders. Genomic editing for disease prevention via phenotypic reversal was a significant challenge prior to the development of clustered regulatory interspaced short tandem repeats (CRISPR) and CRISPR-associated protein (Cas) systems. Gene therapy-editing a patient's DNA to correct a particular mutation-and treating human diseases that have not responded to conventional medicine are two areas where CRISPR/Cas9 technology shows the most promise as a therapeutic tool. This powerful instrument has shown great promise in muscle-related illnesses, offering new insights into muscle biology and developing more effective treatment techniques. Discoveries about the hereditary causes of the majority of inherited myopathies and muscular dystrophies (MDs) have emerged over the last two decades. Additionally, skeletal muscles weaken and degenerate over time due to a group of hereditary disorders known as MDs. The field of skeletal muscle diseases and associated genetic alterations is seeing remarkable progress in developing therapeutic vectors to fix these mutations. Myopathies, MDs, and neuromuscular disorders are just a few examples of the many genetic abnormalities related to muscles that have sparked renewed interest in the potential of genome editing as a therapeutic tool due to its efficiency, adaptability, and relative ease of use in targeted genome editing. Consequently, CRISPR/Cas9 has garnered much interest and is used more often in therapeutic techniques due to its potential capacity to cure various human ailments. To pave the way for more effective and personalized therapies, this review article provides a thorough overview of the revolutionary role of CRISPR/Cas9 in improving our understanding and treatment of genetic disorders related to muscles by combining present knowledge with future perspectives.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing
Animals
*Muscular Diseases/genetics/therapy
*Genetic Diseases, Inborn/genetics
Genetic Therapy
Muscular Dystrophies/genetics

@article {pmid41349515,
year = {2025},
author = {Kim, I and Suh, JY},
title = {Capture first, then deliver!.},
journal = {Structure (London, England : 1993)},
volume = {33},
number = {12},
pages = {2008-2009},
doi = {10.1016/j.str.2025.11.001},
pmid = {41349515},
issn = {1878-4186},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/metabolism ; DNA/metabolism/chemistry ; *Integrases/chemistry/metabolism ; Protein Binding ; },
abstract = {In this issue of Structure, Henriques et al.[1] present structural snapshots that capture distinct conformational states of the type I-F Cas1-Cas2/3 integrase complex, illustrating that foreign DNA binding triggers a large-scale domain rearrangement that enables prespacer delivery to the CRISPR array.},
}

*CRISPR-Cas Systems
*CRISPR-Associated Proteins/chemistry/metabolism
DNA/metabolism/chemistry
*Integrases/chemistry/metabolism
Protein Binding

@article {pmid41349512,
year = {2025},
author = {Kosaka, Y and Lopez, B and Kishimoto, N and Jacob, S and Montenont, E and Huallanca, R and Coughenour, G and Di Paola, J and Ross, J and Lee, K and Rondina, MT and Bray, PF and Rowley, JW},
title = {Functional classification of platelet gene variants using CRISPR HDR in CD34[+] cell-derived megakaryocytes.},
journal = {American journal of human genetics},
volume = {112},
number = {12},
pages = {2888-2901},
doi = {10.1016/j.ajhg.2025.11.004},
pmid = {41349512},
issn = {1537-6605},
mesh = {Humans ; *Megakaryocytes/metabolism ; *Blood Platelets/metabolism ; *CRISPR-Cas Systems/genetics ; *Antigens, CD34/metabolism/genetics ; Integrin beta3/genetics ; Gene Editing/methods ; Integrin alpha2/genetics ; *Genetic Variation ; Hematopoietic Stem Cells/metabolism ; Thrombasthenia/genetics ; },
abstract = {The interpretation of genetic variants in inherited diseases, such as inherited platelet disorders (IPDs), remains a major clinical challenge, as most are classified as variants of uncertain significance (VUSs). A key barrier to functional evaluation is the lack of accessible, lineage-appropriate assays that reliably reflect native gene regulation and cell-specific biology. To address this gap, we developed CRIMSON HD (CRISPR-edited megakaryocytes [MKs] for surveying platelet variant functions through homology-directed repair [HDR]), a CRISPR-Cas9 HDR-based genome-editing platform applicable to CD34[+] cell-derived blood lineages and optimized for evaluating platelet-associated variants. Using this system, we modeled known and candidate disease-associated variants in integrin alpha 2b (ITGA2B) and integrin beta 3 (ITGB3), which encode the platelet αIIb/β3 integrin and are causative in Glanzmann thrombasthenia (GT). We introduced precise variants into primary human MKs derived from CD34[+] hematopoietic stem and progenitor cells, achieving >90% editing efficiency. Edited MKs faithfully recapitulated both expression and functional phenotypes of known type I, II, and III GT variants. CRIMSON HD enabled functional evaluation and reclassification of several GT VUSs, including αIIb Gly201Ala, a population variant now shown to cause near-complete loss of αIIb/β3 expression; αIIb Ala777Asp, which results in intermediate αIIb/β3 expression and impaired agonist-induced integrin binding; and β3 Arg119Gln, previously linked to the loss of anti-HPA1a antibody binding in fetal and neonatal alloimmune thrombocytopenia (FNAIT), now shown to impair integrin surface expression. These findings demonstrate the importance of lineage-specific, physiologically relevant assays for the functional classification of platelet-related variants, providing mechanistic information and clinically meaningful insights for individuals with IPDs.},
}

Humans
*Megakaryocytes/metabolism
*Blood Platelets/metabolism
*CRISPR-Cas Systems/genetics
*Antigens, CD34/metabolism/genetics
Integrin beta3/genetics
Gene Editing/methods
Integrin alpha2/genetics
*Genetic Variation
Hematopoietic Stem Cells/metabolism
Thrombasthenia/genetics

@article {pmid41348871,
year = {2025},
author = {Puppala, AK and Nielsen, AC and Regan, M and Mancinelli, GE and De Pooter, RF and Arnovitz, S and Harding, C and McGregor, M and Balanis, NG and Clarke, R and Merrill, BJ},
title = {Programmable multistep CRISPR gene activation via control of RNA polymerase III termination.},
journal = {Science advances},
volume = {11},
number = {49},
pages = {eadt1532},
pmid = {41348871},
issn = {2375-2548},
mesh = {Humans ; *RNA Polymerase III/metabolism/genetics ; *CRISPR-Cas Systems ; *Transcriptional Activation ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Induced Pluripotent Stem Cells/metabolism/cytology ; HEK293 Cells ; },
abstract = {Although genomes encode instructions for mammalian cell differentiation with rich syntactic relationships, existing methods for genetically programming cells have only modest capabilities for stepwise gene regulation. Here, we develop a sequential genetic system that transcriptionally activates endogenous genes in a preprogrammed, stepwise manner. This system uses the removal of an RNA polymerase III termination sequence to trigger both the transcriptional activation and DNA endonuclease activities of a Cas9-VPR protein, driving progression through a cascade of gene activation events. The system's functionality in human cells, including iPSCs, enables the development of a path for cellular programming by controlling the sequential order of gene activation to influence cellular states.},
}

Humans
*RNA Polymerase III/metabolism/genetics
*CRISPR-Cas Systems
*Transcriptional Activation
Gene Editing/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Induced Pluripotent Stem Cells/metabolism/cytology
HEK293 Cells

@article {pmid41348151,
year = {2025},
author = {Cheng, Y and Gao, W and Shi, S and Han, F and Dong, H},
title = {Identification of the orange pigment in Nonomuraea gerenzanensis and development of a pigment-free mutant with high yield of A40926.},
journal = {AMB Express},
volume = {},
number = {},
pages = {},
doi = {10.1186/s13568-025-01993-4},
pmid = {41348151},
issn = {2191-0855},
support = {2024TSGC0896//Shandong Province Science and Technology-based Small and Medium-sized Enterprises Innovation Capacity Enhancement Project/ ; },
abstract = {The secondary metabolite A40926, a precursor to the glycopeptide antibiotic dalbavancin, is synthesized by the rare actinomycete Nonomuraea gerenzanensis (N. gerenzanensis) within the pharmaceutical industry. The biosynthesis of A40926 is accompanied by the production of an orange pigment, which poses significant challenges and incurs high costs in the purification process of A40926. To identify this orange pigment, a comprehensive analysis was conducted, including the examination of the biosynthetic gene cluster, potential biosynthetic pathways, purification processes, and structural identification. Additionally, the ispF gene, which encodes the enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and is implicated in the biosynthesis of orange pigment, was deleted using the CRISPR/Cas9 system. To enhance A40926 production in the ΔIspF mutant, the overexpression of the cyclic AMP receptor protein (Crp) was implemented to assess its regulatory impact on A40926 biosynthesis. Consequently, the orange pigment produced by N. gerenzanensis was identified as lycopene, synthesized via the methylerythritol phosphate (MEP) pathway. Although the ΔIspF mutant was unable to biosynthesize the orange pigment, its production of A40926 was adversely affected and was lower than that of the original strain. Consequently, the overexpression of the global regulator Crp significantly enhanced A40926 production, achieving a yield of 841.1 mg/L. The investigation of pigment-free mutants presented in this study offers valuable insights for effectively reducing production costs within the microbial pharmaceutical industry.},
}

@article {pmid41271111,
year = {2026},
author = {Huang, Q and Zhao, T and Su, W and Li, S and Liu, J and Ge, Z and Zhang, B and Ren, X and Zhang, X and Wei, J},
title = {Screening of monoclonal vaccine strains based on real-time live-cell imaging technology.},
journal = {Journal of virological methods},
volume = {340},
number = {},
pages = {115305},
doi = {10.1016/j.jviromet.2025.115305},
pmid = {41271111},
issn = {1879-0984},
mesh = {Animals ; *Vaccinia virus/genetics/immunology/isolation & purification ; *Viral Vaccines/immunology/genetics/isolation & purification ; Viral Plaque Assay/methods ; Vaccines, Attenuated/immunology/genetics ; Antibodies, Viral/blood/immunology ; Humans ; Green Fluorescent Proteins/genetics ; CRISPR-Cas Systems ; Vaccines, Synthetic/immunology/genetics ; Antibodies, Neutralizing/blood ; },
abstract = {The plaque purification is a critical step in the screening of traditional live-attenuated vaccines and recombinant viral vaccines, aiming to acquire vaccine clones with homogeneous characteristics and desirable immunogenicity to address outbreaks of emerging diseases such as monkeypox, chikungunya fever, and dengue fever. The traditional plaque purification process to screen out a vaccine strain with genetically consistent stability from a mixed pool of viral clones generally requires laborsome work. We utilized live-cell imaging technique enabling us to isolate monoclonal vaccine strains to simplify and improve the efficiency of this process. Here, we genetically engineered the vaccinia virus TianTan (VTT) using CRISPR/Cas9 system to generate recombinant VTT viruses (VTT-WS01-EGFP) that expressed enhanced green fluorescent protein (EGFP). Initially, we performed 9 rounds of plaque purification using traditional plaque assay, yielding 50 candidate clones. The Incucyte Live-Cell Imaging and Analysis system was subsequently performed to conduct a rigorous, high-resolution screening of these candidates in a more automated, sensitive and high-throughput way. Through this screening process, we ultimately obtained 31 pure viral clones that were free of parental strain contamination, followed by the analysis of plaque formation, fluorescent plaque size, and plaque morphology, and 11 candidate clones were selected for immunological evaluation. Furthermore, we found that clone 49 induced a relatively high titer of anti-VTT neutralizing antibodies and elicited the production of cross-reactive IgG against monkeypox virus antigens, thereby validating its potential as a candidate strain as a monkeypox virus vaccine. Taken together, our data demonstrates that live-cell imaging technique significantly accelerates the screening process for the isolation of monoclonal viral clones as recombinant viral vaccines, and holds considerable potential in attenuated strain selection as well as investigations into biological characteristics of viruses, including viral replication.},
}

