@article {pmid41810060,
year = {2026},
author = {Shen, Z and Liu, Y and Hao, Y and Bo, Y and Dai, X and Wang, S and Xia, T and Su, X and Liu, H},
title = {Advances in Double-Stranded DNA Targeting Technologies.},
journal = {Exploration (Beijing, China)},
volume = {6},
number = {1},
pages = {20250065},
pmid = {41810060},
issn = {2766-2098},
abstract = {Double-stranded DNA (dsDNA) serves as a fundamental repository of genetic information and plays a pivotal role in the diagnosis and therapeutic management of diseases. However, the inherent stability of the DNA double helix under physiological conditions presents a challenge in accessing internal bases. To address this, various molecular targeting technologies have been developed, offering high specificity while destabilizing the DNA structure. This review provides a comprehensive overview of current dsDNA targeting tools, such as hybridization probes, modified nucleic acid probes, zinc finger proteins (ZFPs), transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system, Argonaute proteins (Agos), and the lambda exonuclease-pDNA system (λ Exo-pDNA), and some cutting-edge molecular tools. It delves into the mechanisms behind these technologies. It highlights their applications in diverse areas, including in vitro detection, in situ imaging, gene editing, and their integration with artificial intelligence (AI)-driven tools. Additionally, the review compares these techniques, discusses future technological opportunities, and identifies challenges in integrating these tools into diagnostic and therapeutic practices. By providing a holistic view of these rapidly evolving technologies, this review aims to fill a gap in the current literature and explore the future potential of dsDNA targeting innovations.},
}
@article {pmid41809894,
year = {2025},
author = {Yang, P and Khoshandam, M and Bhia, I and Raji, S and Soltaninejad, H and Hosseinkhani, S and Sani, M and Hamidieh, AA and Sheykhhasan, M},
title = {Integrating CRISPR/Cas technology with clinical trials: Principles, progress and challenges.},
journal = {Asian journal of pharmaceutical sciences},
volume = {20},
number = {6},
pages = {101068},
pmid = {41809894},
issn = {2221-285X},
abstract = {CRISPR represent a groundbreaking genome-editing technology that has revolutionized genetic modification. This innovative tool offers an unparalleled revolution in the future treatment of genetic disorders, neurological diseases, infectious diseases and cancer. Despite the rapid expansion of CRISPR applications, its clinical use in humans is still relatively limited, with only 69 active clinical trials and 6 completed studies reported so far. This review examined current clinical trials and their processes in addressing various diseases via the CRISPR/Cas system. While earlier literatures have focused mainly on delivery methods and materials for CRISPR/Cas9, our review emphasized innovative targeting conditions and approaches for novel and functional therapeutic designs. In addition, we reviewed recent research to increase the efficiency of CRISPR editing in the management of genetic disorders and cancer, while exploring their future challenges and potential. This review provided a unique perspective on the advancement of CRISPR technology. By addressing these aspects, we aim to contribute to ongoing efforts to improve CRISPR-based therapies and expand their clinical applications, ultimately striving to transform the future of medical treatment.},
}
@article {pmid41808396,
year = {2026},
author = {Zargul, A and Liu, H and Zhang, W and Wang, H and Liu, J and Chen, C},
title = {Advances in Pathogen Detection by Nanosensors: Biorecognition Strategies, Signal Amplification, and Platform Engineering.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.5c22148},
pmid = {41808396},
issn = {1936-086X},
abstract = {The escalating global threat of infectious diseases, compounded by antimicrobial resistance (AMR), calls for improved diagnostic strategies. Conventional pathogen detection techniques─culture, enzyme-linked immunosorbent assay (ELISA), and microscopy─remain hindered by prolonged turnaround times, suboptimal sensitivity for low-abundance analytes, and operational intricacy. Nanosensor technologies have emerged as powerful enablers of rapid, ultrasensitive, and field-deployable diagnostics. This review delineates the convergence of three transformative domains: (1) advanced biorecognition strategies─including monoclonal antibodies, aptamers, bacteriophages, antimicrobial peptides, molecularly imprinted polymers, and lectins─that confer high-fidelity molecular selectivity within complex biological matrices; (2) multimodal signal amplification technologies, encompassing nanomaterial-enhanced mechanisms, enzymatic cascades, and isothermal nucleic acid amplification that drive detection down to the single-cell and femtomolar regimes; and (3) integrated platform engineering, uniting clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems, artificial intelligence (AI), and microfluidics to achieve multiplexed, real-time, point-of-care deployment. Advances are critically evaluated through standardized performance metrics─limit of detection, assay time, specificity, and operational simplicity─to reveal both synergistic opportunities and enduring translational bottlenecks. Collectively, these developments define a strategic framework for next-generation nanosensor diagnostics poised to revolutionize infectious disease surveillance and enable precision-guided therapeutic intervention.},
}
@article {pmid41807728,
year = {2026},
author = {Godsil, M and Wei, N and Meeske, AJ},
title = {Conditional activation of Cas13 enforces lysogeny in a native type VI-A CRISPR host.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {41807728},
issn = {2058-5276},
support = {R35 GM142460/GM/NIGMS NIH HHS/United States ; FAIN2235762//NSF | BIO | Division of Molecular and Cellular Biosciences (MCB)/ ; R35GM142460//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) systems present a barrier to prophage acquisition by restricting invading phages or by inducing autoimmune cleavage of integrated prophage DNA. The RNA-sensing type VI CRISPR nuclease Cas13 mediates non-specific RNA cleavage upon recognition of phage lytic transcripts, but how this system influences the temperate phage life cycle remains unknown. Here we report that the Listeria seeligeri type VI-A CRISPR system restricts the lytic cycle of temperate phages but tolerates prophage acquisition and interferes with prophage induction through a non-abortive mechanism. During attempts at induction, Cas13 activation forces prophage re-integration, thus maintaining lysogeny. We also find that during polylysogenic induction, Cas13 acts specifically, restricting only the targeted phage, in contrast to its behaviour during lytic replication. Our findings show that Cas13 elicits a unique response to each stage of the temperate phage life cycle, enabling type VI CRISPR hosts to acquire potentially beneficial prophages while mitigating lysis.},
}
@article {pmid41807051,
year = {2026},
author = {Gur Dedeoglu, B and Noyan, S and İlhan, KNK},
title = {Non-coding RNAs regulation in breast cancer pathogenesis.},
journal = {Epigenomics},
volume = {},
number = {},
pages = {1-20},
doi = {10.1080/17501911.2026.2642583},
pmid = {41807051},
issn = {1750-192X},
abstract = {Breast cancer represents a molecularly heterogeneous disease shaped by complex genetic, epigenetic, and transcriptional dysregulation. Non-coding RNAs (ncRNAs) including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) as well as small nucleolar RNAs (snoRNAs), piwi-interacting RNAs (piRNAs), and small nuclear RNAs (snRNAs), have emerged as key epigenetic regulators that integrate multiple layers of gene control. Through interactions with chromatin-modifying enzymes, RNA-binding proteins, and signaling effectors, ncRNAs modulate transcriptional activity, chromatin accessibility, and post-transcriptional stability of target genes. miRNAs predominantly act as post-transcriptional repressors, whereas lncRNAs and circRNAs exert transcriptional and epigenetic control via scaffolding, miRNA sponging, and chromatin remodeling; some circRNAs even encode functional peptides. Aberrant ncRNA expression contributes to proliferation, metastasis, metabolic reprogramming, immune evasion, and therapeutic resistance, with distinct expression signatures associated with triple-negative, HER2-positive, and hormone receptor - positive breast cancers. Owing to their stability and detectability in plasma and exosomes, ncRNAs hold promise as minimally invasive biomarkers for early detection and disease monitoring. Moreover, therapeutic strategies targeting ncRNAs, such as antisense oligonucleotides, RNA interference, CRISPR/Cas-based editing, and ncRNA-derived vaccines, are advancing toward clinical translation. Collectively, ncRNAs redefine the epigenetic landscape of breast cancer, offering a framework for integrated diagnostic and therapeutic approaches in precision oncology.},
}
@article {pmid41806830,
year = {2026},
author = {Escobar, M and Malik, SA and Srinivasa, MA and Mendez-Sosa, MA and Miller, JM and Lydon, SL and Luong, SN and Mathew, PR and Abouleisa, RRE and Chakravarty, S and Pathan, S and Mohamed, TMA and Ghanta, RK and Hilton, IB},
title = {CRISPR-Cas-based activation of PPARGC1A boosts endogenous mitochondria and enhances cardiac function after myocardial infarction.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2026.02.027},
pmid = {41806830},
issn = {1525-0024},
support = {R01 HL147921/HL/NHLBI NIH HHS/United States ; R01 HL178610/HL/NHLBI NIH HHS/United States ; R61 HL177472/HL/NHLBI NIH HHS/United States ; },
abstract = {Insufficient energy supply due to impaired mitochondria has emerged as a key pathological factor in the development of heart failure (HF) after myocardial infarction (MI). Unfortunately, no current therapeutic strategies directly augment myocardial energy production. While mitochondrial biogenesis is orchestrated by the activity of multiple genes, activation of PPARGC1A, a key regulator, can increase cellular mitochondria; however, supraphysiological levels of PPARGC1A result in adverse tissue remodeling and heart dysfunction. CRISPR activation (CRISPRa) technologies present a unique opportunity to address these shortcomings, as they enable tunable control over endogenous target gene expression. Here, we demonstrate that transcriptional activation of PPARGC1A using CRISPRa increases cellular mitochondria in human cell types. This effect is mediated through the activation of transcriptional programs driving mitochondrial biogenesis, mitochondrial function, and cellular bioenergetics. These activated transcriptional programs synergize to increase ATP production and reserve capacity in human cardiomyocytes. CRISPRa targeting of PPARGC1A in vivo increases cardiac mitochondria to recover heart ejection fraction in an acute MI model. Furthermore, CRISPRa acts on the adult human heart to increase PPARGC1A protein and cellular mitochondria, elevating mitochondrial function in both normal and HF-diagnosed hearts. These results provide the first proof of concept that endogenous gene activation via CRISPRa can improve heart function after MI.},
}
@article {pmid41713565,
year = {2026},
author = {Shashikala, T and Yogi, D and Akshay, K and Nagesh, SN and Manamohan, M and Venkataravanappa, V and Jha, GK and Ashok, K and Asokan, R},
title = {First report of CRISPR/Cas13a-based rapid detection of groundnut bud necrosis virus without amplification.},
journal = {Methods (San Diego, Calif.)},
volume = {249},
number = {},
pages = {9-22},
doi = {10.1016/j.ymeth.2026.02.010},
pmid = {41713565},
issn = {1095-9130},
mesh = {*CRISPR-Cas Systems/genetics ; *Plant Diseases/virology ; RNA, Viral/genetics/isolation & purification ; *Tospovirus/genetics/isolation & purification ; Vigna/virology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Globally, the groundnut bud necrosis virus (GBNV) (Bunyaviridae), pose a serious threat to solanaceous and leguminous crops causing serious crop loss. This requires a rapid and sensitive diagnostics for initially identifying at the earliest stage and further to initiate disease management. This study presents the first report of a CRISPR/Cas13a-based diagnostic assay for GBNV detection without amplification. GBNV was maintained in cowpea cv. C152 through mechanical inoculation, further viral RNA was isolated to clone the two target genes viz. nucleocapsid (NP) and movement protein (MP) genes. These genes were ligated to pTZ57R/T vector and sequenced. Similarly, the LshCas13a gene was cloned from pUC19 into pET28a, expressed in E. coli BL21, and purified using Ni-NTA affinity chromatography. Guide RNAs targeting conserved regions of NP and MP genes were synthesized by in vitro transcription and mixed with Cas13a protein to form ribonucleoprotein (RNP) complex. Target RNA, obtained either by in vitro-transcription or crude extract of infected tomato was used to detect GBNV, using a fluorescence-based reporter assay. This method found to be highly sensitive that could detect GBNV at as low as 0.01 ng. From the field perspective, GBNV could be detected from the crude extract of the GBNV infected tomato leaves using an alkaline PEG buffer. Thus CRISPR/Cas13a-based assay provides a rapid, amplification-free, and field-deployable diagnostic platform for GBNV. This lays the groundwork for a field adoptable CRISPR diagnostics for other plant RNA viruses also.},
}
@article {pmid41678668,
year = {2026},
author = {Kim, J and Yoon, J and Chen, J and Lee, J and Lee, HJ and Whitworth, K and Redel, B and Prather, RS and Lee, K},
title = {Enhancing the specificity of gene editing outcomes by using Cas9 variants in porcine embryos.},
journal = {Journal of animal science},
volume = {104},
number = {},
pages = {},
doi = {10.1093/jas/skag030},
pmid = {41678668},
issn = {1525-3163},
mesh = {Animals ; *Gene Editing/veterinary/methods ; Swine/embryology/genetics ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Embryo, Mammalian ; Female ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas9 technology has improved the ability to introduce targeted modifications in cells and embryos in diverse species. The use of this technology enables the establishment of genetically modified livestock models to study human diseases or improve food production. However, one of the main concerns with employing this technology is the possibility of introducing unintended genome modifications induced by the Streptococcus pyogenes Cas9 (SpCas9), a commonly used Cas9 protein. Recent advancements in CRISPR/Cas9 technology offer Cas9 variants that are designed to improve gene editing specificity. Here, three high-fidelity SpCas9 variants (eSpCas9, HiFi Cas9, and LZ3 Cas9) were employed to examine their efficacy and specificity in pig embryos. To introduce targeted modifications, mRNA coding for each Cas9 variant was mixed with IGH single guide RNA (sgRNA) and were injected into fertilized pig zygotes. The frequency of on- and off-targeting was calculated by amplifying IGH, AR, and RBFOX1 regions from genomic DNA derived from the injected embryos at the blastocyst stage and sent for Sanger sequencing. The sgRNA targeting IGH locus resulted in a 100% on-target editing rate using SpCas9. However, SpCas9 introduced off-targeting events in AR and RBFOX1 at a high frequency (> 60%) in embryos. Injecting each Cas9 variant at 20 ng/µl could modify the target gene (IGH) at 100% efficiency except for LZ3 Cas9 (59.1%). Importantly, off-target events on AR and RBFOX1 were not detected in any Cas9 variant groups. Gradually reducing the concentration of Cas9 mRNAs lowered the efficacy of on-targeting in all groups; however, the reduction was more dramatic in HiFi Cas9 and LZ3 Cas9 injected embryos. No embryonic toxicity was identified in embryo injected with Cas9 variants and more embryos reached blastocyst stage when injected with either eSpCas9 or HiFiCas9 mRNA. In vivo competency of embryos receiving eSpCas9 was examined by embryo transfer and fetuses recovered from a pregnant sow presented 100% on-target editing efficiency without any detectable off-target events. In summary, among the Cas9 variants examined, eSpCas9 presented the highest specificity with no detectable off-target events and supported the development of gene-edited fetuses. Our findings indicate that the use of Cas9 variants can advance the field of gene editing in livestock models.},
}
@article {pmid41644661,
year = {2026},
author = {Davydova, S and Liu, J and Kandul, NP and Antoshechkin, I and Mann, J and Braswell, WE and Akbari, OS and Meccariello, A},
title = {Generating cisgenic sexing strains in insect pests.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41644661},
issn = {2399-3642},
support = {101059523//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; AP23PPQS&T00C108//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; },
mesh = {Animals ; Male ; Female ; *Ceratitis capitata/genetics ; CRISPR-Cas Systems ; *Pest Control, Biological/methods ; Gene Editing ; Introns ; },
abstract = {Insect pest population control via sterile insect technique markedly benefits from separation by sex prior to release. To simplify this process, traditional genetics has been deployed to develop genetic sexing strains (GSSs) for several disease vectors and agricultural pests of vast economic significance, although very few are applied in the field due to associated fitness costs and instability. In this study, we generated a method to engineer cisgenic GSS (CGSS) in insects. We use CRISPR/Cas9-mediated homology-directed repair to seamlessly translocate a sex-specific alternatively spliced intron into a dominant phenotypic gene generating a genetically stable strain that enables sex-sorting by eye. To achieve this feat, we use Ceratitis capitata as our model and relied on the sex-specifically spliced intron of its endogenous transformer gene, which we seamlessly inserted a copy into the pupal colouration white pupae gene. This minimal modification resulted in the generation of a homozygous strain we term IMPERIAL that was genetically and phenotypically stable where all female pupae are brown while male pupae are white with overall good fitness. By minimally editing the genome, our novel CGSS approach can be applied to other pests that may aid more efficient and economically suitable pest control.},
}
@article {pmid41454745,
year = {2026},
author = {Mehnath, S},
title = {Engineering stimuli-responsive nanocarriers for CRISPR/Cas9 genome editing: next-generation cancer therapeutics.},
journal = {The Journal of pharmacy and pharmacology},
volume = {78},
number = {3},
pages = {},
doi = {10.1093/jpp/rgaf127},
pmid = {41454745},
issn = {2042-7158},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Neoplasms/genetics/therapy ; Animals ; *Nanoparticles ; Drug Delivery Systems/methods ; Drug Carriers ; Genetic Therapy/methods ; },
abstract = {OBJECTIVES: To highlight recent developments in CRISPR/Cas9 genome-editing strategies for cancer therapy and to evaluate how nanocarrier-based delivery systems enable controlled, spatiotemporal manipulation of genetic information to overcome off-target effects, cytotoxicity, and limitations in clinical translation.<br><br>KEY FINDINGS: CRISPR/Cas9 has emerged as a simple and programmable tool for correcting cancer-associated mutations and regulating adaptive immune responses; however, challenges such as off-target effects, unintended mutations in healthy cells, and cytotoxicity hinder its clinical application. Nanocarriers address these limitations through refined spatiotemporal delivery of Cas9 nuclease and sgRNA using internal and external stimuli-responsive functional groups. These systems improve cancer-cell specificity by engineering guide RNAs, prevent premature clearance, enhance systemic circulation and intracellular delivery, enable nuclear targeting, and regulate Cas9 activity. Stimuli such as light, heat, ultrasound, magnetic fields, pH, redox conditions, glutathione, and oxygen play key roles in controlled activation and release.<br><br>SUMMARY: This review critically evaluates the structural design of nanocarriers, advanced spatiotemporal regulation strategies, and safety and efficacy concerns in CRISPR/Cas9-based cancer therapeutics. It discusses the role of cell-specific promoters, small-molecule stimulation, and stimuli-responsive delivery systems in improving genome-editing precision and therapeutic outcomes. The review also outlines future opportunities for exploiting CRISPR/Cas9 in advanced biomedical applications to enhance the effectiveness of next-generation cancer therapy.},
}
@article {pmid41235827,
year = {2026},
author = {Chen, W and Zhang, X and Fan, R and Li, X and Guan, F and Wan, G and Kong, W and Qi, X and Pan, S and Shi, S and Su, Y and Gao, S and Huang, W and Xian, X and Liu, J and Wang, Y and Ma, Y},
title = {Generating golden Syrian hamsters with conditional alleles via zygote microinjection of CRISPR/Cas9.},
journal = {Animal models and experimental medicine},
volume = {9},
number = {2},
pages = {308-318},
doi = {10.1002/ame2.70107},
pmid = {41235827},
issn = {2576-2095},
support = {2060204//State Key Laboratory Special Fund/ ; Peking University, 202411//Open Research Project in State Key Laboratory of Vascular Homeostasis and Remodeling/ ; 2023-PT180-01//The Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences/ ; HH24KYZX0007//Haihe Laboratory of Cell Ecosystem Innovation Fund/ ; 2021-I2M-1-024//CAMS Innovation Fund for Medical Sciences/ ; 2022-I2M-1-020//CAMS Innovation Fund for Medical Sciences/ ; 2023-I2M-2-001//CAMS Innovation Fund for Medical Sciences/ ; 2021YFF0702802//the National Key Research and Development Program of China from the Ministry of Science and Technology/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Mesocricetus/genetics ; *Microinjections ; Cricetinae ; *Zygote ; Alleles ; Gene Knockout Techniques ; Female ; Male ; Animals, Genetically Modified ; },
abstract = {BACKGROUND: The golden Syrian hamster is a valuable animal model for studying carcinogenesis, metabolic disorders, cardiovascular diseases, and viral infections due to its biological and pathological similarities to humans. However, the development of genetically engineered hamsters has lagged behind that of mice and rats, largely because of an embryonic development block at the two-cell stage in vitro. Although CRISPR/Cas9-mediated gene knockout has been achieved in hamsters, precise DNA fragment insertion or conditional knockout (cKO) models have not previously been reported, likely due to technical limitations in embryo manipulation and insufficient efficiency of homology-directed repair (HDR).<br><br>METHODS: In this study, we generated conditional alleles of the ApoF gene in golden Syrian hamsters. A two-cut strategy was applied using Cas9 protein, two sgRNAs, and a single donor plasmid containing exon 2 flanked by loxP sites and two ~0.8&#x2009;kb homology arms. A mixture of Cas9 protein, sgRNAs, and the donor plasmid was microinjected into the pronuclei of one-cell stage hamster embryos.<br><br>RESULTS: The efficiency of CRISPR/Cas9-mediated loxP knock-in reached up to 27%, and the genetically modified floxed alleles were successfully transmitted through the germline. The functionality of the inserted loxP sites was validated by in&#xa0;vivo Cre-mediated recombination following local administration of AAV vectors, including AAV-cTnT-Cre in the heart and AAV-CMV-Cre in the brain.<br><br>CONCLUSIONS: To our knowledge, this work represents the first successful establishment of a conditional knockout model in the golden Syrian hamster, providing a valuable tool for mechanistic studies of gene function and disease modeling.},
}
@article {pmid41191919,
year = {2026},
author = {Liu, C and Xu, F and Wu, Y and Li, J and Ni, M and Xia, S and Chen, L and Zhao, H and Yu, M and Zhou, Y and Zhang, M and Li, J and Wu, X and Huang, Y and Zhu, T and Chen, X},
title = {Genome-wide CRISPR-Cas9 screening identifies CLK1 inhibition as a strategy to restore PARP inhibitor sensitivity via ERCC1 isoform switching.},
journal = {Protein &amp; cell},
volume = {17},
number = {3},
pages = {248-262},
doi = {10.1093/procel/pwaf091},
pmid = {41191919},
issn = {1674-8018},
support = {82403208//National Natural Science Foundation of China/ ; 82303660//National Natural Science Foundation of China/ ; 82272898//National Natural Science Foundation of China/ ; 82272682//National Natural Science Foundation of China/ ; 82503636//National Natural Science Foundation of China/ ; SACA-AX202215//Shanghai Anti-Cancer Association/ ; 22YF1408100//Science and Technology Commission of Shanghai Municipality/ ; 16411950200//Science and Technology Commission of Shanghai Municipality/ ; KW1711//Science and Technology Commission of Shanghai Municipality/ ; 17411963000//Science and Technology Commission of Shanghai Municipality/ ; 22YF1409100//Science and Technology Commission of Shanghai Municipality/ ; CORP-252-1//Chinese Anti-Cancer Association-Hengrui PARP Inhibitor Cancer Research Fund/ ; 2025ZD0545600//Noncommunicable Chronic Diseases-National Science and Technology Major/ ; LGF21H160008//Social Development Project of Public Welfare Technology Research in Zhejiang Province/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Female ; *Protein-Tyrosine Kinases/genetics/metabolism ; *Endonucleases/metabolism/genetics ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; *Ovarian Neoplasms/drug therapy/genetics/pathology/metabolism ; Animals ; *DNA-Binding Proteins/metabolism/genetics ; Cell Line, Tumor ; *Protein Serine-Threonine Kinases/antagonists &amp; inhibitors/genetics/metabolism ; Mice ; Phthalazines/pharmacology ; Piperazines/pharmacology ; Drug Resistance, Neoplasm/drug effects ; *Carcinoma, Ovarian Epithelial/drug therapy/genetics/pathology/metabolism ; Protein Isoforms/metabolism/genetics ; Apoptosis/drug effects ; },
abstract = {Epithelial ovarian cancer (EOC) is an aggressive malignancy with limited therapeutic options. Poly(ADP-ribose) polymerase inhibitors (PARPi) have shown remarkable efficacy, especially in BRCA-mutant patients, and are approved as maintenance therapy to prevent recurrence after initial response to chemotherapy. However, the development of PARPi resistance poses a major clinical challenge. This study utilized a whole-genome CRISPR-Cas9 genetic screening to identify genes associated with PARPi sensitivity upon knockout. Based on the screening and validated through further experiments, we confirmed that CLK1 knockdown is synthetically lethal with PARPi in ovarian cancer. The combination of the PARPi Olaparib and CLK1 inhibitor TG003 exhibited potent anti-proliferative effects both in vitro and in vivo. Mechanistically, CLK1 inhibition downregulated the functional ERCC1-202 isoform, resulting in enhanced DNA damage and apoptosis. Our findings reveal a novel mechanism underlying PARPi sensitivity and suggest that targeting CLK1 in combination with PARPi may represent a promising therapeutic strategy for PARPi-resistant ovarian cancer.},
}
@article {pmid41806414,
year = {2026},
author = {Kim, H and Kim, D and Han, H and Lee, C and Roh, YH and Han, TS and Lim, EK and Park, J and Ahn, JK and Kang, T and Jung, J and Lee, CY},
title = {On-site microRNA detection with 'off-the-shelf' glucose meter empowered by chimeric probe connecting CRISPR/Cas13a activation to kinases-driven glucose phosphorylation.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118568},
doi = {10.1016/j.bios.2026.118568},
pmid = {41806414},
issn = {1873-4235},
abstract = {MicroRNAs (miRNAs) are promising biomarkers for cancer diagnosis due to their stability in body fluids and disease-specific expression profiles. However, current detection methods suffer from limitations including cumbersome workflows, heavy instrumentation for signal readout, or vulnerability in minimizing instrumentation. To address these challenges, we describe a novel point-of-care miRNA detection platform executable with "off-the-shelf", personal glucose meter (PGM), termed 'KEY-FACT (Kinases Ensemble-driven glucose phosphorYlation upon Fuel-Aided CRISPR acTivation)'. Upon recognition of target miRNA, a fuel-assisted toehold-mediated strand displacement reactions liberate guide RNAs (gRNAs) to activate Cas13a to cleave a chimeric reporter probe, producing 2',3'-cyclic adenosine monophosphates (cAMP). Subsequent dephosphorylation and kinases ensemble-mediated phosphorylation/dephosphorylation cycles lead cAMP to consume a large amount of glucose. A user can immediately measure resulting glucose level change with PGM on the spot. This strategy allows sensitive, prompt detection of miR-135b, a gastric cancer (GC) biomarker, with a limit of detection (LOD) of 1.4 pM within 2 h. KEY-FACT is specific to the target miRNA and is applicable to body fluids such as human serum with dilution (95.2% < recovery rates <104.3%, coefficients of variation ≤13%). Owing to its simple probe design, KEY-FACT was readily expanded to detect another GC biomarker, miR-21, with comparable sensitivity (LOD = 1.5 pM). The proposed platform fulfills minimal instrumentation and thus enables cost-effective, field-deployable analysis, paving the way for practical, on-demand miRNA diagnostics.},
}
@article {pmid41806413,
year = {2026},
author = {Jeong, Y and Lee, J and Choi, S and Shin, D and Jang, S and Son, SU and Kang, T and Jung, J and Hwang, J and Lim, EK},
title = {On-site detection of airborne foodborne pathogens using a field-deployable recombinase polymerase amplification and CRISPR/Cas12a cleavage activity assay.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118571},
doi = {10.1016/j.bios.2026.118571},
pmid = {41806413},
issn = {1873-4235},
abstract = {With the global increase in single-person households, the demand for meal kits is increasing, leading to the development of large-scale food production systems and complex supply chains. However, under the influence of global warming, these systems can be susceptible to food contamination, particularly by airborne foodborne bacteria. Conventional methods for detecting airborne bacteria involve complex, time-consuming, and labor-intensive processes, which limit their applicability for field use and rapid food hygiene surveillance. In the present study, we developed a field-deployable diagnostic platform by combining recombinase polymerase amplification with CRISPR/Cas12a cleaVage Activity (RCCVA assay) for the rapid and sensitive identification of airborne foodborne bacteria. Airborne bacteria were collected using a self-developed electrostatic air sampler and analyzed using a portable isothermal amplification device. The RCCVA assay was designed to detect four major foodborne pathogens: Staphylococcus aureus, Salmonella enteritidis, Listeria monocytogenes, and Bacillus cereus. The limit of detection was measured as 274.9, 4.5, 9.5, and 28.5 culture-forming units (CFU)/mL, respectively, within 45 min. This platform enables early on-site detection of airborne pathogens within approximately 1 h (for the analytical phase) and shows potential for real-time monitoring in food processing environments, thereby contributing to improved public health and food safety.},
}
@article {pmid41805869,
year = {2026},
author = {Moghe, AS and Nandi, SS and Bhonde, RR and Kamyab, SS and Sawant, SA and Karandikar, MN},
title = {Engineering of cell line assembled enteric organoid for enterovirus infection.},
journal = {Archives of virology},
volume = {171},
number = {4},
pages = {},
pmid = {41805869},
issn = {1432-8798},
support = {5/3/8/57/2020-ITR Date:11.03.2021//Indian Council of Medical Research/ ; },
mesh = {Humans ; *Organoids/virology ; Virus Replication ; Cell Line ; *Enterovirus Infections/virology ; *Enterovirus A, Human/physiology ; Epithelial Cells/virology ; *Enterovirus/physiology ; CRISPR-Cas Systems ; Coculture Techniques ; },
abstract = {The non-polio-enteroviruses are ubiquitous pathogens infecting over a billion people in the world. An alarming number of enterovirus-associated acute flaccid paralysis, encephalitis, hand, foot, and mouth disease, conjunctivitis and diarrhoea cases are reported worldwide. Despite their clinical significance, vaccine development has been hindered due to lack of suitable in vitro models for preclinical investigations. The present study was undertaken to develop a cell line assembled organotypic model of human intestine for replication of enteroviruses. An enterovirus specific PSGL1 receptor was introduced in intestinal epithelial HCT-8 cell line employing CRISPR/cas9 gene editing. It was co-cultured with human colon (CCD-18) and endothelial (HUVEC) cell lines with peripheral blood mononuclear cells in hanging drops and rotating wall vessel bioreactor to yield three-dimensional organoids. Histological analysis of the organoids showed presence of columnar epithelium cells with prominent intracytoplasmic mucin, hyperchromatic nuclei and presence of CK, CK20, MUC 2 and Villin markers characteristic of epithelial cells. Infection with Enterovirus A71 (EV-A71) demonstrated significantly higher viral titre in organoids compared to individual cell lines. Collectively, these findings determine, for the first time, a cell line-derived enteric organoid model that supports robust enterovirus replication, offering a cost-effective and physiologically relevant system for virology research and preclinical applications.},
}
@article {pmid41805349,
year = {2026},
author = {Han, F and Xu, Y and Wang, W and Li, Z and Zhang, Z and Du, R and Xu, Q},
title = {Methylobacterium as a Dual-Function Platform: Advances in C1-Based Biomanufacturing and Plant-Associated Applications.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c08026},
pmid = {41805349},
issn = {1520-5118},
abstract = {One-carbon (C1) substrates are promising feedstocks for microbial bioproduction. Methylobacterium, known for its exceptional C1 utilization capacity, has emerged as a model microbial chassis for sustainable biomanufacturing. In this review, we first outline the C1 assimilation pathways in Methylobacterium and underscore its potential for producing valuable native metabolites. Furthermore, we then survey the genetic tools available for engineering this genus, including plasmid-based methods, transposon mutagenesis, homologous recombination, and CRISPR/Cas systems. Notably, recent advances in metabolic engineering have significantly expanded its biosynthetic scope, enabling the biosynthesis of diverse non-native compounds. Beyond its biomanufacturing potential, Methylobacterium also serves as a versatile plant growth-promoting bacterium, enhancing plant health and productivity through hormone synthesis, nutrient mobilization, stress mitigation, and induced systemic resistance. Collectively, this work highlights the dual potential of Methylobacterium as a sustainable microbial cell factory for biomanufacturing and a beneficial bioinoculant for agriculture.},
}
@article {pmid41805130,
year = {2026},
author = {Yu, W and Yuan, L and Zhou, W and He, L and Huang, X and Yu, J and Deng, J and Zhang, T and Hu, Y and Zhang, Y and Chen, S},
title = {Orn-mediated c-di-GMP regulates the CRISPR-Cas system to confer stress response in Mycobacterium tuberculosis.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41805130},
issn = {1362-4962},
support = {2021YFA1300901//National Key R&amp;D Program of China/ ; 2022YFA1303500//National Key R&amp;D Program of China/ ; GZNL2024A01024//Guangzhou National Laboratory/ ; //National Key Research and Development Program of China/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/metabolism/drug effects ; *CRISPR-Cas Systems/genetics ; *Cyclic GMP/analogs &amp; derivatives/metabolism ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Oxidative Stress/genetics ; *Stress, Physiological/genetics ; Promoter Regions, Genetic ; CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {Mycobacterium tuberculosis (Mtb) possesses a type III-A CRISPR-Cas system and has anti-plasmid immune activity. However, whether this system exerts other additional functions remains to be characterized. Here, we investigated the in vivo roles of the Mtb CRISPR-Cas system. We show that this system is transcriptionally dependent and exhibits limited ability to counteract exogenous nucleic acids, primarily through the Csm6 protein rather than the Cas10 HD domain. We further demonstrate that this system plays a role in mitigating oxidative stress and antibiotic treatment, a function mainly mediated by the Cas10 HD domain. Importantly, through transposon library screening, we identified oligoribonuclease (Orn) as a regulatory protein of the Mtb CRISPR-Cas system. Deletion of the orn gene resulted in elevated c-di-GMP levels. A subsequent biotin-labeled c-di-GMP pull-down assay identified the transcriptional regulator Rv3058. Knockdown of rv3058 significantly increased cas6 promoter activity, and its transcriptional repressor function was directly modulated by c-di-GMP. This regulatory pathway enhances stress defense by activating multiple protective pathways, including DNA repair, cell envelope maintenance, and iron homeostasis regulation. Together, we conclude that the regulation of the CRISPR-Cas system by Orn-mediated c-di-GMP contributes to oxidative and antibiotic stress responses in Mtb.},
}
@article {pmid41804827,
year = {2026},
author = {Parada, F and Cabedo-Díaz, P and Cerda, A and Osorio-Navarro, C and Toledo, JA and Villalobos-González, L and Handford, M and Pimentel, P},
title = {CRISPR/dCas9-Mediated BRL3 Activation Enhances Growth and Metabolic Resilience Under Osmotic Stress in Nicotiana tabacum.},
journal = {Physiologia plantarum},
volume = {178},
number = {2},
pages = {e70816},
doi = {10.1111/ppl.70816},
pmid = {41804827},
issn = {1399-3054},
support = {3240290//ANID-FONDECYT Postdoctoral Project/ ; 3210631//ANID-FONDECYT Postdoctoral Project/ ; 1231417//ANID-FONDECYT Regular Project/ ; RF23F0002//ANID Fortalecimiento de Centros Regionales Project/ ; NCN2024_047//ANID-Millennium Science Initiative Program/ ; },
mesh = {*Nicotiana/genetics/growth &amp; development/metabolism/physiology ; Osmotic Pressure/physiology ; Gene Expression Regulation, Plant ; *Plant Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Promoter Regions, Genetic/genetics ; Brassinosteroids/metabolism ; Plant Leaves/genetics ; },
abstract = {Brassinosteroids (BRs) are crucial plant hormones that influence growth and stress adaptation. However, the specific function of the BR receptor BRL3 under osmotic stress remains largely unexplored outside Arabidopsis thaliana. In this study, we used a CRISPR/dCas9-based transcriptional activation (CRISPRa) system to upregulate the Nicotiana tabacum BRASSINOSTEROID INSENSITIVE-LIKE 3 receptor (NtBRL3) and assessed its impact on osmotic stress tolerance. Synthetic activation vectors were constructed using Loop Assembly, featuring dCas9-6TAL-VP128 modules driven by either a constitutive (CaMV35S) or ABA-inducible (SlAREB) promoter, paired with dual sgRNAs targeting the NtBRL3 promoter. Transient Agrobacterium-mediated transformation followed by PEG treatment was used to impose osmotic stress. RT-qPCR confirmed a 3- to 4-fold activation of NtBRL3 transcripts in CRISPRa-infiltrated leaves. The stress-inducible SlAREB promoter produced the strongest improvements, yielding nearly four-fold higher leaf biomass and a five-fold increase in root biomass relative to PEG-stressed controls. Both constructs reduced malondialdehyde (MDA) accumulation, indicating diminished oxidative damage, and modulated osmoprotectant balance, including reduced root proline and increased total soluble solids, particularly under SlAREB-driven activation. Histological segmentation revealed promoter-dependent anatomical remodeling, with NtBRL3-activated plants exhibiting a higher frequency of enlarged leaf cells and expanded tissue domains, consistent with brassinosteroid-mediated structural plasticity. Collectively, these findings demonstrate that CRISPR/dCas9-mediated transcriptional activation of NtBRL3 enhances osmotic stress resilience in tobacco through coordinated biomass recovery, oxidative stress mitigation, osmolyte homeostasis, and tissue remodeling. This transient, non-integrative CRISPRa approach provides a robust synthetic biology framework for dissecting BR signaling and engineering stress-tolerant crops.},
}
@article {pmid41803497,
year = {2026},
author = {Perez Taboada, V and Wu, Y and Cassidy, R and Medvedev, KE and Loeff, L and Nemudraia, A and Nemudryi, A},
title = {Bacterial Schlafen proteins mediate phage defence.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {41803497},
issn = {2058-5276},
support = {R00AI171893//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 1T32GM156737-01//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
abstract = {Human Schlafen proteins restrict viral replication by cleaving tRNA, thereby suppressing protein synthesis. Although the ribonuclease domain of Schlafen proteins is conserved across all domains of life, its function in prokaryotes has remained unclear. Here we demonstrate that prokaryotic Schlafen nucleases are widespread antiviral effectors that protect bacteria from bacteriophages and are fused to a diverse array of phage-sensing domains. We expressed seven Enterobacterales Schlafen systems in Escherichia coli, identifying two that confer defence against coliphages. We focused on a system where Schlafen nuclease is fused to a previously unknown immunoglobulin-like sensor domain and demonstrated that it recognizes tail assembly chaperones of T5-like phages. Upon activation, the Schlafen nuclease cleaves both E. coli and phage-encoded tRNAs and restricts T5 phage by reducing its burst size. Our findings redefine Schlafens as an ancient, mechanistically conserved family of immune effectors, revealing the deep evolutionary origin of tRNA-targeting antiviral immunity in humans.},
}
@article {pmid41803127,
year = {2026},
author = {Zhou, R and Liu, Y and Zhang, Q and Yin, Z and Tong, J and Zhang, C and Zhang, L and Li, X and Zhao, Y and Zhang, S and Liu, Z and Chen, W and Ji, N and Zhang, H and Li, Z and Yin, H and Zuo, S and Wei, Y},
title = {Structural and mechanistic insights into the dual-nuclease defense protein Upx as an anti-phage system.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-70435-x},
pmid = {41803127},
issn = {2041-1723},
support = {2022YFC3400400//National Science Foundation of China | Key Programme/ ; 23ZYCGSY00750//Tianjin Science and Technology Committee (Tianjin Municipal Science and Technology Commission)/ ; },
abstract = {Nucleic acid degradation is a common strategy for prokaryotic anti-phage systems, as exemplified by the CRISPR-Cas system. The PD-(D/E)-XK nucleases constitute a widely distributed family in these defenses. Notably, most members exhibit a single nuclease domain, while variants containing dual nuclease domains within a single polypeptide remain underexplored, and their molecular mechanisms largely obscure. Here, we biochemically and functionally study a single-protein system containing an uncharacterized PD-(D/E)-XK defense protein (Upx). As revealed by single-particle electron cryo-microscopy (cryo-EM) structure, the C-terminal domain (CTD) harboring the conserved PD-(D/E)XK catalytic core is buttressed by the N-terminal domain (NTD) and the middle domain (MD). Functional assays demonstrate that the nucleic acid binding capability of the CTD is enhanced by the MD. The NTD also displays a noncanonical, basal exonuclease activity that is auto-inhibited by MD. IP-MS experiments identify Upx-interacting phage proteins, and substrate profiling defines its physiological preferences, collectively pointing to its potential physiological targets. Notably, the phage protein gp16 was found to relieve MD-mediated inhibition of the NTD, suggesting a virus-triggered mechanism for activating Upx's dual nuclease activity. Together, these findings establish Upx as a single-protein dual-nuclease anti-phage system, expanding our understanding of bacterial immunity and informing antiviral strategy development.},
}
@article {pmid41802999,
year = {2026},
author = {Hao, M and Zhou, M and Pan, F and Liu, T and Li, Y and Su, N and Ashfaq, A and Song, M and Wang, H and Wang, W and Liu, J and Li, C and Fu, L and He, P and Hu, Q and Mei, D and Cheng, H},
title = {Efficient CRISPR/Cas-SF01 genome editing tools with high editing efficiency in allotetraploid oilseed rape.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.70221},
pmid = {41802999},
issn = {1744-7909},
support = {2025BEA003//the Major Program (JD) of Hubei Province/ ; CAAS-CSNCB-202303//Innovation Program of Chinese Academy of Agricultural Sciences/ ; 2025AFB468//Hubei Provincial Natural Science Foundation of China/ ; CARS-12//Earmarked Fund for China Agriculture Research System/ ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 has been widely utilized for plant genome editing, but the protospacer adjacent motif (PAM) requirement limits its editing scope. CRISPR/Cas12i3 belongs to the type-VI Cas system that has gained extensive attention due to its smaller size and less restricted canonical TTN PAM sequence. In this study, we explored the newly developed Cas-SF01 system (Cas12i3 variant) for genome editing in oilseed rape. We established an efficient protoplast transformation system in oilseed rape to compare editing efficiency between Cas-SF01 and Cas9. Cas-SF01 shows cleavage activities at the tested 5'-TTN-3' PAM sites with editing outcomes sharing considerable similarities with the CRISPR-Cas9 system in protoplast. Cas-SF01 also induces high efficiency mutagenesis for multiple target sites in stable transformed oilseed rape lines, generating mutants with multilocular silique and male sterile phenotypes. Furthermore, Cas-SF01-derived cytosine base editors (CBEs) were developed to produce targeted C-to-T base edits. Compared to SpCas9, Cas-SF01 has an expanded PAM range and effectively recognizes TTN PAMs, which has substantially broadened the scope of editable sites within the rapeseed genome. No mutations were identified at the putative off-target sites among the edited plants. This study developed a robust, first-of-its-kind Cas12 system in the allotetraploid Brassica napus, expanding the scope of editing and enriching genome-editing toolkits for biological research and genetic improvement.},
}
@article {pmid41747377,
year = {2026},
author = {Pan, MX and Lv, MM and Nie, YG and Su, M and Zha, CJ and Mei, RY and Ying, ZM},
title = {Ultrasensitive miRNA detection via magnetic bead-confined catalytic hairpin assembly enabling transcription-driven crRNA assembly and CRISPR/Cas12a activation.},
journal = {Biosensors &amp; bioelectronics},
volume = {302},
number = {},
pages = {118559},
doi = {10.1016/j.bios.2026.118559},
pmid = {41747377},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation &amp; purification/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/genetics/chemistry ; DNA/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; Limit of Detection ; *Bacterial Proteins/chemistry/genetics ; Nucleic Acid Hybridization ; Transcription, Genetic ; },
abstract = {The integration of CRISPR/Cas12a with catalytic hairpin assembly (CHA), a strategy that predominantly relies on CHA to generate dsDNA activators for direct Cas12a activation, has emerged as a powerful tool in molecular diagnostics. However, two major challenges remain: the strict protospacer adjacent motif (PAM) dependence of the dsDNA and background leakage from hairpin hybridization. Herein, we report a bead-confined platform that transcription mediates crRNA reassembly and template activation of Cas12a for ultrasensitive miRNA detection. The target-triggered CHA assembly dynamically constructed a T7 transcription template from three initially locked hairpins (H1, H2, and H3), which not only transcribed scaffold RNA but also hybridized with its own product to form a DNA/RNA complex that activates Cas12a. The integration of the split T7 promoter with CHA effectively suppressed background suppression and enhanced detection sensitivity. Additionally, the magnetic beads increase local concentration and reaction kinetics, collectively contributing to a substantially enhanced detection sensitivity. Moreover, a crRNA assembly strategy designed for transcription-powered Cas12a not only circumvents the conventional PAM-dependent dsDNA activation pathway of Cas12a but also enables self-supplied crRNA without requiring additional activators. We demonstrated that the biosensor exhibits exceptional sensitivity for miRNA-21 detection, achieving a limit of 65.3 aM. Furthermore, the practicality of this method was preliminarily confirmed through accurately quantifying target levels in cell lines and human serum. Our method presents a viable solution with transformative potential, designed to address complex challenges in contemporary diagnostic applications.},
}
@article {pmid41710969,
year = {2026},
author = {Xu, Z and Wei, M and Jiang, M and Wang, Y and He, K},
title = {Single-step duplex CRISPR coupled with lateral flow assay for point-of-care detection of human immunodeficiency virus and Treponema pallidum.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {10},
pages = {1996-2004},
doi = {10.1039/d5ay02168e},
pmid = {41710969},
issn = {1759-9679},
mesh = {Humans ; *Treponema pallidum/genetics/isolation &amp; purification ; *Point-of-Care Systems ; *HIV Infections/diagnosis/virology ; *Syphilis/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; *HIV/genetics/isolation &amp; purification ; Point-of-Care Testing ; Sensitivity and Specificity ; },
abstract = {Continued acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) and syphilis caused by Treponema pallidum (TP) pose great challenges for global health, highlighting the need for rapid and sensitive diagnostics. Here, we introduced DIAL (Duplex Integrated All-in-one CRISPR Lateral flow assay), a simplified CRISPR-based diagnostic system for simultaneous detection of HIV and TP. We developed a single-step duplex CRISPR assay for dual-target recognition, which incorporated reverse transcription, multiplex recombinase polymerase amplification, transcription, CRISPR/Cas12a detection, and CRISPR/Cas13a detection in one pot. Then, we combined CRISPR detection assay with a "line-elimination" dual-line lateral flow assay for low-cost and equipment-free visual readout. We further developed reagent lyophilization and extraction-free sample lysis techniques to meet the point-of-care testing. The optimized DIAL system detected as low as 100 copies per µL of both HIV RNA and TP DNA within 45 minutes. In a clinical evaluation of 46 samples, it demonstrated 100% sensitivity and 97.5% specificity for HIV and 90% sensitivity and 100% specificity for TP, relative to quantitative PCR. The DIAL provided a scalable, accessible approach for decentralized screening of sexually transmitted infections or other pathogens in resource-limited settings.},
}
@article {pmid41707429,
year = {2026},
author = {Nong, J and Pan, Z and Li, Y and Wei, J and Gong, Y and Li, J and Zhang, K and Liao, X},
title = {COF-confined CsPbBr3 nanocomposite with CRISPR/Cas12a-driven DNA walking for ultrasensitive electrochemiluminescent detection of circulating tumor DNA.},
journal = {Biosensors &amp; bioelectronics},
volume = {302},
number = {},
pages = {118536},
doi = {10.1016/j.bios.2026.118536},
pmid = {41707429},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Nanocomposites/chemistry ; Luminescent Measurements/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Limit of Detection ; *Circulating Tumor DNA/blood/isolation &amp; purification/genetics ; Endodeoxyribonucleases/chemistry ; DNA Probes/chemistry ; Metal-Organic Frameworks/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {A highly sensitive electrochemiluminescence biosensor was developed for circulating tumor DNA detection by integrating a covalent organic framework-confined CsPbBr3 nanocomposite with a CRISPR/Cas12a-driven amplification strategy. The covalent organic framework not only stabilizes CsPbBr3 nanocrystals in aqueous environments but also regulates interfacial charge transfer and enables programmable immobilization of DNA probes. Upon recognition of the target sequence, Cas12a is activated and progressively cleaves surface-tethered quencher-modified DNA strands, leading to stepwise restoration of electrochemiluminescence emission. This surface-confined signal amplification eliminates the need for polymerase chain reaction or isothermal preamplification. Under optimized conditions, the biosensor exhibited a linear response over a concentration range from 10 fM to 10 nM, with a detection limit of 5.4 fM. The method demonstrated good selectivity toward single-base mismatches and satisfactory performance in diluted serum and clinical plasma samples. These results highlight a synergistic material-enzyme strategy for sensitive and robust nucleic acid detection and provide a proof-of-concept platform for electrochemiluminescence-based circulating tumor DNA analysis.},
}
@article {pmid41707427,
year = {2026},
author = {Tu, Z and Wang, Y and Qian, X and Chen, J and Li, L and Wang, T and Chen, H and Wei, H and Yang, P and Pan, J and Wang, S and Rong, Z},
title = {Plasmonic magnetic nanoparticles-enabled universal enrichment, photothermal lysis, and duplex CRISPR detection of bacteria in urine samples.},
journal = {Biosensors &amp; bioelectronics},
volume = {302},
number = {},
pages = {118518},
doi = {10.1016/j.bios.2026.118518},
pmid = {41707427},
issn = {1873-4235},
mesh = {Humans ; *Escherichia coli/isolation &amp; purification/genetics ; *Magnetite Nanoparticles/chemistry ; *Enterococcus faecalis/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *Urinary Tract Infections/microbiology/urine/diagnosis ; CRISPR-Cas Systems ; Surface Plasmon Resonance/methods ; *Escherichia coli Infections/urine/microbiology/diagnosis ; Limit of Detection ; Boronic Acids/chemistry ; },
abstract = {Urinary tract infections (UTIs), exacerbated by antibiotic resistance and evolving pathogen diversity, demand rapid and sensitive diagnostics. This study introduces an integrated platform combining magnetic enrichment, photothermal lysis, and CRISPR-based detection (ME-CRISPR) for simultaneous identification of Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) in UTIs. We engineered plasmonic magnetic nanoparticles functionalized with 4-mercaptophenylboronic acid for broad-spectrum bacterial capture within 10 min. Subsequent near-infrared laser irradiation leveraged localized surface plasmon resonance to lyse captured bacteria in situ. The released nucleic acids were directly analyzed in a single-step duplex RPA-CRISPR/Cas12a-Cas13a assay. This 40-min workflow achieved a limit of detection of 10 CFU/mL for both pathogens. Validation with 90 clinical samples (39 E. coli, 26 E. faecalis, and 25 negative controls) demonstrated 100% sensitivity and specificity, matching qPCR performance while significantly reducing turnaround time. The platform overcomes limitations of centrifugation and Gram-class-dependent lysis, offering a rapid and ultrasensitive point-of-care testing tool to curb antibiotic misuse.},
}
@article {pmid41707424,
year = {2026},
author = {Zhao, L and Liu, Z and Ding, G and Zhu, Y and Wang, H and Liu, R and Qu, F and Ao, Q and Zhu, X and Zhang, Y and Yang, G and Wang, Z},
title = {Dual-readout aptasensor based on CRISPR/Cas12a and nanozyme for accurate detection of KIM-1 and its application in kidney transplant prognosis.},
journal = {Biosensors &amp; bioelectronics},
volume = {302},
number = {},
pages = {118533},
doi = {10.1016/j.bios.2026.118533},
pmid = {41707424},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Hepatitis A Virus Cellular Receptor 1/isolation &amp; purification ; *Aptamers, Nucleotide/chemistry ; CRISPR-Cas Systems ; *Kidney Transplantation ; Limit of Detection ; Prognosis ; Metal Nanoparticles/chemistry ; Silver/chemistry ; Colorimetry/methods ; Metal-Organic Frameworks/chemistry ; SELEX Aptamer Technique ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Kidney injury molecule-1 (KIM-1) has emerged as a pivotal prognostic biomarker for renal allograft function. However, its detection remains challenging due to sensitivity and accuracy limitations. An innovative biosensing platform synergizing aptamer recognition, CRISPR trans-cleavage and nanozyme amplification for dual-readout KIM-1 detection has been presented in this work. This platform employs a meticulously selected high-affinity aptamer with capillary electrophoresis SELEX for specific target recognition, leverages the CRISPR/Cas12a system for signal transduction and cascade amplification, and utilizes engineered FeNi MOF@AgNPs nanozyme for dual-signal output. With the presence of KIM-1, whose binding with aptamer, effectively inhibits the trans-cleavage activity of the CRISPR/Cas12a system, and uninhibited Cas12a subsequently cleaves nanozyme-conjugated magnetic probes, releasing FeNi MOF@AgNPs nanozymes that catalyze a TMB-based reaction to generate intense colorimetric and fluorescent dual-readout signals. The as developed aptasensor demonstrates satisfied sensitivity achieving detection limits of 58.7 pg/mL (colorimetric) and 34.4 pg/mL (fluorometric), and dependable accuracy achieving average relative deviation of -2.7% (colorimetric) and 3.2% (fluorometric) with commercial ELISA kit in urine samples from patients with acute kidney injury. Moreover, longitudinally track the dynamic changes in urinary KIM-1 concentrations over the first 8 days following renal transplantation was successfully realized. This work not only provides a robust analytical tool for KIM-1 detection but also establishes a generic research approach for extending CRISPR-based systems to the precise detection of proteins.},
}
@article {pmid41702192,
year = {2026},
author = {Lu, L and Zhang, Y and Liu, B and Zhou, N and Yu, DJ and Wang, Y},
title = {Magnetic Fe3O4-Au@UIO-66-NH2@toehold probe mediated fluorescent sensor for detecting ovarian cancer-specific circRNA coupled with hybridization chain reaction and the CRISPR-Cas12a system.},
journal = {Biosensors &amp; bioelectronics},
volume = {302},
number = {},
pages = {118535},
doi = {10.1016/j.bios.2026.118535},
pmid = {41702192},
issn = {1873-4235},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *RNA, Circular/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; Female ; *Ovarian Neoplasms/genetics/diagnosis ; Gold/chemistry ; Fluorescent Dyes/chemistry ; Nucleic Acid Hybridization ; Limit of Detection ; Magnetite Nanoparticles/chemistry ; Ferrosoferric Oxide/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Circular RNAs (circRNAs) represent an emerging family of noncoding transcripts defined by closed-loop architecture, which are now established as key participants in the etiology of tumorigenesis and malignant progression. While circRNAs show potential as therapeutic targets and biomarkers, the accurate detection of circRNAs remains challenging due to interference from homologous linear RNAs. In this study, an ultrasensitive method for detecting circ_0051240 based on the sulfhydrated toehold capture probe-initiator assembly-mediated hybridization chain reaction (HCR) and the CRISPR-Cas12a system was developed. The capture probe (with a toehold domain)-initiator strand duplex was ingeniously fabricated to identify the back-splice junction (BSJ) of circRNA. After magnetic enrichment and separation by the Fe3O4-Au@UIO-66-NH2 nanocomposite, the initiator strand (H0) was delivered to trigger HCR. The HCR product dsDNA concatemers contained multiply repeated CRISPR-targetable DNA sites that were readily recognized by the CRISPR RNA (crRNA). This specific recognition and binding activated the CRISPR-Cas12a system's collateral endonuclease activity, leading to cleavage of the fluorophore-quencher (FQ) reporters and fluorescence emission at a characteristic wavelength. This design eliminated linear RNA-related interference and enhanced the detection of fluorescence intensity (FI). Under optimal conditions, the proposed HCR/CRISPR-Cas12a method exhibited a wide quantitative measurement range spanning from 45 pM up to 180 nM, achieving a notable limit of detection (LOD) of 0.03 pM. In this study, a novel circRNA sensing strategy capable of accurate and highly sensitive quantification of ovarian cancer-specific circRNA was reported. The proposed method exhibits acceptable performance when compared to present approaches.},
}
@article {pmid41677799,
year = {2026},
author = {Boubakri, H},
title = {CRISPR-Cas9-mediated genome editing in fungi: applications, challenges, and future directions.},
journal = {Journal of applied microbiology},
volume = {137},
number = {3},
pages = {},
doi = {10.1093/jambio/lxag046},
pmid = {41677799},
issn = {1365-2672},
mesh = {*Gene Editing/methods/trends ; *CRISPR-Cas Systems ; *Fungi/genetics ; *Genome, Fungal ; },
abstract = {The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been shown to be an effective genome-editing tool in many organisms, including fungi. It enables precise modifications to the DNA of fungal species, facilitating advancements in research, agriculture, and biotechnology. CRISPR-Cas9-edited non-pathogenic antagonists have emerged as a promising alternative for biocontrol. Several filamentous fungi have been engineered to produce secondary metabolites. Furthermore, the CRISPR-Cas9 system has been used to improve the quality of several edible fungi. However, the application of CRISPR-Cas9 technology for fungal genome editing is still facing some challenges that researchers must address. This review highlights the major approaches and applications of genome editing in fungi, as well as the associated challenges.},
}
@article {pmid41670347,
year = {2026},
author = {Sullivan, JR and Ferrara, KM and Barrick, R and Romano, KP and Warrier, T and Hung, DT},
title = {An inducible CRISPRi system for phenotypic analysis of essential genes in Pseudomonas aeruginosa.},
journal = {mBio},
volume = {17},
number = {3},
pages = {e0276725},
pmid = {41670347},
issn = {2150-7511},
support = {U19 AI142780/AI/NIAID NIH HHS/United States ; U19AI142780/NH/NIH HHS/United States ; },
mesh = {*Pseudomonas aeruginosa/genetics/drug effects/growth &amp; development ; *Genes, Essential ; Phenotype ; *CRISPR-Cas Systems ; Plasmids ; Gene Expression Regulation, Bacterial ; Rhamnose/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Promoter Regions, Genetic ; },
abstract = {Precise and tunable genetic tools are essential for high-throughput functional genomics. To address this need in the important gram-negative pathogen Pseudomonas aeruginosa, we developed and characterized a tightly regulated CRISPR-interference (CRISPRi) system that enables precise and tunable repression of essential genes. The system utilizes a rhamnose-inducible promoter to control both the Streptococcus pasteurianus-derived dCas9 and gene-specific sgRNAs, each encoded on separate plasmids for modularity and efficiency. The combination of tight regulation and high conjugation efficiency facilitated the rapid and facile construction of strains with regulated depletion of 16 essential genes spanning diverse pathways. Comparison of phenotypes across the different genetically depleted strains, including growth rate, susceptibility to antibiotics, and changes in transcriptional programs, revealed novel aspects of gene function or small-molecule mechanism of action. Finally, the rhamnose-inducible CRISPRi system supports the generation and stable maintenance of pooled mutant libraries, thereby paving the way for future genome-wide, systematic assessment of individual gene vulnerabilities, which will provide critical insights for target prioritization in antibiotic discovery efforts against this recalcitrant pathogen.IMPORTANCECRISPR-interference (CRISPRi) has become an invaluable tool for studying genetics. In particular, the ability to knockdown (KD) genes enables the study of essential genes and their role in cell survival. However, a tightly regulated gene KD system is required to gain valuable insights into these vulnerable genes by virtue of their essentiality. We report a tightly regulated CRISPRi system to study the biology of essential gene perturbations in Pseudomonas aeruginosa, an important gram-negative pathogen that causes severe infections and is increasingly resistant to current antibiotics. This system enables characterization of both chemical genetic interactions between small molecules and specific gene depletions and the impact of genetic perturbations on transcriptional networks. Genetic perturbations using CRISPRi can thus further our understanding of basic biology with translation toward future antimicrobial development.},
}
@article {pmid41642947,
year = {2026},
author = {Pelea, O and Tálas, A and Carrera, JF and Mathis, N and van de Venn, L and Yeh, CD and Kulcsár, PI and Marquart, KF and Weber, Y and Gerecke, SE and Harvey-Seutcheu, IF and Mailänder, D and Pfleiderer, MM and Chanez, C and Corn, JE and Schwank, G and Jinek, M},
title = {Programmable genome editing in human cells using RNA-guided bridge recombinases.},
journal = {Science (New York, N.Y.)},
volume = {391},
number = {6790},
pages = {eadz1884},
doi = {10.1126/science.adz1884},
pmid = {41642947},
issn = {1095-9203},
mesh = {Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; Plasmids ; Recombination, Genetic ; Genome, Human ; *DNA Nucleotidyltransferases/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; DNA/genetics ; },
abstract = {Site-specific insertion of gene-sized DNA fragments remains an unmet need in the field of genome editing. IS110-family serine recombinases have recently been shown to mediate programmable DNA recombination in bacteria by using a bispecific RNA guide (bridge RNA) that simultaneously recognizes target and donor sites. In this work, we have shown that the bridge recombinase ISCro4 is highly active in human cells and provided structural insights into its enhanced activity. Using plasmid- or all-RNA-based delivery, ISCro4 supports programmable multikilobase excisions and inversions and facilitates donor DNA insertion at genomic sites with efficiencies that exceed 6%. Last, we assessed ISCro4 specificity and off-target activity. These results establish a framework for the development of bridge recombinases as next-generation tools for editing modalities that are beyond the capabilities of current technologies.},
}
@article {pmid41524478,
year = {2026},
author = {Kim, M and Kim, K and Lee, J and Lee, S and Choi, S and Park, SA and Jeong, E and Choi, SY and Park, HH and Park, TE and Kwon, T and Myung, K and Yoo, J and Cho, SW and Joo, J},
title = {FAST-CRISPR: Fusogenic Association and Secured Transfection of CRISPR/Cas9 Ribonucleoproteins Using Lipid-Silica Hybrid Nanoparticles for Therapeutic Genome Editing.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {15},
pages = {e11362},
pmid = {41524478},
issn = {1613-6829},
support = {RS-2024-00512120//Korean ARPA-H Project through the KHIDI/ ; 22A0102L1-11//Korean Fund for Regenerative Medicine (KFRM)/ ; RS-2024-00509412//National Research Foundation (NRF)/ ; RS-2023-00209822//National Research Foundation (NRF)/ ; RS-2023-00207746//National Research Foundation (NRF)/ ; IBS-R022-D1//Institute for Basic Science/ ; 1.250006.01//UNIST research fund/ ; RS-2024-00403508//Korea Basic Science Institute/ ; 1.250006.01//Ulsan National Institute of Science and Technology/ ; },
mesh = {*Nanoparticles/chemistry ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/metabolism/genetics ; *Silicon Dioxide/chemistry ; Humans ; *Lipids/chemistry ; Animals ; *Transfection/methods ; Mice ; Cell Line, Tumor ; },
abstract = {Clinical translation of CRISPR/Cas9 therapeutics is challenged by inefficient cytosolic delivery and toxicity issues associated with viral vectors and nanoparticle-based carriers. To overcome these concerns, herein we report a lipid-silica hybrid nanoparticle platform for fusogenic association and secured transfection of CRISPR/Cas9 (FAST-CRISPR), designed for rapid cytosolic delivery of CRISPR/Cas9 ribonucleoproteins, followed by efficient gene editing. Through direct fusion with the plasma membrane and bypassing conventional endocytic barriers, FAST-CRISPR nanoparticles displayed superior intracellular delivery efficacy. Optimizing lipid compositions, we discovered that a 1:1 weight mixture of cationic DOTAP and ionizable DODMA lipids, combined with tailored large-pore silica nanoparticles, enables enhanced loading capacity, rapid cytosolic dispersion, and significant nuclear transport of Cas9/gRNA complexes. FAST-CRISPR nanoparticles efficiently delivered multiplex genome-targeting ribonucleoproteins to induce targeted double-strand DNA breaks, triggering apoptosis in cancer cells and significantly suppressing tumor growth in a mouse xenograft model without systemic toxicity. Our findings demonstrate the therapeutic efficacy and translational potential of FAST-CRISPR nanoparticles as a safe and versatile non-viral delivery platform for precision genome editing.},
}
@article {pmid41270742,
year = {2026},
author = {Sun, J and Atiş, IS and Empke, SLL and Khokha, MK and Breslow, DK},
title = {A microscopy-based CRISPR screening platform enables organellar functional genomics and illuminates ciliary biology.},
journal = {Developmental cell},
volume = {61},
number = {3},
pages = {687-705.e9},
pmid = {41270742},
issn = {1878-1551},
support = {S10 OD023651/OD/NIH HHS/United States ; R35 GM137956/GM/NIGMS NIH HHS/United States ; S10 OD018034/OD/NIH HHS/United States ; R01 HD102186/HD/NICHD NIH HHS/United States ; S10 OD030363/OD/NIH HHS/United States ; },
mesh = {*Cilia/metabolism/genetics ; Humans ; Animals ; *Genomics/methods ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Microscopy, Fluorescence/methods ; Xenopus ; },
abstract = {Microscopy offers an indispensable technique for visualizing biological processes and for defining cytological abnormalities characteristic of disease. However, combining microscopy with the power of pooled CRISPR screening presents considerable technical challenges, hindering application of systematic genetic analysis to imaging-defined phenotypes. Here, we establish a fluorescence microscopy-based CRISPR screening platform that combines ease of implementation with flexible analysis of live-cell or antibody-based molecular markers, including post-translational modifications. Applying this methodology, we systematically identify regulators of primary cilium structure and function in human cells through targeted and genome-wide screens. We further show that integration of screens focused on distinct ciliary phenotypes yields multi-dimensional profiles that delineate precise gene functions. Among the identified hits, TZMP1 (SMIM27) encodes a microprotein at the ciliary transition zone that is required for ciliogenesis in human cells and for ciliary function in Xenopus embryos. More broadly, our approach provides a technological and conceptual strategy for microscopy-based functional genomics.},
}
@article {pmid37751223,
year = {2023},
author = {Ji, S and Wang, X and Wang, Y and Sun, Y and Su, Y and Lv, X and Song, X},
title = {Advances in Cas12a-Based Amplification-Free Nucleic Acid Detection.},
journal = {The CRISPR journal},
volume = {6},
number = {5},
pages = {405-418},
doi = {10.1089/crispr.2023.0023},
pmid = {37751223},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; *Endodeoxyribonucleases/genetics/metabolism ; *Nucleic Acids/genetics/analysis ; Biosensing Techniques/methods ; *Bacterial Proteins/genetics ; Electrochemical Techniques/methods ; },