Animals
*Vaccinia virus/genetics/immunology/isolation & purification
*Viral Vaccines/immunology/genetics/isolation & purification
Viral Plaque Assay/methods
Vaccines, Attenuated/immunology/genetics
Antibodies, Viral/blood/immunology
Humans
Green Fluorescent Proteins/genetics
CRISPR-Cas Systems
Vaccines, Synthetic/immunology/genetics
Antibodies, Neutralizing/blood

@article {pmid41260396,
year = {2026},
author = {Zhong, Z and Li, G and Liang, G and Ren, T and Teng, C and Xiong, J and Ji, G and Zheng, M and Pan, Y and Qin, Y and Ouyang, K and Yin, Y and Chen, Y and Huang, W and Wei, Z},
title = {Establishment of a nucleic acid detection method for foot-and-mouth disease virus serotype O utilizing RPA-CRISPR/Cas12a technology.},
journal = {Journal of virological methods},
volume = {340},
number = {},
pages = {115304},
doi = {10.1016/j.jviromet.2025.115304},
pmid = {41260396},
issn = {1879-0984},
mesh = {*Foot-and-Mouth Disease Virus/genetics/isolation & purification/classification ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Foot-and-Mouth Disease/diagnosis/virology ; Animals ; Serogroup ; Recombinases/metabolism/genetics ; DNA Primers/genetics ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {This study aimed to develop a rapid and visually interpretable nucleic acid detection assay for Foot-and-Mouth Disease Virus serotype O (FMDV-O) by integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Specific RPA primers and CRISPR RNA (crRNA) sequences were designed and optimized based on the conserved 3D gene region of FMDV-O. An assay combining RPA pre-amplification with Cas12a-mediated cleavage was subsequently established. The sensitivity and specificity of the RPA-CRISPR/Cas12a method were systematically evaluated, and its diagnostic utility was further assessed using clinical samples. The results demonstrated that the primer set RPA-F1/R1 paired with crRNA1 constituted the optimal combination, with an ideal reaction system comprising 50 nM Cas12a protein and 200 nM crRNA. This system exhibited a detection limit of 2.60 × 10[2] copies/μL for target plasmid DNA following a 20-minute incubation at 37°C. Specificity analysis confirmed positive detection exclusively for FMDV-O plasmids, with no cross-reactivity observed with other tested pathogens. When applied to clinical samples, the proposed method demonstrated a superior detection rate relative to conventional PCR. In conclusion, a novel diagnostic platform for FMDV-O was successfully developed based on RPA-CRISPR/Cas12a. This method is characterized by its rapidity, operational simplicity, high sensitivity, and excellent specificity, holding significant promise for application in clinical diagnostics, epidemiological surveillance, and field-based testing.},
}

*Foot-and-Mouth Disease Virus/genetics/isolation & purification/classification
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*Foot-and-Mouth Disease/diagnosis/virology
Animals
Serogroup
Recombinases/metabolism/genetics
DNA Primers/genetics
RNA, Viral/genetics
*Molecular Diagnostic Techniques/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41231539,
year = {2025},
author = {Jia, N and Zhou, YJ and Gao, J},
title = {Engineering recombination machinery facilitates the construction of yeast cell factories.},
journal = {FEMS yeast research},
volume = {25},
number = {},
pages = {},
doi = {10.1093/femsyr/foaf066},
pmid = {41231539},
issn = {1567-1364},
support = {22478382//National Natural Science Foundation of China/ ; E411040705//Energy Revolution S&T Program of Yulin Innovation Institute of Clean Energy/ ; },
mesh = {*Metabolic Engineering/methods ; *Gene Editing/methods ; *DNA End-Joining Repair ; *Saccharomyces cerevisiae/genetics/metabolism ; *Homologous Recombination ; *Recombination, Genetic ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; },
abstract = {Advances in genome editing have been promoted by programmable nucleases like CRISPR-Cas9, which triggers endogenous DNA repair mechanisms by inducing double-strand break (DSB). Cellular responses to DSBs are governed by competing repair pathways: error-prone non-homologous end joining (NHEJ) and high-fidelity homologous recombination (HR). This review systematically compares the molecular mechanisms and key regulators of NHEJ and HR, with a focus on recent breakthroughs in recombination engineering in non-conventional yeasts. These advances address challenges in precise genome editing, enabling robust metabolic engineering of yeast cell factories for sustainable bioproduction.},
}

*Metabolic Engineering/methods
*Gene Editing/methods
*DNA End-Joining Repair
*Saccharomyces cerevisiae/genetics/metabolism
*Homologous Recombination
*Recombination, Genetic
CRISPR-Cas Systems
DNA Breaks, Double-Stranded

@article {pmid41347244,
year = {2025},
author = {Daraghmeh, DN and AbuIriban, RW and Nawawreh, N and Abuamro, AM and Alassar, MM and Daraghma, SN and Alhajahmed, NM and Thandar, Y},
title = {Advancements in alternative approaches to address antimicrobial resistance in bacterial pneumonia: a comprehensive review.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1704931},
pmid = {41347244},
issn = {1664-302X},
abstract = {PURPOSE: This review explores both current and emerging alternative treatment approaches to combat AMR specifically in the context of bacterial pneumonia, highlighting therapies that extend beyond conventional antibiotics.

METHODS: PubMed, Embase, and Google Scholar were searched for full-text, English-language articles, with emphasis on publications from 2020 to 2025. Earlier seminal studies were also included when necessary to provide historical, mechanistic, or conceptual context. The review focuses was on alternative strategies that have shown effectiveness in preclinical or clinical settings to combat AMR in relation to bacterial pneumonia.

RESULTS: Emerging strategies to tackle AMR in bacterial pneumonia involve several innovative approaches including stem cells, bacteriophage therapy, metal based nanoparticles (e.g., silver, copper, and gold). The adjunctive use of probiotics and herbal medicine has demonstrated potential in enhancing clinical outcomes and modulating host immunity. Moreover, gene editing technologies like CRISPR-CAS and various vaccination programs are being investigated for their roles in prevention and resistance management. While these methods show promise, many are still in the early stages of development and encounter challenges related to standardization, safety, and regulatory approval.

CONCLUSION: Alternative therapies present exciting possibilities for addressing AMR in bacterial pneumonia. However, to effectively translate these innovations into clinical practice, we need thorough research, international collaboration, and supportive policy frameworks. By combining these strategies with antimicrobial stewardship initiatives, we can help maintain antibiotic effectiveness and enhance patient outcomes.},
}

@article {pmid41346702,
year = {2026},
author = {Patra, C and Hussein, Z and Ace, VD and Misnik, EV and Rybalko, DS and Salimova, AA and Ereshko, DS and Dubovichenko, MV and Nour, MAY and Drozd, VS and Kolpashchikov, DM},
title = {The efficacy of oligonucleotide-based gene therapeutics in gene silencing.},
journal = {Theranostics},
volume = {16},
number = {2},
pages = {599-616},
pmid = {41346702},
issn = {1838-7640},
mesh = {Humans ; *Genetic Therapy/methods ; *Gene Silencing ; *Oligonucleotides, Antisense/therapeutic use/genetics ; RNA, Small Interfering/genetics/therapeutic use ; Animals ; *Oligonucleotides/genetics/therapeutic use ; },
abstract = {Oligonucleotide-based gene therapeutics (OGTs) have emerged as a promising strategy for treating a variety of diseases, offering a tool for gene modulation at the mRNA level. Despite significant progress in OGTs development, their efficacy in both experimental and clinical settings has often fallen short of expectations. Current estimates suggest that less than 1% of transfected OGTs are released into the cytosol, significantly limiting the interaction with target RNA. Moreover, data suggests that only about 2% of the tested siRNAs achieve the expected 70% target gene knockdown in vitro. Clinically approved OGTs appear to be effective only against genetic disorders that lack effective alternative treatment, and even in these cases their therapeutic contribution remains marginal. Notably, the majority of approved OGTs, as well as those currently in clinical trials, are antisense oligonucleotides (ASOs) despite cell culture data showing that small interfering RNAs (siRNAs) exhibit greater potency. The delayed commercialization of siRNAs, despite high research interest, may be attributed to passenger stand-dependent off target effect and the immaturity of their design and modification strategies. This review critically evaluates the factors influencing therapeutic efficacy of OGTs and highlights the persistent gap between theoretical promise and clinical reality.},
}

Humans
*Genetic Therapy/methods
*Gene Silencing
*Oligonucleotides, Antisense/therapeutic use/genetics
RNA, Small Interfering/genetics/therapeutic use
Animals
*Oligonucleotides/genetics/therapeutic use