abstract = {In biomedicine, rapid and sensitive nucleic acid detection technology plays an important role in the early detection of infectious diseases. However, most traditional nucleic acid detection methods require the amplification of nucleic acids, resulting in problems such as long detection time, complex operation, and false-positive results. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) systems have been widely used in nucleic acid detection, especially the CRISPR-Cas12a system, which can trans cleave single-stranded DNA and can realize the detection of DNA targets. But, amplification of nucleic acids is still required to further improve detection sensitivity, which makes Cas12a-based amplification-free nucleic acid detection methods a great challenge. This article reviews the recent progress of Cas12a-based amplification-free detection methods for nucleic acids. These detection methods apply electrochemical detection methods, fluorescence detection methods, noble metal nanomaterial detection methods, and lateral flow assay. Under various optimization strategies, unamplified nucleic acids have the same sensitivity as amplified nucleic acids. At the same time, the article discusses the advantages and disadvantages of each method and further discusses the current challenges such as off-target effects and the ability to achieve high-throughput detection. Amplification-free nucleic acid detection technology based on CRISPR-Cas12a has great potential in the biomedical field.},
}
@article {pmid37739193,
year = {2024},
author = {Mori, AA and Malaquias, VB and Bonjour, K and Ferreira, GM and Bortolin, RH and Borges, JB and Oliveira, VF and Gonçalves, RM and Faludi, AA and Bastos, GM and Thurow, H and Sampaio, MF and Ciconelli, RM and Cury, AN and Fajardo, CM and Hirata, RDC and Hirata, MH},
title = {Effects of LDLR variants rs5928, rs750518671 and rs879254797 on protein structure and functional activity in HepG2 cells transfected with CRISPR/Cas9 constructs.},
journal = {Gene},
volume = {890},
number = {},
pages = {147821},
doi = {10.1016/j.gene.2023.147821},
pmid = {37739193},
issn = {1879-0038},
mesh = {Humans ; Hep G2 Cells ; *Receptors, LDL/genetics/chemistry/metabolism ; *CRISPR-Cas Systems/genetics ; Mutation, Missense ; *Hyperlipoproteinemia Type II/genetics ; Female ; Male ; Polymorphism, Single Nucleotide ; Transfection ; Middle Aged ; },
abstract = {Familial Hypercholesterolemia (FH) is a genetic disorder associated with premature atherosclerosis and increased risk of cardiovascular diseases. LDLR deleterious mutations are associated with FH, however the role of some missense variants in FH pathogenicity remains to be elucidated. This study explored the predictive impact of LDLR missense variants on protein structure and investigated their functional effects on LDLR expression in HepG2 cells transfected with CRISPR/Cas9 constructs. FH (n = 287) and non-FH patients (n = 45) were selected, and lipid profile was obtained from medical records. LDLR variants were identified using an exon-targeted gene sequencing strategy, considering its cost-effective to increase accuracy in the identification step of the most likely FH-related variants in a less laborious process. LDLR variants were selected based on conflicting pathogenicity results found in Clinvar, in silico prediction tools, affected LDLR domains, and less common variants considering minor allele frequency < 0.05. Molecular modeling studies were used to predict the effects of LDLR missense variants on protein structure. Recombinant LDLR variants were constructed using CRISPR/Cas9 system and were used to transfect HepG2 cells. Functional assays in transfected cells were performed to assess LDLR expression using flow cytometry and western blotting, and LDLR activity using flow cytometry and confocal microscopy. The variants rs121908039 (c.551G>A, p.C184Y), rs879254797 (c.1118G>A, p.G373D), rs28941776 (c.1646G>A, p.G549D), rs750518671 (c.2389G>C, p.V797L), rs5928 (c.2441G>A, p.R814Q) and rs137853964 (c.2479G>A, p.V827I) were selected for molecular docking analysis. The p.C184Y exhibited a favorable energy change for protein stability due to its interaction with EGF-A/EGF-B regions; p.G373D and p.G549D displayed intermediate energy changes; and p.R814Q and p.V827I showed smaller energy changes. The results of functional assays showed that p.G373D, p.V797L and p.R814Q reduced LDLR expression and activity (p < 0.05). Microscopic analysis of the p.V797L and p.G373D variants revealed altered lipid localization and accumulation in transfected HepG2 cells. Carriers of p.G549D, p.V797L and p.R814Q had higher LDL cholesterol levels than non-FH group, and (p < 0.05). p.G373D and p.G549D were associated with clinical manifestations of FH. In conclusion, the p.C184Y, p.G373D, p.G549D and p.R814Q variants alter protein stability and intramolecular interactions, while p.V797L has a minimal impact on protein stability, and p.V827I has no significant intramolecular interactions. p.G373D, p.V767L and p.R814Q are associated with impaired LDLR expression and activity.},
}
@article {pmid41802204,
year = {2026},
author = {de Paula, JA and de Araújo, MRB and Guimarães Sousa, E and Prates, FD and Castro, DLC and Fonseca, PAS and Brenig, B and Felice, AG and Pacheco, LGC and Viana, MVC and Azevedo, VAC and Soares, S},
title = {Clonal clusters of multidrug-resistant Brazilian Corynebacterium striatum strains reveal putative virulence traits.},
journal = {Journal of applied microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jambio/lxag070},
pmid = {41802204},
issn = {1365-2672},
abstract = {CORYNEBACTERIUM STRIATUM: Has been increasingly associated with nosocomial outbreaks and antimicrobial resistance.<br><br>OBJECTIVES: This study presents the comparative analysis of 26 multidrug-resistant (MDR) C. striatum strains isolated in Brazil.<br><br>METHODS: Additional genomes from international sources were incorporated. The analyses encompassed in vitro antimicrobial susceptibility testing and an in silico workflow for genomic similarity comparison, phylogenetic reconstruction, genomic clustering, pangenome analysis, mobilome content, virulence prediction, and functional annotation of unique proteins and putative virulence clusters.<br><br>RESULTS: Strong in silico evidence of clonality among several Brazilian isolates was obtained at the same time that some strains consistently indicated a divergent genomic profile. There are 196 unique coding sequences (CDSs) across the Brazilian IHPs. Of particular interest, strain IHP2030 carried an exclusive fimbria, sharing less than 50% similarity with other fimbriae in the dataset. Yet, structural predictions suggested conservation of key structural domains typically associated with fimbrial proteins. Mobilome content analysis revealed that IHPs strains were overall similar, differing primarily in the number of insertion sequences and in the presence or absence of CRISPR-Cas defense systems. Regarding virulence, an exclusive cluster in IHP2050 and IHP2060 suggests adaptive advantages associated with their respective environments of isolation.<br><br>CONCLUSION: This study reveals a complex genomic landscape among Brazilian MDR C. striatum strains, marked by clonal dissemination alongside strain-level genetic variation in accessory genomes, mobilome composition, and virulence-associated gene repertoires, providing genomic evidence of diversification within hospital-associated lineages.},
}
@article {pmid41799755,
year = {2026},
author = {Vadrot, N and Moulin, M and Ferreiro, A and Richard, P and Buendia, B},
title = {LAP2 Isoform Profile in Heart Ageing and in Cardiac Cell Proliferation and Differentiation: Input From CRISPR-Cas9-mediated LAP2a Knockdown in H9C2.},
journal = {International journal of medical sciences},
volume = {23},
number = {3},
pages = {741-757},
pmid = {41799755},
issn = {1449-1907},
mesh = {Animals ; Cell Differentiation/genetics ; *Myocytes, Cardiac/metabolism ; Cell Proliferation/genetics ; CRISPR-Cas Systems/genetics ; Mice ; *Membrane Proteins/genetics/metabolism ; MEF2 Transcription Factors/genetics/metabolism ; Protein Isoforms/genetics/metabolism ; Rats ; *Aging/genetics ; Cell Line ; Gene Knockdown Techniques ; Myocardium/metabolism ; Humans ; Heart/growth &amp; development/physiology ; DNA-Binding Proteins ; },
abstract = {Haploinsufficiency of Lap2 alpha (LAP2a), a nuclear partner of Lamins A/C, has been associated with cardiac disease in rare cases, but LAP2a function remains largely unknown. To investigate the functional role of LAP2a in cardiomyocytes, we generated clones of embryonic myocardium-derived H9C2 cells in which LAP2a expression was specifically reduced through gene editing of the LAP2a gene Tmpo by CRISPR-Cas9. Downregulation (+/-) and absence (-/-) of LAP2a expression led to a decreased proliferation capacity of cardiomyocytes in vitro. Upon differentiation, the expression of myocardial markers (alpha cardiac Actin 1/Actc1, cardiac Troponin T2/Tnnt2, Myosin-2/Myh2 and Myosin-7/Myh7) was higher in LAP2a -/- cells compared to LAP2a +/- or LAP2a +/+ cells, with consistently higher expression of their upstream regulator Mef2c in LAP2a-devoid cells. These results suggest that LAP2a promotes cardiomyocyte proliferation and negatively modulates cardiomyocyte differentiation, through mechanisms including Mef2c regulation. Accordingly, normal protein expression of LAP2a was downregulated upon cardiomyocyte differentiation, contrary to LAP2b and a LAP2b-related shorter isoform. The latter tended to increase upon differentiation in all cells, most significantly in the LAP2a -/- clone. In postnatal mouse hearts, LAP2a levels were higher in the right than in the left ventricle, and lowest in the septum. The LAP2a:LAP2b ratio was much lower in murine hearts than in H9C2 cells, and decreased significantly upon ageing, specifically in the left ventricle. Finally, our data show that expression of the nuclear envelope proteins LEMD2 and Lamin A might be influenced by LAP2a upon cardiac differentiation. Our results show that LAP2 expression is finely regulated upon cardiac differentiation in vitro and is dependent on age and heart compartment in vivo. They contribute to clarifying the potential impact of genetic LAP2a defects and their connection with heart disease, possibly including reduced cardiomyoblast proliferation, increased cardiomyocyte differentiation and altered nuclear envelope remodelling.},
}
@article {pmid41799190,
year = {2026},
author = {Zhao, P and Li, H and Cai, Z and Zhang, X and Wen, X and Liu, Z and Jiang, S and Jiang, X and Wang, J and Dang, Z and Liu, M and Xie, F and Ma, X},
title = {Molecular hydrogen triggers TRPC4-TRPC4AP-dependent reversible calcium transients via extracellular influx.},
journal = {Theranostics},
volume = {16},
number = {9},
pages = {4843-4864},
pmid = {41799190},
issn = {1838-7640},
mesh = {Animals ; Mice ; *TRPC Cation Channels/metabolism/genetics ; *Calcium/metabolism ; Mice, Inbred C57BL ; *Calcium Signaling/drug effects ; *Hydrogen/metabolism/pharmacology ; Humans ; Cell Movement/drug effects ; Molecular Dynamics Simulation ; Molecular Docking Simulation ; Brain/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {RATIONALE: Hydrogen gas (H2) produces pleiotropic therapeutic actions, but the exact molecular targets and ion-channel-based signaling cascades that underlie these benefits remain elusive. H2 may regulate calcium ion (Ca[2+])-dependent processes, but the direct involvement of H2 in Ca[2+] signaling and its underlying molecular mechanisms are unknown. We propose that H2 functions as a gaseous messenger that selectively opens a plasma-membrane Ca[2+] channel to evoke Ca[2+] transients ([Ca[2+] i]t) while avoiding cytotoxic overload, thereby offering a mechanism for its diverse biological effects.<br><br>METHODS: This study employed real-time calcium imaging and CRISPR-Cas9 gene editing, with live-cell imaging to monitor real-time calcium signal intensity in living cells. Two-photon in vivo imaging was applied to detect real-time Ca[2+] signals in the brain and dorsal skin of C57BL/6 mice carrying adeno-associated virus-delivered calcium sensors. Live-cell F-actin staining and a wound healing (scratch) assay were used to assess the effects of H2 on cell motility. Protein-protein docking and molecular dynamics simulations were performed to analyze the interaction interface and binding forces between TRPC4 and TRPC4AP in three-dimensional space. Additionally, RNA sequencing was performed to validate downstream biological effects and transcriptional regulation triggered by H2.<br><br>RESULTS: H2 elicited rapid and reversible [Ca[2+] i]t across multiple cell types in a Ca[2+]- and concentration-dependent manner, an effect that was absent in TRPC4&#x207b;/&#x207b; or TRPC4AP&#x207b;/&#x207b; cells. In vivo imaging in mice expressing a genetically encoded Ca&#xb2;&#x207a; sensor showed that H2 inhalation elevated Ca[2+] signals in the motor cortex (M1 region) and dorsal skin. Functionally, live-cell imaging and wound-healing assays confirmed that H2-induced Ca[2+] transients enhanced cell motility. Mechanistically, protein&#x202f;docking revealed a dual-arginine cluster within the CIRB domain of TRPC4; its interaction with TRPC4AP was essential for H2-evoked Ca[2+] influx. Mutating these arginines to alanine residues completely abolishing the response. H2 triggered proton efflux and increased intracellular pH. Molecular dynamics simulations indicated that altered pH modulates the binding force between TRPC4 Arg730/Arg731 and TRPC4AP. Transcriptomic analysis further demonstrated that H2 activates calcium-related channels and promotes cytoskeletal remodeling and cell migration.<br><br>CONCLUSIONS: This study identifies H2 as a novel gaseous signaling molecule that can regulate Ca[2+] channels via the TRPC4-TRPC4AP axis. The 730Arg-731Arg motif in TRPC4 serves as a critical H2-sensitive site, enabling dynamic calcium homeostasis without overload. These findings provide a mechanistic framework for developing gas-controlled H2 regenerative therapeutics.},
}
@article {pmid41797538,
year = {2026},
author = {Fang, M and Yap, J and Fei, M and Gong, M and Li, N and Lu, Y and Yu, M and Xu, Y and Wu, F and Gao, H and Sun, D},
title = {LysR-type regulator LrhA promotes CRISPR-Cas immunity in Escherichia coli.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41797538},
issn = {1362-4962},
support = {32170083//National Natural Science Foundation of China/ ; 31670084//National Natural Science Foundation of China/ ; 31930003//National Natural Science Foundation of China/ ; 2020C02031//Key Research and Development Program of Zhejiang Province/ ; LHDMY23H160003//Natural Science Foundation of China/ ; YS2022005//Natural Science Foundation of China/ ; 2026C02A1080//Zhejiang Lingyan Research and Development Program/ ; LMRY26H200010//Joint Funds of the Zhejiang Provincial Natural Science Foundation of China/ ; },
mesh = {*Escherichia coli/genetics/immunology/virology ; *CRISPR-Cas Systems/genetics ; *Escherichia coli Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Promoter Regions, Genetic ; *Transcription Factors/genetics/metabolism ; Operon ; Plasmids/genetics ; Trans-Activators/genetics ; },
abstract = {The CRISPR-Cas defense system safeguards prokaryotes against foreign genetic elements. Its activity is determined by the combined effects of adaptation and interference. However, the dynamic regulation of these two processes remains not fully understood. In this study, we identify the LysR-type transcriptional regulator LrhA, which is differentially expressed in various Escherichia coli strains, as a novel CRISPR-Cas activator that plays a critical role in modulating host defense levels. In a representative strain expressing a high level of LrhA, the regulator enhances CRISPR-Cas-mediated adaptive immunity against bacteriophage infection by promoting cas gene transcription through direct interaction with the promoter of the cas operon. Moderate activation of cas genes by weakly expressed LrhA in another representative strain efficiently accelerates the clearance of horizontally transferred CRISPR-targeted plasmids by enhancing spacer acquisition via interference-driven adaptation. This divergence, likely a result of genome evolution, suggests that adaptive immunity is optimized with intermediate transcription levels of cas genes by triggering positive feedback between adaptation and interference. Collectively, our findings highlight the crucial role of LrhA in fine-tuning host defense responses.},
}
@article {pmid41796733,
year = {2026},
author = {Lin, J and Wang, Y and Zeng, B and Chen, Z and Lin, X and Zeng, T},
title = {CRISPR-Cas12a/Cas13a in cancer molecular diagnosis.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {},
number = {},
pages = {120934},
doi = {10.1016/j.cca.2026.120934},
pmid = {41796733},
issn = {1873-3492},
abstract = {Cancer remains a leading cause of global mortality, with early diagnosis being pivotal for improving treatment outcomes. Traditional tissue biopsy is limited by its invasiveness, inability to capture tumor heterogeneity, and failure to support dynamic monitoring. Liquid biopsy has emerged as a non-invasive alternative, enabling the analysis of circulating tumor biomarkers (e.g., ctDNA, miRNAs, exosomes) in bodily fluids. However, current liquid biopsy technologies (e.g., NGS, ddPCR) suffer from high costs, complex workflows, poor standardization, and insufficient sensitivity for low-abundance biomarkers. The CRISPR-Cas systems, particularly Cas12a and Cas13a, have revolutionized molecular diagnostics due to their programmable sequence recognition, robust signal amplification via trans-cleavage/collateral cleavage activity, and compatibility with point-of-care testing (POCT). Cas12a targets DNA molecules, enabling sensitive detection of gene mutations and DNA methylation, while Cas13a specifically recognizes RNA, facilitating direct analysis of miRNAs and viral RNAs. Additionally, these systems have been extended to non-nucleic acid biomarkers (e.g., proteins, exosomes) through signal conversion strategies. This review summarizes the latest advances in CRISPR-Cas12a/Cas13a-based biosensors for cancer molecular diagnosis, including the detection of gene mutations, epigenetic modifications, miRNAs, tumor-associated viruses, and non-nucleic acid biomarkers. We critically analyze current challenges (e.g., PAM dependence, matrix interference, multiplexing limitations, clinical validation gaps) and discuss future perspectives, such as engineering PAM-less Cas variants, integrating nanotechnology, microfluidics, and artificial intelligence/artificial intelligence (AI), and advancing clinical standardization. This review aims to provide a comprehensive reference for the development and clinical translation of CRISPR-based cancer diagnostic technologies.},
}
@article {pmid41795439,
year = {2026},
author = {Zhu, L and Yang, C and Bernards, R and Wang, C},
title = {CLIM-TIME links genetic cancer drivers to immune landscapes.},
journal = {Cell},
volume = {189},
number = {5},
pages = {1263-1265},
doi = {10.1016/j.cell.2026.01.014},
pmid = {41795439},
issn = {1097-4172},
mesh = {Humans ; *Neoplasms/genetics/immunology/therapy ; Immunotherapy ; Tumor Microenvironment/immunology/genetics ; Animals ; CRISPR-Cas Systems ; Genes, Tumor Suppressor ; },
abstract = {Immunotherapy resistance is associated with immune-privileged microenvironments, yet the interacting role of tumor-intrinsic genetics remains unclear. In this issue of Cell, Wang et al. introduce CLIM-TIME, a spatially resolved in vivo CRISPR screening platform linking loss of tumor suppressor genes to distinct metastatic immune architectures and divergent responses to immunotherapy.},
}
@article {pmid41794473,
year = {2026},
author = {Safenkova, IV and Kamionskaya, MV and Serchenya, TS and Sviridov, OV and Dzantiev, BB and Zherdev, AV},
title = {CRISPR/Cas12a and fork-shaped probe enhance LAMP-LFT integration for equipment-free detection of Listeria monocytogenes.},
journal = {Food research international (Ottawa, Ont.)},
volume = {230},
number = {},
pages = {118592},
doi = {10.1016/j.foodres.2026.118592},
pmid = {41794473},
issn = {1873-7145},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Listeria monocytogenes/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Food Microbiology/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Limit of Detection ; DNA, Bacterial/genetics ; Food Contamination/analysis ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Listeria monocytogenes is an important foodborne pathogen associated with high mortality rates, especially among vulnerable populations, and therefore requires diagnostic methods that are not only highly sensitive and rapid but also suitable for use in resource-limited settings. In this study, we developed an isothermal amplification assay integrated with a lateral flow test (LFT) for reliable detection of L.monocytogenes. Two assay formats were designed and compared: (1) loop-mediated isothermal amplification (LAMP) with LFT detection of fluorescein- and biotin-labeled amplicons, and (2) LAMP combined with CRISPR/Cas12a, using LFT to detect a cleaved fork-shaped enhanced probe labeled with three fluoresceins. Both LFT formats utilized a common conjugate of gold nanoparticles and anti-fluorescein antibodies (anti-FAM), but differed in the test zone immobilization strategy: streptavidin for LAMP, and anti-FAM for LAMP-CRISPR/Cas12a. Among 12 tested (primer - label) combinations, the most effective was identified, but the sensitivity of the LAMP-LFT format was limited by high signal variability. In contrast, the LAMP-CRISPR/Cas12a assay, targeting LAMP amplicons with guide RNA, achieved a detection limit of 0.9 copies/reaction-representing > 20,000-fold improvement in detectable DNA concentration compared with LAMP-LFT-and comparable to fluorescence-based detection techniques. The LAMP-CRISPR/Cas12a-LFT assay was first reported to detect L.monocytogenes cells following thermal lysis (10 min at 95 °C), with a single-cell detection limit (0.2 cells/reaction in buffer, 1 cells/reaction in spiked milk) and an analysis time of 80 min. These results demonstrate the potential of the approach for sensitive, equipment-free detection of foodborne pathogens in complex food matrices.},
}
@article {pmid41793913,
year = {2026},
author = {Li, X and Liu, L and Luo, C and Chen, Z and Shu, B},
title = {Efficient CRISPR/Cas9 system established via co-cultivation of plantlets and Agrobacterium tumefaciens for positive transgenic calluses generation and regeneration in cultivated strawberry (Fragaria × ananassa).},
journal = {Plant physiology and biochemistry : PPB},
volume = {232},
number = {},
pages = {111195},
doi = {10.1016/j.plaphy.2026.111195},
pmid = {41793913},
issn = {1873-2690},
abstract = {Recently, an Agrobacterium-mediated CRISPR/Cas9 editing system was successfully applied in a gene function analysis, highlighting its great value for improving strawberry genetics. However, the resulting low transformation rates and long regeneration cycles have limited its extensive application. Based on the biological characteristics of crown branching, an Agrobacterium tumefaciens-mediated CRISPR/Cas9 gene editing system was developed to increase the transformation rate and decrease the regeneration time of cultivated strawberry. Two single guide (sg)RNAs were designed for the strawberry anthracnose-related transcription factor, WRKY (FxaC_17g55530), and its alleles. These sgRNAs were inserted into pKSE401G using pCBC-DT1T2; sgRNAs for subtilisin-like protease (FxaC_22g21540) were designed and cloned in a similar manner. After 10 days of co-cultivating plantlets (without media supply of carbon) and GV3101, 65 (61.9%) and 72 (68.6%) GFP-positive calluses for the two genes were respectively obtained from the crown of 105 plantlets. The positive calluses were removed from the crown and placed on Murashige and Skoog media containing 3 mg/L thidiazuron and 0.2 mg/L indole-3-butyric acid. After 50-80 days, 3-5 positive shoots were obtained from different positive calluses for each gene. The three T0 lines for FxaC_17g55530 and FxaC_22g21540 were found to be successfully edited at the target sites of both sgRNA1 and sgRNA2 or either sgRNA1 or sgRNA2. Overall, a quick and effective CRISPR-Cas 9 gene editing system was developed for cultivated strawberry, highlighting the applicability of gene editing in breeding and gene function analysis.},
}
@article {pmid41791397,
year = {2026},
author = {Low, SJ and O'Neill, MT and Fernando, JA and Kerry, WJ and Prestedge, J and Wild, N and Chahal, S and Pollock, GL and Papadakis, G and Krysiak, M and Williams, E and Azzato, F and Tran, T and Fairley, C and Bradshaw, C and Chen, MY and Lim, CK and Williamson, DA and Pasricha, S},
title = {CRISPR-Cas-based diagnostics for point-of-care detection of sexually transmitted infections: a laboratory development and evaluation study.},
journal = {The Lancet. Microbe},
volume = {},
number = {},
pages = {101289},
doi = {10.1016/j.lanmic.2025.101289},
pmid = {41791397},
issn = {2666-5247},
abstract = {BACKGROUND: Timely, point-of-care diagnosis of sexually transmitted infections (STIs) is crucial for enabling prompt treatment and reducing transmission. We aimed to develop a portable, multiplexed, CRISPR-based assay panel for the detection of Neisseria gonorrhoeae (including the ciprofloxacin resistance marker gyrA S91F), Chlamydia trachomatis, Treponema pallidum, and herpes simplex virus (HSV).<br><br>METHODS: In this laboratory development and evaluation study, we developed and optimised four multiplexed, CRISPR-based, diagnostic STI assays for point-of-care use. The complete assay panel comprised a CRISPR TP-HSV (cTP-HSV) panel for the detection of T pallidum and pan-HSV, with reflex testing to distinguish HSV-1 from HSV-2, and a CRISPR NG-CT (cNG-CT) panel for the detection of N gonorrhoeae and C trachomatis, with reflex testing to detect N gonorrhoeae using two additional genome regions and to identify the gyrA S91F mutation. Each pathogen was targeted at two independent genomic regions by isothermal amplification and CRISPR-Cas reaction using Cas12a and Cas13a, each with distinct fluorescent reporters. Analytical specificity and limits of detection (LODs) were determined, and a retrospective, masked concordance study was conducted on genomic DNA from 900 clinical samples (400 for cTP-HSV and reflex testing and 500 for cNG-CT and reflex testing), using quantitative PCR as the reference standard. The diagnostic accuracy of the test was assessed by analysis of receiver operating characteristic curves.<br><br>FINDINGS: The overall sensitivity of the TP-HSV CRISPR assay was 82&#xb7;5% (95% CI 74&#xb7;0-88&#xb7;7) for T pallidum and 94&#xb7;4% (90&#xb7;2-97&#xb7;0) for pan-HSV; LODs were 6&#xb7;2 copies per &#x3bc;L for T pallidum and 7&#xb7;8 copies per &#x3bc;L for HSV. Reflex testing gave sensitivities of 97&#xb7;0% (91&#xb7;1-99&#xb7;3) for HSV-1 and 96&#xb7;0% (89&#xb7;7-98&#xb7;7) for HSV-2. The NG-CT CRISPR assay had an overall sensitivity of 80&#xb7;0% (74&#xb7;0-84&#xb7;9) for N gonorrhoeae and 73&#xb7;0% (65&#xb7;5-79&#xb7;3) for C trachomatis, with a LOD of 3&#xb7;9 copies per &#x3bc;L for both pathogens. Reflex testing for the detection of the gyrA S91F mutation in N gonorrhoeae showed an overall sensitivity of 63&#xb7;1% (55&#xb7;1-70&#xb7;4); however, this was dependent on sample type, with a sensitivity of 85&#xb7;7% (46&#xb7;7-99&#xb7;5) in genital samples and 61&#xb7;2% (52&#xb7;8-68&#xb7;9) in extragenital samples. For all pathogens, assay sensitivity was positively correlated with pathogen load. Area under the curve (AUC) values were 0&#xb7;90 for T pallidum and 0&#xb7;99 for pan-HSV in the TP-HSV assay, with values of 0&#xb7;99 for HSV-1 and 0&#xb7;97 for HSV-2 obtained in the reflex HSV-1-HSV-2 assay. For the cNG-CT assay, AUC values were 0&#xb7;90 for N gonorrhoeae and 0&#xb7;85 for C trachomatis, with a value of 0&#xb7;72 obtained for gyrA S91F in the reflex cNG-gyrA assay.<br><br>INTERPRETATION: Our multiplexed, CRISPR-based, point-of-care platform achieved performance consistent with WHO target product profiles for N gonorrhoeae and T pallidum. Proof-of-concept detection of the gyrA S91F resistance marker highlights its potential for resistance-guided therapy. Although optimisation is required before large-scale deployment, this suite offers a promising approach for rapid, decentralised, and resistance-informed STI diagnosis, particularly in resource-limited settings.<br><br>FUNDING: Victorian Government Department of Health, Australian Government Department of Health, Disability and Ageing and Aged Care, and Australian Research Council.},
}
@article {pmid41789142,
year = {2026},
author = {Xu, C and Zeng, C and Wang, M and Wei, X and Song, M and Liu, X and Wang, W and Chen, Q and Ji, X and Luo, P and Ma, L and Sun, Y and Gou, H and Zhu, Z and Li, X and Lv, YX and Liu, P and Zhu, JK},
title = {mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo.},
journal = {Innovation (Cambridge (Mass.))},
volume = {7},
number = {3},
pages = {101151},
pmid = {41789142},
issn = {2666-6758},
abstract = {Programmable epigenetic editors (EEs) that achieve long-term gene expression modulation without altering the DNA sequence hold immense therapeutic potential. However, the clinical translation of current CRISPR-based epigenome editors is impeded by substantial challenges, particularly their large molecular size, which limits efficient in vivo delivery. Here, we report the rational design and engineering of compact, mRNA-delivered EEs (CRISPR OFF-EE) using Streptococcus pyogenes Cas9 (SpCas9), intein-split-SpCas9, or the smaller Cas-SF01 (a Cas12i3 variant). Combined with optimized mRNA architecture and lipid nanoparticle (LNP) delivery, a single intravenous LNP administration of the optimized OFF-EE V2 mRNA, along with selected guide RNAs (gRNAs) targeting Pcsk9 in mice, resulted in an ∼83.2% reduction in circulating PCSK9 levels and a corresponding ∼51.4% reduction in low-density lipoprotein cholesterol (LDL-C) levels, persisting for at least 180 days. SF01-based EEs showed higher specificity with fewer off-target methylation events than SpCas9-based counterparts. Our optimized LNP formulation also demonstrated a favorable safety profile with predominantly liver-tropic activity. These findings establish a robust and versatile platform for advancing in vivo therapeutics based on precise and durable epigenetic silencing using transiently delivered, engineered mRNA editors.},
}
@article {pmid41785880,
year = {2026},
author = {Petersen, A&#xd8; and Damholt, B and Grove, M and Hink, J and Marotte-Hurbon, T and Söderqvist, J and Troy, A and Zdravkovic, M and Bayer, L and Brunner, K and Bryde, T and Clube, J and Gencay, YE and Gram, A and Haaber, JK and Hallström, B and Jasinskytė, D and Pascal, R and Petersen, M and Semsey, S and Torio, AS and Turcu, IC and Smrekar, F and Taur, Y and Satlin, MJ and Sommer, MOA and van der Helm, E and Grøndahl, C},
title = {Safety, recovery, and pharmacodynamics of CRISPR-Cas therapeutic SNIPR001: a phase 1, randomised, double-blind, first-in-human, dose-escalation study.},
journal = {The Lancet. Microbe},
volume = {},
number = {},
pages = {101257},
doi = {10.1016/j.lanmic.2025.101257},
pmid = {41785880},
issn = {2666-5247},
abstract = {BACKGROUND: Patients with haematological cancer who receive stem-cell transplantation are at risk of bloodstream infections, often caused by multidrug resistant gut pathogens such as Escherichia coli. SNIPR001 is a cocktail of four CRISPR-Cas-armed bacteriophages that reduce colonisation of E coli in the gastrointestinal tract in animal models and is designed to not affect other members of the commensal microbiota. We aimed to investigate the safety and tolerability of SNIPR001 in healthy participants.<br><br>METHODS: In this randomised, placebo-controlled, double-blind, first-in-human, dose-escalation trial conducted at a single centre (Medpace Clinical Pharmacology Unit; Cincinnati, OH, USA), we sequentially enrolled healthy participants (aged 18-65 years) with more than 10[7]E coli colony-forming units per gram of stool into cohorts 1, 2, and 3, pending a safety review of the previous enrolment group where applicable. Participants in each cohort were randomly assigned to treatment or placebo using a unique three-digit participant identification number. Participants were orally administered 10[8] plaque-forming units (PFU) per dose (cohort 1), 10[10] PFU per dose (cohort 2), and 10[12] PFU per dose (cohort 3) of SNIPR001 or placebo (phosphate-buffered saline buffer), twice daily for 7 days. All personnel, except for a pharmacy staff member who prepared both SNIPR001 and placebo vials, were masked to the administered dose and assignment; masking was ensured by fully covering the surface of each vial. Participants were followed up to day 187. The primary outcome was the incidence and severity of adverse events and medically attended adverse events from the first administration of the study drug until 4 weeks after the last dose administration on day 35 of the study. Recovery and biodistribution of SNIPR001 in faeces, blood, and urine; pharmacodynamics, including the ability of SNIPR001 to reduce E coli levels in stool (assessed using a linear mixed-effects model); and microbiome composition (using Bray-Curtis dissimilarity) were secondary outcomes. Primary safety analyses were assessed per-protocol (ie, all enrolled participants who received at least one administration of the study drug). This trial was conducted under an Investigational New Drug application from the US Food and Drug Administration, is registered with ClinicalTrials.gov (NCT05277350), and is closed to new participants.<br><br>FINDINGS: The trial was carried out between March 24, 2022, and Nov 30, 2022. 36 eligible participants were randomly assigned to receive SNIPR001 or placebo in cohorts 1 (six assigned to 10[8] PFU per dose and two assigned to placebo), 2 (six to 10[10] PFU per dose and two to placebo), and 3 (12 to 10[12] PFU per dose and eight to placebo). The mean age of participants was 42&#xb7;1 years (SD 13&#xb7;8), with 14 (39%) female participants and 22 (61%) male participants. During the trial and 4-week follow-up period, only mild and moderate adverse events were observed, with most adverse events occurring in the placebo group (13, six, one, and nine for participants receiving either placebo or SNIPR001 at 10[8], 10[10], and 10[12] PFU twice a day, respectively). The number of participants who had adverse events was not significantly higher in treatment groups than in the placebo group (p=0&#xb7;94, one-sided Fisher's exact test). The most frequently reported adverse events were headaches and diarrhoea. No grade 3-4 adverse events were reported and no serious adverse events were reported in the SNIPR001 dose groups. During and after the dosing period, the gut microbiota composition did not significantly differ between the treatment and placebo groups (p>0&#xb7;05, two-sided Mann-Whitney U test of Bray-Curtis distances, false discovery rate [FDR]-corrected). Functional SNIPR001 was recovered from stool samples in concentrations proportional to the administered dose but was not meaningfully detected in plasma (only one sample) or urine (only one sample). SNIPR001 was undetected in all samples 6 months after the last dosing, which is a favourable pharmacokinetic property and meets regulatory expectations. We observed the largest reduction in E coli levels compared with placebo 2 weeks after treatment initiation at day 14 (78%; -0&#xb7;65 log10 [SE 0&#xb7;64] for 10[12] PFU SNIPR001 twice-daily group), according to a linear mixed-effects model for the highest dose population; however, this change was not statistically significant (p=0&#xb7;811, linear mixed-effects model, FDR-corrected).<br><br>INTERPRETATION: This first-in-human study of SNIPR001 supported its safety, tolerability, and restriction to the gastrointestinal tract, while not systemically disrupting the gut microbiome. These results justify further clinical development of SNIPR001 in an ongoing phase 1b/2a trial.<br><br>FUNDING: Combating Antibiotic-Resistant Bacteria Biopharmaceutical Accelerator (CARB-X) and SNIPR Biome.},