@article {pmid41275796,
year = {2025},
author = {Gao, Y and Zhao, L and Shi, J and Wang, B},
title = {NtQPT2 plays critical roles in nicotine biosynthesis and development of tobacco plant.},
journal = {Biochemical and biophysical research communications},
volume = {793},
number = {},
pages = {153030},
doi = {10.1016/j.bbrc.2025.153030},
pmid = {41275796},
issn = {1090-2104},
mesh = {*Nicotiana/growth & development/genetics/metabolism/enzymology ; *Nicotine/biosynthesis ; *Pentosyltransferases/genetics/metabolism ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Plants, Genetically Modified ; },
abstract = {The enzyme quinolinate phosphoribosyltransferase (QPT), encoded by a small gene family in tobacco plant, plays a critical role in the biosynthesis of nicotine, a defensive pyridine alkaloid in Nicotiana species, in addition to its vital function in the NAD(P)(H) synthesis. Previous studies have demonstrated that two NtQPT genes (NtQPT1 and NtQPT2) are present in N. tabacum genome, and it has been believed that NtQPT1 is responsible for NAD(P)(H) synthesis and thus essential for primary metabolism, while NtQPT2 is specifically involved in nicotine biosynthesis. In this study, we generated knockout tobacco lines for NtQPT1 and NtQPT2 respectively using the CRISPR/Cas9-based genome-editing technology and found that knockout of NtQPT2 caused both dramatic reduction of nicotine biosynthesis and a retardation of plant development, indicating that NtQPT2 is important not only to nicotine biosynthesis, but also to the development of tobacco plant. Like NtQPT2, NtQPT1 was also found to contribute to nicotine biosynthesis although to a much lesser extent than NtQPT2. Meanwhile, knockout of NtQPT1 did not significantly affect plant growth. Together with the observation that NtQPT2's expression is remarkably higher than that of NtQPT1 in root, leaf, stem and flower of tobacco plant, it is reasonable to infer that their functional diversification on nicotine biosynthesis and tobacco plant growth may be attributed largely to their markedly different transcript abundance.},
}

*Nicotiana/growth & development/genetics/metabolism/enzymology
*Nicotine/biosynthesis
*Pentosyltransferases/genetics/metabolism
Gene Expression Regulation, Plant
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems
Gene Knockout Techniques
Plants, Genetically Modified

@article {pmid41274245,
year = {2025},
author = {Shimura, R and Yamamoto, K and Chang, YH and Otaki, A and Goyama, S},
title = {Development of a CRISPR/Cas9-degron system that enables in vivo specific gene depletion in leukemia models.},
journal = {Biochemical and biophysical research communications},
volume = {793},
number = {},
pages = {153002},
doi = {10.1016/j.bbrc.2025.153002},
pmid = {41274245},
issn = {1090-2104},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Humans ; *Leukemia, Myeloid, Acute/genetics/pathology ; Histone-Lysine N-Methyltransferase/genetics ; Mice ; *Gene Editing/methods ; Cell Line, Tumor ; Disease Models, Animal ; CRISPR-Associated Protein 9 ; Degrons ; },
abstract = {The CRISPR/Cas9 system has transformed genome editing, yet precise temporal control of Cas9 activity remains challenging. We developed a Cas9-degron platform that couples degron-tagged Cas9 with a dTAG-based chemical degradation strategy. In the presence of dTAG, Cas9 is rapidly and near-completely degraded, preventing editing; upon dTAG withdrawal, Cas9 activity is restored, enabling precise temporal control. Using this system, we achieved selective in vivo gene depletion in acute myeloid leukemia (AML) models and confirmed that SETDB1, a histone H3K9 methyltransferase, is essential for the in vivo growth of both human (MOLM13) and murine (cSAM) AML cells. By maintaining SETDB1 intact prior to transplantation and depleting it afterward, we avoided culture-induced pre-selection bias inherent to sgRNA transduction and validated its critical role in AML progression within the in vivo context. The Cas9-degron retains activity and delivery efficiency comparable to conventional Cas9 in the absence of dTAG. Thus, this versatile system provides a superior alternative to conventional Cas9 and a powerful platform for in vivo CRISPR screening, gene function studies, and potentially temporally controlled gene therapy.},
}

*CRISPR-Cas Systems/genetics
Animals
Humans
*Leukemia, Myeloid, Acute/genetics/pathology
Histone-Lysine N-Methyltransferase/genetics
Mice
*Gene Editing/methods
Cell Line, Tumor
Disease Models, Animal
CRISPR-Associated Protein 9
Degrons

@article {pmid41086995,
year = {2025},
author = {Jang, SH and Song, HG and Jung, J and Gee, HY},
title = {Recent preclinical and clinical advances in gene therapy for hereditary hearing loss.},
journal = {Molecules and cells},
volume = {48},
number = {12},
pages = {100285},
pmid = {41086995},
issn = {0219-1032},
mesh = {*Genetic Therapy/methods ; Humans ; *Hearing Loss/therapy/genetics ; Animals ; Gene Editing ; Genetic Vectors/genetics ; CRISPR-Cas Systems ; Dependovirus/genetics ; Gene Transfer Techniques ; Clinical Trials as Topic ; },
abstract = {Hereditary hearing loss is a genetically heterogeneous condition that affects millions of people worldwide and has limited curative treatment options. Recent advancements in gene therapy have opened promising avenues for correcting the underlying genetic defects in the inner ear. This review summarizes the key developments in vector platforms, delivery strategies, target genes, preclinical models, and clinical trials relevant to both gene supplementation and gene editing approaches, as well as future directions. Adeno-associated virus vectors have emerged as the leading platform for inner ear gene transfer, owing to their safety and efficacy. Clinical programs, such as those targeting OTOF variants, are currently underway and are supported by robust preclinical data. Additionally, genome editing technologies, including CRISPR/Cas9-mediated nonhomologous end joining, base editing, and prime editing, offer variant-specific therapeutic potential. Despite these advances, challenges remain in expanding the therapeutic window, ensuring long-term safety, and establishing ethical and regulatory frameworks for their use.},
}

*Genetic Therapy/methods
Humans
*Hearing Loss/therapy/genetics
Animals
Gene Editing
Genetic Vectors/genetics
CRISPR-Cas Systems
Dependovirus/genetics
Gene Transfer Techniques
Clinical Trials as Topic

@article {pmid41346247,
year = {2025},
author = {Singh, V and Mishra, M and Singla-Pareek, SL and Roy, JK and Pareek, A},
title = {Lysine Matters: Genetic and Biotechnological Innovations to Combat Protein Malnutrition.},
journal = {Plant, cell & environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.70316},
pmid = {41346247},
issn = {1365-3040},
support = {//This study was supported by Department of Biotechnology, Ministry of Science and Technology, India./ ; },
abstract = {Lysine deficiency in staple crops like maize, rice, and wheat remains a major cause for global protein malnutrition, underscoring the urgent need for effective biofortification strategies. This review critically examines recent advances in enhancing lysine content, spanning conventional breeding and metabolic engineering to cutting-edge precision genome editing. While conventional breeding, exemplified by Quality Protein Maize, has improved lysine levels, it is often constrained by yield and quality trade-offs. Metabolic engineering strategies, including overexpression of lysine biosynthetic genes, suppression of catabolic genes, and modification of storage proteins, have achieved substantial lysine enrichment but face regulatory and consumer acceptance challenges due to their transgenic nature. The advent of CRISPR/Cas technology now enables precise, transgene-free editing of key enzymes such as DHDPS, AK, and LKR/SDH offering a powerful alternative, though concerns regarding off-target effects and pleiotropy remain. While integrating multi-omics with AI-driven predictive modelling can optimise metabolic flux for higher lysine yield, coupling next-generation genome editing with speed breeding offers a transformative route to develop high-lysine, high-yielding crops for sustainable nutritional security.},
}

@article {pmid41345283,
year = {2025},
author = {Saydam, S and Dinçer, P},
title = {Precision rewriting of muscle genetics: therapeutic horizons of base and prime editing in skeletal muscle disorders.},
journal = {Gene therapy},
volume = {},
number = {},
pages = {},
pmid = {41345283},
issn = {1476-5462},
abstract = {Base Editing (BE) and Prime Editing (PE), novel precision tools of the CRISPR/Cas toolbox, have emerged as transformative technologies that enable highly specific genetic modifications. Their compatibility with post-mitotic cell types makes them invaluable for treating genetic skeletal muscle disorders. Despite their severity and progressive nature, monogenic muscle diseases remain without definitive treatments. They are caused by diverse mutations in critical muscle proteins, for which gene editing offers a promising therapeutic avenue. However, traditional CRISPR/Cas9 applications face challenges such as genotoxicity and inefficiency in post-mitotic tissues. BE and PE technologies overcome these limitations by enabling safe and efficient modifications without causing double-strand breaks or requiring homology-directed repair. Their therapeutic potential comes from two key features: their ability to work in non-dividing cells such as myotubes and cardiomyocytes, and their capacity to target a broad range of mutations found in genetic muscle diseases. In this review, we explore mechanisms of BE and PE and summarize their current applications in monogenic skeletal muscle disorders. We discuss the challenges of in vivo application in skeletal muscle and highlight innovations to bypass them. Collectively, both systems offer flexible precision solutions with immense potential for mutation-specific and personalized gene therapy approaches for monogenic skeletal muscle disorders.},
}

@article {pmid41344769,
year = {2026},
author = {Liu, X and Zheng, Y and Chen, Z and Wang, S and Liao, H and Jia, J and Wang, G and Wang, J and Yuan, C and Guo, X and Yin, Y and Hu, Q},
title = {Rapid and visual detection of Listeria monocytogenes by combining one-pot LAMP-CRISPR/Cas12b with lateral flow assay.},
journal = {Food microbiology},
volume = {135},
number = {},
pages = {104977},
doi = {10.1016/j.fm.2025.104977},
pmid = {41344769},
issn = {1095-9998},
mesh = {*Listeria monocytogenes/isolation & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Animals ; Food Contamination/analysis ; Food Microbiology/methods ; Swine ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Limit of Detection ; },
abstract = {Listeria monocytogenes, the leading cause of fatalities worldwide among foodborne pathogens, poses serious risks to food safety and public health. Therefore, a rapid and accurate detection method is crucial for early interception and effective management. In this study, a one-pot LAMP-CRISPR/Cas12b detection system based on the lmo0753 gene was developed for rapid detection of L. monocytogenes by combining loop-mediated isothermal amplification (LAMP) with a CRISPR/Cas12b assay. Further integration of a lateral flow assay (LFA) to develop a LAMP-CRISPR/Cas12b-LFA assay enabled direct detection of the results on the strips with the naked eye. Nine L. monocytogenes strains belonging to eight serotypes tested positive with both the one-pot LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA assays. Two assays did not show cross-reactivity with L. innocua and eight other foodborne bacteria. The limits of detection were 10 CFU/mL for pure culture and 20 CFU/g for spiked pork samples. Moreover, the enrichment time was substantially shortened to 3 h for pork samples spiked with only L. monocytogenes F2365, and 4-5 h for pork samples spiked with mixed bacteria. In addition, with one-pot LAMP-CRISPR/Cas12b detection, 5 of 66 fresh pork samples, 1 of 20 ready-to-eat food samples, and 2 of 24 raw milk samples tested positive for L. monocytogenes, in agreement with the results obtained through a culture based standard method. Thus, this study established one-pot LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA assays for rapid, visual detection of L. monocytogenes in food samples.},
}