}
@article {pmid41721525,
year = {2026},
author = {Chen, N and Gao, M and Bai, Y and Wang, M and Liu, M and Xiong, W and Mo, B},
title = {5'UTR Editing of the ribosomal protein UL3Z gene unveils its critical roles in pre-rRNA processing and global mRNA translation dynamics.},
journal = {Plant physiology},
volume = {200},
number = {3},
pages = {},
doi = {10.1093/plphys/kiag073},
pmid = {41721525},
issn = {1532-2548},
support = {32470592//National Natural Science Foundation of China/ ; 32270595//National Natural Science Foundation of China/ ; 2023B001//SZU 2035 Excellence Research Program/ ; },
mesh = {*5' Untranslated Regions/genetics ; *Ribosomal Proteins/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; *RNA Precursors/metabolism/genetics ; *Arabidopsis Proteins/genetics/metabolism ; *Protein Biosynthesis/genetics ; RNA, Messenger/metabolism/genetics ; Gene Expression Regulation, Plant ; *RNA Processing, Post-Transcriptional/genetics ; Gene Editing ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {The EMBRYO DEFECTIVE 2207 (EMB2207) gene, encoding ribosomal protein UL3Z, is critical for embryonic development in Arabidopsis, with loss of function resulting in embryo lethality. Despite its importance, the role of UL3Z in the complicated protein translation machinery in plants remains poorly understood due to the lack of viable hypomorphic alleles. In this study, we utilized CRISPR/Cas9 to edit the 5' untranslated region (5'UTR) of UL3Z, generating 5 ul3z mutants with varying degrees of reduced expression levels of UL3Z proteins. The mutant with the lowest expression exhibited the most severe developmental defects. In contrast, null mutants of its paralog UL3Y displayed no observable phenotypes. Interestingly, expression of UL3Y driven by the UL3Z/EMB2207 promoter successfully rescued the phenotypes of ul3z, demonstrating that these 2 paralogous ribosomal proteins actually possess functionally interchangeable roles. GUS staining results showed that UL3Z was constitutively expressed in all examined tissues, while UL3Y was only appreciably expressed in specific tissues. Molecular analysis further revealed the accumulation of ribosomal RNA (rRNA) maturation intermediates and increased polysome levels in ul3z mutants, indicating compromised pre-rRNA processing and disturbed global mRNA translation. Interestingly, 3' ends of many rRNA precursors in ul3z had higher frequency of non-encoded tails compared with Col-0. This study demonstrates that CRISPR/Cas9-mediated 5'UTR editing is an effective tool for generating viable hypomorphic alleles of lethal genes and uncovers the critical roles of UL3Z/EMB2207 in pre-rRNA processing and the maintenance of appropriate mRNA translation on ribosomes, underscoring its importance in plant development.},
}
@article {pmid41650957,
year = {2026},
author = {Shi, B and Li, J and Wang, X and Liu, D and Xiang, J and Wang, H and Xu, C and Zou, X and Wang, Z and Huang, T and Min, Q and Wang, K and Yang, Y and Li, J and Wang, B and Zhao, C and Pei, D},
title = {Generating high-quality porcine iPSCs with the new medium cocktail LACID.},
journal = {Stem cell reports},
volume = {21},
number = {3},
pages = {102790},
doi = {10.1016/j.stemcr.2026.102790},
pmid = {41650957},
issn = {2213-6711},
mesh = {Animals ; *Induced Pluripotent Stem Cells/cytology/metabolism/drug effects ; Swine ; Cellular Reprogramming ; *Culture Media/pharmacology/chemistry ; Blastocyst/cytology/metabolism ; *Cell Culture Techniques/methods ; CRISPR-Cas Systems ; Nuclear Transfer Techniques ; Cells, Cultured ; Cell Differentiation ; },
abstract = {Pigs are important for disease model generation, xenotransplantation, and interspecies organogenesis. Porcine induced pluripotent stem cells (piPSCs) should enable these efforts, but have not been generated to meet the attributes, such as feeder-free culture, robust development potential, and blastocyst generation through nuclear transfer. We report an improved strategy to generate such piPSCs. We show that chemically defined medium 3 promotes the formation of epithelium-like colonies in porcine reprogramming, which allows further reprogramming under the new medium cocktail LACID. The resulting piPSCs have key features, including flat morphology with feeder-free culture, generating robust teratoma and blastoids, forming chimeric blastocysts, and readily edited with CRISPR-Cas9. Lastly, nuclear transfer with piPSCs can develop into blastocysts. Despite maintaining a primed pluripotent state, our results suggest that the newly established LACID piPSCs may be ideal for applications in regenerative medicine. This method may be further improved to generate naive or totipotent stem cells.},
}
@article {pmid41517973,
year = {2026},
author = {Jin, G and Li, H and Yu, H and Gu, Z and Cui, H and Chen, J and Li, X},
title = {Asp-2078-Gly mutation in ACCase confers quizalofop-p-ethyl resistance in Eleusine indica and establishment of a LAMP-CRISPR/Cas12a visual genotyping assay for the target mutation.},
journal = {Pest management science},
volume = {82},
number = {4},
pages = {3374-3383},
doi = {10.1002/ps.70468},
pmid = {41517973},
issn = {1526-4998},
support = {//the Key Project in Soybean Bio breeding and Commercialization/ ; //the China Agriculture Research System/ ; 2022ZD04021//the Sci-Tech Innovation 2030 Agenda/ ; },
mesh = {*Herbicide Resistance/genetics ; *Acetyl-CoA Carboxylase/genetics/metabolism ; *Eleusine/genetics/drug effects/enzymology ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; *Herbicides/pharmacology ; Mutation ; *Genotyping Techniques/methods ; *Plant Proteins/genetics/metabolism ; Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Eleusine indica is a widespread, competitive weed causing yield losses in major crops. Repeated use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in cotton fields has led to the evolution of resistant populations, posing a growing threat to cotton production in China. This study aimed to elucidate the target-site resistance mechanism of E. indica to quizalofop-p-ethyl and establish a rapid visual detection method based on the identified mutation.<br><br>RESULTS: The NJC-R population showed resistance to quizalofop-p-ethyl (resistance index&#x2009;=&#x2009;5.5). Gene sequencing revealed that an Asp-2078-Gly mutation in ACCase was one of the mechanisms underlying resistance. Loop-mediated isothermal amplification (LAMP) combined with the CRISPR/Cas12a system was developed to detect this mutation in E. indica. This method not only enabled genotype discrimination (wild-type, heterozygous, homozygous mutant), but also provided visual results within 70&#x2009;min, exhibiting superior performance compared with the derived cleaved amplified polymorphic sequences assay. In addition, this method eliminated false positives from nonspecific LAMP amplification, was ~100-fold more sensitive than a polymerase chain reaction, and the assay results were 100% concordant with Sanger sequencing for the 50 samples tested.<br><br>CONCLUSION: This study confirmed that the Asp-2078-Gly mutation confers quizalofop-p-ethyl resistance in E. indica from the cotton field in China, and LAMP-CRISPR/Cas12a was first applied for detecting ACCase target-site mutations in E. indica. Given its rapidity and high accuracy, this technique has the potential to be applied for resistance monitoring and to guide rational herbicide application. &#xa9; 2026 Society of Chemical Industry.},
}
@article {pmid41445368,
year = {2026},
author = {Wei, J and Jiang, C and Chen, Y and Yang, X and Li, Q},
title = {Functional characterization of Hsk1 and Chit1 genes in the virulence of Metarhizium guizhouense Xct1 via CRISPR-Cas9-mediated gene editing.},
journal = {Pest management science},
volume = {82},
number = {4},
pages = {3625-3639},
doi = {10.1002/ps.70484},
pmid = {41445368},
issn = {1526-4998},
support = {//SCCXTD-2024-04/ ; },
mesh = {*CRISPR-Cas Systems ; Virulence/genetics ; Gene Editing ; Animals ; *Metarhizium/genetics/pathogenicity ; *Fungal Proteins/genetics/metabolism ; Spodoptera/growth &amp; development/microbiology ; *Protein Serine-Threonine Kinases/genetics/metabolism ; Larva/growth &amp; development/microbiology ; Pest Control, Biological ; },
abstract = {BACKGROUND: The entomopathogenic fungus Metarhizium guizhouense Xct1 exhibits high virulence against early-instar Spodoptera frugiperda larvae (>90% mortality in preliminary studies), yet the molecular mechanisms, particularly the roles of key genes such as the chitin-degrading enzyme (Chit1) and serine/threonine kinase (Hsk1) are poorly understood. Functional studies using CRISPR-Cas9 are lacking, limiting its biocontrol application.<br><br>RESULTS: Chit1 and Hsk1 genes were amplified from M.&#x2009;guizhouense Xct1. Chit1 showed high homology to M.&#x2009;anisopliae, whereas Hsk1 exhibited greater genetic diversity. Expression analysis revealed peak Chit1 expression on Day (D)4 and peak Hsk1 expression on D2. A CRISPR-Cas9 system was established, and knockout of Chit1 resulted in thickened cell walls [119&#x2009;nm versus 87&#x2009;nm in wild-type (WT)] and reduced virulence [median lethal time (LT50)&#x2009;=&#x2009;7.4&#x2009;days versus 4.8&#x2009;days in WT]. Overexpression of Chit1 improved virulence (LT50&#x2009;=&#x2009;3.3&#x2009;days). Hsk1 knockout was lethal, confirming its essential role, while overexpression did not alter virulence (LT50&#x2009;=&#x2009;4.8&#x2009;days).<br><br>CONCLUSIONS: Chit1 is a critical virulence factor, influencing cell-wall integrity and insecticidal activity, while Hsk1 is essential for fungal viability. This study presents the first CRISPR-Cas9-mediated functional analysis of these genes, revealing that Chit1 overexpression enhances biocontrol efficacy against S.&#x2009;frugiperda. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid41057363,
year = {2025},
author = {Li, J and Pan, Z and Peng, X and Feng, Y and Wu, J and Liang, F and Feng, Q and Yu, X and Deng, H},
title = {Ecdysone signaling-induced dumpless1 expression controls nurse cell dumping in Drosophila oogenesis.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8917},
pmid = {41057363},
issn = {2041-1723},
support = {32170494//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Oogenesis/genetics/physiology ; *Ecdysone/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; Animals ; Signal Transduction ; *Drosophila melanogaster/genetics/metabolism ; rho GTP-Binding Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Ovarian Follicle/cytology/metabolism ; Integrin beta Chains/genetics/metabolism ; Transcription Factors/genetics/metabolism ; Oocytes/cytology/metabolism ; Female ; },
abstract = {Nurse cell (NC) dumping, a process essential for oocyte development, involves the rapid cytoplasmic transfer from germline-derived NCs into the oocyte. However, its regulatory mechanism remains unclear. Here, we report that ecdysone signaling in stretch follicle cells (SFCs) regulates NC dumping through dumpless1, a ZAD-C2H2 zinc finger transcription factor, in Drosophila. Ecdysone induced dumpless1 expression in SFCs, and CRISPR/Cas9-mediated knockout of dumpless1 or its functional domain ZAD suppresses NC dumping. Depletion of dumpless1 upregulates integrin βPS expression in SFC plasma membrane, while reducing cortical enrichment of Rho1 signaling-dependent phosphorylated myosin light chain (p-MLC) and disrupting actin cables organization in NCs. SFC-specific overexpression of integrin βPS reduces p-MLC enrichment in the NC cortex, whereas its knockdown in SFCs of dumpless1[-/-] mutants partially rescues NC dumping defect. Our findings identify dumpless1 as a critical effector of ecdysone signaling, bridging somatic-germline communication through the integrin βPS-Rho1-p-MLC axis, revealing a multicellular regulatory mechanism in Drosophila oogenesis.},
}
@article {pmid37766223,
year = {2023},
author = {Zhang, H and Duan, K and Du, Y and Xiao, S and Fang, L and Zhou, Y},
title = {One-Step Assembly of a PRRSV Infectious cDNA Clone and a Convenient CRISPR/Cas9-Based Gene-Editing Technology for Manipulation of PRRSV Genome.},
journal = {Viruses},
volume = {15},
number = {9},
pages = {},
pmid = {37766223},
issn = {1999-4915},
support = {32130103//National Natural Science Foundation of China/ ; 32002279//National Natural Science Foundation of China/ ; },
mesh = {*Porcine respiratory and reproductive syndrome virus/genetics ; Animals ; *CRISPR-Cas Systems ; *Genome, Viral ; *Gene Editing/methods ; Swine ; *DNA, Complementary/genetics ; Porcine Reproductive and Respiratory Syndrome/virology ; Reverse Genetics ; Cell Line ; },
abstract = {Porcine reproductive and respiratory syndrome (PRRS) has been a persistent challenge for the swine industry for over three decades due to the lack of effective treatments and vaccines. Reverse genetics systems have been extensively employed to build rapid drug screening platforms and develop genetically engineered vaccines. Herein, we rescued recombinant PRRS virus (rPRRSV) WUH3 using an infectious cDNA clone of PRRSV WUH3 acquired through a BstXI-based one-step-assembly approach. The rPRRSV WUH3 and its parental PRRSV WUH3 share similar plaque sizes and multiple-step growth curves. Previously, gene-editing of viral genomes depends on appropriate restrictive endonucleases, which are arduous to select in some specific viral genes. Thus, we developed a restrictive endonucleases-free method based on CRISPR/Cas9 to edit the PRRSV genome. Using this method, we successfully inserted the exogenous gene (EGFP gene as an example) into the interval between ORF1b and ORF2a of the PRRSV genome to generate rPRRSV WUH3-EGFP, or precisely mutated the lysine (K) at position 150 of PRRSV nsp1α to glutamine (Q) to acquire rPRRSV WUH3 nsp1α-K150Q. Taken together, our study provides a rapid and convenient method for the development of genetically engineered vaccines against PRRSV and the study on the functions of PRRSV genes.},
}
@article {pmid37722405,
year = {2023},
author = {Low, SJ and O'Neill, MT and Kerry, WJ and Krysiak, M and Papadakis, G and Whitehead, LW and Savic, I and Prestedge, J and Williams, L and Cooney, JP and Tran, T and Lim, CK and Caly, L and Towns, JM and Bradshaw, CS and Fairley, C and Chow, EPF and Chen, MY and Pellegrini, M and Pasricha, S and Williamson, DA},
title = {Rapid detection of monkeypox virus using a CRISPR-Cas12a mediated assay: a laboratory validation and evaluation study.},
journal = {The Lancet. Microbe},
volume = {4},
number = {10},
pages = {e800-e810},
doi = {10.1016/S2666-5247(23)00148-9},
pmid = {37722405},
issn = {2666-5247},
mesh = {*CRISPR-Cas Systems ; Humans ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *Monkeypox virus/isolation &amp; purification/genetics ; *Molecular Diagnostic Techniques/methods ; *Mpox, Monkeypox/diagnosis/virology ; Limit of Detection ; Proof of Concept Study ; Point-of-Care Systems ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: The 2022 outbreak of mpox (formerly known as monkeypox) led to the spread of monkeypox virus (MPXV) in over 110 countries, demanding effective disease management and surveillance. As current diagnostics rely largely on centralised laboratory testing, our objective was to develop a simple rapid point-of-care assay to detect MPXV in clinical samples using isothermal amplification coupled with CRISPR and CRISPR-associated protein (Cas) technology.<br><br>METHODS: In this proof-of-concept study, we developed a portable isothermal amplification CRISPR-Cas12a-based assay for the detection of MPXV. We designed a panel of 22 primer-guide RNA sets using pangenome and gene-agnostic approaches, and subsequently shortlisted the three sets producing the strongest signals for evaluation of analytical sensitivity and specificity using a fluorescence-based readout. The set displaying 100% specificity and the lowest limit of detection (LOD) was selected for further assay validation using both a fluorescence-based and lateral-flow readout. Assay specificity was confirmed using a panel of viral and bacterial pathogens. Finally, we did a blind concordance study on genomic DNA extracted from 185 clinical samples, comparing assay results with a gold-standard quantitative PCR (qPCR) assay. We identified the optimal time to detection and analysed the performance of the assay relative to qPCR using receiver operating characteristic (ROC) curves. We also assessed the compatibility with lateral-flow strips, both visually and computationally, where strips were interpreted blinded to the fluorescence results on the basis of the presence or absence of test bands.<br><br>FINDINGS: With an optimal run duration of approximately 45 min from isothermal amplification to CRISPR-assay readout, the MPXV recombinase polymerase amplification CRISPR-Cas12a-based assay with the selected primer-guide set had an LOD of 1 copy per &#x3bc;L and 100% specificity against tested viral pathogens. Blinded concordance testing of 185 clinical samples resulted in 100% sensitivity (95% CI 89&#xb7;3-100) and 99&#xb7;3% specificity (95% CI 95&#xb7;7-100) using the fluorescence readout. For optimal time to detection by fluorescence readout, we estimated the areas under the ROC curve to be 0&#xb7;98 at 2 min and 0&#xb7;99 at 4 min. Lateral-flow strips had 100% sensitivity (89&#xb7;3-100) and 98&#xb7;6% specificity (94&#xb7;7-100) with both visual and computational assessment. Overall, lateral-flow results were highly concordant with fluorescence-based readouts (179 of 185 tests, 96&#xb7;8% concordant), with discrepancies associated with low viral load samples.<br><br>INTERPRETATION: Our assay for the diagnosis of mpox displayed good performance characteristics compared with qPCR. Although optimisation of the assay will be required before deployment, its usability and versatility present a potential solution to MPXV detection in low-resource and remote settings, as well as a means of community-based, on-site testing.<br><br>FUNDING: Victorian Medical Research Accelerator Fund and the Australian Government Department of Health.},
}
@article {pmid37721041,
year = {2023},
author = {Gentili, HG and Pignataro, MF and Olmos, J and Pavan, MF and Ibañez, LI and Santos, J and Velazquez Duarte, F},
title = {CRISPR/Cas9-based edition of frataxin gene in Dictyostelium discoideum.},
journal = {The Biochemical journal},
volume = {480},
number = {19},
pages = {1533-1551},
doi = {10.1042/BCJ20230244},
pmid = {37721041},
issn = {1470-8728},
mesh = {*Dictyostelium/genetics/metabolism/growth &amp; development ; Frataxin ; *Iron-Binding Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; *Protozoan Proteins/genetics/metabolism ; Mutation ; },
abstract = {In this paper, we describe the development of a Dictyostelium discoideum strain deficient in frataxin protein (FXN). We investigated the conservation of function between humans and D. discoideum and showed that DdFXN can substitute the human version in the interaction and activation of the Fe-S assembly supercomplex. We edited the D. discoideum fxn locus and isolated a defective mutant, clone 8, which presents landmarks of frataxin deficiency, such as a decrease in Fe-S cluster-dependent enzymatic functions, growth rate reduction, and increased sensitivity to oxidative stress. In addition, the multicellular development is affected as well as growing on bacterial lawn. We also assessed the rescuing capacity of DdFXN-G122V, a version that mimics a human variant present in some FA patients. While the expression of DdFXN-G122V rescues growth and enzymatic activity defects, as DdFXN does, multicellular development defects were only partially rescued. The results of the study suggest that this new D. discoideum strain offers a wide range of possibilities to easily explore diverse FA FXN variants. This can facilitate the development of straightforward drug screenings to look for new therapeutic strategies.},
}
@article {pmid37718190,
year = {2023},
author = {Kalinna, BH},
title = {Programming schistosomes - a crisper approach to transgenesis.},
journal = {Trends in parasitology},
volume = {39},
number = {11},
pages = {896-897},
doi = {10.1016/j.pt.2023.09.003},
pmid = {37718190},
issn = {1471-5007},
mesh = {Animals ; *Schistosoma mansoni/genetics ; *Gene Transfer Techniques ; CRISPR-Cas Systems ; *Schistosoma/genetics ; Transgenes ; },
abstract = {Ittiprasert and colleagues identified genomic safe harbour (GSH) sites in Schistosoma mansoni using computational methods and inserted a transgene into one of the sites through clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-assisted homology-directed repair. This study outlines a promising strategy for functional genomics to study this parasite that causes a debilitating and neglected tropical disease.},
}
@article {pmid37702151,
year = {2023},
author = {van Schie, JJM and de Lint, K and Molenaar, TM and Moronta Gines, M and Balk, JA and Rooimans, MA and Roohollahi, K and Pai, GM and Borghuis, L and Ramadhin, AR and Corazza, F and Dorsman, JC and Wendt, KS and Wolthuis, RMF and de Lange, J},
title = {CRISPR screens in sister chromatid cohesion defective cells reveal PAXIP1-PAGR1 as regulator of chromatin association of cohesin.},
journal = {Nucleic acids research},
volume = {51},
number = {18},
pages = {9594-9609},
pmid = {37702151},
issn = {1362-4962},
support = {10701//Dutch Cancer Society/ ; },
mesh = {Cohesins ; Humans ; *Cell Cycle Proteins/metabolism/genetics ; *Chromosomal Proteins, Non-Histone/metabolism/genetics ; *Chromatin/metabolism/genetics ; *Chromatids/genetics/metabolism ; CRISPR-Cas Systems ; DNA-Binding Proteins/genetics/metabolism ; Mitosis/genetics ; DNA Replication/genetics ; Nuclear Proteins/genetics/metabolism ; Sister Chromatid Exchange/genetics ; Cell Line ; Acetyltransferases ; DNA Helicases ; DEAD-box RNA Helicases ; },
abstract = {The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.},
}
@article {pmid37695673,
year = {2023},
author = {Zhang, H and Li, X and Wang, Y and Liu, X and Guo, J and Wang, Z and Zhang, L and Xiong, S and Dong, C},
title = {Genome-Wide CRISPR/Cas9 Screening Identifies That Mitochondrial Solute Carrier SLC25A23 Attenuates Type I IFN Antiviral Immunity via Interfering with MAVS Aggregation.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {211},
number = {9},
pages = {1406-1417},
doi = {10.4049/jimmunol.2300187},
pmid = {37695673},
issn = {1550-6606},
mesh = {Animals ; Mice ; *Interferon Type I/immunology/metabolism ; CRISPR-Cas Systems/genetics ; *Adaptor Proteins, Signal Transducing/metabolism/immunology/genetics ; Humans ; Ubiquitin-Protein Ligases/metabolism ; *RNA Virus Infections/immunology ; Signal Transduction ; Tripartite Motif Proteins/metabolism ; Ubiquitination ; Immunity, Innate ; HEK293 Cells ; Mice, Inbred C57BL ; *Mitochondrial Membrane Transport Proteins/genetics/metabolism ; Mitochondria/metabolism ; },
abstract = {Activation of the mitochondrial antiviral signaling (MAVS) adaptor, also known as IPS-1, VISA, or Cardif, is crucial for antiviral immunity in retinoic acid-inducible gene I (RIG-I)-like receptor signaling. Upon interacting with RIG-I, MAVS undergoes K63-linked polyubiquitination by the E3 ligase Trim31, and subsequently aggregates to activate downstream signaling effectors. However, the molecular mechanisms that modulate MAVS activation are not yet fully understood. In this study, the mitochondrial solute carrier SLC25A23 was found to attenuate type I IFN antiviral immunity using genome-wide CRISPR/Cas9 screening. SLC25A23 interacts with Trim31, interfering with its binding of Trim31 to MAVS. Indeed, SLC25A23 downregulation was found to increase K63-linked polyubiquitination and subsequent aggregation of MAVS, which promoted type I IFN production upon RNA virus infection. Consistently, mice with SLC25A23 knockdown were more resistant to RNA virus infection in vivo. These findings establish SLC25A23 as a novel regulator of MAVS posttranslational modifications and of type I antiviral immunity.},
}
@article {pmid37689217,
year = {2023},
author = {Colaco, JC and Chandrasekaran, AP and Karapurkar, JK and Gowda, DAA and Birappa, G and Rajkumar, S and Suresh, B and Ko, N and Hong, SH and Oh, SJ and Kim, KS and Ramakrishna, S},
title = {E3 ubiquitin ligase APC/C[Cdh1] regulates SLC35F2 protein turnover and inhibits cancer progression in HeLa cells.},
journal = {Biochimica et biophysica acta. General subjects},
volume = {1867},
number = {11},
pages = {130454},
doi = {10.1016/j.bbagen.2023.130454},
pmid = {37689217},
issn = {1872-8006},
mesh = {Humans ; HeLa Cells ; Ubiquitination ; *Ubiquitin-Protein Ligases/metabolism/genetics ; Disease Progression ; *Neoplasms/metabolism/pathology/genetics ; CRISPR-Cas Systems ; Cell Proliferation ; Adenomatous Polyposis Coli Protein ; },
abstract = {BACKGROUND: The solute carrier family 35 F2 (SLC35F2), belongs to membrane-bound carrier proteins that control various physiological functions and are activated in several cancers. However, the molecular mechanism regulating SLC35F2 protein turnover and its implication in cancer progression remains unexplored. Therefore, screening for E3 ligases that promote SLC35F2 protein degradation is essential during cancer progression.<br><br>METHODS: The immunoprecipitation and Duolink proximity ligation assays (PLA) were used to determine the interaction between APC/C[Cdh1] and SLC35F2 proteins. A CRISPR/Cas9-mediated knockdown and rescue experiment were used to validate the functional significance of APC/C[Cdh1] on SLC35F2 protein stabilization. The ubiquitination function of APC/C[Cdh1] on SLC35F2 protein was validated using in vitro ubiquitination assay and half-life analysis. The role of APC/C[Cdh1] regulating SLC35F2-mediated tumorigenesis was confirmed by in vitro oncogenic experiments in HeLa cells.<br><br>RESULTS: Based on the E3 ligase screen and in vitro biochemical experiments, we identified that APC/C[Cdh1] interacts with and reduces SLC35F2 protein level. APC/C[Cdh1] promotes SLC35F2 ubiquitination and decreases the half-life of SLC35F2 protein. On the other hand, the CRISPR/Cas9-mediated depletion of APC/C[Cdh1] increased SLC35F2 protein levels. The mRNA expression analysis revealed a negative correlation between APC/C[Cdh1] and SLC35F2 across a panel of cancer cell lines tested. Additionally, we demonstrated that depletion in APC/C[Cdh1] promotes SLC35F2-mediated cell proliferation, colony formation, migration, and invasion in HeLa cells.<br><br>CONCLUSION: Our study highlights that APC/C[Cdh1] is a critical regulator of SLC35F2 protein turnover and depletion of APC/C[Cdh1] promotes SLC35F2-mediated tumorigenesis. Thus, we envision that APC/C[Cdh1]-SLC35F2 axis might be a therapeutic target in cancer.},
}
@article {pmid37688599,
year = {2023},
author = {Minnaar, L and den Haan, R},
title = {Engineering natural isolates of Saccharomyces cerevisiae for consolidated bioprocessing of cellulosic feedstocks.},
journal = {Applied microbiology and biotechnology},
volume = {107},
number = {22},
pages = {7013-7028},
pmid = {37688599},
issn = {1432-0614},
support = {137967//National Research Foundation/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism/isolation &amp; purification/growth &amp; development ; *Cellulose/metabolism ; Ethanol/metabolism ; Fermentation ; *Cellulases/genetics/metabolism ; CRISPR-Cas Systems ; *Metabolic Engineering ; },
abstract = {Saccharomyces cerevisiae has gained much attention as a potential host for cellulosic bioethanol production using consolidated bioprocessing (CBP) methodologies, due to its high-ethanol-producing titres, heterologous protein production capabilities, and tolerance to various industry-relevant stresses. Since the secretion levels of heterologous proteins are generally low in domesticated strains of S. cerevisiae, natural isolates may offer a more diverse genetic background for improved heterologous protein secretion, while also displaying greater robustness to process stresses. In this study, the potential of natural and industrial S. cerevisiae strains to secrete a core set of cellulases (CBH1, CBH2, EG2, and BGL1), encoded by genes integrated using CRISPR/Cas9 tools, was evaluated. High levels of heterologous protein production were associated with a reduced maximal growth rate and with slight changes in overall strain robustness, compared to the parental strains. The natural isolate derivatives YI13_BECC and YI59_BECC displayed superior secretion capacity for the heterologous cellulases at high incubation temperature and in the presence of acetic acid, respectively, compared to the reference industrial strain MH1000_BECC. These strains also exhibited multi-tolerance to several fermentation-associated and secretion stresses. Cultivation of the strains on crystalline cellulose in oxygen-limited conditions yielded ethanol concentrations in the range of 4-4.5 g/L, representing 35-40% of the theoretical maximum ethanol yield after 120 h, without the addition of exogenous enzymes. This study therefore highlights the potential of these natural isolates to be used as chassis organisms in CBP bioethanol production. KEY POINTS: • Process-related fermentation stresses influence heterologous protein production. • Transformants produced up to 4.5 g/L ethanol, ~ 40% of the theoretical yield in CBP. • CRISPR/Cas9 was feasible for integrating genes in natural S. cerevisiae isolates.},
}
@article {pmid37683439,
year = {2024},
author = {Liu, B and Li, Y and Du, L and Zhang, F and Liu, Y and Sun, J and Zhang, Q and Li, C and Li, X and Xue, Q},
title = {"One-to-many" signal-output strategy-based CRISPR/Cas12a system for sensitive label-free fluorescence detection of HBV-DNA.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {304},
number = {},
pages = {123338},
doi = {10.1016/j.saa.2023.123338},
pmid = {37683439},
issn = {1873-3557},
mesh = {*CRISPR-Cas Systems/genetics ; *DNA, Viral/analysis/genetics ; *Hepatitis B virus/genetics ; *Biosensing Techniques/methods ; Spectrometry, Fluorescence/methods ; Limit of Detection ; Dendrimers/chemistry ; CRISPR-Associated Proteins/metabolism ; Fluorescence ; Nucleic Acid Amplification Techniques ; Humans ; Benzothiazoles ; },
abstract = {Although CRISPR/Cas12a systems significantly enhance the analytical accuracy and flexibility of fluorescent biosensors, their sensitivity is limited by traditional "one-to-one" mediation types and ineffective signal-output turnover routes. Herein, we demonstrate a "one-to-many" signal-output strategy-based CRISPR/Cas12a systems resembling a "seaweed" to enhance the sensitivity. Based on dendrimer DNA from high-dimensional hybridization chain (HCR) of three hairpin-free DNA building blocks, the 3D magnetic DNA machine was created. The HBV-DNA initiates the rolling circle amplification (RCA) reaction and produces DNA nanowires to activate the CRISPR/Cas12a system. The trans-cleavage of the "seaweed root" by CRISPR/Cas12a system left dendrimer DNA in solution, thus, adding SYBR Green I (SG I) to the high-density DNA duplexes, achieving multiple-turnover label-free fluorescence signal output demonstrated and a low LOD (1.502 pM). However, in the absence of target, the blocked RCA failed to activate the CRISPR/Cas12a system, resulting in complete separation from substrate and negligible fluorescence signals. Moreover, the mandatory RCA-based pre-amplification of the DNA activator could efficiently trigger the multiple-turnover trans-cleavage activity of Cas12a. it can cleave one single-stranded linker of "seaweed-like" DNA machine, thereby releasing massive DNA duplex-enriched dendrimer DNA with a "one-to-many" signal-output turnover. By coupling the periodically extended Cas12a activator generated by RCA with hyperbranched DNA duplex by high-dimensional HCR, compact 3D extension structures were formed, achieving high-density fluorescence distribution in focal volume, avoiding signal dilution and ensuring high enhancement. Additionally, spiked recoveries in physiological media exceeded 95%, demonstrating the potential application of such platforms in clinical diagnosis.},
}
@article {pmid37673694,
year = {2023},
author = {Awan, MJA and Awan, MRA and Amin, I and Mansoor, S},
title = {Fanzor: a compact programmable RNA-guided endonuclease from eukaryotes.},
journal = {Trends in biotechnology},
volume = {41},
number = {11},
pages = {1332-1334},
doi = {10.1016/j.tibtech.2023.08.003},
pmid = {37673694},
issn = {1879-3096},
mesh = {*Endonucleases/genetics/metabolism ; *Gene Editing/methods ; *Eukaryota/genetics/enzymology ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; DNA Transposable Elements/genetics ; },
abstract = {The IS200/605 transposons in prokaryotes are known to harbor programmable endonucleases. Despite carrying their own transposable elements, no such effector has been characterized in eukaryotes. Saito et al. recently reported compact and programmable RNA-guided eukaryotic endonucleases, called Fanzors, that can induce targeted genetic modifications, thus expanding the genome-editing toolbox.},
}
@article {pmid37667014,
year = {2023},
author = {Hooghvorst, I and Altpeter, F},
title = {dCas9-3xSRDX-mediated transcriptional repression in sugarcane.},
journal = {Plant cell reports},
volume = {42},
number = {11},
pages = {1837-1840},
pmid = {37667014},
issn = {1432-203X},
support = {DE-SC0018420//Biological and Environmental Research/ ; },
mesh = {*Saccharum/genetics/metabolism ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; 5' Untranslated Regions/genetics ; *Transcription, Genetic ; CRISPR-Cas Systems/genetics ; },
abstract = {Targeting dCas9 fused with the 3xSRDX effector to the 5'UTR leads to strong repression of magnesium chelatase in highly polyploid sugarcane.},
}
@article {pmid37666660,
year = {2023},
author = {Agostini, V and Tessier, A and Djaziri, N and Khonsari, RH and Galliani, E and Kurihara, Y and Honda, M and Kurihara, H and Hidaka, K and Tuncbilek, G and Picard, A and Konas, E and Amiel, J and Gordon, CT},
title = {Biallelic truncating variants in VGLL2 cause syngnathia in humans.},
journal = {Journal of medical genetics},
volume = {60},
number = {11},
pages = {1084-1091},
doi = {10.1136/jmg-2022-109059},
pmid = {37666660},
issn = {1468-6244},
mesh = {Humans ; Zebrafish/genetics ; Animals ; Male ; Female ; *Transcription Factors/genetics ; Alleles ; Pedigree ; Homozygote ; *Craniofacial Abnormalities/genetics/pathology ; *Jaw Abnormalities/genetics/pathology ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Syngnathia is an ultrarare craniofacial malformation characterised by an inability to open the mouth due to congenital fusion of the upper and lower jaws. The genetic causes of isolated bony syngnathia are unknown.<br><br>METHODS: We used whole exome and Sanger sequencing and microsatellite analysis in six patients (from four families) presenting with syngnathia. We used CRISPR/Cas9 genome editing to generate vgll2a and vgll4l germline mutant zebrafish, and performed craniofacial cartilage analysis in homozygous mutants.<br><br>RESULTS: We identified homozygous truncating variants in vestigial-like family member 2 (VGLL2) in all six patients. Two alleles were identified: one in families of Turkish origin and the other in families of Moroccan origin, suggesting a founder effect for each. A shared haplotype was confirmed for the Turkish patients. The VGLL family of genes encode cofactors of TEAD transcriptional regulators. Vgll2 is regionally expressed in the pharyngeal arches of model vertebrate embryos, and morpholino-based knockdown of vgll2a in zebrafish has been reported to cause defects in development of pharyngeal arch cartilages. However, we did not observe craniofacial anomalies in vgll2a or vgll4l homozygous mutant zebrafish nor in fish with double knockout of vgll2a and vgll4l. In Vgll2 [-/-] mice, which are known to present a skeletal muscle phenotype, we did not identify defects of the craniofacial skeleton.<br><br>CONCLUSION: Our results suggest that although loss of VGLL2 leads to a striking jaw phenotype in humans, other vertebrates may have the capacity to compensate for its absence during craniofacial development.},
}
@article {pmid37650631,
year = {2023},
author = {Mohammad, N and Talton, L and Hetzler, Z and Gongireddy, M and Wei, Q},
title = {Unidirectional trans-cleaving behavior of CRISPR-Cas12a unlocks for an ultrasensitive assay using hybrid DNA reporters containing a 3' toehold.},
journal = {Nucleic acids research},
volume = {51},
number = {18},
pages = {9894-9904},
pmid = {37650631},
issn = {1362-4962},
support = {1944167//National Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; *DNA/metabolism/chemistry/genetics ; DNA, Single-Stranded/metabolism/genetics ; DNA Cleavage ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {CRISPR-Cas12a can induce nonspecific trans-cleavage of dsDNA substrate, including long and stable λ DNA. However, the mechanism behind this is still largely undetermined. In this study, we observed that while trans-activated Cas12a didn't cleave blunt-end dsDNA within a short reaction time, it could degrade dsDNA reporters with a short overhang. More interestingly, we discovered that the location of the overhang also affected the susceptibility of dsDNA substrate to trans-activated Cas12a. Cas12a trans-cleaved 3' overhang dsDNA substrates at least 3 times faster than 5' overhang substrates. We attributed this unique preference of overhang location to the directional trans-cleavage behavior of Cas12a, which may be governed by RuvC and Nuc domains. Utilizing this new finding, we designed a new hybrid DNA reporter as nonoptical substrate for the CRISPR-Cas12a detection platform, which sensitively detected ssDNA targets at sub picomolar level. This study not only unfolded new insight into the trans-cleavage behavior of Cas12a but also demonstrated a sensitive CRISPR-Cas12a assay by using a hybrid dsDNA reporter molecule.},
}
@article {pmid37642940,
year = {2023},
author = {Schramm, T and Lubrano, P and Pahl, V and Stadelmann, A and Verhülsdonk, A and Link, H},
title = {Mapping temperature-sensitive mutations at a genome scale to engineer growth switches in Escherichia coli.},
journal = {Molecular systems biology},
volume = {19},
number = {10},
pages = {e11596},
pmid = {37642940},
issn = {1744-4292},
support = {715650//European Research Council (ERC)/ ; },
mesh = {*Escherichia coli/genetics/growth &amp; development/metabolism ; *Mutation ; Temperature ; Escherichia coli Proteins/genetics/metabolism ; Genome, Bacterial ; Metabolomics/methods ; CRISPR-Cas Systems ; Metabolome ; DNA Polymerase III/genetics/metabolism ; Metabolic Engineering/methods ; },
abstract = {Temperature-sensitive (TS) mutants are a unique tool to perturb and engineer cellular systems. Here, we constructed a CRISPR library with 15,120 Escherichia coli mutants, each with a single amino acid change in one of 346 essential proteins. 1,269 of these mutants showed temperature-sensitive growth in a time-resolved competition assay. We reconstructed 94 TS mutants and measured their metabolism under growth arrest at 42°C using metabolomics. Metabolome changes were strong and mutant-specific, showing that metabolism of nongrowing E. coli is perturbation-dependent. For example, 24 TS mutants of metabolic enzymes overproduced the direct substrate metabolite due to a bottleneck in their associated pathway. A strain with TS homoserine kinase (ThrB[F267D]) produced homoserine for 24 h, and production was tunable by temperature. Finally, we used a TS subunit of DNA polymerase III (DnaX[L289Q]) to decouple growth from arginine overproduction in engineered E. coli. These results provide a strategy to identify TS mutants en masse and demonstrate their large potential to produce bacterial metabolites with nongrowing cells.},
}
@article {pmid37642675,
year = {2023},
author = {Guo, WF and Guo, DD and Li, F and Shang, SZ and Li, TW and Tang, YC and Jiang, M and Xu, FC and Gao, W},
title = {Efficient genome editing in cotton using the virus-mediated CRISPR/Cas9 and grafting system.},
journal = {Plant cell reports},
volume = {42},
number = {11},
pages = {1833-1836},
pmid = {37642675},
issn = {1432-203X},
support = {202203021222310//Shanxi Basic Research Program/ ; },
mesh = {*Gossypium/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; *Genome, Plant/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Plant Viruses/genetics ; },
abstract = {The extensive application of CRISPR in cotton was limited due to the labor-intensive transformation process. Thus, we here established a convenient method of CRISPR in cotton by CLCrV-mediated sgRNA delivery.},
}
@article {pmid37634280,
year = {2023},
author = {Han, X and Abdallah, MOE and Breuer, P and Stahl, F and Bakhit, Y and Potthoff, AL and Pregler, BEF and Schneider, M and Waha, A and Wüllner, U and Evert, BO},
title = {Downregulation of MGMT expression by targeted editing of DNA methylation enhances temozolomide sensitivity in glioblastoma.},
journal = {Neoplasia (New York, N.Y.)},
volume = {44},
number = {},
pages = {100929},
pmid = {37634280},
issn = {1476-5586},
mesh = {Humans ; *Glioblastoma/genetics/drug therapy/pathology ; *Temozolomide/pharmacology ; *DNA Methylation ; *DNA Modification Methylases/genetics/metabolism ; *Tumor Suppressor Proteins/genetics/metabolism ; *DNA Repair Enzymes/genetics/metabolism ; Drug Resistance, Neoplasm/genetics ; Cell Line, Tumor ; Promoter Regions, Genetic ; Gene Expression Regulation, Neoplastic/drug effects ; *Gene Editing/methods ; Antineoplastic Agents, Alkylating/pharmacology ; Down-Regulation ; *Brain Neoplasms/genetics/drug therapy ; CRISPR-Cas Systems ; },
abstract = {Glioblastoma is the most common and aggressive primary tumor of the central nervous system with poor outcome. Current gold standard treatment is surgical resection followed by a combination of radio- and chemotherapy. Efficacy of temozolomide (TMZ), the primary chemotherapeutic agent, depends on the DNA methylation status of the O6-methylguanine DNA methyltransferase (MGMT), which has been identified as a prognostic biomarker in glioblastoma patients. Clinical studies revealed that glioblastoma patients with hypermethylated MGMT promoter have a better response to TMZ treatment and a significantly improved overall survival. In this study, we thus used the CRISPRoff genome editing tool to mediate targeted DNA methylation within the MGMT promoter region. The system carrying a CRISPR-deactivated Cas9 (dCas9) fused with a methyltransferase (Dnmt3A/3L) domain downregulated MGMT expression in TMZ-resistant human glioblastoma cell lines through targeted DNA methylation. The reduction of MGMT expression levels reversed TMZ resistance in TMZ-resistant glioblastoma cell lines resulting in TMZ induced dose-dependent cell death rates. In conclusion, we demonstrate targeted RNA-guided methylation of the MGMT promoter as a promising tool to overcome chemoresistance and improve the cytotoxic effect of TMZ in glioblastoma.},
}
@article {pmid37625131,
year = {2023},
author = {Ueki, K and Nishida, Y and Aoyama, S and Uzawa, H and Kanai, A and Ito, M and Ikeda, K and Iida, H and Miyatsuka, T and Watada, H},
title = {Establishment of Pancreatic β-Cell-Specific Gene Knockout System Based on CRISPR-Cas9 Technology With AAV8-Mediated gRNA Delivery.},
journal = {Diabetes},
volume = {72},
number = {11},
pages = {1609-1620},
doi = {10.2337/db23-0445},
pmid = {37625131},
issn = {1939-327X},
support = {20H03735//Japan Society for the Promotion of Science/ ; },
mesh = {Animals ; *Insulin-Secreting Cells/metabolism ; *CRISPR-Cas Systems/genetics ; *Dependovirus/genetics ; Mice ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Homeodomain Proteins/genetics ; Trans-Activators/genetics ; Mice, Transgenic ; Mice, Knockout ; },
abstract = {The Cre-loxP system provides valuable resources to analyze the importance of tissue-specific gene knockout (KO), including pancreatic β-cells associated with the pathogenesis of diabetes. However, it is expensive and time consuming to generate transgenic mice harboring floxed genes of interest and cross them with cell-specific Cre expression mice. We establish a βCas9 system with mice expressing Cas9 in pancreatic β-cells and adeno-associated virus 8 (AAV8)-mediated guide RNA (gRNA) delivery based on CRISPR-Cas9 technology to overcome those shortcomings. Interbreeding CAG-loxP-STOP-loxP (LSL)-Cas9 with Ins1-Cre mice generates normal glucose-tolerant βCas9 mice expressing Cas9 with fluorescent reporter EGFP specifically in β-cells. We also show significant β-cell-specific gene KO efficiency with AAV8-mediated delivery of gRNA for EGFP reporter by intraperitoneal injection in the mice. As a proof of concept, we administered AAV8 to βCas9 mice for expressing gRNA for Pdx1, a culprit gene of maturity-onset diabetes of the young 4. As reported previously, we demonstrate that those mice show glucose intolerance with transdifferentiation of Pdx1 KO β-cells into glucagon-expressing cells. We successfully generated a convenient β-cell-specific gene KO system with βCas9 mice and AAV8-mediated gRNA delivery.},
}
@article {pmid37517884,
year = {2023},
author = {Ahmar, S and Gruszka, D},
title = {CRISPR/Cas9 boosts wheat yield by reducing brassinosteroid signaling.},
journal = {Trends in biochemical sciences},
volume = {48},
number = {11},
pages = {917-919},
doi = {10.1016/j.tibs.2023.07.005},
pmid = {37517884},
issn = {0968-0004},
mesh = {*Brassinosteroids/metabolism ; *CRISPR-Cas Systems/genetics ; *Triticum/genetics/metabolism/growth &amp; development ; *Signal Transduction ; },
abstract = {A modern green revolution is needed to ensure global food security. Recently, Song et al. reported a new strategy to create high-yielding, semi-dwarf wheat varieties with improved nitrogen-use efficiency by inhibiting brassinosteroid (BR) signaling through clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9)-mediated knockout of the ZnF-B gene encoding a zinc-finger RING-type E3 ligase.},
}
@article {pmid37463293,
year = {2023},
author = {Deng, W and Feng, S and Stejskal, V and Opit, G and Li, Z},
title = {An advanced approach for rapid visual identification of Liposcelis bostrychophila (Psocoptera: Liposcelididae) based on CRISPR/Cas12a combined with RPA.},
journal = {Journal of economic entomology},
volume = {116},
number = {5},
pages = {1911-1921},
doi = {10.1093/jee/toad139},
pmid = {37463293},
issn = {1938-291X},
mesh = {Animals ; *Insecta/genetics/classification ; *CRISPR-Cas Systems ; Insect Proteins/genetics ; NADH Dehydrogenase/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Liposcelis bostrychophila Badonnel (Psocoptera: Liposcelididae) is a booklouse pest that is a threat to commodity storage security worldwide. Accurate and sensitive methods of L. bostrychophila on-site identification are essential prerequisites for its effective management. Evidence suggests that L. bostrychophila contains 3 intraspecific biotypes that are morphologically indistinguishable but can be discriminated at the level of mitochondrial genome organization and sequences. The traditional molecular identification methods, such as DNA barcoding and PCR-RFLP, are instrumentally demanding and time-consuming, limiting the application of the identification in the field. Therefore, this study developed a new CRISPR/Cas12a-based visual nucleic acid system based on the mitochondrial gene coding for NADH dehydrogenase subunit 2 (nad2), combined with recombinase polymerase amplification (RPA) to accurately identify L. bostrychophila from 4 other common stored-product booklice, and also differentiate 3 biotypes of this species at the same time. The entire identification process could be completed at 37 °C within 20 min with high sensitivity. The system could stably detect at least 1 ng/μl of DNA template. The green fluorescence signal produced by the trans-cleaving of the single-stranded DNA reporter could be observed by the naked eye under blue light. Additionally, the suggested system combined with the crude DNA extraction method to extract DNA rapidly, enabled identification of all developmental stages of L. bostrychophila. With crude DNA, this novel diagnostic system successfully identified an unknown booklouse by holding the reaction tubes in the hand, thus can be considered as an accurate, rapid, highly sensitive, and instrument-flexible method for on-site visual identification of L. bostrychophila.},
}
@article {pmid37258389,
year = {2023},
author = {Zaman, QU and Raza, A and Gill, RA and Hussain, MA and Wang, HF and Varshney, RK},
title = {New possibilities for trait improvement via mobile CRISPR-RNA.},
journal = {Trends in biotechnology},
volume = {41},
number = {11},
pages = {1335-1338},
doi = {10.1016/j.tibtech.2023.05.001},
pmid = {37258389},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR/Cas9 gene technology is transported as RNA from transgenic roots to distal parts of unmodified grafted scion, where it is translated into proteins to induce heritable mutagenesis at desired loci. This technique has the potential to produce transgene-free and genetically stable plants in difficult-to-propagate and near-extinct species.},
}
@article {pmid41785636,
year = {2026},
author = {Xue, S and Sun, H and Hou, X and Li, N and Xue, L and Dai, E and Wan, J},
title = {An off-target exonuclease activity in AsCpf1 undermines CRISPR diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {303},
number = {},
pages = {118578},
doi = {10.1016/j.bios.2026.118578},
pmid = {41785636},
issn = {1873-4235},
abstract = {The extensive utilization of CRISPR-Cas systems in molecular diagnostics stems from their crRNA-guided trans-cleavage capabilities. However, AsCpf1-based detection systems frequently exhibit unexplained sensitive variations. This research reveals that AsCpf1 maintains a crRNA-independent function, similar to exonuclease I, when utilized in standard buffers containing Mg[2+]. From a structural perspective, this exonuclease activity is independent of the RuvC domain-mediated canonical trans-cleavage activity. It is predicted by structural modeling to be potentially localized within the WED-PI domain. In the context of diagnostics, the effective target concentration is diminished by AsCpf1-mediated degradation of the free 3' ends of target DNA, which impairs detection sensitivity. To mitigate this interference, we demonstrate that 3' end capping effectively restores detection performance. This approach was validated in a CRISPR-EXPAR-based microRNA biosensor, which exhibited approximately 10-fold improvement in sensitivity following 3' end capping. Overall, this investigation characterizes a previously unidentified exonuclease activity within the AsCpf1 system and establishes practical design criteria to improve the robustness and accuracy of CRISPR-based diagnostic tools.},
}
@article {pmid41785320,
year = {2026},
author = {Zhang, X and Richart, D and McFarlin, S and Cheng, F and Park, SY and Zhang-Chen, A and McFarlane, R and Xiao, C and Yan, Z and Qiu, J},
title = {Identification of CD164 as an essential entry receptor for divergent adeno-associated viruses.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2525865123},
doi = {10.1073/pnas.2525865123},
pmid = {41785320},
issn = {1091-6490},
support = {AI166293//HHS | National Institutes of Health (NIH)/ ; AI180416//HHS | National Institutes of Health (NIH)/ ; HL174593//HHS | National Institutes of Health (NIH)/ ; AI182645//HHS | National Institutes of Health (NIH)/ ; HL174593//HHS | National Institutes of Health (NIH)/ ; AI182645//HHS | National Institutes of Health (NIH)/ ; GM129525//HHS | National Institutes of Health (NIH)/ ; YAN23G0//Cystic Fibrosis Foundation (CFF)/ ; AH-2126-20220331//Welch Foundation (The Welch Foundation)/ ; },
mesh = {*Dependovirus/genetics/physiology/metabolism ; Animals ; Mice ; Humans ; Mice, Knockout ; *Virus Internalization ; *Receptors, Cell Surface/metabolism/genetics ; HEK293 Cells ; *Antigens, CD/metabolism/genetics ; Genetic Vectors/genetics ; Transduction, Genetic ; CRISPR-Cas Systems ; *Receptors, Virus/metabolism/genetics ; Capsid/metabolism ; },
abstract = {Recombinant adeno-associated viruses (rAAVs) are widely used for in vivo gene delivery. While KIAA0319L, known as AAV receptor (AAVR), is essential for the transduction of multiserotype AAVs, it is dispensable for AAV4-related (Clade G) AAVs. We conducted a genome-wide CRISPR/Cas9 screen and identified CD164, a type I transmembrane sialomucin, as an essential entry receptor for Clade G AAVs. Ablation of CD164 expression substantially impaired both entry and transduction of Clade G AAVs. CD164-targeting antibodies and soluble CD164 ectodomain effectively blocked transduction. AAV4 capsids colocalized with CD164 at the plasma membrane and in endosomal compartments. In vitro, CD164 interacted with AAV4 or AAVrh32.33 capsids at high affinity. Importantly, systemic administration of rAAV4 or rAAVrh32.33 in CD164 knockout (KO) mice resulted in nearly complete loss of transgene expression. These findings establish CD164 as an essential entry receptor for Clade G AAV vectors and uncover a distinct AAVR-independent mechanism of AAV tropism.},
}
@article {pmid41785318,
year = {2026},
author = {Weber, LI and Timpen, LE and Egger-Hörschinger, AS and Schöpf, P and Ayhan, ND and Demmel, D and Hotze, M and Zhang, Y and Mehrabi, M and Puglisi, K and Stefan, E and Ghaffari-Tabrizi-Wizsy, N and Ramos-Pittol, JM and Kwiatkowski, M and Hartl, M},
title = {Reactivation of the silenced BASP1 gene suppresses oncogenic WNT signaling in human colorectal cancer cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2524159123},
doi = {10.1073/pnas.2524159123},
pmid = {41785318},
issn = {1091-6490},
support = {P33662//Austrian Science Fund (FWF)/ ; },
mesh = {Humans ; *Wnt Signaling Pathway/genetics ; *Colorectal Neoplasms/genetics/metabolism/pathology ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; *Nerve Tissue Proteins/genetics/metabolism ; *Membrane Proteins/genetics/metabolism ; beta Catenin/metabolism/genetics ; Promoter Regions, Genetic ; Gene Silencing ; Cell Movement ; },
abstract = {Starting from human colon cancer cells showing aberrant WNT/β-catenin/TCF signaling, hyperactivated MYC, and silenced BASP1, we generated stable cell lines overexpressing BASP1, either ectopically, or by reactivating the dormant BASP1 promoter using a lentiviral CRISPR-based system. BASP1 encodes a neuronal signaling protein and transcriptional corepressor, from which tumor-suppressive functions have been described in avian cell systems and in multiple human cancer cell types. Proteome and transcriptome analyses revealed activation of several tumor and metastasis suppressors in BASP1-expressing cells, which also show strong repression of the transformed phenotype in terms of contact inhibition, anchorage-independent growth, and tumor formation. Cells with reactivated BASP1 display a flat and differentiated morphology with enhanced migratory potential, accompanied by expression of multiple genes implicated in actin polymerization, focal adhesion, and neuronal migration. Furthermore, MYC protein expression is substantially repressed due to BASP1-mediated transcriptional MYC downregulation involving BASP1 interaction with β-catenin and binding to the MYC promoter. Upon BASP1 activation, multiple key proteins of the canonical WNT signaling pathway become suppressed. One of these BASP1 targets is the protein kinase TNIK catalyzing phosphorylation of TCF7L2, the latter required for transcriptional MYC activation. Results obtained with a preclinical TNIK inhibitor in human colorectal cancer cells show efficient abrogation of MYC expression and consequently impaired dimerization with its interaction partner MAX. The antagonistic BASP1 effect on MYC and the MYC dependency on TNIK could enhance the development of strategies to interfere with oncogenic functions of the cancer driver MYC.},
}
@article {pmid41784822,
year = {2026},
author = {Eweda, MA and Li, J and Hassan, U and Jalil, S and Jin, X},
title = {Peroxisomal acyl-CoA oxidase OsACX4 negatively regulates salt and drought stress tolerance by modulating cellular redox homeostasis in rice.},
journal = {Plant cell reports},
volume = {45},
number = {3},
pages = {},
pmid = {41784822},
issn = {1432-203X},
support = {517000-N12502ZJ//National Natural Science Foundation of China/ ; 2025ZDXT01-4//The Major Agricultural Technology Collaborative Extension Project/ ; LZ25C130006//Natural Science Foundation of Zhejiang Province/ ; 2021C02063-6//Science and Technology Department of Zhejiang Province/ ; },
mesh = {*Oryza/genetics/physiology/enzymology/metabolism ; Homeostasis ; Oxidation-Reduction ; *Plant Proteins/metabolism/genetics ; Droughts ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; Peroxisomes/metabolism/enzymology ; *Acyl-CoA Oxidase/metabolism/genetics ; Reactive Oxygen Species/metabolism ; Stress, Physiological/genetics ; *Salt Tolerance/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Oxidative Stress ; },
abstract = {OsACX4 knockout reduces peroxisomal oxidative stress, enhancing rice drought and salt tolerance through metabolic-redox rebalancing for climate-resilient breeding. Climate change is intensifying the frequency and severity of abiotic stress, such as salt and drought stresses, which severely limit rice productivity worldwide, necessitating the identification of molecular targets for crop improvement. This study provides the first comprehensive functional characterization of the peroxisomal acyl-CoA oxidase OsACX4 in rice (Oryza sativa L.) drought and salinity tolerance, revealing its unexpected role as a negative regulator of stress tolerance through modulation of cellular redox homeostasis. Through genome editing using CRISPR/Cas9-mediated knockout and overexpression approaches, we generated transgenic lines to investigate the function of OsACX4 under salt and drought stress. Knockout lines exhibited superior stress tolerance compared to the wild-type (WT) and overexpression lines, demonstrating significantly higher survival rates under severe stress conditions. Enhanced tolerance correlated with coordinated upregulation of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities. Knockout lines accumulated substantially higher proline (Pro) levels while maintaining markedly reduced reactive oxygen species (ROS) compared to overexpression lines. Transcriptional analysis revealed that OsACX4 disruption triggered upregulation of stress-responsive genes, including OsSOD1, OsDREB2A, OsDREB1B, and OsAPX1 under severe stress. Subcellular localization confirmed peroxisomal targeting of OsACX4, where its β-oxidation activity generates hydrogen peroxide (H2O2) as a metabolic by-product. Our results reveal a metabolic trade-off whereby stress-induced OsACX4 expression mobilizes energy reserves but compromises cellular redox homeostasis. The superior performance of knockout lines under both stresses demonstrates that strategic OsACX4 disruption enhances plant resilience, identifying this gene as a promising breeding target for developing climate-resilient rice varieties through precision genome editing.},
}
@article {pmid41784340,
year = {2026},
author = {Zhou, C and Cheng, T and Zhou, J and Zhang, B and Liu, L and Jiang, G and Li, W and Wang, C},
title = {Synthetic Biofilms for Green Membranes: Engineering Low-Energy Filtration Systems.},
journal = {Environmental science &amp; technology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.est.5c15661},
pmid = {41784340},
issn = {1520-5851},
abstract = {Membrane filtration is a key technology to modern water purification, yet its sustainability is compromised by biofouling, which increases energy consumption and ecological impacts. Conventional control strategies often struggle to balance efficacy and environmental footprint. In this study, an inducible, engineered quorum-quenching (QQ) bacterium was constructed via a genomic integration strategy, thereby achieving control over the biofilm structure in membrane filtration biofouling layers. By using the clustered regularly interspaced short palindromic repeat (CRISPR-Cas) targeted gene editing technology, the engineered bacteria that were constructed to express aiiO under l-ribose induction have achieved the regulation of biofilms. Validation using a gravity-driven membrane ultrafiltration system (UF-GDM) model showed that the engineered bacterium effectively reduced extracellular polymeric substances (EPS) components, increased the hydrophilic porosity of the residual biofilm, and decreased its stickiness. This approach reduced transmembrane pressure by 64.5%, increased total organic carbon (TOC) removal by 13.2%, and extended membrane lifespan by 16.1%. A technical-economic analysis indicates that the 100,000 m[3]/day treatment plant achieves an annual net profit increase of 31.52%, reaching 1.55 × 10[7] CNY, while reducing its net carbon footprint by 27.43%, with an annual net reduction of 2.96 × 10[5] kg CO2eq. This study provides a novel solution strategy for achieving biofouling resistance and sustainable, low-energy operation in membrane filtration processes, which contributes to the broader application and adoption of this technology.},
}
@article {pmid41784267,
year = {2026},
author = {Wang, Z and Wu, Y and Wang, Z and Zhang, S and Liu, H and Nie, Y and Chen, K and Huang, Y and Zhou, Y and Cao, Y and Sun, L and Hao, R},
title = {crRNA scaffold remodeling controls CRISPR-Cas12a activity for enhanced performance.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41784267},
issn = {1362-4962},
support = {//Prevention and Control of Emerging/ ; 2025ZD01903403//National Science and Technology Major Project/ ; L234051//Beijing Municipal Natural Science Foundation/ ; L246011//Beijing Municipal Natural Science Foundation/ ; 2024-03-18//Training Plan for High-Level Public Health Technical Talents of Beijing Municipal Health Commission/ ; 2025ZD01903403//Prevention and Control of Emerging and Major Infectious Diseases-National Science/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Mycobacterium tuberculosis/genetics/isolation &amp; purification ; Klebsiella pneumoniae/genetics/isolation &amp; purification ; Nucleic Acid Conformation ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {CRISPR-Cas12a has transformative potential in molecular diagnostics owing to its robust signal amplification, but its sustained activity state severely limits temporal programmability and precise nuclease control in complex detection workflows. Here, we demonstrate that the conserved crRNA scaffold secondary structure itself can be repurposed as a reversible and programmable conformational switch to regulate Cas12a activity. By introducing short complementary DNA blockers of tunable length, we achieved length-dependent disruption and remodeling of scaffold secondary structure, shifting LbCas12a into an inactive conformation. Scaffold structure was subsequently reinstated through either single or cooperative strand displacement activation, enabling time-resolved and on-demand restoration of Cas12a activity. The conserved scaffold ensures intrinsic assay universality, while its programmable rewiring markedly improves SNVs discrimination and enables compatibility with one-pot isothermal amplification assays, delivering analytical sensitivity comparable to conventional two-step assays. This regulatory framework was further demonstrated in the detection of Klebsiella pneumoniae and Mycobacterium tuberculosis. By validating the crRNA scaffold as a practical and programmable switch for Cas12a activity control, this work establishes a universal and reversible framework for scaffold rewiring to modulate CRISPR nucleases and offers mechanistic insight to guide future assay engineering.},
}
@article {pmid41783940,
year = {2026},
author = {Berti, M and Ceriotti, S and Santi, L and Alberti, G and Beretta, S and Degl'Innocenti, S and Ruatti, C and Savoia, EO and Jofra-Hernandez, R and De Ponti, G and Bolamperti, S and Villa, I and Galeotti, F and Romano, A and Visigalli, I and Norata, R and Rocchi, M and Cristofori, P and Cossutta, M and Consiglieri, G and Tucci, F and Santorelli, L and Grumati, P and Ronfani, L and D'Adamo, P and Giustina, A and Angelozzi, M and Settembre, C and Mortellaro, A and Scala, S and Sanvito, F and Volpi, N and Aiuti, A and Bernardo, ME and Crippa, S},
title = {Development and characterization of a model of mucopolysaccharidosis type IVA for evaluating therapies targeting bone disease.},
journal = {Disease models &amp; mechanisms},
volume = {19},
number = {2},
pages = {},
doi = {10.1242/dmm.052540},
pmid = {41783940},
issn = {1754-8411},
support = {20228H9T82//Ministero dell'Istruzione, dell'Universit&#xe0; e della Ricerca/ ; P20223MF7X_001//Ministero dell'Istruzione, dell'Universit&#xe0; e della Ricerca/ ; CN_00000041 - CUP G83C22000270001//NextGenerationEU/ ; TTAGTXEKFA//Else Kr&#xf6;ner-Fresenius-Zentrum f&#xfc;r Ern&#xe4;hrungsmedizin/ ; TELE-MB//Fondazione Telethon/ ; TELE-AA//Fondazione Telethon/ ; //Ospedale San Raffaele/ ; },
mesh = {Animals ; Disease Models, Animal ; *Mucopolysaccharidosis IV/therapy/pathology/blood/complications ; *Bone Diseases/therapy/pathology/complications ; Chondroitinsulfatases/metabolism/deficiency ; Glycosaminoglycans/metabolism ; Keratan Sulfate/metabolism/urine ; Chondroitin Sulfates/metabolism/urine ; Mice ; Humans ; Genetic Therapy ; Mice, Knockout ; Bone and Bones/pathology ; CRISPR-Cas Systems/genetics ; },
abstract = {Mucopolysaccharidosis type IVA (MPSIVA) is a lysosomal storage disease (LSD) caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), which causes the accumulation of keratan sulphate (KS) and chondroitin sulphate (CS). Patients with MPSIVA typically present with severe skeletal and joint disorders, which are not addressed by conventional therapies. Currently, no animal model accurately replicates the human disease, hindering the development of novel therapeutic interventions. To overcome this limitation, we established, by CRISPR-Cas9 technology, a Galns-/- mouse model that expresses a non-functional enzyme and accumulates CS and KS in the urine, plasma and distinct tissues, and glycosaminoglycans in the spleen. The mice exhibit shortened long bones, trabecular bone alterations and skeletal abnormalities in the growth plate. Additionally, we observed increased levels of inflammatory and oxidative markers in visceral organs and plasma. Our newly developed model of MPSIVA demonstrates clear and quantifiable signs of skeletal alterations, providing novel means of assessment of the safety and efficacy of innovative therapies, including hematopoietic stem and progenitor cell gene therapy, which has recently been shown to provide a beneficial effect on skeletal alterations in Hurler syndrome.},
}
@article {pmid41781627,
year = {2026},
author = {Hoffmann, FT and Wiegand, T and Palmieri, AI and Glass-Klaiber, J and Xiao, R and Tang, S and Le, HC and Meers, C and Lampe, GD and Chang, L and Sternberg, SH},
title = {Exapted CRISPR-Cas12f homologues drive RNA-guided transcription.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41781627},
issn = {1476-4687},
abstract = {Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP)[1]. Although the seven σ factors in Escherichia coli have been extensively characterized[2], Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ[E]) whose precise roles are unknown, pointing to additional layers of regulatory potential[3]. Here we uncover a mechanism of RNA-guided gene activation involving the coordinated action of σ[E] factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically linked dCas12f and σ[E] homologues in E. coli using RNA and chromatin immunoprecipitation experiments, revealing systems that exhibit robust guide RNA enrichment and DNA target binding with a minimal 5'-G target-adjacent motif. Recruitment of σ[E] was dependent on dCas12f and guide RNA, suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σ[E] ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σ[E] complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription that embodies natural features reminiscent of CRISPR activation (CRISPRa) technology[4,5].},
}
@article {pmid41781609,
year = {2026},
author = {Xiao, R and Hoffmann, FT and Xie, D and Wiegand, T and Palmieri, AI and Sternberg, SH and Chang, L},
title = {Structural basis of RNA-guided transcription by a dCas12f-σ[E]-RNAP complex.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41781609},
issn = {1476-4687},
abstract = {In both natural and engineered biological systems, RNA-guided proteins have emerged as critical transcriptional regulators by modulating RNA polymerase (RNAP) and its associated factors[1-3]. In bacteria, diverse clades of repurposed TnpB and CRISPR-associated proteins repress gene expression by blocking transcription initiation or elongation, enabling non-canonical modes of regulatory control and adaptive immunity[1,4,5]. A distinct class of nuclease-dead Cas12f homologues (dCas12f) instead activates gene expression through its association with unique extracytoplasmic function sigma factors (σ[E])[6], although the molecular basis has remained elusive. Here we reveal a new mode of RNA-guided transcription initiation by determining the cryo-electron microscopy structures of the dCas12f-σ[E] system from Flagellimonas taeanensis. We captured multiple conformational and compositional states, including the DNA-bound dCas12f-σ[E]-RNAP holoenzyme complex, revealing how RNA-guided DNA binding leads to σ[E]-RNAP recruitment and nascent mRNA synthesis at a precisely defined distance downstream of the R-loop. Rather than following the classical paradigm of σ[E]-dependent promoter recognition, these studies show that recognition of the -35 element is largely supplanted by CRISPR-Cas targeting, whereas the melted -10 element is stabilized through unusual stacking interactions rather than insertion into the typical recognition pocket. Collectively, this work provides high-resolution insights into an unexpected mechanism of RNA-guided transcription, expanding our understanding of bacterial gene regulation and opening new avenues for programmable transcriptional control.},
}
@article {pmid41747766,
year = {2026},
author = {Cai, Y and Zhuang, L and Wang, Z and He, L and Li, X and Liu, BF and Li, T and Zhang, G and Zhou, H and Huang, X and Li, Y},
title = {Gravity-Driven Formation of Water-in-Wax Spheres for Efficient One-Pot CRISPR Diagnostics.},
journal = {ACS nano},
volume = {20},
number = {9},
pages = {8055-8067},
doi = {10.1021/acsnano.6c01232},
pmid = {41747766},
issn = {1936-086X},
mesh = {*Gravitation ; *Water/chemistry ; Humans ; *Waxes/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Microspheres ; CRISPR-Cas Systems ; Human papillomavirus 16/genetics/isolation &amp; purification ; },