*Listeria monocytogenes/isolation & purification/genetics
*Nucleic Acid Amplification Techniques/methods
Animals
Food Contamination/analysis
Food Microbiology/methods
Swine
*CRISPR-Cas Systems
*Molecular Diagnostic Techniques/methods
Sensitivity and Specificity
Limit of Detection

@article {pmid41341583,
year = {2025},
author = {Jiang, Z and Jia, B and Hu, N and Zhang, M and Xiao, H and Chen, G and Yu, J and Li, X and Shen, B and Feng, J and Wang, J},
title = {In Vivo engineering of transgenic mice for systemic human neutralizing antibody production against staphylococcal enterotoxin B.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1679421},
pmid = {41341583},
issn = {1664-3224},
mesh = {Animals ; Mice, Transgenic ; Humans ; Mice ; *Enterotoxins/immunology ; *Antibodies, Neutralizing/immunology/genetics/biosynthesis ; Female ; CRISPR-Cas Systems ; Genetic Engineering ; Antibodies, Monoclonal/immunology/genetics ; Glycosylation ; },
abstract = {Transgenic animal bioreactors provide a complementary strategy to traditional mammalian cell culture systems for the production of therapeutic human monoclonal antibodies (mAbs). Here we present a CRISPR/Cas9-mediated breakthrough in creating two novel genetically engineered (GE) mouse models with species-specific chromosomal integration of human anti-staphylococcal enterotoxin B (SEB) mAb genes at either the ROSA26 or Hipp11 (H11) safe-harbor loci - evolutionarily conserved genomic safe harbors (GSH). These genetically optimized animals demonstrated broad tissue capability for glycosylation-competent human antibodies, achieving exceptional secretion levels reaching 208 mg/L in serum, 43 mg/L in mammary secretions, 24 mg/L in saliva on average. The transgenic lines maintained this antibody production stability for >140 weeks without compromising animal viability, while preserving germline transmission fidelity through six successive generations. Furthermore, the highly glycosylated human antibodies derived from these genetic engineered mice exhibited high binding affinity to SEB (KD=0.108 nM for ROSA26; 0.154 nM for H11), providing comprehensive protection against SEB intoxication in vivo. This study opens avenues for utilizing transgenic animal bioreactors for large-scale production of fully human antibodies or disease-resistant livestock in the foreseeable future.},
}

Animals
Mice, Transgenic
Humans
Mice
*Enterotoxins/immunology
*Antibodies, Neutralizing/immunology/genetics/biosynthesis
Female
CRISPR-Cas Systems
Genetic Engineering
Antibodies, Monoclonal/immunology/genetics
Glycosylation

@article {pmid41330077,
year = {2026},
author = {Fathpour, H and Fouladi, M and Jafarpour, F and Moradi-Hajidavaloo, R and Izadi, T and Shiralian-Esfahani, H and Kues, W and Nasr-Esfahani, MH and Hajian, M and Eghbalsaied, S},
title = {Crosstalk between myostatin and callipyge in CRISPR/Cas9-edited goat fibroblast cells.},
journal = {Research in veterinary science},
volume = {198},
number = {},
pages = {105992},
doi = {10.1016/j.rvsc.2025.105992},
pmid = {41330077},
issn = {1532-2661},
mesh = {Animals ; *Myostatin/genetics/metabolism ; *Goats/genetics ; *Fibroblasts/metabolism ; *CRISPR-Cas Systems ; Gene Editing/veterinary ; },
abstract = {Myostatin (MSTN) and Callipyge (CLPG) genes are key regulators of muscle growth. While MSTN inhibits muscle development, the CLPG mutation induces muscle hypertrophy through a specific imprinted genetic mechanism. The interaction between these genes remains of interest for improving livestock muscle traits. In this study, CRISPR/Cas9 was employed to edit MSTN and CLPG genes in goat fibroblast cells via electrotransfection. Cells were selected using puromycin antibiotic, and gene-editing efficiency was evaluated through Sanger sequencing. Gene expression changes were analyzed using RT-qPCR analysis. MSTN gene knockout resulted in significant downregulation of MSTN and CLPG, while GTL2 expression was upregulated by more than 50-fold. Additionally, myosin heavy chain genes (MYH1, MYH3, MYH4) were strongly upregulated, with MYH3 13-fold and MYH4 30-fold increase in the expression. In CLPG-edited cells, the expression of MSTN, TRIM28, and CLPG was reduced, while GTL2 was upregulated by 6-fold. MYH3 and MYH4 expression increased 4-fold in CLPG-edited cells, though the increase was less pronounced compared to MSTN-edited cells. DLK1 expression was undetectable in both non-edited control and gene-edited fibroblast cells. Our findings support the interaction between MSTN and CLPG, contributing to the regulation of muscle growth. Notably, the study also highlights the challenges associated with editing imprinted genes like CLPG and suggests that TRIM28 may play a role downstream of CLPG regulation. These results provide valuable insights into muscle development regulation, offering potential applications in livestock genetic improvement.},
}

Animals
*Myostatin/genetics/metabolism
*Goats/genetics
*Fibroblasts/metabolism
*CRISPR-Cas Systems
Gene Editing/veterinary

@article {pmid41285661,
year = {2026},
author = {Gu, X and Zhang, T and Yao, H and Guo, F and Yang, C and Xu, H and He, X and Ma, Z and Zhang, X and Yu, S and An, R and Wang, F},
title = {CRISPR-Cas12a-integrated pregnancy test strip biosensors: Visual detection of telomerase and miRNA let-7a in cervical cancer diagnostics.},
journal = {Biosensors & bioelectronics},
volume = {294},
number = {},
pages = {118241},
doi = {10.1016/j.bios.2025.118241},
pmid = {41285661},
issn = {1873-4235},
mesh = {Humans ; Female ; *MicroRNAs/genetics/isolation & purification/analysis ; *Uterine Cervical Neoplasms/diagnosis/genetics ; *Biosensing Techniques ; *Telomerase/isolation & purification/genetics/analysis ; CRISPR-Cas Systems/genetics ; HeLa Cells ; Pregnancy ; Limit of Detection ; Reagent Strips/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Cervical cancer is a leading cause of female cancer-related mortality globally, and early screening based on reliable biomarkers is critical for improving prognosis. Telomerase (a key driver of cellular immortalization) and microRNA let-7a (a tumor suppressor with downregulated expression in cervical cancer) are well-validated diagnostic targets, but existing detection methods are hindered by complex procedures, high instrumentation costs, and reliance on specialized technical expertise-limiting their accessibility in resource-constrained settings. To address these limitations, we developed two novel CRISPR-Cas12a-integrated biosensors using commercially available pregnancy test strips (PTS) for instrument-free, visual readout. Both biosensors leverage a core signal mediator, probe 1 ("MB-ssDNA1-hCG"), which links CRISPR-Cas12a activation to visible color development on the PTS. The first Biosensor CRISPR-PTS-Telo detects telomerase activity in one-step without PCR: telomerase-generated (TTAGGG)n repeats activate Cas12a-crRNA1 complex, cleaving the probe 1 to release hCG, achieving a detection limit of 18 HeLa cells-comparable to sensitive laboratory assays. The second Biosensor CRISPR-PTS-let7a detects miRNA let-7a by first converting miRNA signals to Trigger DNA via Assister DNA and probe 2 ("MB-ssDNA2+Trigger"), activating Cas12a-crRNA2 complex, cleaving the probe 1 and inducing PTS coloration. This achieves a detection limit of 25.1 fM for let-7a. Validation with clinical samples (24 cervical tissues and 26 blood samples) confirmed their concordance with gold-standard methods (ELISA for telomerase, RT-qPCR for let-7a). These versatile tools hold significant potential as point-of-care testing (POCT) solutions to facilitate early, accessible cervical cancer screening.},
}

Humans
Female
*MicroRNAs/genetics/isolation & purification/analysis
*Uterine Cervical Neoplasms/diagnosis/genetics
*Biosensing Techniques
*Telomerase/isolation & purification/genetics/analysis
CRISPR-Cas Systems/genetics
HeLa Cells
Pregnancy
Limit of Detection
Reagent Strips/analysis
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41093250,
year = {2025},
author = {Buck-Wiese, M and Liechocki, S and Erfle, H and Starkuviene, V},
title = {Comparative analysis of antibody-mediated loss-of-function versus gene knock-out and knock-down.},
journal = {SLAS discovery : advancing life sciences R & D},
volume = {37},
number = {},
pages = {100283},
doi = {10.1016/j.slasd.2025.100283},
pmid = {41093250},
issn = {2472-5560},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; *Gene Knockdown Techniques/methods ; *Antibodies ; Talin/genetics/metabolism ; RNA Interference ; RNA, Small Interfering/genetics ; Gene Expression Profiling ; Cell Adhesion/genetics ; },
abstract = {In this study we compare three methods for manipulating cell function: RNA interference (RNAi), CRISPR-Cas9 gene knock-out, and antibody-mediated loss-of-function. We have focused on analyzing changes in cell-matrix adhesion via targeting two key regulators, Talin1 (TLN1) and Kindlin-2 (KD2). Adhesion-relevant phenotypic assays revealed distinct temporal onset dynamics for each method. RNAi and CRISPR-Cas9 effectively reduced target mRNA and protein levels. In contrast, antibody transfection induced phenotypic changes without altering target expression, suggesting direct intracellular antibody-target interaction. Transcriptome analysis demonstrated that antibody transfection and CRISPR-Cas9 induced fewer deregulated mRNAs than RNAi. Furthermore, transfected antibodies and sgRNAs shared 30 % and 70 % of deregulated transcripts to their negative controls, respectively. Whereas only 10 % of overlap was recorded between targeting and control siRNAs. Our findings emphasize the importance of considering method-specific temporal dynamics of on-target phenotype appearance and off-target manifestation. Additionally, they highlight intracellular delivered antibodies as a valuable alternative for validating and complementing genetic approaches.},
}

Humans
CRISPR-Cas Systems/genetics
*Gene Knockout Techniques/methods
*Gene Knockdown Techniques/methods
*Antibodies
Talin/genetics/metabolism
RNA Interference
RNA, Small Interfering/genetics
Gene Expression Profiling
Cell Adhesion/genetics