abstract = {Rapid, decentralized molecular diagnostics are urgently needed for effective infectious disease control. Here, we present "Wax-Sphere CRISPR" (WS-CRISPR), a paradigm-shifting platform for CRISPR-based diagnostics centered on a gravity-driven, interfacial phase-change self-encapsulation mechanism. This system fundamentally decouples bioreagent engineering from specific reaction vessels, transforming conventional, labor-intensive manual encapsulation into a standardized, physics-driven assembly process that generates discrete wax microspheres. Guided by fluid mechanics and interfacial thermodynamics, aqueous CRISPR droplets spontaneously traverse air/molten wax/ethanol to self-encapsulate and solidify, enabling standardized, high-throughput fabrication without manual wax handling. Upon temperature modulation, the wax phase change triggers sequential recombinase polymerase amplification (RPA) and CRISPR detection within a sealed, one-pot vessel. As a clinically oriented demonstration, WS-CRISPR enables multiplexed detection and risk stratification of all 14 high-risk HPV genotypes (HPV16/18 vs others) with a detection limit of 1 × 10[-18] M in under 30 min. Integrated with widely accessible devices─including a thermocycler, hand-held fluorescence reader, and microfluidic platform─the system demonstrated 97.4% sensitivity and 100% specificity across 70 clinical samples. By solving the engineering bottlenecks of scalability and universality, WS-CRISPR offers a robust tool for decentralized, large-scale pathogen surveillance.},
}
@article {pmid41742365,
year = {2026},
author = {Yao, W and Li, Y and Du, Y and Gao, R and Tan, J and Fan, J and Liu, Y},
title = {Aptamer-Mediated Protein Corona In Situ Multiplex Microfluidic Detection for Cancer Diagnosis.},
journal = {Analytical chemistry},
volume = {98},
number = {9},
pages = {7123-7135},
doi = {10.1021/acs.analchem.6c00170},
pmid = {41742365},
issn = {1520-6882},
mesh = {Humans ; *Aptamers, Nucleotide/chemistry ; *Neoplasms/diagnosis/blood ; *Protein Corona/chemistry ; *Microfluidic Analytical Techniques ; *Biomarkers, Tumor/blood ; *Lab-On-A-Chip Devices ; CRISPR-Cas Systems ; Silicon Dioxide/chemistry ; },
abstract = {Liquid biopsy faces critical limitations in detecting low-abundance protein biomarkers due to serum complexity, single-target constraints, and insufficient sensitivity. Here, we present an integrated microfluidic protein aptamer corona-CRISPR (PACC) platform that synergizes nanoparticle protein corona (PC) enrichment, multiplexed aptamer recognition, and CRISPR-Cas12a-driven signal generation for facile and rapid point-of-care testing. Fe3O4@SiO2 nanoparticles capable of streamlining PC isolation via magnetic separation were employed to concentrate cancer-associated proteins from serum. A 6-plex aptamer panel, which selectively bound target proteins via forming protein-aptamer coronas (PACs), was used to initiate the in situ activation of CRISPR-Cas12a via complementary crRNAs for fluorescence signal generation. This entire workflow was miniaturized into a 3D-printed microfluidic chip and portable reader, achieving a fast sample-to-answer process. Clinical validation with 112 serum specimens demonstrated excellent diagnostic performance, achieving an area under the curve (AUC) of 0.95 and 88.24% accuracy. By integrating these modalities, the PACC platform overcomes serum matrix interference and biomarker scarcity, providing a scalable and low-cost avenue for detecting low-abundance protein biomarkers and advancing liquid biopsy toward precision oncology.},
}
@article {pmid41565063,
year = {2026},
author = {Wu, Q and Du, F and Zhang, X and Lu, Z and Zheng, X and Li, A and Zhang, X and Zhang, R and Wang, Q},
title = {Development and evaluation of a one-pot CRISPR/Cas13-based assay for syphilis detection across multiple clinical sample types.},
journal = {International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases},
volume = {164},
number = {},
pages = {108414},
doi = {10.1016/j.ijid.2026.108414},
pmid = {41565063},
issn = {1878-3511},
mesh = {Humans ; Sensitivity and Specificity ; *Treponema pallidum/genetics/isolation &amp; purification ; *Syphilis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; },
abstract = {OBJECTIVES: To develop and evaluate a rapid, one-pot molecular assay for the detection of Treponema pallidum subspecies pallidum (TPA), addressing the limitations of current diagnostic methods influenced by sample type and pathogen load.<br><br>METHODS: A one-pot assay integrating recombinase polymerase amplification (RPA) and Cas13a-based collateral cleavage activity was established for isothermal detection of TPA. The assay targeted the tpp47/tp0574 gene and was validated using 186 clinical specimens, including whole blood, lesion exudate, and cerebrospinal fluid (CSF) samples.<br><br>RESULTS: The one-pot RPA-Cas13a assay demonstrated high analytical sensitivity and specificity for TPA detection. Clinical sensitivities were 58.97% in whole blood, 84.21% in lesion exudate, and 57.14% in CSF, with 100% specificity across all sample types.<br><br>CONCLUSION: This one-pot isothermal assay enables rapid and accurate detection of T. pallidum directly from diverse clinical samples. Its high specificity and field-friendly format make it a promising complementary tool to conventional diagnostic approaches, particularly for point-of-care testing and screening in resource-limited or high-risk settings.},
}
@article {pmid41491239,
year = {2026},
author = {Amelan, A and Collins, SC and Damseh, NS and Hamada, N and Salim, A and Dvir, E and Monderer-Rothkoff, G and Harel, T and Nagata, KI and Yalcin, B and Shifman, S},
title = {CRISPR knockout screens reveal genes and pathways essential for neuronal differentiation and implicate PEDS1 in neurodevelopment.},
journal = {Nature neuroscience},
volume = {29},
number = {3},
pages = {592-603},
pmid = {41491239},
issn = {1546-1726},
support = {466/21//Israel Science Foundation (ISF)/ ; 1863/24//Israel Science Foundation (ISF)/ ; ANR-18-CE12-0009//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {Animals ; Mice ; *Cell Differentiation/genetics ; *Neurons/physiology ; *Neurogenesis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; *Neurodevelopmental Disorders/genetics ; Gene Knockout Techniques/methods ; Mice, Knockout ; CRISPR-Cas Systems ; Male ; },
abstract = {Neurodevelopmental disorders (NDDs) arise from disruptions in brain development, yet the underlying pathways remain incompletely understood. Here we demonstrate that genome-wide CRISPR knockout screens in mouse embryonic stem cells differentiating into neural lineages identify hundreds of essential genes, only a minority of which are currently implicated in NDDs. Dominant NDD genes were enriched for transcriptional regulators, whereas recessive NDD genes were predominantly involved in metabolic processes. Mouse models for eight genes (Eml1, Dusp26, Dynlrb2, Mta3, Peds1, Sgms1, Slitrk4 and Vamp3) revealed marked neuroanatomical abnormalities, including microcephaly in half of the cases. Focusing on PEDS1, a key enzyme in plasmalogen biosynthesis, we identified a bi-allelic variant in individuals with microcephaly, global developmental delay and congenital cataracts. In mice, Peds1 deficiency led to accelerated cell-cycle exit and impaired neuronal differentiation and migration. These pathways required for neural differentiation provide a genetic framework for discovering additional NDD genes.},
}
@article {pmid41413662,
year = {2026},
author = {Green, NFO and Sutton, GJ and Pérez-Burillo, J and Wang, J and Bagot, S and Danon, HG and Walsh, K and Gokool, A and Miles, SA and Yang, G and Herring, CA and Liang, Y and Pfundstein, G and Sytnyk, V and Alinejad-Rokny, H and Lister, R and Rosenbluh, J and Gagnon-Bartsch, JA and Voineagu, I},
title = {CRISPRi screening in cultured human astrocytes uncovers distal enhancers controlling genes dysregulated in Alzheimer's disease.},
journal = {Nature neuroscience},
volume = {29},
number = {3},
pages = {703-716},
pmid = {41413662},
issn = {1546-1726},
support = {2020814//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {Humans ; *Astrocytes/metabolism ; *Alzheimer Disease/genetics/metabolism ; *Enhancer Elements, Genetic/genetics ; Cells, Cultured ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Analysis ; Machine Learning ; },
abstract = {Genetic variants associated with complex traits often lie in distal enhancers. While candidate enhancers have been mapped genome wide, their functional state and gene targets in specific cell types remain unclear. Here we present AstroREG, a resource of enhancer-gene interactions in human primary astrocytes, generated by combining CRISPR inhibition (CRISPRi), single-cell RNA-seq and machine learning. By functionally testing nearly 1,000 PsychENCODE enhancers, we identified more than 150 regulatory interactions, revealing enhancers that control key astrocyte functions and genes implicated in Alzheimer's disease. The CRISPRi screen also provided valuable ground-truth data from a primary cell type for training and benchmarking prediction models of enhancer activity. We thus developed EGrf, a random forest (RF) model trained on these data, and applied it genome wide to predict regulatory interactions with high specificity. Together, our data provide a comprehensive functional map of enhancer-mediated regulation in a key glial cell type, shedding light on brain function and disease.},
}
@article {pmid37762360,
year = {2023},
author = {Mak, MCE and Gurung, R and Foo, RSY},
title = {Applications of Genome Editing Technologies in CAD Research and Therapy with a Focus on Atherosclerosis.},
journal = {International journal of molecular sciences},
volume = {24},
number = {18},
pages = {},
pmid = {37762360},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; Humans ; *Coronary Artery Disease/therapy/genetics ; Animals ; *Atherosclerosis/genetics/therapy ; CRISPR-Cas Systems ; Genetic Therapy/methods ; },
abstract = {Cardiovascular diseases, particularly coronary artery disease (CAD), remain the leading cause of death worldwide in recent years, with myocardial infarction (MI) being the most common form of CAD. Atherosclerosis has been highlighted as one of the drivers of CAD, and much research has been carried out to understand and treat this disease. However, there remains much to be better understood and developed in treating this disease. Genome editing technologies have been widely used to establish models of disease as well as to treat various genetic disorders at their root. In this review, we aim to highlight the various ways genome editing technologies can be applied to establish models of atherosclerosis, as well as their therapeutic roles in both atherosclerosis and the clinical implications of CAD.},
}
@article {pmid37762332,
year = {2023},
author = {Akhtar, J and Imran, M and Wang, G},
title = {CRISPR/Cas9-Mediated CtBP1 Gene Editing Enhances Chemosensitivity and Inhibits Metastatic Potential in Esophageal Squamous Cell Carcinoma Cells.},
journal = {International journal of molecular sciences},
volume = {24},
number = {18},
pages = {},
pmid = {37762332},
issn = {1422-0067},
support = {32070681//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Alcohol Oxidoreductases/genetics/metabolism ; *Esophageal Squamous Cell Carcinoma/genetics/pathology/drug therapy/metabolism ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/genetics ; *CRISPR-Cas Systems/genetics ; *Esophageal Neoplasms/genetics/pathology/drug therapy/metabolism ; Paclitaxel/pharmacology ; *DNA-Binding Proteins/genetics/metabolism ; Cell Proliferation/drug effects/genetics ; *Gene Editing/methods ; Gene Expression Regulation, Neoplastic/drug effects ; Cell Survival/drug effects ; Neoplasm Metastasis ; },
abstract = {Innovative therapeutic strategies for esophageal squamous cell carcinoma (ESCC) are urgently required due to the limited effectiveness of standard chemotherapies. C-Terminal Binding Protein 1 (CtBP1) has been implicated in various cancers, including ESCC. However, the precise expression patterns and functional roles of CtBP1 in ESCC remain inadequately characterized. In this study, we aimed to investigate CtBP1 expression and its role in the resistance of ESCC to paclitaxel, an effective chemotherapeutic agent. Western blotting and immunofluorescence were applied to assess CtBP1 expression in the TE-1 and KYSE-50 cell lines. We observed the marked expression of CtBP1, which was associated with enhanced proliferation, invasion, and metastasis in these cell lines. Further, we successfully generated paclitaxel resistant ESCC cell lines and conducted cell viability assays. We employed the CRISPR/Cas9 genome editing system to disable the CtBP1 gene in ESCC cell lines. Through the analysis of the drug dose-response curve, we assessed the sensitivity of these cell lines in different treatment groups. Remarkably, CtBP1-disabled cell lines displayed not only improved sensitivity but also a remarkable inhibition of proliferation, invasion, and metastasis. This demonstrates that CtBP1 may promote ESCC cell malignancy and confer paclitaxel resistance. In summary, our study opens a promising avenue for targeted therapies, revealing the potential of CtBP1 inhibition to enhance the effectiveness of paclitaxel treatment for the personalized management of ESCC.},
}
@article {pmid37755318,
year = {2023},
author = {Ravichandran, K and Khargonkar, T and Samaddar, S and Banerjee, S},
title = {CRISPR Based Programmable RNA Editing in Primary Hippocampal Neurons.},
journal = {Current protocols},
volume = {3},
number = {9},
pages = {e886},
doi = {10.1002/cpz1.886},
pmid = {37755318},
issn = {2691-1299},
mesh = {*Hippocampus/cytology/metabolism ; *Neurons/metabolism/cytology ; *RNA Editing/genetics ; Animals ; Mice ; *CRISPR-Cas Systems/genetics ; Cells, Cultured ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Investigating the RNA regulation landscape primarily relies on understanding how RNA-protein interactions are governed in various cell types, including neurons. Analysis of RNA-protein interactions in physiological environments warrants the development of new tools that rely on RNA manipulation. Recently, a CRISPR-based RNA-editing tool (dCas13b-ADAR2DD) was developed to mitigate disease-associated point mutations in cell lines. Here, we explored the targeted sequence editing potential of the tool (dCas13b-ADAR2DD system) by adapting it to manipulate RNA function to visualize RNA editing in primary hippocampal neurons. This two-component system includes a programmable guide RNA (gRNA) complementary to the target RNA and a catalytically dead version of the Cas13b enzyme fused to ADAR. The RNA editing protocol outlined in this article relies on gRNA-dependent targeting of the dCas13b-ADAR fusion protein to the mutant form of the Dendra2 transcript. Dendra2 is not required for intrinsic cellular functioning. It was ectopically expressed for fluorescent detection as a proof-of-principle demonstration of targeted RNA editing. We first abrogated the fluorescence of Dendra2 by introducing a nonsense mutation that precludes the formation of the functional protein. To visualize the efficacy of the RNA editing in neurons, we used the dCas13b-ADAR2DD system to edit specific nucleotides within the Dendra2 mRNA to restore the amino acid codes critical for Dendra2 fluorescence. This method lays the foundation for future studies on the dynamics of activity-induced RNA-protein interactions in neurons and can be extended to manipulate the endogenous RNome in diverse neuronal subtypes. Furthermore, this methodology will enable investigators to visualize the spatial and temporal resolution of RNA-protein interactions without altering the genomes via conventional methods. © 2023 Wiley Periodicals LLC. Support Protocol: Preparation of mouse primary hippocampal culture Basic Protocol: Targeted editing of RNA.},
}
@article {pmid37741117,
year = {2023},
author = {Lee, J and Kim, DH and Lee, K},
title = {Research Note: Injection of adenoviral CRISPR/Cas9 system targeting melanophilin gene into different sites of embryos induced regional feather color changes in posthatch quail.},
journal = {Poultry science},
volume = {102},
number = {11},
pages = {103087},
pmid = {37741117},
issn = {1525-3171},
mesh = {Animals ; *CRISPR-Cas Systems ; *Feathers/physiology ; *Gene Editing/veterinary/methods ; *Coturnix/genetics/physiology ; Adenoviridae/genetics ; *Pigmentation/genetics ; *Avian Proteins/genetics/metabolism ; },
abstract = {Poultry species is an important animal model in both avian research and the poultry industry. To advance our understanding of genetic factors and benefit both fields, a gene of interest can be genetically edited, and consequential phenotypic changes can be investigated. Injection of adenovirus containing the CRISPR/Cas9 system into avian blastoderm induced genome editing in blastodermal cells randomly, including primordial germ cells, which results in generation of whole-body knockout in the offspring of the virus-injected quail. However, to observe phenotypic and functional changes in whole-body, homozygous knockout of genes using this genome editing technology requires at least 2 generations of breeding of chimeric, and heterozygotes birds. In the current study, we developed a strategy to investigate the gene function in 1-generation by inducing regional genome editing around the injection sites with CRISPR/Cas9 adenovirus. The adenoviral CRISPR/Cas9 vector targeting the melanophilin (Mlph) gene, regulating feather pigmentation, was injected into 2 different regions of embryos, the cervical flexure of quail embryos at HH stage 13 to 15 and the tip of the upper limb bud of embryos at HH stage 22 to 24, to induce genome editing in those regions. Indel mutations in the target loci of the Mlph gene were detected by extracting genomic DNA from the embryonic tissues, and consequential phenotypes, feather color changes, were analyzed at 1 mo after hatch. Injection of the adenovirus into the cervical flexure and the tip of the upper limb bud of embryos resulted in 8 to 21% efficiency of indel mutation in the embryonic cells of the injected regions. In the posthatch quail, gray feathers were shown on their upper back and primary wing feathers, corresponding to the injection sites at embryos. Successful validation of this strategy for inducing genome editing in parts of tissues within 1-generation will accelerate studies on genetic functions with advantages of less time and cost, facilitating avian research and providing foundations for future application for the poultry industry.},
}
@article {pmid37718698,
year = {2023},
author = {Kumar, A and Stirling, PC},
title = {Turning up the heat on essential E. coli genes.},
journal = {Molecular systems biology},
volume = {19},
number = {10},
pages = {e11933},
pmid = {37718698},
issn = {1744-4292},
support = {RGPIN 2020-04360//Gouvernement du Canada | Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; },
mesh = {*Escherichia coli/genetics ; *Genes, Essential ; CRISPR-Cas Systems ; Alleles ; Escherichia coli Proteins/genetics ; Hot Temperature ; Gene Expression Regulation, Bacterial ; Gene Editing/methods ; },
abstract = {Temperature-sensitive (TS) alleles create tunable thermoswitches to deplete essential cellular activities and are used to dissect gene function. In their recent study, Link and colleagues (Schramm et al 2023) use a CRISPR-based approach to systematically create TS alleles across essential genes in E. coli.},
}
@article {pmid37702604,
year = {2023},
author = {Pescod, P and Bevivino, G and Anthousi, A and Shelton, R and Shepherd, J and Lombardo, F and Nolan, T},
title = {Measuring the Impact of Genetic Heterogeneity and Chromosomal Inversions on the Efficacy of CRISPR-Cas9 Gene Drives in Different Strains of Anopheles gambiae.},
journal = {The CRISPR journal},
volume = {6},
number = {5},
pages = {419-429},
doi = {10.1089/crispr.2023.0029},
pmid = {37702604},
issn = {2573-1602},
mesh = {*Anopheles/genetics ; Animals ; *Chromosome Inversion/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Drive Technology/methods ; Gene Editing/methods ; *Genetic Heterogeneity ; Mosquito Vectors/genetics ; },
abstract = {The human malaria vector Anopheles gambiae is becoming increasingly resistant to insecticides, spurring the development of genetic control strategies. CRISPR-Cas9 gene drives can modify a population by creating double-stranded breaks at highly specific targets, triggering copying of the gene drive into the cut site ("homing"), ensuring its inheritance. The DNA repair mechanism responsible requires homology between the donor and recipient chromosomes, presenting challenges for the invasion of laboratory-developed gene drives into wild populations of target species An. gambiae species complex, which show high levels of genome variation. Two gene drives (vas2-5958 and zpg-7280) were introduced into three An. gambiae strains collected across Africa with 5.3-6.6% variation around the target sites, and the effect of this variation on homing was measured. Gene drive homing across different karyotypes of the 2La chromosomal inversion was also assessed. No decrease in gene drive homing was seen despite target site heterology, demonstrating the applicability of gene drives to wild populations.},
}
@article {pmid37493514,
year = {2023},
author = {Wei, H and Li, J and Liu, Y and Cheng, W and Huang, H and Liang, X and Huang, W and Lin, L and Zheng, Y and Chen, W and Wang, C and Chen, W and Xu, G and Wei, W and Chen, L and Zeng, Y and Lu, Z and Li, S and Lin, Z and Wang, J and Lin, M},
title = {Rapid and Ultrasensitive Detection of Plasmodium spp. Parasites via the RPA-CRISPR/Cas12a Platform.},
journal = {ACS infectious diseases},
volume = {9},
number = {8},
pages = {1534-1545},
doi = {10.1021/acsinfecdis.3c00087},
pmid = {37493514},
issn = {2373-8227},
mesh = {Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Plasmodium/genetics/isolation &amp; purification ; Sensitivity and Specificity ; *Malaria/diagnosis/parasitology ; Recombinases/metabolism/genetics ; Plasmodium falciparum/genetics/isolation &amp; purification ; *Molecular Diagnostic Techniques/methods ; Point-of-Care Testing ; Malaria, Falciparum/diagnosis ; },
abstract = {Microscopic examination of thick and thin blood smears stained with Giemsa dye is considered the primary diagnostic tool for the confirmation and management of suspected clinical malaria. However, detecting gametocytes is relatively insensitive, particularly in asymptomatic individuals with low-density Plasmodium infections. To complement existing diagnostic methods, a rapid and ultrasensitive point-of-care testing (POCT) platform for malaria detection is urgently needed and necessary. A platform based on recombinase polymerase amplification (RPA) followed by CRISPR/Cas12a (referred to as RPA-CRISPR/Cas12a) was developed and optimized for the determination of Plasmodium spp. parasites, particularly Plasmodium falciparum, using a fluorescence-based assay (FBDA), lateral flow test strips (LFTS), or naked eye observation (NEO). Then, the established platform was assessed with clinical malaria isolates. Under optimal conditions, the detection threshold was 1 copy/μL for the plasmid, and the limit of detection was 3.11-7.27 parasites/μL for dried blood spots. There was no cross-reactivity against blood-borne pathogens. For the accuracies of RPA-CRISPR/Cas12a, Plasmodium spp. and P. falciparum testing were 98.68 and 94.74%, respectively. The method was consistent with nested PCR results and superior to the qPCR results. RPA-CRISPR/Cas12a is a rapid, ultrasensitive, and reliable platform for malaria diagnosis. The platform requires no or minimal instrumentation for nucleic acid amplification reactions and can be read with the naked eye. Compared with similar diagnostic methods, this platform improves the reaction speed while reducing detection requirements. Therefore, this platform has the potential to become a true POCT for malaria parasites.},
}
@article {pmid37482112,
year = {2023},
author = {Liu, L and Zhang, S and Zhi, F and Song, Y and Li, B and Gao, P and Zhang, Y and Ma, K and Xu, J and Jiang, B and Chu, Y and Li, Y and Qin, J},
title = {RNA helicase DExD/H-box 5 modulates intestinal microbiota in mice.},
journal = {Microbial pathogenesis},
volume = {182},
number = {},
pages = {106265},
doi = {10.1016/j.micpath.2023.106265},
pmid = {37482112},
issn = {1096-1208},
mesh = {Animals ; *Gastrointestinal Microbiome/genetics ; *DEAD-box RNA Helicases/genetics/metabolism ; Mice, Knockout ; Mice ; RNA, Ribosomal, 16S/genetics ; Ileum/microbiology/pathology ; Feces/microbiology ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Bacteria/classification/genetics/isolation &amp; purification ; },
abstract = {The RNA helicase DExD/H-box (DDX) family of proteins plays a central role in host cellular RNA metabolism, including mRNA regulation, microRNA biogenesis, and ribosomal processing. DDX5, also known as p68, promotes viral replication and tumorigenesis. However, there have been no studies on the regulation of the intestinal microbiota by DDX family proteins. We constructed DDX5 knockout mice (Ddx5[+/-]) using CRISPR/CAS9 technology. Subsequently, DDX5 knockout mice were analyzed for PCR products, mRNA levels, protein expression, immunohistochemistry, and histopathological lesions. Fecal (n = 12) and ileum (n = 12) samples were collected from the Ddx5[+/-] and wild-type (Ddx5[+/+]) mice. The diversity, richness, and structural separation of the intestinal microbiota of the Ddx5[+/-] and Ddx5[+/+] mice were determined by 16S rRNA sequencing and analysis. Ddx5[+/-] mice were successfully established, and the ileum had normal morphology, a clear layer of tissue structures, and neatly arranged cupped cells. DDX5 knockout mice did not exhibit adverse effects on the ileal tissue. Microbial diversity and abundance were not significantly different, but the microbial structure of the intestinal microbiota was clustered separately between Ddx5[+/+] and Ddx5[+/-] mice. Furthermore, we found that the relative abundance of Akkermansia and Clostridium_sensu_stricto_1 in the Ddx5[+/-] mice was significantly lower than in the Ddx5[+/+] mice. These analyses indicated specific interactions between the intestinal microbiota and DDX5 protein. Our results indicate that DDX5 has a significant effect on the composition of the intestinal microbiota in mice, suggesting its potential as a promising novel target for the treatment of inflammation and tumorigenesis in the intestine.},
}
@article {pmid37477766,
year = {2023},
author = {Hu, J and Guan, X and Zhao, M and Xie, P and Guo, J and Tan, J},
title = {Genome-wide CRISPR-Cas9 Knockout Screening Reveals a TSPAN3-mediated Endo-lysosome Pathway Regulating the Degradation of α-Synuclein Oligomers.},
journal = {Molecular neurobiology},
volume = {60},
number = {11},
pages = {6731-6747},
pmid = {37477766},
issn = {1559-1182},
mesh = {Humans ; *Lysosomes/metabolism ; *alpha-Synuclein/metabolism ; HEK293 Cells ; *Tetraspanins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *Proteolysis ; *Endosomes/metabolism ; *Gene Knockout Techniques ; *Protein Multimerization ; },
abstract = {Misfolding and aggregation of α-Synuclein (α-Syn), which are hallmark pathological features of neurodegenerative diseases such as Parkinson's disease (PD) and dementia with Lewy Bodies, continue to be significant areas of research. Among the diverse forms of α-Syn - monomer, oligomer, and fibril, the oligomer is considered the most toxic. However, the mechanisms governing α-Syn oligomerization are not yet fully understood. In this study, we utilized genome-wide CRISPR/Cas9 loss-of-function screening in human HEK293 cells to identify negative regulators of α-Syn oligomerization. We found that tetraspanin 3 (TSPAN3), a presumptive four-pass transmembrane protein, but not its homolog TSPAN7, significantly modulates α-Syn oligomer levels. TSPAN3 was observed to interact with α-Syn oligomers, regulate the amount of α-Syn oligomers on the cell membrane, and promote their degradation via the clathrin-AP2 mediated endo-lysosome pathway. Our findings highlight TSPAN3 as a potential regulator of α-Syn oligomers, presenting a promising target for future PD prevention and treatment strategies.},
}
@article {pmid37451121,
year = {2023},
author = {Hu, Y and Qiao, Y and Li, XQ and Xiang, Z and Wan, Y and Wang, P and Yang, Z},
title = {Development of an inducible Cas9 nickase and PAM-free Cas12a platform for bacterial diagnostics.},
journal = {Talanta},
volume = {265},
number = {},
pages = {124931},
doi = {10.1016/j.talanta.2023.124931},
pmid = {37451121},
issn = {1873-3573},
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *Deoxyribonuclease I/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Methicillin-Resistant Staphylococcus aureus/isolation &amp; purification/genetics ; CRISPR-Cas Systems ; DNA, Single-Stranded/genetics ; *Bacterial Proteins/genetics/metabolism ; Humans ; },
abstract = {Rapid, efficient, specific and sensitive diagnostic techniques are critical for selecting appropriate treatments for drug-resistant bacterial infections. To address this challenge, we have developed a novel diagnostic method, called the Dual-Cas Tandem Diagnostic Platform (DTDP), which combines the use of Cas9 nickase (Cas9n) and Cas12a. DTDP works by utilizing the Cas9n-sgRNA complex to create a nick in the target strand's double-stranded DNA (dsDNA). This prompts DNA polymerase to displace the single-stranded DNA (ssDNA) and leads to cycles of DNA replication through nicking, displacement, and extension. The ssDNA is then detected by the Cas12a-crRNA complex (which is PAM-free), activating trans-cleavage and generating a fluorescent signal from the fluorescent reporter. DTDP exhibits a high sensitivity (1 CFU/mL or 100 ag/μL), high specificity (specifically to MRSA in nine pathogenic species), and excellent accuracy (100%). The dual RNA recognition process in our method improves diagnostic specificity by decreasing the limitations of Cas12a in detecting dsDNA protospacer adjacent motifs (PAMs) and leverages multiple advantages of multi-Cas enzymes in diagnostics. This novel approach to pathogenic microorganism detection has also great potential for clinical diagnosis.},
}
@article {pmid37421327,
year = {2023},
author = {Yang, M and Wan, S and Chen, J and Chen, W and Wang, Y and Li, W and Wang, M and Guan, R},
title = {Mutation to a cytochrome P450 -like gene alters the leaf color by affecting the heme and chlorophyll biosynthesis pathways in Brassica napus.},
journal = {The Plant journal : for cell and molecular biology},
volume = {116},
number = {2},
pages = {432-445},
doi = {10.1111/tpj.16382},
pmid = {37421327},
issn = {1365-313X},
support = {32171974//National Natural Science Foundation of China/ ; 2022YFF1003500//the National Key R&amp;D Program of China/ ; },
mesh = {*Chlorophyll/biosynthesis/metabolism ; *Brassica napus/genetics/metabolism ; *Plant Leaves/genetics/metabolism/physiology ; *Cytochrome P-450 Enzyme System/genetics/metabolism ; *Heme/biosynthesis/metabolism ; *Plant Proteins/genetics/metabolism ; Mutation ; CRISPR-Cas Systems ; Photosynthesis ; Biosynthetic Pathways ; Gene Expression Regulation, Plant ; Gene Editing ; },
abstract = {The regulated biosynthesis of chlorophyll is important because of its effects on plant photosynthesis and dry biomass production. In this study, a map-based cloning approach was used to isolate the cytochrome P450 -like gene BnaC08g34840D (BnCDE1) from a chlorophyll-deficient mutant (cde1) of Brassica napus obtained by ethyl methanesulfonate (EMS) mutagenization. Sequence analyses revealed that BnaC08g34840D in the cde1 mutant (BnCDE1[I320T]) encodes a substitution at amino acid 320 (Ile320Thr) in the conserved region. The over-expression of BnCDE1[I320T] in ZS11 (i.e., gene-mapping parent with green leaves) recapitulated a yellow-green leaf phenotype. The CRISPR/Cas9 genome-editing system was used to design two single-guide RNAs (sgRNAs) targeting BnCDE1[I320T] in the cde1 mutant. The knockout of BnCDE1[I320T] in the cde1 mutant via a gene-editing method restored normal leaf coloration (i.e., green leaves). These results indicate that the substitution in BnaC08g34840D alters the leaf color. Physiological analyses showed that the over-expression of BnCDE1[I320T] leads to decreases in the number of chloroplasts per mesophyll cell and in the contents of the intermediates of the chlorophyll biosynthesis pathway in leaves, while it increases heme biosynthesis, thereby lowering the photosynthetic efficiency of the cde1 mutant. The Ile320Thr mutation in the highly conserved region of BnaC08g34840D inhibited chlorophyll biosynthesis and disrupted the balance between heme and chlorophyll biosynthesis. Our findings may further reveal how the proper balance between the chlorophyll and heme biosynthesis pathways is maintained.},
}
@article {pmid36960824,
year = {2023},
author = {Makrogkikas, S and Cheng, RK and Lu, H and Roy, S},
title = {A conserved function of Pkhd1l1, a mammalian hair cell stereociliary coat protein, in regulating hearing in zebrafish.},
journal = {Journal of neurogenetics},
volume = {37},
number = {3},
pages = {85-92},
doi = {10.1080/01677063.2023.2187792},
pmid = {36960824},
issn = {1563-5260},
mesh = {Animals ; Zebrafish ; *Zebrafish Proteins/genetics/metabolism/physiology ; *Hair Cells, Auditory/metabolism/physiology ; *Hearing/genetics/physiology ; *Stereocilia/metabolism ; Mice ; Hearing Loss/genetics ; Animals, Genetically Modified ; CRISPR-Cas Systems ; },
abstract = {Pkhd1l1 is predicted to encode a very large type-I transmembrane protein, but its function has largely remained obscure. Recently, it was shown that Pkhdl1l1 is a component of the coat that decorates stereocilia of outer hair cells in the mouse ear. Consistent with this localization, conditional deletion of Pkhd1l1 specifically from hair cells, was associated with progressive hearing loss. In the zebrafish, there are two paralogous pkhd1l1 genes - pkhd1l1α and pkhd1l1β. Using CRISPR-Cas9 mediated gene editing, we generated loss-of-function alleles for both and show that the double mutants exhibit nonsense-mediated-decay (NMD) of the RNAs. With behavioural assays, we demonstrate that zebrafish pkhd1l1 genes also regulate hearing; however, in contrast to Pkhd1l1 mutant mice, which develop progressive hearing loss, the double mutant zebrafish exhibited statistically significant hearing loss even from the larval stage. Our data highlight a conserved function of Pkhd1l1 in hearing and based on these findings from animal models, we postulate that PKHD1L1 could be a candidate gene for sensorineural hearing loss (SNHL) in humans.},
}
@article {pmid36948605,
year = {2023},
author = {Deng, Y and Han, X and Chen, H and Zhao, C and Chen, Y and Zhou, J and de The, H and Zhu, J and Yuan, H},
title = {Ypel5 regulates liver development and function in zebrafish.},
journal = {Journal of molecular cell biology},
volume = {15},
number = {3},
pages = {},
pmid = {36948605},
issn = {1759-4685},
support = {31371479//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Zebrafish/genetics/metabolism/embryology ; *Zebrafish Proteins/metabolism/genetics ; *Liver/metabolism/embryology/growth &amp; development ; Hepatocyte Nuclear Factor 4/metabolism/genetics ; Cell Proliferation/genetics ; Hepatocytes/metabolism ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; Signal Transduction ; },
abstract = {YPEL5 is a member of the Yippee-like (YPEL) gene family that is evolutionarily conserved in eukaryotic species. To date, the physiological function of YPEL5 has not been assessed due to a paucity of genetic animal models. Here, using CRISPR/Cas9-mediated genome editing, we generated a stable ypel5-/- mutant zebrafish line. Disruption of ypel5 expression leads to liver enlargement associated with hepatic cell proliferation. Meanwhile, hepatic metabolism and function are dysregulated in ypel5-/- mutant zebrafish, as revealed by metabolomic and transcriptomic analyses. Mechanistically, Hnf4a is identified as a crucial downstream mediator that is positively regulated by Ypel5. Zebrafish hnf4a overexpression could largely rescue ypel5 deficiency-induced hepatic defects. Furthermore, PPARα signaling mediates the regulation of Hnf4a by Ypel5 through directly binding to the transcriptional enhancer of the Hnf4a gene. Herein, this work demonstrates an essential role of Ypel5 in hepatocyte proliferation and function and provides the first in vivo evidence for a physiological role of the ypel5 gene in vertebrates.},
}
@article {pmid41779860,
year = {2026},
author = {Zhou, X and Pan, D and Zhou, J and Chen, W and Han, G and Zhang, R and Wang, C and Mao, Y and Du, Z and Zhang, F and Yue, H and Ma, J and Li, Z and Shen, RJ and Wang, B and Zhu, W and Peng, Y and Jin, K and Wu, DD and Wang, W and Zhou, B and Jin, ZB and Chen, L},
title = {Cis-regulatory evolution reveals sensory trade-offs as a genetic basis for temporal niche evolution in tapirs.},
journal = {Science advances},
volume = {12},
number = {10},
pages = {eadz4758},
pmid = {41779860},
issn = {2375-2548},
mesh = {Animals ; *Evolution, Molecular ; Mice ; *Regulatory Sequences, Nucleic Acid ; Biological Evolution ; CRISPR-Cas Systems ; Humans ; },
abstract = {Evolutionary shifts in diel activity patterns shape sensory remodeling across mammals, yet the genetic basis remains poorly understood. Tapirs represent a unique natural experiment, having reverted from a cathemeral ancestor to a nocturnal niche characterized by reduced vision but enhanced hearing and olfaction. Here, we investigate the genetic basis of this phenomenon by generating high-quality chromosome-level genomes for Tapirus terrestris and Tapirus indicus. Comparative analyses revealed extensive lineage-specific remodeling of genes and cis-regulatory elements linked to sensory pathways. Notably, functional validation via CRISPR-Cas9 editing of a tapir-specific conserved noncoding element (CNE74) upstream of the FLT1 gene in mice revealed coordinated sensory effects, including retinal degeneration and reduced visual acuity, yet enhanced auditory sensitivity. These findings suggest that regulatory element evolution may induce pleiotropic effects on competing sensory modalities, offering genetic insights into sensory evolution during temporal niche adaptation and potential relevance to human retinal vascular diseases.},
}
@article {pmid41779781,
year = {2026},
author = {Ju, X and Dong, L and Liu, T and Zhang, F and Sun, X and Schwoerer, MP and Ren, W and Gong, M and Ploss, A and Qin, W and Wu, X and Wang, L and Ding, Q},
title = {EIF4H and YBX1 are essential host factors for hepatitis E virus replication and pathogenesis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2529289123},
doi = {10.1073/pnas.2529289123},
pmid = {41779781},
issn = {1091-6490},
support = {2023YFC2306900//National Key Research and Development Plan of China/ ; 82341084//MOST | National Natural Science Foundation of China (NSFC)/ ; 82272302//MOST | National Natural Science Foundation of China (NSFC)/ ; 82522053//MOST | National Natural Science Foundation of China (NSFC)/ ; 20251080029//Tsinghua University Dushi Program/ ; Not applicable//SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine/ ; Not applicable//High Meadows Environmental Institute/ ; T32 GM007388/GM/NIGMS NIH HHS/United States ; },
mesh = {*Hepatitis E virus/physiology/pathogenicity/genetics ; *Virus Replication ; Humans ; *Hepatitis E/virology/metabolism/pathology/genetics ; Animals ; Rats ; *Y-Box-Binding Protein 1/metabolism/genetics ; *Eukaryotic Initiation Factors/metabolism/genetics ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; Hepatocytes/virology ; RNA-Binding Proteins ; },
abstract = {Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Despite the availability of a vaccine in China, no direct-acting antivirals are approved, and host factors required for HEV replication remain poorly defined. Here, using a genome-wide CRISPR/Cas9 knockout screen in a replicon system, we identified Eukaryotic Translation Initiation Factor 4H (EIF4H) and Y-Box Binding Protein 1 (YBX1) as essential host factors for HEV replication and pathogenesis. Knockout of either factor markedly impaired replication of HEV genotypes 1, 3, and 4, as well as HEV infection and production in hepatocellular carcinoma cells and human induced pluripotent stem cell-derived hepatocyte-like cells, while leaving SARS-CoV-2, hepatitis B virus, hepatitis C virus, and Zika virus unaffected, underscoring their HEV-specific roles. Mechanistically, EIF4H interacts with ORF1 via its methyltransferase-Y-papain-like protease region, and EIF4H deficiency alters the composition of the ORF1-associated replication complex. By contrast, YBX1 is dispensable for ORF1 translation and RNA binding but is specifically required for ORF1 proteolytic processing, a prerequisite for assembling a functional replication machinery. EIF4H knockout rats and liver-specific YBX1 knockout rats were largely resistant to rat HEV-C1 infection, showing profound reductions in viral shedding, suppressed hepatic and intestinal viral loads, and protection from liver pathology. Together, our findings establish EIF4H and YBX1 as essential host factors for HEV infection and pathogenesis and reveal potential targets for antiviral intervention.},
}
@article {pmid41777069,
year = {2026},
author = {Zamperin, G and Palumbo, E and Castellan, M and Marciano, S and Fusaro, A and Monne, I},
title = {Metagenomic sequencing of zoonotic viruses: evaluation of a CRISPR-Cas-based rRNA depletion system.},
journal = {Veterinaria italiana},
volume = {62},
number = {2},
pages = {},
doi = {10.12834/VetIt.3908.38985.2},
pmid = {41777069},
issn = {1828-1427},
mesh = {*CRISPR-Cas Systems ; Animals ; *Metagenomics/methods ; *RNA, Ribosomal/genetics ; *Zoonoses/virology ; Genome, Viral ; },
abstract = {Pathogen-agnostic diagnostics are crucial for the early detection of emerging viruses. Shotgun metagenomic sequencing enables unbiased detection of viral genomes but is frequently constrained by the abundance of host and microbial ribosomal RNA (rRNA), which reduces sensitivity and increases sequencing costs. CRISPR-Cas9-based rRNA depletion has emerged as an alternative to enzymatic methods; however, its performance for the characterization of zoonotic viruses across diverse animal hosts and tissues remains underexplored. We compared CRISPR-Cas9 (Jumpcode CRISPRclean™ Plus) and RNase H-based enzymatic depletion (Ribo-Zero Plus, Illumina) using 12 samples positive for rabies lyssavirus, influenza A virus, West Nile virus or norovirus, from multiple host species and tissues, including both high-quality and degraded RNA. CRISPR-Cas9 efficiently reduced rRNA content (14.5%) but recovered fewer viral reads than Ribo-Zero, which achieved up to 60.7× enrichment. Both methods produced complete viral consensus genomes when RNA quality and viral load were sufficient. However, based on the data generated here, enzymatic depletion currently remains more efficient and cost-effective for viral metagenomics. Further optimization of CRISPR-Cas9 workflows could enhance its utility for viral surveillance and diagnostics.},
}
@article {pmid41773018,
year = {2026},
author = {Yang, T and Tang, M and Xu, L and Jiang, L and Jiang, L and Zou, Y and Wang, J and Liu, Z and Chen, F and Ban, Y and Ren, W and Cheng, W},
title = {A tailored phosphorothioate coordinator enables CRISPR/Cas in-situ amplification.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41773018},
issn = {1362-4962},
support = {U24A20751//National Natural Science Foundation of China/ ; 82372334//National Natural Science Foundation of China/ ; 82502827//National Natural Science Foundation of China/ ; CSTB2023NSCQ-LZX0022//Chongqing Education Commission/ ; CSTB2024NSCQ-QCXMX0006//New Chongqing Youth Innovative Talents Project/ ; //National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Phosphorothioate Oligonucleotides/chemistry/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Human papillomavirus 16/genetics ; CRISPR-Associated Proteins/metabolism/genetics/chemistry ; RNA, Viral/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Uterine Cervical Neoplasms/virology/diagnosis ; Female ; HeLa Cells ; Endodeoxyribonucleases/metabolism/genetics ; },
abstract = {The CRISPR/Cas system is a powerful tool for molecular diagnostics, but its reliance on linear amplification constrains sensitivity, particularly for in situ imaging. Here, we discovered that phosphorothioate (PS)-modified activators can modulate Cas enzyme conformation via hydrophobic anchoring. By adjusting the PS modification sites, we achieved precise control over Cas activation and trans-cleavage resistance. Guided by this mechanism, we proposed a tailored design strategy featuring a "scattered" PS modification to engineer a linear "Coordinator" probe. This design effectively decouples Cas enzyme activation from substrate trans-cleavage resistance, enabling the construction of a Scattered PS Nucleic Acid-driven Cas Autocatalytic system (SACA). SACA achieves exponential amplification without external enzymes, enhancing Cas12a and Cas13a sensitivity by 50 000-fold and 10 000-fold, respectively. Furthermore, the superior biostability and structural simplicity of these linear probes endow SACA with excellent compatibility, facilitating precise in situ imaging of HPV16 and HPV18 mRNA in cervical cancer cells. This study not only advances the understanding of Cas enzyme regulation by chemically modified nucleic acids but also establishes a new paradigm for precise and efficient molecular diagnostics.},
}
@article {pmid41773016,
year = {2026},
author = {Park, H and Yun, J and Lee, K and Kim, JH and Park, JH and Park, YJ and Park, JH and Lee, H and Kim, MG},
title = {Functional decoupling of crRNA enables customizable CRISPR diagnostics.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41773016},
issn = {1362-4962},
support = {//National Research Foundation of Korea/ ; RS-2025-16063091//National Research Council of Science and Technology/ ; RS-2024-00411137//National Research Council of Science and Technology/ ; CRC22024-500//National Research Council of Science and Technology/ ; //National Research Foundation of Korea/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nucleic Acid Amplification Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {One-pot CRISPR-based diagnostics have transformed nucleic acid testing, yet their design customizability remains constrained. Because target programming and cis-cleavage activity are simultaneously determined during CRISPR RNA (crRNA) design, optimizing cleavage activity to match isothermal amplification inevitably requires altering the programmed crRNA sequence. This requirement fundamentally constrains the range of compatible target sequences, imposing limitations on the flexible design of diagnostic assays. Here, we establish a customizable one-pot system by decoupling the dual functions inherent in crRNA design to enable their independent control. In this strategy, target programming remains defined by the crRNA sequence, whereas cis-cleavage activity is regulated by the reaction energy barrier. We selectively modulate this energy barrier through the introduction of a crRNA-complementary RNA oligonucleotide, achieving cleavage regulation without altering the crRNA sequence. Consequently, this approach ensures that cis-cleavage activity matches isothermal amplification conditions independent of the programmed target sequence, thereby realizing a customizable CRISPR diagnostic system. We validated the clinical applicability of this system using 120 patient-derived samples, achieving sensitivity and specificity comparable to quantitative polymerase chain reaction. Collectively, this work resolves a fundamental constraint of CRISPR diagnostics and establishes a customizable and clinically deployable platform for next-generation nucleic acid testing.},
}
@article {pmid41772759,
year = {2026},
author = {Donega, S and Gorospe, M and Harries, LW and Ferrucci, L},
title = {Loss of Splicing Homeostasis as a Hallmark of Aging.},
journal = {Molecular and cellular biology},
volume = {},
number = {},
pages = {1-19},
doi = {10.1080/10985549.2026.2627235},
pmid = {41772759},
issn = {1098-5549},
abstract = {Alternative splicing is a fundamental mechanism that ensures accurate gene expression, supports cellular adaptability, and expands protein diversity beyond the limits of a fixed gene pool. With aging, splicing fidelity weakens, contributing to decline in RNA homeostasis and disrupting essential cellular functions, including mitochondrial oxidative phosphorylation, genome stability, and immune regulation, and in turn accelerating tissue and organ dysfunction. Evidence from senescent cells, aged tissues, and model organisms shows that altered levels of splicing factors and increased RNA polymerase II elongation rates impair co-transcriptional splicing and promote mis-spliced isoforms that reinforce senescence and drive pathology. Dysfunction of RNA-binding proteins further contributes to aberrant splicing, linking splicing defects to age-related diseases such as atherosclerosis, osteoarthritis, sarcopenia, and neurodegenerative disorders like Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Therapeutic strategies to correct splicing defects, such as antisense oligonucleotides, RNA interference, CRISPR-Cas systems, ADAR-mediated editing, and RNA aptamers, can restore a homeostatic balance of mRNA isoforms. However, major challenges remain, including distinguishing adaptive physiological from pathological splicing 'noise' and achieving targeted delivery to tissues. Despite these obstacles, RNA splicing dysregulation represents a promising avenue to extend health span by reestablishing homeostatic RNA programs, and reinforces the idea that "transcriptomic instability" is a hallmark of aging.},
}
@article {pmid41771871,
year = {2026},
author = {Becerra, B and Wittibschlager, S and Patel, ZM and Kutschat, AP and Delano, J and Che, E and Tauber, A and Wu, T and Starrs, M and Horstmann, CS and Müller, S and Whittaker, MN and Sylvander, E and Lehner, M and Love, MI and Kleinstiver, BP and Jankowiak, M and Bauer, DE and Seruggia, D and Pinello, L},
title = {Nucleotide-resolution mapping of regulatory elements via allelic readout of tiled base editing.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-69918-8},
pmid = {41771871},
issn = {2041-1723},
abstract = {CRISPR tiling screens have enabled the characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing and enrichment analysis. This study introduces an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. As a proof of concept, we studied a putative CD19 enhancer, an immunotherapy target in leukemia, and identified alleles and single nucleotides crucial for CD19 regulation. Our visualization tools revealed transcription factor motifs corresponding to the top-ranked nucleotides. Validation experiments confirmed that mutations in MYB, PAX5, and EBF1 binding sites reduce CD19 expression. Critically, editing MYB and PAX5 motifs conferred resistance to CD19 CAR-T cell therapy, revealing how non-coding variants can drive immunotherapy escape. Taken together, this approach achieves nucleotide-resolution genotype-phenotype mapping at regulatory elements beyond conventional gRNA-based screens.},
}
@article {pmid41771837,
year = {2026},
author = {Schoger, E and Kim, R and Bleckwedel, F and Peralta, T and Priesmeier, L and Fischer, J and Stengel, L and Rocha, C and Santos, GL and Lutz, S and Boileau, E and Baumgarten, N and Schulz, MH and Dieterich, C and Müller, OJ and Cyganek, L and Cabrera-Orefice, A and Eberl, H and Maack, C and Streckfuss-Bömeke, K and Pavez-Giani, M and Doroudgar, S and Sossalla, ST and Zelarayán, LC},
title = {Enhancing KLF15 activity in cardiomyocytes: a novel approach to prevent pathological reprogramming and fibrosis via nuclease-deficient dCas9VPR.},
journal = {Signal transduction and targeted therapy},
volume = {11},
number = {1},
pages = {},
pmid = {41771837},
issn = {2059-3635},
mesh = {*Myocytes, Cardiac/metabolism/pathology ; *Kruppel-Like Transcription Factors/genetics/metabolism ; Animals ; CRISPR-Cas Systems/genetics ; *Fibrosis/genetics/pathology ; *Cellular Reprogramming/genetics ; Humans ; Mice ; Fibroblasts/metabolism/pathology ; Gene Expression Regulation/genetics ; *Transcription Factors/genetics ; },
abstract = {Transcriptional activity perturbation holds promise for selectively modulating harmful transcriptional networks, but its therapeutic potential remains largely unexplored. We employed a network-based analysis of single-cell heart transcriptomes to identify transcription factor activities linked to pathological cardiomyocytes in vivo. This analysis revealed that transcriptional activity of Krüppel-like factor 15 (KLF15) exhibited the most significant change in pathological cardiomyocytes, characterized by less effective repression of disease-associated genes in stressed hearts, which correlated with reduced KLF15 expression. To restore KLF15 activity, we utilized CRISPR/nuclease-dead (d)Cas9-based transcriptional enhancement (CRISPRa) in cardiomyocytes, which effectively abolished fetal reprogramming by simultaneously suppressing pathological gene expression and restoring metabolic homeostasis under sustained stress conditions. Furthermore, we identified a novel cell-nonautonomous anti-fibrotic effect mediated by cardiomyocyte-fibroblast crosstalk, and revealed the contribution of KLF15-dependent Alpha-2-glycoprotein 1, zinc-binding (AZGP1) regulation in this process. We also elucidated the upstream mechanisms of KLF15 regulation, highlighting its role as a cell-specific downstream target of the broad TGF-β canonical signaling pathway, along with its downstream-dependent mechanisms in human cardiomyocytes. Finally, to enhance the therapeutic potential of this approach, we engineered and validated an adeno-associated viral (AAV) vector with a small CRISPRa system for endogenous regulation in human cardiomyocytes suitable for clinical applications. Overall, we elucidated a regulatory circuit involving TGF-β, KLF15, and AZGP1, which coordinates critical pathological responses through cellular crosstalk between cardiomyocytes and fibroblasts. Importantly, we demonstrated the efficacy of CRISPRa as an epigenetic intervention restoring a critical transcriptional function disrupted in non-genetic heart failure. This approach provides a promising blueprint for future adaptation targeting additional non-hereditary pathologies.},
}
@article {pmid41769381,
year = {2026},
author = {Mandal, S and Baloch, AR and Yuan, X and Chen, J and Saribas, AS and Zhu, Y and Zhang, D and Jaijyan, D and Xu, J and Hossain, R and Sisto, I and Wang, H and Yang, X and Li, Q and Hu, W},
title = {Bipolar CD4-targeted dual-DARPin-55/57 lipid nanoparticle enables efficient CRISPR/Cas-mediated HIV-1 DNA excision and reactivation blockade in latent CD4 T cell lines.},
journal = {Materials today. Bio},
volume = {37},
number = {},
pages = {102939},
pmid = {41769381},
issn = {2590-0064},
abstract = {The persistence of HIV-1 latent reservoirs remains the principal barrier to a cure, as viral rebound occurs upon interruption of antiretroviral therapy. CRISPR/Cas genome editing offers a promising strategy to excise proviruses from host genome; however, the absence of a targeted and clinically viable delivery platform has hindered its translational application. Here, we report a chemistry-driven, CD4-targeted lipid nanoparticle (LNP) delivery platform employing a unique bipolar conjugation strategy to decorate dual CD4-targeted Designed Ankyrin Repeat Proteins (DARPins-55 and -57) on LNP (dual-DARPin-LNP). The N- and C-terminally modified DARPin-55/57 was thiolated stepwise, then bipolar maleimide-thiol coupling conjugated the thiolates to the maleimide-functionalized LNP surface. This coupling strategy ensured DARPin proper orientation on the LNP surface for efficient uptake by resting CD4 T cells. This dual-DARPin-LNP system was engineered for selective and efficient co-delivery of spCas9-GFP mRNA (Sp9m) and HIV-1-specific single-guide RNAs (sgRNAs) targeting LTR and Gag (LGsg) into HIV-1 latently infected CD4 T cells. In widely used HIV-1 latency models with defined proviral modifications (J-Lat 10.6 and 2D10 cell lines), dual-DARPin-LNP loaded with Sp9m/LGsg efficiently excised integrated HIV-1 proviral DNA, as confirmed by standard PCR genotyping, absolute digital PCR quantification, confocal microscopy, and flow cytometry. Importantly, proviral excision functionally blocked HIV-1 reactivation following stimulation with latency-reversing agents suberoylanilide hydroxamic acid (SAHA) and TNFα. Together, these findings establish a modular, non-viral, receptor-guided delivery platform for CD4 T cell targeting and provide proof-of-concept for precise HIV-1 DNA excision and reactivation blockade in established latency models. This new strategy represents a step toward next-generation curative interventions against persistent HIV-1 infection.},
}
@article {pmid41766888,
year = {2026},
author = {Li, X and Zhao, Y and Guo, X and Bai, Y and Wang, J},
title = {Characterization and diversity of defense systems in Providencia pathogen.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1755933},
pmid = {41766888},
issn = {1664-3224},
mesh = {*Providencia/genetics/immunology/virology ; Phylogeny ; Genome, Bacterial ; *Enterobacteriaceae Infections/microbiology/immunology ; CRISPR-Cas Systems ; Bacteriophages ; Humans ; },
abstract = {INTRODUCTION: Providencia species are emerging opportunistic pathogens associated with multidrug-resistant infections, yet their molecular defense mechanisms against phage or mobile genetic elements remain poorly characterized.<br><br>METHODS: We present a comprehensive pan-genomic analysis of antiviral defense systems across 73 complete genomes (or chromosomes) of Providencia stuartii (n = 31) and Providencia rettgeri (n = 42), using DefenseFinder and CRISPRCasFinder. We further expanded analysis of contig/scaffold assemblies to confirm conservation of core defense profiles across assembly types. BacMGEnet was employed to derive spacer-MGE interaction networks. Phylogenetic reconstruction and gene gain and loss modeling were performed to assess evolutionary patterns. To validate functionality, we experimentally tested the anti-phage activity of Gabija and Septu in heterologous E. coli assays, including point mutation analysis of conserved residues.<br><br>RESULTS: We reveal a diverse and complex defense repertoire dominated by restriction-modification systems and CRISPR-Cas Class 1 Type I-F, with significant contributions from toxin-antitoxin, GAPS2, PsyrTA, and Mokosh systems. Notably, defense genes are non-randomly distributed, often clustering into genomic islands suggestive of horizontal acquisition. Expanded analysis confirms conservation of core defense profiles across assembly types, supporting the utility of lower-quality data when complete genomes are scarce. Comparative analysis uncovers species-specific differences, with P. rettgeri harboring a higher abundance of non-CRISPR systems. BacMGEnet-derived spacer-MGE interaction networks further highlight species-specific dynamics, dense, hub-driven networks in P. stuartii versus sparser networks in P. rettgeri. Correlation analysis indicates potential associations between specific defense systems and virulence or antibiotic resistance genes. Phylogenetic reconstruction and gene gain and loss modeling further highlight dynamic evolutionary patterns. Both Gabija and Septu systems conferred robust, phage-specific protection; point mutations in conserved residues (GajA E465K and PtuB H53K) abolished defense.<br><br>DISCUSSION: Our findings unveil a multi-layered, modular immune architecture in Providencia, providing crucial insights into its genome plasticity, phage resistance, and adaptation in clinical environments. This work establishes a foundation for understanding the role of defense systems in the evolution and pathogenicity of the Providencia genus.},
}
@article {pmid41764730,
year = {2026},
author = {D'Souza, LJ and Young, JN and Coffman, H and Petrow, EP and Bhattacharya, D},
title = {A genome-wide CRISPR screen reveals novel determinants of long-lived plasma cell secretory capacity.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {215},
number = {2},
pages = {},
doi = {10.1093/jimmun/vkaf354},
pmid = {41764730},
issn = {1550-6606},
support = {R01AI129945//National Institutes of Health (NIH)/ ; P30CA023074//Research, Innovation &amp; Impact (RII) of the University of Arizona and National Cancer Institute/ ; S10 OD028466/GF/NIH HHS/United States ; },
mesh = {Animals ; *Plasma Cells/immunology/metabolism ; Mice ; Humans ; Myeloid Differentiation Factor 88/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Multiple Myeloma/immunology/genetics ; Mice, Inbred C57BL ; Mice, Knockout ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Plasma cell subsets vary in their lifespans and ability to sustain humoral immunity. We conducted a genome-wide CRISPR-Cas9 screen in myeloma cells for factors that promote surface expression of CD98, a marker of longevity in mouse plasma cells. A large fraction of genes found to promote CD98 expression in this screen are involved in secretory and other vesicles, including subunits of the V-type ATPase complex. Genetic ablation and chemical inhibition of V-type ATPases in myeloma cells and primary plasma cells, respectively, reduced antibody secretion. Mouse and human long-lived plasma cells had greater numbers of acidified vesicles than their short-lived counterparts, and this correlated with increased antibody secretory capacity. The screen also revealed a requirement for the signaling adapter MYD88 in CD98 expression. Plasma cell-specific deletion of Myd88 led to reduced survival and antibody secretion by antigen-specific cells in vivo and unresponsiveness to BAFF and APRIL ex vivo. These data reveal novel regulators that link plasma cell secretory capacity and lifespan.},
}
@article {pmid41628535,
year = {2026},
author = {Xie, K and Ren, H and Ban, D and Chen, L and Xin, X and Zhang, J and Tang, Q and Huang, L and Wei, J and Zhang, K and Liao, X},
title = {Silica-detoxified perovskite ECL: Cas13a-triggered signal-on sensing with CsPbBr3@SiO2@Au.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {170},
number = {},
pages = {109243},
doi = {10.1016/j.bioelechem.2026.109243},
pmid = {41628535},
issn = {1878-562X},
mesh = {*Silicon Dioxide/chemistry ; *Gold/chemistry ; *Biosensing Techniques/methods ; *Titanium/chemistry ; *MicroRNAs/analysis/blood ; *Oxides/chemistry ; *Calcium Compounds/chemistry ; Electrochemical Techniques/methods ; Limit of Detection ; Humans ; Luminescent Measurements/methods ; CRISPR-Cas Systems ; Nanocomposites/chemistry ; },
abstract = {Perovskite nanocrystals are attractive ECL emitters but suffer from poor water stability and potential toxicity. Here we report a signal-on electrochemiluminescent biosensor that integrates CsPbBr3@SiO2@Au nanocomposites with a CRISPR/Cas13a-Nb.BbvCI amplification cascade for ultrasensitive microRNA detection. The CsPbBr3 core provides bright emission, a conformal SiO2 shell enhances water compatibility and suppresses ion leakage, and surface Au nanoparticles offer abundant sites for thiolated ferrocene-hairpin (Fc-HP) immobilization. In the resting state, proximal Fc efficiently quenches the CsPbBr3 ECL. Target miRNA activates Cas13a to cleave a dumbbell probe and release an intermediate strand that hybridizes with Fc-HP; subsequent Nb.BbvCI nicking removes Fc from the electrode and is recycled, producing robust signal restoration. Morphology (TEM), composition (EDS/XPS), and stepwise electrochemistry (CV/EIS) verify a core-shell-Au architecture and a reliably assembled interface that follows the expected quench→restore behavior. Under optimized conditions (0.5 mg mL[-1] CsPbBr3@SiO2@Au, 2.0 μM Fc-HP, 40 min target incubation, 100 mM TPrA, 120 s pre-reaction), the assay affords a 1 aM-1.0 × 10[9] aM linear range with an estimated limit of detection (LOD) of 1.86 aM. The sensor shows high specificity against homologous sequences and achieves 95.22%-104.61% recoveries with RSD < 5% in spiked serum. Pilot measurements distinguish patient serum samples from healthy controls, underscoring clinical potential. This modular platform couples stable perovskite ECL emission with programmable CRISPR chemistry, offering a sensitive, selective, and water-compatible route for microRNA analysis and readily extensible nucleic-acid diagnostics.},
}
@article {pmid41534449,
year = {2026},
author = {Kirino, Y and Takeno, M},
title = {A critical look at animal and cellular models in autoinflammatory diseases.},
journal = {Current opinion in immunology},
volume = {99},
number = {},
pages = {102719},
doi = {10.1016/j.coi.2025.102719},
pmid = {41534449},
issn = {1879-0372},
mesh = {Animals ; Humans ; Disease Models, Animal ; *Autoimmune Diseases/immunology/genetics/etiology ; Gene Editing ; Induced Pluripotent Stem Cells ; *Inflammation/immunology ; CRISPR-Cas Systems ; *Hereditary Autoinflammatory Diseases ; },
abstract = {Autoinflammatory diseases (AIDs) comprise a diverse group of conditions arising from dysregulated immune control due to congenital or acquired genetic abnormalities in innate immune pathways, and patients typically require lifelong treatment. Owing to their rarity, access to patient samples is limited, making animal and cellular models indispensable for elucidating pathogenesis and advancing therapeutic development. Nevertheless, robust animal and cell-based models remain scarce. Recent advances in precision genome editing now enable lineage- and cell type-specific modeling of autoinflammation, steadily improving the fidelity with which disease phenotypes are recapitulated. In this review, we survey the current landscape of CRISPR-enabled knock-in/knock-out animal models, engineered cell lines, and patient-derived induced pluripotent stem cells for AIDs, and discuss how these platforms can be leveraged to dissect disease mechanisms and accelerate drug discovery.},
}
@article {pmid41506441,
year = {2026},
author = {Mokhles, F and Moosavi, MA and Gutierrez-Uzquiza, A and Velasco, G and Li, M and Cordani, M},
title = {Unraveling stress-adaptation pathways in cancer: Functional dissection through CRISPR-based genetic screens.},
journal = {Cancer letters},
volume = {644},
number = {},
pages = {218246},
doi = {10.1016/j.canlet.2026.218246},
pmid = {41506441},
issn = {1872-7980},
mesh = {Humans ; *Neoplasms/genetics/pathology/metabolism ; *CRISPR-Cas Systems ; Animals ; Endoplasmic Reticulum Stress/genetics ; *Genetic Testing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Signal Transduction ; Tumor Microenvironment ; Unfolded Protein Response/genetics ; *Stress, Physiological ; Adaptation, Physiological/genetics ; },
abstract = {Cancer cells face a hostile microenvironment characterized by hypoxia, nutrient deprivation, endoplasmic reticulum (ER) stress, and oxidative imbalance. To cope with these challenges, they activate an interconnected network of adaptive pathways including autophagy, the unfolded protein response, metabolic reprogramming, and the integrated stress response., which promote cell survival, therapy resistance, immune evasion, and metastasis. CRISPR-based functional genomics has emerged as a powerful strategy to systematically dissect these stress-adaptive networks, enabling the identification of key regulators and vulnerabilities across diverse contexts. In this review, we first summarize tumor progression in major stress conditions and then highlight how CRISPR screening strategies ranging from genome-wide loss-of-function studies to single-cell and combinatorial platforms, are unraveling critical stress regulators. We further discuss emerging tools, model systems, and translational perspectives, underscoring how the integration of CRISPR technologies with multi-omics, artificial intelligence, and advanced preclinical models is reshaping our understanding of cancer stress biology and guiding the development of novel therapeutic strategies. Finally, we addressed how these novel dissection technologies influence translational opportunities, specifically in the context of combining stress-pathway modulators with immunotherapy and targeted therapy drugs.},
}
@article {pmid41763755,
year = {2026},
author = {Ma, C and French, N and Wu, X and Gupta, SK and Gupta, TB},
title = {Molecular detection of Clostridium and Bacillus species in foods: recent advances and applications.},
journal = {Food research international (Ottawa, Ont.)},
volume = {229},
number = {},
pages = {118370},
doi = {10.1016/j.foodres.2026.118370},
pmid = {41763755},
issn = {1873-7145},
mesh = {*Clostridium/isolation &amp; purification/genetics ; *Bacillus/isolation &amp; purification/genetics ; *Food Microbiology/methods ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction ; CRISPR-Cas Systems ; In Situ Hybridization, Fluorescence ; Humans ; Food Contamination/analysis ; },
abstract = {Spore-forming bacteria, especially Clostridium spp. and Bacillus spp., are ubiquitous in food systems, and their ingestion can cause serious diseases in humans and animals. Their persistence in diverse food matrices and resistance to conventional treatments make rapid and accurate detection essential for effective monitoring and control. Traditional culture-based and biochemical assays remain the standard for identifying these bacteria but are often time-consuming, labor-intensive and limited in sensitivity. In contrast, nucleic acid-based methods provide rapid, specific and sensitive alternatives by directly targeting genetic markers of pathogenic or spoilage strains. This review summarizes how nucleic acid methods, including PCR, FISH, LAMP, RPA, WGS, and the emerging CRISPR/Cas systems, have been applied specifically to detect Clostridium spp. and Bacillus spp. in food systems. Each method offers unique advantages and limitations. PCR-based methods enable accurate quantification but require thermal cycling. FISH-based methods are simple but require microscopy and have limited validation in food. WGS-based methods provide strain-level characterization but depend on informatics and specialized equipment. Isothermal techniques such as LAMP- and RPA-based methods allow rapid field detection but involve complex primer design or poor discrimination of closely related genes. CRISPR/Cas-based platforms further enhance simplicity, specificity, sensitivity for on-site detection, though the validation for spore-forming bacteria remains limited. Overall, this review provides an overview of gene targets, methodological adaptations, and analytical performance of nucleic acid-based assays for detecting Clostridium spp. and Bacillus spp., highlighting current progress and future opportunities for improving food safety monitoring.},
}
@article {pmid41762821,
year = {2026},
author = {Golla, DA and Sun, C and Haugh, L and Straub, N and Gao, X},
title = {Advances in multiplex precision genome editing in eukaryotic and prokaryotic systems.},
journal = {Current opinion in biotechnology},
volume = {99},
number = {},
pages = {103470},
doi = {10.1016/j.copbio.2026.103470},
pmid = {41762821},
issn = {1879-0429},
abstract = {Multiplex genome editing (MGE) enables coordinated modification of multiple genomic loci and is foundational for engineering complex biological traits. Traditional CRISPR-Cas nuclease-based strategies rely on DNA double-strand breaks (DSBs), which limit precision and pose scaling challenges for incorporating simultaneous edits across different loci. Recent advances in genome editing technologies that operate without generating DSBs have expanded the accuracy and feasibility of multiplexed genomic manipulation. This review focuses on emerging strategies for precise MGE, including base editing, prime editing, and related genome rewriting platforms. We highlight key engineering principles that impact the success of scalable multiplexing, including the choice of editing platform, edit size, and guide RNA architecture, and discuss applications across mammalian, plant, fungal, and bacterial systems. Together, these technologies establish MGE as a versatile framework for precise multigene control in biotechnology and agriculture.},
}