@article {pmid40392482,
year = {2025},
author = {Huang, S and Wu, J and Yang, Y and Zhu, M and Chen, L and Zhang, S and Yang, Y and Sun, X and Xie, Y},
title = {Investigate the Effect of ZFP64 on mRNA Expression of HBG Based on Bioinformatics and Experimental Validation.},
journal = {Cell biochemistry and biophysics},
volume = {83},
number = {4},
pages = {4427-4437},
pmid = {40392482},
issn = {1559-0283},
support = {2020A0505100062, 2023A1515010872, 2024A1515012233//the Natural Science Foundation of Guangdong Province/ ; 32070582//National Natural Science Foundation of China/ ; 2023HLLH01//the Joint Foundation of He Lin Academical Workstation of the Third Affiliated Hospital of Guangzhou Medical University/ ; 2023A03J0386, 2023A03J0395//Guangdong Municipal Department of Science and Technology, Municipal Schools (Institutes) Jointly Funded Project/ ; .02-408-2203-2059//Guangzhou Medical University for the First-class Professional Construction Project in 2022-Enhancement of Undergraduates' Scientific Research and Innovation Ability Project/ ; 2024SRP119//plan on enhancing scientific research in GMU/ ; },
mesh = {Humans ; *RNA, Messenger/metabolism/genetics ; *Transcription Factors/genetics/metabolism ; *Computational Biology ; K562 Cells ; *gamma-Globins/genetics/metabolism ; Protein Interaction Maps ; *DNA-Binding Proteins/genetics/metabolism ; *Gene Expression Regulation ; CRISPR-Cas Systems ; },
abstract = {γ-globin genes (HBG1 and HBG2) are usually expressed during fetal life, and almost no expression after birth. Therefore, the reactivation of HBG is a key target for the treatment of hemoglobinopathy. ZFP64 is a C2H2 type zinc finger transcription factor, which has been shown to play an important role in the maintenance of gene expression in mixed lineage leukemia, and other C2H2 type zinc finger transcription factors (such as ZFP410 and ZFP644) have been shown to regulate the expression of fetal hemoglobin (HbF) in thalassemia. This study aims to investigate the effect of ZFP64 on mRNA expression of HBG. We performed bioinformatics analyses using the Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and protein-protein interaction (PPI) networks to identify genes and transcription factors associated with ZFP64. ZFP64 was knocked out in K562 and HUDEP-2 cell lines by CRISPR-Cas9 electroporation, and the transcription levels of ZFP64, HBB and HBG were analyzed. In undifferentiated and 7-day differentiated HUDEP-2 cells, knocking down ZFP64 resulted in a 1.5-fold and 2.5-fold increase in HBG mRNA expression, respectively (p < 0.05). These findings suggest that ZFP64 is a potential regulator of HBG expression and warrants further investigation as a therapeutic target in hemoglobinopathies.},
}

Humans
*RNA, Messenger/metabolism/genetics
*Transcription Factors/genetics/metabolism
*Computational Biology
K562 Cells
*gamma-Globins/genetics/metabolism
Protein Interaction Maps
*DNA-Binding Proteins/genetics/metabolism
*Gene Expression Regulation
CRISPR-Cas Systems

@article {pmid41341502,
year = {2025},
author = {Gao, Y and Chen, J},
title = {Fast but accurate: a systematic review and meta-analysis on diagnostic performance of MRSA detection in clinical samples by using CRISPR-based rapid molecular methods.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1703247},
pmid = {41341502},
issn = {1664-302X},
abstract = {BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant global health threat due to its multidrug resistance and association with severe infections. Conventional culture methods are time-consuming, usually requiring 48-72 h to obtain results, while conventional molecular methods such as PCR or qPCR, though faster, still require trained personnel and specialized instruments, which may delay timely clinical treatment and infection control. CRISPR-based methods have emerged as promising alternative tools for MRSA detection, but their real-world performance still requires comprehensive assessment. This meta-analysis aimed to systematically evaluate the diagnostic accuracy and timeliness of CRISPR/Cas systems for MRSA detection in clinical samples.

METHODS: A systematic search of PubMed, Embase, Web of Science, and Cochrane Library was conducted using search terms related to MRSA, CRISPR/Cas, diagnostic accuracy, and rapid detection. Studies reporting sensitivity and specificity with extractable 2 × 2 contingency tables were included. Quality was assessed via QUADAS-2. Meta-disc 1.4.0 and Stata 16.0 were used for statistical analysis, including pooled sensitivity, specificity, likelihood ratios, diagnostic odds ratios (DOR) and summary receiver operating characteristic (SROC). Median detection time and subgroup analyses were also conducted.

RESULTS: Twelve studies were included. The results showed that the CRISPR-based methods showed a pooled sensitivity of 99% (95% CI: 97-100%) and specificity of 100% (95% CI: 99-100%), with a PLR of 32.68 (95% CI: 15.45-69.15), NLR of 0.03 (95% CI: 0.02-0.07), and DOR of 664.25 (95% CI: 234.59-1880.84). The median detection time across included studies was 60 min (IQR: 41.25-98.75 min).

CONCLUSION: CRISPR-based molecular assays demonstrated exceptional accuracy and rapid detection capability for MRSA in clinical settings, significantly outperforming conventional methods. However, potential publication bias and methodological limitations warrant cautious interpretation of these results.

PROSPERO ID: CRD420251115439.},
}

@article {pmid41340056,
year = {2025},
author = {Braun, S and Knackfuß, K and Ziesmann, T and Mlinzk, L and Goerg, A and Frankenheim, J and Walter, A and Schneider-Brachert, W and Distler, U and Fritsch, J},
title = {Loss of ADAM15 prevents necroptosis induction by partial RIPK1 degradation due to enhanced TNF-R1 surface expression and basal caspase-8 activation.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {520},
pmid = {41340056},
issn = {1478-811X},
mesh = {Humans ; *Caspase 8/metabolism ; *ADAM Proteins/metabolism/genetics/deficiency ; *Receptor-Interacting Protein Serine-Threonine Kinases/metabolism ; *Proteolysis ; *Membrane Proteins/metabolism/genetics/deficiency ; *Necroptosis ; *Receptors, Tumor Necrosis Factor, Type I/metabolism ; Enzyme Activation ; Jurkat Cells ; U937 Cells ; Signal Transduction ; },
abstract = {BACKGROUND: Cell death and survival processes must be tightly regulated to ensure proper tissue homeostasis and prevent excessive inflammation and tissue damage. Death receptors, including TNF-R1, can induce either immunogenic (necroptosis) or non-immunogenic (apoptosis) cell death and relay proliferative / cell survival signaling by activating NFκB and MAPK cascades. In a recent report, we identified the metalloproteinase ADAM15 as a possible TNF-responding enzyme, leading to the hypothesis that it regulates either cell survival or death cascades.

METHODS: CRISPR/Cas-9 was used to knock out the adam15 gene. Loss of gene expression was validated by Western blot and flow cytometry in U937 and Jurkat cells. NFκB, MAPK signaling, and cell death cascades were monitored by Western blot, flow cytometry, and enzyme assays. A bottom-up proteome analysis was performed to elucidate cellular processes affected by ADAM15 loss. The subcellular localization of ADAM15 was monitored by microscopy and immuno-magnetic fractionation.

RESULTS: We identified ADAM15 as a regulator of necroptosis, leaving apoptosis and cell survival signaling unaffected. Loss of ADAM15 resulted in abrogated necroptosis, as evidenced by the application of death ligands TNF, TRAIL, FasL, and TL1a, as well as the BH3 mimetic Obatoclax. We observed enhanced basal Caspase-8 activity, which was not cytotoxic, and partial RIPK1 proteolysis. The loss of ADAM15 was verified in a proteome screen, which revealed alterations in various molecular pathways, including autophagy, organelle trafficking, and sorting. We observed ADAM15 in intracellular compartments, which in part have a lysosomal protein signature. We observed enhanced surface expression of TNF-R1, proposing it as a possible ADAM15 substrate.

CONCLUSIONS: ADAM15 is a previously unknown regulator of necroptosis, likely due to its role in modulating intracellular organelle sorting processes. Its proteolytic activity and possible scaffolding capacity for recruiting adaptor molecules make it a veritable drug target. The activation or deactivation of ADAM15 may be exploited to modulate various disease conditions.},
}

Humans
*Caspase 8/metabolism
*ADAM Proteins/metabolism/genetics/deficiency
*Receptor-Interacting Protein Serine-Threonine Kinases/metabolism
*Proteolysis
*Membrane Proteins/metabolism/genetics/deficiency
*Necroptosis
*Receptors, Tumor Necrosis Factor, Type I/metabolism
Enzyme Activation
Jurkat Cells
U937 Cells
Signal Transduction

@article {pmid41339642,
year = {2025},
author = {Yang, WJ and Liu, BY and Xue, L},
title = {Knockout of protein arginine methyltransferase 1 inhibited cell growth and promoted cell migration in human bronchial epithelial cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {43069},
pmid = {41339642},
issn = {2045-2322},
support = {2018BFC360//Fund for Key Laboratory Construction of Hubei Province/ ; 31101047//National Natural Science Foundation of China/ ; CZQ22013//"the Fundamental Research Funds for the Central Universities", South-Central MinZu University/ ; PTZ24018//i Medical Biology International Science and Technology Cooperation Base/ ; },
mesh = {Humans ; *Cell Movement/genetics ; *Cell Proliferation/genetics ; *Protein-Arginine N-Methyltransferases/genetics/metabolism ; *Epithelial Cells/metabolism/cytology ; *Bronchi/cytology/metabolism ; Cell Line ; Apoptosis/genetics ; Gene Knockout Techniques ; *Repressor Proteins/genetics/metabolism ; Cell Cycle/genetics ; CRISPR-Cas Systems ; },
abstract = {Previous studies have demonstrated that PRMT1 was involved in the progression of multiple lung diseases. However, its specific function within the bronchial epithelium was still limited and needed further exploration. In the present study, human bronchial epithelial cell line 16HBE was chosen to elucidate the biological role of PRMT1 in lung epithelium. Cell proliferation, cell-cycle distribution, cell apoptosis, and cell motility capacity were systematically evaluated following CRISPR/Cas9-mediated knockout of PRMT1. We showed that knockout of PRMT1 in 16HBE inhibited cell proliferation, redistributed cell cycle, promoted cell apoptosis, and accelerated cell migration via a series of regulated cyclins, cyclin-dependent kinase regulators, and EMT markers. Taken together, these findings identify PRMT1 as a potential modulator of epithelial cell proliferation, survival, and motility in the human bronchial epithelium, offering new insights into its possible role in epithelial remodeling during pulmonary disorders.},
}

Humans
*Cell Movement/genetics
*Cell Proliferation/genetics
*Protein-Arginine N-Methyltransferases/genetics/metabolism
*Epithelial Cells/metabolism/cytology
*Bronchi/cytology/metabolism
Cell Line
Apoptosis/genetics
Gene Knockout Techniques
*Repressor Proteins/genetics/metabolism
Cell Cycle/genetics
CRISPR-Cas Systems

@article {pmid41337296,
year = {2025},
author = {Cimolato, C and Letrari, S and Chiacchiera, AF and Del Favero, S and Schenato, L and Pasotti, L and Bellato, M},
title = {Modeling of Phage-Mediated CRISPRi System to Inhibit Antibiotic Resistances in Bacteria.},
journal = {Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference},
volume = {2025},
number = {},
pages = {1-7},
doi = {10.1109/EMBC58623.2025.11253443},
pmid = {41337296},
issn = {2694-0604},
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems/genetics ; *Bacteria/genetics/drug effects/virology ; *Drug Resistance, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Humans ; },
abstract = {Antimicrobial resistance (AMR) poses a critical threat to global health, rendering traditional antibiotics increasingly ineffective and amplifying the urgency for innovative solutions. Among promising alternatives, synthetic biology emerges as a powerful tool to combat AMR. This work proposes an innovative strategy based on engineering bacteriophages to deliver CRISPR interference (CRISPRi) systems into antibiotic-resistant pathogens to precisely silence target resistance genes. A comprehensive mathematical model is developed and simulated to capture the dynamics of phage-mediated CRISPRi delivery. By explicitly incorporating mutations that affect CRISPRi functionality, the study evaluates system performance and its potential for long-term therapeutic efficacy. This model serves as a critical framework for optimizing future CRISPRi-based interventions and advancing synthetic biology-driven approaches to tackle AMR.Clinical relevance- This paper provides a quantitative modeling framework to evaluate key parameters affecting engineered phage therapy efficiency, supporting rational design and phage posology optimization.},
}

*Bacteriophages/genetics
*CRISPR-Cas Systems/genetics
*Bacteria/genetics/drug effects/virology
*Drug Resistance, Bacterial/genetics
Anti-Bacterial Agents/pharmacology
Humans

@article {pmid41336948,
year = {2025},
author = {Yu, ES and Jang, H and Kwon, J and Jeong, H and Park, J and Kang, T and Jeong, KH},
title = {On-chip Nanoplasmonic RT-RPA and CRISPR/Cas12a Assay for Point-of-care Molecular Diagnostics.},
journal = {Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference},
volume = {2025},
number = {},
pages = {1-4},
doi = {10.1109/EMBC58623.2025.11253682},
pmid = {41336948},
issn = {2694-0604},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics ; *COVID-19/diagnosis ; *Point-of-Care Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; *Lab-On-A-Chip Devices ; *Molecular Diagnostic Techniques ; *Pathology, Molecular/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and accurate nucleic acid detection at point-of-care (POC) is essential for advancing effective disease diagnosis and management. Here, we report a handheld nanoplasmonic all-in-one setup for on-chip recombinase polymerase amplification (RPA) and real-time fluorescence detection by CRISPR/Cas12a reaction. The all-in-one setup consists of AuNIs-based nanoplasmonic cavity (AuNIs-NC), a disposable plastic-on-polymer (PoP) cartridge, and fluorescence microlens array (FMLA) camera. The AuNIs-NC allows uniform and efficient photothermal heating under white LED illumination due to strong broadband light absorption and internal reflection by randomly distributed AuNIs and thin Al film. This setup allows the RPA and CRISPR/Cas 12a reactions in a single chamber of PoP cartridge, with fluorescence signals monitored by a FMLA camera. The experimental result demonstrates rapid SARS-CoV-2 E gene plasmid DNA detection within 20 min, achieving a detection sensitivity of 10 copies/ul. Testing with 16 clinical samples shows a linear trend with RT-qPCR, indicating the platform's reliable sensitivity and specificity. This compact platform offers affordable and reliable molecular diagnosis, facilitating rapid and scalable POC testing for a range of infectious diseases.Clinical Relevance- This on-chip real-time RT-RPA and CRISPR/Cas12a assay provides rapid and precise molecular diagnostics at POC using fully integrated plasmonic system.},
}

Humans
*CRISPR-Cas Systems/genetics
SARS-CoV-2/genetics
*COVID-19/diagnosis
*Point-of-Care Systems
*Nucleic Acid Amplification Techniques/methods/instrumentation
*Lab-On-A-Chip Devices
*Molecular Diagnostic Techniques
*Pathology, Molecular/methods
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41332531,
year = {2025},
author = {Zhang, P and Xue, B and Xie, Y and Li, K and Yang, H and Sun, P and Zhang, L},
title = {OSM-11 modulates salinity-stress tolerance in Caenorhabditis elegans.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.11.20.689412},
pmid = {41332531},
issn = {2692-8205},
abstract = {Most terrestrial animals exhibit narrow salinity tolerance compared to their marine counterparts. Previous studies identified osm-11 (which encodes a Notch co-ligand) mutations as a driver of hyper-saline tolerance in Caenorhabditis elegans , but mechanistic insights remained unclear. This study employs RNA sequencing and CRISPR/Cas-9 genome editing to demonstrate that osm-11 mutations enhance salinity stress resistance through up-regulation of fatty acid metabolism (acdh-12 , acs-17) and cytochrome P450 pathways (ugt-15), while suppressing calcium signaling. Furthermore, we demonstrated that acdh-12 mutation impairs salinity-stress tolerance by activating ferroptosis and mitophagy, accompanied by down-regulated oxidative phosphorylation and up-regulated autophagic pathways. Morphological observations show that mitochondrial fragmentation contributes to wild-type nematode mortality under high salinity, while enlarged lipid droplets in wild-types correlate with reduced β-oxidation gene expression (dhs-28 , daf-22), whose knockout disrupts tolerance in mutants. These findings unravel the multi-pathway regulatory network of osm-11 -mediated salinity tolerance, providing mechanistic insights for developing protective strategies against environmental salinity stressors impacting animal survival.},
}

@article {pmid41331925,
year = {2025},
author = {Matsumoto, D and Kubota, K and Sato, Y and Kato-Inui, T and Nigorikawa, K and Miyaoka, Y and Nomura, W},
title = {Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin.},
journal = {Journal of biomedical science},
volume = {32},
number = {1},
pages = {102},
pmid = {41331925},
issn = {1423-0127},
support = {JP23K13844//KAKENHI/ ; JP24K09445//KAKENHI/ ; JP20H03442//KAKENHI/ ; JP20K21253//KAKENHI/ ; JP22H02201//KAKENHI/ ; JP23K23468//KAKENHI/ ; },
mesh = {Humans ; *Diphtheria Toxin/genetics ; *CRISPR-Associated Protein 9/genetics ; *Gene Editing/methods ; *Recombinational DNA Repair/genetics ; *CRISPR-Cas Systems ; HEK293 Cells ; Mutation ; },
abstract = {BACKGROUND: In gene therapy via genome editing, it is essential to precisely repair disease-associated gene sequences without introducing random mutations. However, achieving highly accurate genome editing remains challenging owing to the low efficiency of homology-directed repair (HDR)-mediated gene repair, which relies on template DNA. Therefore, if Cas9 mutants capable of enhancing HDR can be identified, they could enable more precise gene therapies.

METHOD: In this research project, we developed a screening system that uses the acquisition of diphtheria toxin resistance as an indicator of HDR efficiency in human cells and EGFP disruption as an indicator of off-target effect.

RESULTS: By screening a library of SpCas9 variants with random mutations introduced into its nuclease domain, we identified a novel SpCas9 mutant with higher HDR efficiency than wild-type Cas9.

CONCLUSION: We explored the possibility of obtaining Cas9 mutants with high HDR efficiency via this screening system.},
}

Humans
*Diphtheria Toxin/genetics
*CRISPR-Associated Protein 9/genetics
*Gene Editing/methods
*Recombinational DNA Repair/genetics
*CRISPR-Cas Systems
HEK293 Cells
Mutation

@article {pmid41331675,
year = {2025},
author = {Wang, M and Zhang, Y and Bi, C and Li, M},
title = {CRISPR-Cas9-induced double-strand breaks disrupt maintenance of epigenetic information.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {411},
pmid = {41331675},
issn = {1474-760X},
support = {BAS/1/1080-01-01//KAUST Office of Sponsored Research/ ; 5932//KAUST Center of Excellence for Smart Health/ ; },
mesh = {Humans ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; DNA Methylation ; *Epigenesis, Genetic ; Gene Editing ; MutL Protein Homolog 1/genetics ; Human Embryonic Stem Cells/metabolism ; Genomic Imprinting ; DNA Repair ; },
abstract = {BACKGROUND: CRISPR-Cas9 genome editing enables precise genetic modifications by introducing targeted DNA double-strand breaks (DSBs). While Cas9-induced DSBs are known to cause unintended on-target mutations, their impact on the epigenetic landscape remains unexplored.

RESULTS: Here, we investigate how Cas9-induced DSBs affect DNA methylation patterns in human embryonic stem cells (hESCs). We induce DSBs at differentially methylated regions of imprinted genomic loci and perform high-coverage, long-read native DNA sequencing to simultaneously obtain genetic variant and base-resolution methylation data in a haplotype-resolved manner. Our findings reveal that DSBs cause significant changes in DNA methylation at target sites through mechanisms including homologous recombination, large structural variations, or defective methylation maintenance during DNA repair. Notably, these epigenetic changes can occur either together with or independently of genetic alterations. Beyond imprinted loci, Cas9-induced DSBs significantly disrupt DNA methylation patterns of the MLH1 epimutation alleles in colorectal cancer cells, and hypermethylated heterochromatin loci in hESCs. Clonal analysis indicates that the aberrant methylation changes are stable during in vitro passaging. Intriguingly, significant changes in DNA methylation levels are also detected around endogenous deletions in unedited genomic regions, suggesting that methylation alterations are not unique to Cas9 nuclease activity but represent a general outcome of DSB repair in human cells.

CONCLUSIONS: This study underscores the importance of assessing and mitigating unintended epigenetic consequences in genome editing applications, as such changes can profoundly affect gene regulation and cellular function.},
}

Humans
*DNA Breaks, Double-Stranded
*CRISPR-Cas Systems
DNA Methylation
*Epigenesis, Genetic
Gene Editing
MutL Protein Homolog 1/genetics
Human Embryonic Stem Cells/metabolism
Genomic Imprinting
DNA Repair

@article {pmid41277755,
year = {2025},
author = {Wang, Z and Zhang, D and Wu, Y and Ye, X and Qian, Y and Chen, F},
title = {A capillary SERS sensor based on CRISPR/Cas13a and DS Au-AgNRs for detecting miRNA-221 in serum of hepatocellular carcinoma patients.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {47},
pages = {9627-9637},
doi = {10.1039/d5ay01517k},
pmid = {41277755},
issn = {1759-9679},
mesh = {*MicroRNAs/blood/genetics ; Humans ; *Carcinoma, Hepatocellular/blood/diagnosis/genetics ; *Liver Neoplasms/blood/diagnosis/genetics ; *Spectrum Analysis, Raman/methods ; Gold/chemistry ; Silver/chemistry ; CRISPR-Cas Systems ; Nanotubes/chemistry ; Biomarkers, Tumor/blood ; Limit of Detection ; },
abstract = {The low early diagnosis rate of hepatocellular carcinoma (HCC) severely impacts patient prognosis, making the development of highly sensitive and specific early diagnostic technologies crucial. MicroRNA-221 (miR-221), an aberrantly overexpressed biomarker in HCC, holds significant diagnostic potential. This paper constructed a capillary surface-enhanced Raman scattering (SERS) sensing platform utilizing CRISPR/Cas13a trans-cleavage and double-shell gold-silver nanorods (DS Au-AgNRs) to detect serum miR-221 in HCC patients. DS Au-AgNRs were synthesized and assembled onto aminated capillaries, followed by conjugation of Cy5-labeled single-stranded DNA (ssDNA) to the DS Au-AgNR surface via Au-S bonds. In the presence of miR-221, activated CRISPR/Cas13a trans-cleavage cleaves the ssDNA, releasing Cy5 from the sensor surface and diminishing the SERS signal, enabling miR-221 quantification. The synthesized DS Au-AgNRs exhibit uniform morphology and size, are uniformly distributed on the capillary, and form numerous "hotspots", thereby significantly enhancing the SERS signal. According to the characteristic peak of Cy5 at 1074 cm[-1], a linear relationship is established between the log concentration of miR-221 and the measured SERS intensity (y = -3527.97 × -35369.60, R[2] = 0.97767), with a LOD as low as 4.17 × 10[-17] M. The sensor demonstrated high specificity and high sensitivity, and its capacity to detect miR-221 expression aligned with qRT-PCR results when analyzing serum samples, confirming that hepatocellular carcinoma patients exhibited significantly higher miR-221 levels compared to healthy individuals. The capillary SERS sensor thus provides an accurate and convenient approach for early HCC diagnosis.},
}

*MicroRNAs/blood/genetics
Humans
*Carcinoma, Hepatocellular/blood/diagnosis/genetics
*Liver Neoplasms/blood/diagnosis/genetics
*Spectrum Analysis, Raman/methods
Gold/chemistry
Silver/chemistry
CRISPR-Cas Systems
Nanotubes/chemistry
Biomarkers, Tumor/blood
Limit of Detection

@article {pmid41252908,
year = {2026},
author = {Zhou, F and Zhao, X and Wang, Y and Huang, Y},
title = {Cooperation of CRISPR/Cas12a and exonuclease III-assisted cascade cycling amplification for ultrasensitive electrochemical detection of ciprofloxacin.},
journal = {Talanta},
volume = {299},
number = {},
pages = {129131},
doi = {10.1016/j.talanta.2025.129131},
pmid = {41252908},
issn = {1873-3573},
mesh = {*Ciprofloxacin/analysis ; *Exodeoxyribonucleases/metabolism/chemistry ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Limit of Detection ; *Nucleic Acid Amplification Techniques ; *Anti-Bacterial Agents/analysis ; Food Contamination/analysis ; *CRISPR-Associated Proteins/metabolism ; DNA Probes/chemistry/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Antibiotic residues have been a serious public health concern worldwide, while sensitive and reliable detection of antibiotic residues is significant to control antibiotic contamination, ensure food safety, and safeguard human health. Herein, an ultrasensitive electrochemical biosensor is engineered for the detection of ciprofloxacin (CIP) based on the cooperation of CRISPR/Cas12a and exonuclease III (Exo III)-assisted cascade cycling amplification. The presence of CIP induces the conformational change of DNA probes and further triggers Exo III to catalyze the cascade cycling amplification, enabling propagation and ongoing accumulation of DNA fragments which act as the target strands to activate the trans-cleavage activity of CRISPR/Cas12a. Consequently, the activated CRISPR/Cas12a initiates its trans-cleavage activity to swiftly cleave the signal probes on the surface of electrode, bringing about remarkable change of electrochemical signal and eventually realizing the ultrasensitive detection of CIP. The exceptional enzymatic cycle amplification of Exo III incorporated with the superior trans-cleavage activity of CRISPR/Cas12a synergistically facilitates considerable improvement of analytical performance, resulting in a limit of detection as low as 0.022 ng mL[-1]. Benefiting from the effective amplification capacity, high fidelity and programmability of the designed detection system, the biosensor shows good precision and specificity along with robust stability for CIP detection. Moreover, the proposed electrochemical biosensor dispensing with complicated probe construction is label-free and convenient-operated, which contributes to the credible application for CIP detection in real food samples with satisfactory results, indicating promising practicability in food safety monitoring.},
}

*Ciprofloxacin/analysis
*Exodeoxyribonucleases/metabolism/chemistry
*Biosensing Techniques/methods
*Electrochemical Techniques/methods
*CRISPR-Cas Systems
Limit of Detection
*Nucleic Acid Amplification Techniques
*Anti-Bacterial Agents/analysis
Food Contamination/analysis
*CRISPR-Associated Proteins/metabolism
DNA Probes/chemistry/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41223903,
year = {2025},
author = {Alsultan, A and Karim, SM and Al-Saadi, M and Alsallami, D and Ben Said, M and Belkahia, H},
title = {Rapid and sensitive detection of Theileria equi using a novel integrated RPACRISPR/Cas13a lateral flow assay.},
journal = {Journal of equine veterinary science},
volume = {155},
number = {},
pages = {105732},
doi = {10.1016/j.jevs.2025.105732},
pmid = {41223903},
issn = {0737-0806},
mesh = {Horses ; Animals ; *Theileria/isolation & purification ; *Theileriasis/diagnosis/parasitology ; *Horse Diseases/diagnosis/parasitology ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/veterinary/methods ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Equine piroplasmosis (EP), caused by the intracellular protozoa Theileria equi, Babesia caballi, and Theileria haneyi, represents a major health and economic threat to the equine industry worldwide. Existing diagnostic methods, including PCR, serology, and microscopy, are constrained by their dependence on specialized equipment, lengthy protocols, and the requirement for skilled personnel.

AIM: This study aimed to develop a rapid, accurate, and field-deployable molecular diagnostic assay for T. equi.

METHODS: A nucleic acid-based diagnostic platform combining recombinase polymerase amplification (RPA) with CRISPR/Cas13-mediated detection and lateral flow device (LFD) readout was developed. The assay targets a conserved region of the erythrocyte merozoite antigen 1 (EMA-1) gene of T. equi. Validation was performed using 22 blood samples collected from horses, as well as specificity controls including B. caballi- and Anaplasma phagocytophilum-infected samples, synthetic EMA-1 DNA, and non-template controls. All assay steps were conducted at room temperature.

RESULTS: The integrated RPA-CRISPR/Cas13-LFD assay generated clear visual results within 50 minutes. It demonstrated complete specificity with no false positives across all tested samples. The method effectively differentiated horses infected with T. equi, including both clinically affected and asymptomatic individuals, from healthy, uninfected animals, confirming its high accuracy and reliability.

CONCLUSION: The developed assay provides a rapid, precise, and equipment-free diagnostic platform suitable for both field and clinical environments. Although the current protocol relies on DNA extraction, future optimization will aim to enable direct detection from unprocessed blood samples, thereby further simplifying point-of-care diagnostics for equine piroplasmosis.},
}

Horses
Animals
*Theileria/isolation & purification
*Theileriasis/diagnosis/parasitology
*Horse Diseases/diagnosis/parasitology
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/veterinary/methods
CRISPR-Cas Systems

@article {pmid41221894,
year = {2025},
author = {Wei, R and Wang, S and Pan, Y and Pan, W and Li, N and Tang, B},
title = {CRISPR-coupled triple cascade amplification for simultaneous lateral flow detection of Mycoplasma pneumoniae and H1N1.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {97},
pages = {19241-19244},
doi = {10.1039/d5cc04993h},
pmid = {41221894},
issn = {1364-548X},
mesh = {*Mycoplasma pneumoniae/isolation & purification/genetics ; *Influenza A Virus, H1N1 Subtype/isolation & purification/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Saliva/microbiology/virology ; Limit of Detection ; },
abstract = {We developed a CRISPR-coupled triple cascade system integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) for simultaneous lateral flow detection of Mycoplasma pneumoniae and H1N1 in saliva samples, achieving a LOD of 10 aM for H1N1 RNA and 25 aM for MP DNA on a single LFA.},
}

*Mycoplasma pneumoniae/isolation & purification/genetics
*Influenza A Virus, H1N1 Subtype/isolation & purification/genetics
Humans
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
Saliva/microbiology/virology
Limit of Detection

@article {pmid41202787,
year = {2026},
author = {Xiao, X and Zhong, Y and Xie, H and Liao, H and Wang, H and Liu, H and Liang, F and Chen, J and Zhong, H and Chen, Z and Yu, L},
title = {A reverse transcription-free CRISPR/Cas12a biosensor for ultrasensitive detection of SARS-CoV-2 variants.},
journal = {Talanta},
volume = {299},
number = {},
pages = {129072},
doi = {10.1016/j.talanta.2025.129072},
pmid = {41202787},
issn = {1873-3573},
mesh = {*SARS-CoV-2/genetics/isolation & purification ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *COVID-19/diagnosis/virology ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Mutation ; RNA, Viral/genetics ; Reverse Transcription ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {In this work, we developed a reverse transcription-free self-primer isothermal exponential amplification reaction (RTF-SP-EXPAR) combined with a CRISPR/Cas12a biosensor for the simultaneous detection of single- and double-stranded amplification products of SARS-CoV-2 mutant variants. SP-EXPAR can recognize RNA targets directly, simultaneously generate double-stranded DNA products containing PAM sequences, and single-stranded DNA products without PAM sequences. Both types of SP-EXPAR products can be recognized by the crRNA and produce fluorescent signals, thereby enhancing detection sensitivity. This RTF-SP-EXPAR-CRISPR/Cas12a biosensor enables the detection of SARS-CoV-2 mutations within 1 h, achieving a detection limit of 7.49 aM and a dynamic range of 10 aM to 10 pM. This method shows high specificity in differentiating mutant variants from wild-type sequences. For the detection of 106 clinical samples, this RTF-SP-EXPAR-CRISPR/Cas12a assay demonstrates 100 % sensitivity and 100 % specificity compared with DNA sequencing results. These findings highlight our proposed assay's strong applicability for the application of RNA samples without reverse transcription.},
}

*SARS-CoV-2/genetics/isolation & purification
*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
*COVID-19/diagnosis/virology
Nucleic Acid Amplification Techniques/methods
Limit of Detection
Mutation
RNA, Viral/genetics
Reverse Transcription
CRISPR-Associated Proteins/genetics
Bacterial Proteins
Endodeoxyribonucleases

@article {pmid41175865,
year = {2025},
author = {Ahrens-Nicklas, RC and Musunuru, K},
title = {How to create personalized gene editing platforms: Next steps toward interventional genetics.},
journal = {American journal of human genetics},
volume = {112},
number = {12},
pages = {2826-2829},
doi = {10.1016/j.ajhg.2025.10.006},
pmid = {41175865},
issn = {1537-6605},
mesh = {Humans ; *Gene Editing/methods/trends ; *Precision Medicine/methods/trends ; *Genetic Therapy/methods/trends ; CRISPR-Cas Systems/genetics ; },
abstract = {How do we go from a single individual receiving a personalized gene-editing therapy to a future of "interventional genetics" in which such therapies are the standard of care? First and foremost: regulatory innovation.},
}

Humans
*Gene Editing/methods/trends
*Precision Medicine/methods/trends
*Genetic Therapy/methods/trends
CRISPR-Cas Systems/genetics

@article {pmid41135356,
year = {2026},
author = {Wang, W and Zheng, Y and Zhang, L and Bao, Y and Liu, J and Zang, J and Qu, Y and Zhang, K and Han, R and Ren, H and Zhu, L and Cao, Y and Wang, B and Shen, Q and Sun, W},
title = {A multiplex RPA-CRISPR/Cas12a platform for rapid and accurate toxinotyping of Clostridium perfringens.},
journal = {Talanta},
volume = {298},
number = {Pt B},
pages = {128998},
doi = {10.1016/j.talanta.2025.128998},
pmid = {41135356},
issn = {1873-3573},
mesh = {*Clostridium perfringens/genetics/isolation & purification ; *CRISPR-Cas Systems ; *Bacterial Toxins/genetics/analysis ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; Humans ; Limit of Detection ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Clostridium perfringens (C. perfringens) is a leading cause of foodborne disease worldwide, requiring rapid and accurate toxinotyping for effective outbreak control and surveillance. Herein, we developed C. perfringens-multiplex RPA-CRISPR/Cas12a, an integrated detection platform combing multiplex Recombinase Polymerase Amplification (RPA) with Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 12a (CRISPR/Cas12a)-mediated detection for comprehensive toxinotyping. The system simultaneously identifies six key toxin genes (cpa, cpb, etx, iap, cpe, netB) in two reaction tubes, enabling discrimination of all seven C. perfringens toxinotypes (A-G). The C. perfringens-multiplex RPA-CRISPR/Cas12a assay platform exhibited exceptional analytical performance, achieving a detection limit of ≤10 copies/μL for across all targets while maintaining absolute specificity against the human genomic DNA and 5 common foodborne pathogens. In validation testing with 12 naturally contaminated food samples, the C. perfringens-multiplex RPA-CRISPR/Cas12a assay platform demonstrated superior performance to commercial qPCR kits, accurately identifying eight Type A (cpa-gene-positive) and four Type F (cpa-gene and cpe-gene co-positive) strains. When coupled with a portable detection device, the platform completed the entire diagnostic workflow within 50 min while maintaining laboratory-level accuracy under field conditions. The rapid, cost-effective, and equipment-free system is particularly suited for decentralized toxin surveillance in resource-limited settings. By integrating high sensitivity, multiplex capability, and field applicability, this system significantly advances Point-of-care Testing (POCT) capabilities for food safety monitoring, supporting global food safety initiatives.},
}

*Clostridium perfringens/genetics/isolation & purification
*CRISPR-Cas Systems
*Bacterial Toxins/genetics/analysis
*Nucleic Acid Amplification Techniques/methods
*Recombinases/metabolism
Humans
Limit of Detection
Bacterial Proteins/genetics
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41109522,
year = {2025},
author = {Garcia, AFS and Farinati, S and Draga, S and Riommi, D and Gabelli, G and Vannozzi, A and Barcaccia, G and Palumbo, F},
title = {Establishing a cutting-edge protoplast technology platform for applying new genomic techniques in Cichorium spp.},
journal = {New biotechnology},
volume = {90},
number = {},
pages = {206-222},
doi = {10.1016/j.nbt.2025.10.008},
pmid = {41109522},
issn = {1876-4347},
mesh = {*Protoplasts/metabolism ; *Gene Editing/methods ; *Genomics/methods ; CRISPR-Cas Systems ; *Cichorium intybus/genetics ; },
abstract = {Genome editing technologies, especially those based on the CRISPR/Cas9 system, have revolutionized crop breeding by enabling precise genetic modifications. Specifically, delivering preassembled ribonucleoprotein (RNP) complexes-consisting of the Cas9 endonuclease coupled to specific single guide RNAs (sgRNAs)-into protoplasts offers an effective DNA-free method that prevents the integration of foreign genetic material. Despite the availability of detailed protocols, establishing a standardized and efficient in vitro regeneration procedure-from protoplast isolation to whole plant regeneration-remains challenging due to significant variability in regeneration efficiency across different varieties and biotypes. Therefore, optimizing each step is essential to maximize the recovery of successful edited plants. In this study, we developed an efficient protocol for regenerating whole plants from protoplasts isolated from 12 representative Italian varieties of chicory and endive. We focused on leaf chicory and endive biotypes with high horticultural value, including Radicchio types, which are important targets for quality improvement. Our optimized platform supports protoplast isolation, PEG-mediated transfection, and plant regeneration, demonstrating promising potential for future genome editing applications. Notably, the high responsiveness of protoplasts to PEG-mediated transfection suggests that coupling this method with our regeneration procedure could facilitate the use of advanced biotechnological strategies. The combination of high transient transformation efficiency, versatile encapsulation techniques, and successful plant regeneration establishes chicory and endive as promising candidates for DNA-free genome editing via protoplasts, providing a technically precise approach with reduced environmental and economic impacts compared to conventional breeding methods.},
}

*Protoplasts/metabolism
*Gene Editing/methods
*Genomics/methods
CRISPR-Cas Systems
*Cichorium intybus/genetics

@article {pmid41075867,
year = {2025},
author = {Zhang, D and Liu, H and Zhong, Y},
title = {Monoclonal antibodies production in microbial systems: Current status, challenges and perspectives.},
journal = {New biotechnology},
volume = {90},
number = {},
pages = {163-173},
doi = {10.1016/j.nbt.2025.10.005},
pmid = {41075867},
issn = {1876-4347},
mesh = {*Antibodies, Monoclonal/biosynthesis/genetics ; *Metabolic Engineering ; Humans ; CRISPR-Cas Systems ; Animals ; Gene Editing ; Synthetic Biology ; },
abstract = {Monoclonal antibodies (mAbs) serve as indispensable tools in diagnostics, clinical therapeutics, and biomedical research. However, their large-scale production faces significant challenges due to the high costs and lengthy timelines associated with conventional mammalian cell-based expression systems. Microbial expression platforms have emerged as a transformative alternative, offering cost-effectiveness, rapid cultivation cycles, and superior genetic tractability for industrial-scale monoclonal antibodies production. Recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing enable precise metabolic engineering of host strains to enhance protein folding, secretion efficiency, and translational accuracy. Synthetic biology approaches facilities the reconstruction of mammalian glycosylation pathways in microbial systems, yielding monoclonal antibodies with near-native structural integrity. Furthermore, AI (artificial intelligence)-driven optimization of expression vectors, promoter systems, and culture conditions, combined with high-throughput screening of engineered strains, significantly accelerates the identification of high-yield production clones. This review comprehensively examines current progress in microbial expression systems, strain engineering strategies, and fermentation optimization for enhanced monoclonal antibodies production, while critically discussing existing limitations and potential solutions to advance the field.},
}

*Antibodies, Monoclonal/biosynthesis/genetics
*Metabolic Engineering
Humans
CRISPR-Cas Systems
Animals
Gene Editing
Synthetic Biology

@article {pmid41072226,
year = {2026},
author = {Li, L and Li, M and Wang, S and Dong, Y},
title = {Development of a CRISPR/Cas12a-assisted fluorescent aptasensor for simultaneous detection of zearalenone and ochratoxin A.},
journal = {Talanta},
volume = {298},
number = {Pt B},
pages = {128937},
doi = {10.1016/j.talanta.2025.128937},
pmid = {41072226},
issn = {1873-3573},
mesh = {*Ochratoxins/analysis ; *Zearalenone/analysis ; *Aptamers, Nucleotide/chemistry/genetics/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Limit of Detection ; Food Contamination/analysis ; Spectrometry, Fluorescence ; Fluorescence ; *Fluorescent Dyes/chemistry ; },
abstract = {Mycotoxins, such as zearalenone (ZEN) and ochratoxin A (OTA), represent significant hazards to both human and animal health, necessitating strict monitoring and regulation of mycotoxin levels in food, feed, and environment. In this study, a simple and efficient CRISPR/Cas12a-assisted fluorescent aptasensor is presented for the simultaneous detection of ZEN and OTA. Utilizing a Bifunctional aptamer (B-APT), this biosensor achieves dual recognition of the both targets, subsequently converting their concentrations into observable fluorescent signals through the Cas12a/crRNA cis-cleavage activity. Rational modifications of the complementary strands specific to the two targets enable distinct emission wavelengths under the same excitation light, facilitating simultaneous and independent quantitative determination of ZEN and OTA. Under optimized conditions, the CRISPR/Cas12a-aptasensor demonstrates robust detection capabilities for individual ZEN and OTA targets, as well as their mixture, yielding consistent standard curves. This methodology exhibits reliable detection of ZEN and OTA concentrations spanning from 0.25 nM to 256 nM and 1 nM to 1024 nM, with respective limit of detection (LOD) values of 190 pM and 931 pM. Furthermore, this method showcases exceptional selectivity and considerable recovery rates (89.17 %-109.88 % for ZEN and 101.19 %-106.51 % for OTA) in corn oil samples, underscoring its efficacy as an advanced tool for ZEN and OTA detection and offering valuable insights into the simultaneous detection of diverse targets.},
}

*Ochratoxins/analysis
*Zearalenone/analysis
*Aptamers, Nucleotide/chemistry/genetics/metabolism
*Biosensing Techniques/methods
*CRISPR-Cas Systems
Limit of Detection
Food Contamination/analysis
Spectrometry, Fluorescence
Fluorescence
*Fluorescent Dyes/chemistry