@article {pmid41761908,
year = {2026},
author = {Perry, TN and Mais, CN and Sanchez-Londono, M and Steinchen, W and Plitzko, PA and Randau, L and Pausch, P and Innis, CA and Bange, G},
title = {Structural basis of Cas8-independent Cas3 recruitment in Type I-F2 CRISPR-Cas.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41761908},
issn = {1362-4962},
support = {//Deutsche Forschungsgemeinschaft/ ; 260989694//DFG/ ; 324652314//DFG/ ; 405858511//DFG/ ; 3869//DFG/ ; //Inserm/ ; 5342-2023//Research Council of Lithuania/ ; //Marburg University/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; DNA/chemistry/metabolism/genetics ; Models, Molecular ; DNA Helicases/chemistry/metabolism/genetics ; Protein Domains ; Protein Binding ; *Bacterial Proteins/chemistry/metabolism/genetics ; },
abstract = {CRISPR-Cas systems provide adaptive immunity in prokaryotes by targeting and degrading invasive genetic elements. Among them, the Type I-F2 system represents the most compact Type I CRISPR-Cas variant, distinguished by the complete absence of both large (Cas8) and small (Cas11) subunits. In other Type I systems, Cas8 is essential for protospacer adjacent motif (PAM) recognition and for triggering Cas3 recruitment, while Cas11 stabilizes the Cascade backbone and guides the nontarget DNA strand during R-loop formation. To elucidate how I-F2 executes interference in their absence, we determined the cryo-electron microscopy structure of the I-F2 Cascade bound to target DNA and Cas3. Our structure reveals that Cas5 alone mediates PAM sensing, while Cas7 subunits directly recruit Cas3, which adopts a helicase-loaded conformation compatible with DNA engagement. We show how the helicase and C-terminal domains of Cas3 capture the displaced nontarget strand to initiate directional unwinding and degradation. These findings uncover key mechanistic adaptations that enable efficient interference without canonical large and small subunits and emphasize the mechanistic diversity among closely related Type I systems, including I-E, I-F1, and I-F2. These insights provide a structural basis for engineering the hypercompact I-F2 system for genome editing and biotechnological applications.},
}
@article {pmid41759621,
year = {2026},
author = {Yan, X and Chen, M and Yang, S and Guo, Y and Dai, Y and Chen, Y and Zhong, H and Ma, T and Zha, D and He, Y and Li, B and Jia, X and Guo, L and Hu, J and Wei, Y and Chen, X},
title = {Mitochondrial genome editing tools: prospects in animal breeding.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgg.2026.02.018},
pmid = {41759621},
issn = {1673-8527},
abstract = {Mitochondria are vital organelles responsible for driving cellular energy metabolism and regulating key biological processes. Their circular mitochondrial DNA (mtDNA) encodes 13 subunits of the respiratory chain proteins but is susceptible to mutations due to high levels of reactive oxygen species and limited repair mechanisms. Mutant phenotypes manifest only when heteroplasmy surpasses a critical threshold. Understanding the consequences of mtDNA mutations has long been hampered by the lack of precise editing tools. Recently, CRISPR-free, protein-only mitochondrial base editors have enabled C·G-to-T·A and A·T-to-G·C transitions. These breakthroughs facilitate the creation of relevant disease models and offer unique opportunities for animal breeding, as specific mtDNA variants are known to influence economically important traits in livestock, including production, reproduction, and stress tolerance. This review summarizes recent advances in mitochondrial genome editing technologies, including CRISPR/Cas-based systems, restriction endonucleases, double-stranded DNA deaminase toxin A (DddA)-based cytosine and adenine base editors, and DddA-free base editors, along with their delivery strategies and optimization avenues. Furthermore, we outline the associations between mtDNA polymorphisms, copy number variation, and economic traits in livestock and poultry. Finally, we discuss the potential applications of mitochondrial genome editing in animal breeding and highlight the critical safety and ethical considerations that require careful attention.},
}
@article {pmid41759529,
year = {2026},
author = {Cao, Z and Yu, S and Peng, J and Barrett, DR and Liu, Y and Sussman, JH and Chen, C and Thadi, A and Liu, L and Alikarami, F and Xu, J and Carroll, MP and Tan, K and Bernt, KM and Shi, J},
title = {CRISPR-based functional genomics for dissecting therapeutic dependency in primary acute myeloid leukemia samples.},
journal = {Molecular cell},
volume = {86},
number = {5},
pages = {968-985.e7},
doi = {10.1016/j.molcel.2026.02.003},
pmid = {41759529},
issn = {1097-4164},
support = {R01 CA262260/CA/NCI NIH HHS/United States ; U01 CA243072/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/pathology/drug therapy/therapy ; *CRISPR-Cas Systems ; Animals ; *Genomics/methods ; Mice ; Single-Cell Analysis ; Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; Gene Editing ; Gene Expression Regulation, Leukemic ; Mice, Inbred NOD ; },
abstract = {Cancer functional genomics enables high-throughput target discovery and mechanistic investigation, yet its application has remained largely confined to mouse models and established human cancer cell lines. Direct functional interrogation of heterogeneous primary tumors offers a powerful opportunity to evaluate therapeutic targets and uncover cancer dependencies or resistance mechanisms. Here, we developed an optimized CRISPR-based platform for functional genomics in patient-derived xenograft and primary acute myeloid leukemia (AML) samples harboring diverse pathogenic mutations. Integrated in vitro and in vivo CRISPR-Cas9 knockout and CRISPR interference (CRISPRi) dropout screens validated known AML-biased targets and identified cis-regulatory elements essential for leukemic growth. Coupling pooled CRISPR perturbations with single-cell RNA sequencing (Perturb-seq) further resolved the perturbation-induced alterations in regulatory networks, cell cycle states, and cellular hierarchies in primary AML samples. Together, these studies establish a general and robust framework for leveraging CRISPR-based functional genomics to directly dissect cancer dependencies and cellular heterogeneity in primary AML patient samples.},
}
@article {pmid41742419,
year = {2026},
author = {Zhu, M and Yuan, J and Meng, Q and Yu, J and Xu, X and Xu, M and Ren, X and Hu, Y and Wei, G and Jia, Z and Yuan, G and Zang, L and Liu, S and Yang, Y and Zheng, Y and Wang, J and Cong, T and Xie, W and Lan, X and Cong, L and Ma, T and Ding, S and Guo, W and Zhang, X and Li, Y},
title = {Minimizing far-extending chromatin perturbation in genome editing preserves stem cell identity.},
journal = {Cell stem cell},
volume = {33},
number = {3},
pages = {470-486.e14},
doi = {10.1016/j.stem.2026.01.015},
pmid = {41742419},
issn = {1875-9777},
mesh = {*Gene Editing/methods ; *Chromatin/metabolism/genetics ; Animals ; Mice ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; *Neural Stem Cells/metabolism/cytology ; Mouse Embryonic Stem Cells/metabolism/cytology ; CCCTC-Binding Factor/metabolism ; },
abstract = {Although CRISPR-Cas9 holds therapeutic promise, broader application demands an understanding of complications in vast non-coding regions. We found that CRISPR-Cas9 can cause premature differentiation of neural stem cells in vivo and mouse embryonic stem cells in vitro, even when cleavage occurred at distant sites tens of kilobases away from the nearest regulatory elements. To investigate this, we employed an integrated assay for transposase-accessible chromatin (ATAC)/RNA sequencing (AR-seq) approach and identified editing-induced chromatin accessibility changes, with their scale varying by cell type. Cells with stemness are most affected, experiencing perturbations that extend over a hundred kilobases. Furthermore, even local DNA perturbations can disrupt CTCF- and condensate-associated chromatin architecture, causing distal transcriptional rewiring and, ultimately, loss of stemness identity. To minimize chromatin perturbations and preserve cell identity, we refined gene-editing strategies, including distance-aware sgRNA design, pharmacological attenuation of DNA resection, and alternative editing systems. This work paves the way for the safer and broader application of genome-editing technologies.},
}
@article {pmid41679300,
year = {2026},
author = {Wang, Y and Hu, W and Xia, R and Yang, X and Gu, Y and Ning, Z and Yang, T and Yu, C and Zhang, L and Li, D and Jin, Y and Li, J and Zhang, F and Xu, Y and Xu, C and Wang, Z and Jing, N and Chen, L and Wang, G},
title = {CLIM-TIME identifies metastatic microenvironment modulators for T cell therapy response.},
journal = {Cell},
volume = {189},
number = {5},
pages = {1555-1572.e23},
doi = {10.1016/j.cell.2025.12.042},
pmid = {41679300},
issn = {1097-4172},
mesh = {*Tumor Microenvironment/immunology/genetics ; Animals ; *T-Lymphocytes/immunology ; Mice ; Humans ; Immunotherapy/methods ; Laser Capture Microdissection/methods ; Mice, Inbred C57BL ; Lung Neoplasms/secondary/immunology/therapy ; Cell Line, Tumor ; Extracellular Matrix/metabolism ; CRISPR-Cas Systems ; Neoplasm Metastasis ; Neoplasms/immunology/therapy/pathology ; },
abstract = {The tumor microenvironment (TME) poses a major barrier to effective immunotherapy, yet high-throughput perturbation-mapping approaches to dissect TME spatial complexity and its contextual immune modulators remain lacking. Here, we introduce CRISPR-laser-captured microdissection (LCM) integration mapping of the tumor-immune microenvironment (CLIM-TIME), a scalable platform that integrates CRISPR screening with LCM of metastatic tumors for transcriptomic, deconvolution, and immunofluorescence analyses. CLIM-TIME enables spatially resolved mapping of how tumor suppressor gene (TSG) loss reshapes the TME and modulates immune responses. We identified seven distinct TME subtypes, revealing that DNA repair and Polycomb repressive complex (PRC) TSG loss is linked to immune-infiltrated TMEs sensitive to T cell therapy. In contrast, knockouts of TSGs in the Hippo pathway promoted immune evasion and therapy resistance by fostering myeloid-enriched but T cell-excluded TMEs with elevated extracellular matrix (ECM). Targeting the ECM-crosslinking enzyme LOXL2 effectively remodeled the metastatic TME, enhancing T cell infiltration and improving therapeutic efficacy in lung metastases across multiple cancers.},
}
@article {pmid41636073,
year = {2026},
author = {Zhu, X and Gu, G and Shen, Y and Abdurazik, M and Sun, G},
title = {CRISPR/Cas13a-induced self-priming cyclic amplification enables liquid biopsy of exosomal circular RNA in non-small cell lung cancer.},
journal = {The Analyst},
volume = {151},
number = {5},
pages = {1413-1419},
doi = {10.1039/d5an01345c},
pmid = {41636073},
issn = {1364-5528},
mesh = {*RNA, Circular/genetics/blood ; *Carcinoma, Non-Small-Cell Lung/genetics/diagnosis ; Humans ; *Lung Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; *Exosomes/genetics/chemistry ; Liquid Biopsy/methods ; *Nucleic Acid Amplification Techniques/methods ; DNA Primers/genetics ; },
abstract = {The precise and reliable identification of circular RNA (circRNA) is essential for both biological studies and clinical diagnostics of non-small cell lung cancer (NSCLC), especially the exosomal circRNA. In this study, we utilize a CRISPR/Cas13a system to specifically recognize the unique back-splice junction of target circRNA and develop a novel detection platform termed CRISPR/Cas13a-induced self-priming cyclic amplification. This method enables highly sensitive and specific circRNA detection. A pair of stem-loop DNA primers was carefully designed, each incorporating complementary single-stranded DNA sequences and five ribouridine (rU) residues at the 3' end serving as an overhang. When Cas13a binds to the target circRNA, its trans-cleavage activity is activated, leading to the cleavage of the rU residues. This cleavage permits the 3' ends of the stem-loop primers to extend along one another, generating multiple double stem-loop DNA structures that initiate successive cycles of self-priming chain elongation. By leveraging the sustained trans-cleavage activity of Cas13a and the high amplification efficiency of the self-priming cyclic reaction, the assay achieves sensitive detection of circRNA at concentrations as low as 564 aM within 90 min. In addition, the proposed method has been successfully applied for the analysis of exosomal hsa_circ_0003026 expression level in normal samples and NSCLC samples and demonstrated the potential of exosomal hsa_circ_0003026 in regulating the pathological progression. Owing to the high specificity of Cas13a, the proposed method can be directly applied to detect circRNA in complex biological samples without prior isolation of corresponding linear RNAs.},
}
@article {pmid41622198,
year = {2026},
author = {Speth, ZJ and Pokhrel, V and Featherston, KM and Rehard, DG and Reid, WR and Franz, AWE},
title = {Monoallelic knockout of r2d2 affects the antiviral RNAi response to Mayaro virus and the reproductive potential in Aedes aegypti.},
journal = {Parasites &amp; vectors},
volume = {19},
number = {1},
pages = {},
pmid = {41622198},
issn = {1756-3305},
support = {R01 AI134661/AI/NIAID NIH HHS/United States ; R56 AI167980/AI/NIAID NIH HHS/United States ; R01-AI134661 (awarded to A.W.E.F), R56-AI167980 (awarded to A.W.E.F), and R56-AI180215 (awarded to Dr. Stefan Rothenburg, UC Davis)//National Institutes of Health - National Institute for Allergy and Infectious Diseases (NIH-NIAID)/ ; },
mesh = {Animals ; *Aedes/virology/genetics/physiology/immunology ; *RNA Interference ; Female ; *Alphavirus/physiology/immunology ; Mosquito Vectors/virology/genetics ; Gene Knockout Techniques ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Reproduction ; Fertility ; Alleles ; Argonaute Proteins/genetics ; RNA, Small Interfering/genetics ; },
abstract = {BACKGROUND: Aedes aegypti is an important vector for several human-pathogenic arboviruses. RNAi is the principal antiviral immune pathway in mosquitoes. Key steps of antiviral RNAi are processing of long dsRNAs into siRNA duplexes by dicer-2; loading of the siRNA duplexes onto Argonaute-2 with the help of R2D2; RISC formation via incorporation of Argonaute-2, which contains an siRNA; RISC-mediated targeting and degradation of homologous viral RNAs. Here, we generated an r2d2 knockout mosquito line to reveal how RNAi impairment during RISC loading complex (RLC) formation would affect arbovirus infection of Ae. aegypti.<br><br>METHODS: CRISPR/Cas9 gene editing has been used to knock out r2d2 in Ae. aegypti. Crossing experiments were conducted to reveal the effects of loss of r2d2 function on fecundity and fertility. Mayaro virus (Togaviridae: MAYV) infection and RNAi pathway gene expression levels were monitored using time-course RT-qPCR assays. Small RNA profiling was conducted to determine small RNA abundance in &#x394;R2D2[(+/-)] mosquitoes.<br><br>RESULTS: We show that in Ae. aegypti, the r2d2 allele is linked to the sex determination locus on chromosome 1. It was not possible to generate homozygous &#x394;R2D2[(-/-)] mosquitoes, indicating that complete loss of r2d2 function is lethal to Ae. aegypti. Our observations suggest that r2d2 function is not limited to RNAi but also affects mosquito fecundity/fertility, likely through follicle development. Monoallelic disruption of r2d2 increased the replication of MAYV, and r2d2 expression was also increased in infected mosquitoes. MAYV infection of &#x394;R2D2[(+/-)] mosquitoes was associated with an increase in abundance of putative vpiRNAs. However, impairment of r2d2 did not affect the function of dicer-2, as there was no difference in the 21&#xa0;nt siRNA profiles between the &#x394;R2D2[(+/-)] mosquitoes and the non-transgenic control.<br><br>CONCLUSIONS: The RNAi pathway gene, r2d2, is an essential gene, and it is not possible to generate mosquitoes with biallelic (complete) loss of r2d2 function. Monoallelic impairment of r2d2 compromises the siRNA pathway downstream of dicer-2 function, at the point of RLC formation. In MAYV-infected mosquitoes, this defect in siRNA pathway function is compensated for by an increased piRNA pathway activity, which moderates increases in viral replication over a 10-day period.},
}
@article {pmid41610849,
year = {2026},
author = {Samelson, AJ and Ariqat, N and McKetney, J and Rohanitazangi, G and Parra Bravo, C and Bose, RS and Travaglini, KJ and Lam, VL and Goodness, D and Ta, T and Dixon, G and Marzette, E and Jin, J and Tian, R and Tse, E and Abskharon, R and Pan, HS and Carroll, EC and Lawrence, RE and Gestwicki, JE and Rexach, JE and Eisenberg, DS and Kanaan, NM and Southworth, DR and Gross, JD and Gan, L and Swaney, DL and Kampmann, M},
title = {CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis.},
journal = {Cell},
volume = {189},
number = {5},
pages = {1517-1534.e19},
doi = {10.1016/j.cell.2025.12.038},
pmid = {41610849},
issn = {1097-4172},
support = {U54 NS123746/NS/NINDS NIH HHS/United States ; R01 AG075802/AG/NIA NIH HHS/United States ; K99 AG080116/AG/NIA NIH HHS/United States ; R01 AG085357/AG/NIA NIH HHS/United States ; U19 AG060909/AG/NIA NIH HHS/United States ; R01 AG070895/AG/NIA NIH HHS/United States ; R00 AG080116/AG/NIA NIH HHS/United States ; R01 AG082141/AG/NIA NIH HHS/United States ; U54 NS100717/NS/NINDS NIH HHS/United States ; F32 AG063487/AG/NIA NIH HHS/United States ; R01 AG062359/AG/NIA NIH HHS/United States ; P30 CA082103/CA/NCI NIH HHS/United States ; R01 AG060477/AG/NIA NIH HHS/United States ; U24 AG072458/AG/NIA NIH HHS/United States ; U54 AI170792/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *tau Proteins/metabolism/genetics ; *Neurons/metabolism/cytology ; *Proteostasis ; Tauopathies/metabolism/pathology/genetics ; Ubiquitin-Protein Ligases/metabolism ; *CRISPR-Cas Systems/genetics ; Ubiquitination ; Mitochondria/metabolism ; Proteasome Endopeptidase Complex/metabolism ; },
abstract = {Aggregation of the protein tau defines tauopathies, the most common age-related neurodegenerative diseases, which include Alzheimer's disease and frontotemporal dementia. Specific neuronal subtypes are selectively vulnerable to tau aggregation, dysfunction, and death. However, molecular mechanisms underlying cell-type-selective vulnerability are unknown. To systematically uncover the cellular factors controlling the accumulation of tau aggregates in human neurons, we conducted a genome-wide CRISPRi screen in induced pluripotent stem cell (iPSC)-derived neurons. The screen uncovered both known and unexpected pathways, including UFMylation and GPI anchor biosynthesis, which control tau oligomer levels. We discovered that the E3 ubiquitin ligase CRL5[SOCS4] controls tau levels in human neurons, ubiquitinates tau, and is correlated with resilience to tauopathies in human disease. Disruption of mitochondrial function promotes proteasomal misprocessing of tau, generating disease-relevant tau proteolytic fragments and changing tau aggregation in vitro. These results systematically reveal principles of tau proteostasis in human neurons and suggest potential therapeutic targets for tauopathies.},
}
@article {pmid41431195,
year = {2026},
author = {Spezzani, E and Capelli, L and Di Lena, D and Chamorro-Garcia, A and Ippodrino, R and Porchetta, A and Bertucci, A},
title = {MARPLE: A Proximity-Triggered CRISPR-Cas13 Platform for Ultrasensitive Antibody Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {13},
pages = {e17799},
pmid = {41431195},
issn = {2198-3844},
support = {//National Recovery and Resilience Plan/ ; MUR 2023-2027//'Departments of Excellence' program of the Italian Ministry for University and Research/ ; 31108//Fondazione AIRC per la ricerca sul cancro ETS/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Antibodies/analysis ; Immunoassay/methods ; },
abstract = {Monitoring clinically relevant antibodies-as biomarkers of disease or therapeutic response-is essential for informed clinical decision-making. Traditional immunoassays like ELISA offer reliable quantification but often involve multistep workflows and limited point-of-care utility. New approaches coupling antibody recognition with signal amplification are therefore highly desirable. The CRISPR-Cas13 system, known for its potent collateral cleavage activity, has emerged as a powerful diagnostic tool for nucleic acid detection. However, its application to protein biomarkers such as antibodies remains underdeveloped. Here, we introduce MARPLE (Modular Antibody Recognition via Proximity-triggered Linker Exchange), a modular CRISPR-Cas13-based platform for ultrasensitive antibody detection. MARPLE harnesses antibody-induced proximity to trigger a strand displacement reaction that releases a sequestered RNA target, activating Cas13-mediated collateral cleavage of fluorescent RNA reporters. This cascade enables detection of antibodies at femtomolar concentrations. We demonstrate MARPLE's versatility across diverse targets-including anti-digoxigenin, anti-cholesterol, anti-HA, trastuzumab, and anti-MUC1-highlighting applications in infectious disease monitoring, cancer diagnostics, and therapeutic drug tracking. The assay is isothermal, one-pot, and retains robust performance in complex matrices such as human serum. These features establish MARPLE as a promising tool for immunodiagnostics, extending CRISPR-based sensing beyond nucleic acids to protein biomarker detection.},
}
@article {pmid40898426,
year = {2026},
author = {Sahin, U},
title = {Cas9 beyond CRISPR - SUMOylation, effector-like potential and pathogenic adaptation.},
journal = {The FEBS journal},
volume = {293},
number = {5},
pages = {1285-1296},
doi = {10.1111/febs.70256},
pmid = {40898426},
issn = {1742-4658},
support = {223Z048//T&#xfc;rkiye Bilimsel ve Teknolojik Ara&#x15f;t&#x131;rma Kurumu/ ; 121C230//T&#xfc;rkiye Bilimsel ve Teknolojik Ara&#x15f;t&#x131;rma Kurumu/ ; YIN IG3336//European Molecular Biology Organization/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Sumoylation/genetics ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism ; Gene Editing ; Protein Processing, Post-Translational ; *Bacteria/pathogenicity/genetics ; Host-Pathogen Interactions/genetics ; Animals ; },
abstract = {The CRISPR/Cas9 system has revolutionized molecular biology and gene editing, yet key aspects of its regulation, especially within eukaryotic environments, remain enigmatic. In this Viewpoint article, I will speculate on and explore the provocative hypothesis that Cas9 may possess previously unrecognized effector-like functions when expressed in host cells, potentially shaped by host-mediated post-translational modifications (PTMs). Of particular interest is SUMOylation at lysine 848, a key residue for DNA binding within the catalytic site, raising the possibility that this modification is not incidental, but functionally significant and precisely regulated. SUMOylation, a eukaryotic PTM, is increasingly recognized as a mechanism that also targets bacterial and viral effector proteins and virulence factors during infection, exerting context-dependent effects that may either enhance or hinder pathogen replication. Could Cas9, beyond its canonical role in bacterial CRISPR immunity, act as a host-modulating effector during infection, akin to known bacterial nucleomodulins such as transcription activator-like (TAL) effectors? If so, this would imply that certain pathogenic bacteria may have evolved Cas9 variants capable of exploiting host PTM machinery and targeting the host genome-an adaptation with potential implications for microbial virulence, host-pathogen interactions, and co-evolutionary dynamics. This perspective underscores the importance of systematically mapping Cas9 PTMs and examining their evolutionary conservation, functional significance, and pharmacological tunability, not only for basic biological insight and to deepen our understanding of microbial strategies, but also to refine the precision and safety of Cas9-based therapeutic platforms.},
}
@article {pmid41759295,
year = {2026},
author = {Molina, C and Knight, AL and Lisi, GP},
title = {Comparative thermodynamic and kinetic properties governing the nucleic acid interactions of CRISPR-Cas9 and Cas12a.},
journal = {Physical biology},
volume = {},
number = {},
pages = {},
doi = {10.1088/1478-3975/ae4b7f},
pmid = {41759295},
issn = {1478-3975},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeat-associated proteins (CRISPR-Cas) biochemistry has been leveraged in genome editing applications in biochemical research and therapeutics. CRISPR-Cas9 and CRISPR-Cas12a are the two most widely used RNA-guided endonucleases and while Cas9 and Cas12a have a shared function, both have unique biophysical properties that alter their specificity and efficiency. The thermodynamic and kinetic properties governing their molecular interactions, recognition and binding of target DNA, and R-loop formation can differ. In some cases, these critical biophysical metrics have not been resolved. Distinctions between Cas9 and Cas12a enzymes are also prevalent in RNA:DNA hybrid binding affinities, DNA localization relative to the preferred PAM site and DNA cleavage mechanism. In this review, we examine the biophysical properties of both endonucleases, focused on the nucleic acid interactions that confer specificity and function. Complementing this biophysical overview, we discuss case studies in disparate model organisms that compare the genome editing and fidelity of Cas9 and Cas12a.},
}
@article {pmid41758946,
year = {2026},
author = {Saito, R and Umemura, Y and Makino, S and Fukaya, T},
title = {Decoding the molecular logic of rapidly evolving ZAD zinc finger proteins in Drosophila.},
journal = {Science advances},
volume = {12},
number = {9},
pages = {eady7568},
pmid = {41758946},
issn = {2375-2548},
mesh = {Animals ; *Drosophila Proteins/metabolism/genetics/chemistry ; *Zinc Fingers ; *Transcription Factors/metabolism/genetics ; *Evolution, Molecular ; Protein Binding ; *Drosophila/genetics/metabolism ; *Drosophila melanogaster/genetics/metabolism/embryology ; DNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; CCCTC-Binding Factor ; Microtubule-Associated Proteins ; Nuclear Proteins ; },
abstract = {The zinc finger-associated domain (ZAD)-containing C2H2 zinc finger proteins (ZAD-ZnFs) represent the most abundant class of transcription factors that emerged during insect evolution, yet their molecular diversity and biological functions remain largely unclear. Here, we established a systematic CRISPR-based protein-tagging approach that enables direct, unambiguous comparison of nuclear localization and genome-wide binding profiles of endogenous ZAD-ZnFs in developing Drosophila embryos. Evidence is provided that a subset of ZAD-ZnFs forms nuclear condensates through the stacking of the N-terminal ZAD dimerization surface. Disruption of condensation activity leads to misregulation of genome-wide binding profiles and lethality, underscoring its functional and physiological significance in development. Integrative chromatin immunoprecipitation sequencing and Micro-C analyses reveal that many ZAD-ZnFs colocalize with core insulator proteins such as CCCTC-binding factor and Centrosomal protein 190 kD to control the formation of topological boundaries. We suggest that the diverse molecular functions of ZAD-ZnFs have evolutionarily arisen from their ancestral role as insulator-binding proteins.},
}
@article {pmid41758452,
year = {2026},
author = {Bharti S, AK and Mukherjee, AG and Gopalakrishnan, AV and Gajendran, B and Vashishth, R and Prince, SE},
title = {From bench to bedside: stem cell therapy as a transformative approach against HIV.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {41758452},
issn = {1573-4978},
mesh = {Humans ; *HIV Infections/therapy/virology/immunology ; *Hematopoietic Stem Cell Transplantation/methods ; Gene Editing/methods ; HIV-1 ; *Stem Cell Transplantation/methods ; Induced Pluripotent Stem Cells ; Translational Research, Biomedical ; Receptors, CCR5/genetics ; CRISPR-Cas Systems ; Virus Latency ; },
abstract = {Human immunodeficiency virus (HIV) remains a persistent global health burden, as combination antiretroviral therapy (ART) achieves sustained viral suppression but fails to eliminate long-lived latent reservoirs. Stem cell-based therapeutic strategies have emerged as transformative approaches with the potential to induce durable remission and, ultimately, a functional cure. Clinical proof-of-concept has been established through allogeneic hematopoietic stem cell transplantation (HSCT) using CCR5Δ32/Δ32 donor cells, demonstrating that durable resistance to viral entry can result in prolonged HIV remission. Building on these landmark observations, recent advances in autologous gene-edited hematopoietic stem and progenitor cells and induced pluripotent stem cell (iPSC)-derived immune effectors have accelerated the development of scalable, patient-specific interventions. The convergence of stem cell biology with precision genome-editing platforms, including CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs), has enabled targeted disruption of viral entry pathways and host dependency factors, while offering new strategies to address viral latency and immune reconstitution. Despite significant challenges related to treatment-associated toxicity, manufacturing complexity, long-term safety, and ethical considerations, rapid progress in cellular engineering and translational immunology continues to advance the field toward curative outcomes. This review critically synthesizes recent progress in stem cell-based HIV therapeutics, elucidates the underlying mechanistic frameworks, evaluates emerging clinical and preclinical evidence, and outlines future directions required to achieve a durable functional cure.},
}
@article {pmid41758321,
year = {2026},
author = {Pathak, A and Singh, J and Swati, and Dwibedi, V},
title = {Deciphering microbial biofilm: mechanism, infection, and advanced approaches for control.},
journal = {Folia microbiologica},
volume = {},
number = {},
pages = {},
pmid = {41758321},
issn = {1874-9356},
abstract = {Microbial biofilms are densely organised microbial communities that adhere to biotic and abiotic surfaces, encased within an extracellular polymeric substance (EPS). Microorganisms within these biofilm structures gain enhanced protection, versatility, and resistance to external stresses, antibiotics, and host immune systems. The biofilm formation follows a series of steps, including initial microbial adherence, microcolony establishment, EPS production, regulation by quorum sensing (QS), and dispersal. This flexibility enables biofilm survival in multiple environments, such as medical devices and natural systems, posing serious challenges in healthcare, agricultural, and industrial sectors. The review focuses on the mechanisms involved in biofilm formation and discusses the role of EPS in promoting biofilm stability and resistance to antimicrobials. It addresses biofilm-associated infections in medical environments, such as chronic wounds, cystic fibrosis, urinary tract infections (UTIs), and complications with implanted medical devices. The capacity of biofilm-forming microorganisms to evade immune responses and persist through extended antibiotic use highlights the urgent demand for novel therapeutic approaches. The discussion includes emerging strategies for biofilm control, including anti-biofilm agents, QS inhibitors, enzymatic treatments, and innovative combination therapies combining antibiotics with biofilm-disrupting agents. Emerging technologies, like antimicrobial peptides (AMPs), CRISPR-Cas systems, nanotechnology, and bioelectric therapies, present innovative biofilm disruption and removal approaches. This paper discusses the effectiveness of natural products, plant-derived compounds, and bacteriophage therapies for mitigating biofilm-associated infections linked to biofilms. The review examines the dynamic challenges posed by biofilms, particularly their role in chronic and device-related infections, which contribute to significant healthcare complications. The study highlights the significance of adopting new approaches to overcome biofilm-induced antimicrobial resistance (AMR) and improve therapeutic outcomes. Furthermore, this paper discusses the promising potential of emerging technologies, such as nanomaterials, QS interference, and biofilm-specific antimicrobial agents, in enhancing biofilm control and prevention measures across clinical, industrial, and environmental domains.},
}
@article {pmid41757824,
year = {2026},
author = {Chen, J and Davison, CW and Ellis, J and Blevins, B and Presley, W and Myers, MT and Kong, D and Hou, Z and Mian, SI and Prasov, L and Zhang, Y},
title = {CRISPR Base Editing Correction of TGFBI Mutations in Autosomal Dominant Corneal Dystrophies.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {67},
number = {2},
pages = {60},
pmid = {41757824},
issn = {1552-5783},
mesh = {Humans ; *Corneal Dystrophies, Hereditary/genetics/therapy/metabolism ; *Gene Editing/methods ; *Mutation ; *Transforming Growth Factor beta/genetics/metabolism ; *Genetic Therapy/methods ; Epithelium, Corneal/metabolism/pathology ; *Extracellular Matrix Proteins/genetics ; *CRISPR-Cas Systems ; Dependovirus/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {PURPOSE: Lattice and granular corneal dystrophy comprise two common TGFBI-associated autosomal dominant corneal disorders. Existing therapies are only temporizing and carry significant morbidity. Here, we develop a novel therapeutic approach using an adenine base editor (ABE) to correct common TGFBI mutations.<br><br>METHOD: We generated two human corneal epithelial (HCE) cell models harboring a copy of the most common disease-causing TGBFI mutations, R124C or R555W. These lines were electroporated with an ABE8e-NG-encoding mRNA and guide RNAs targeting the mutations. The resulting A&#x2022;T-to-G&#x2022;C editing efficiencies and off-target (OT) effects were assessed by amplicon sequencing. GFP-expressing adeno-associated viruses (AAVs) with different capsid types were transduced into HCE cells and healthy human corneal donor tissues, and GFP fluorescence was evaluated.<br><br>RESULTS: Using all-RNA delivery for ABE8e-NG, we achieved 91% and 62% correction of the pathogenic adenines in HCE TGFBIR124C/WT and TGFBIR555W/WT cells, without editing the wild-type allele. Indel formation was negligible (<0.2%), bystander adenine editing was minimal (<0.7%), and editing at top computationally predicted OT sites was modest (<1.2% at all but 1 of the 20 OT sites analyzed), suggesting minimal safety concerns. Correction of TGFBIR124C/WT in HCEs rescued the aberrant lysosomal localization of TGFBI. We further identified AAV1 as the most effective serotype for gene delivery into both human corneal donor tissue and HCE cells.<br><br>CONCLUSIONS: Our study demonstrates the feasibility and safety of CRISPR adenine base editing as a new therapeutic strategy for correcting common TGFBI mutations in corneal dystrophies, paving the way for further preclinical testing.},
}
@article {pmid41757451,
year = {2026},
author = {Molina-Márquez, A and Kelterborn, S and Hegemann, P and Pérez-Rodríguez, M and Vigara, J and León, R},
title = {Characterization of Phytoene Desaturase Knockout Carotenoid-Deficient Microalgal Mutants Generated by Cas9-Ribonucleoprotein Complexes.},
journal = {Physiologia plantarum},
volume = {178},
number = {2},
pages = {e70811},
pmid = {41757451},
issn = {1399-3054},
support = {2019-110438RB-C22//Agencia Estatal de Investigaci&#xf3;n-MCIN/AEI/10.13039/501100011033/ ; PID2022-140995OB-C21//Agencia Estatal de Investigaci&#xf3;n-MCIN/AEI/10.13039/501100011033/ ; 426566805//German Research Foundation (DFG)/ ; //Hertie Foundation/ ; },
mesh = {*Carotenoids/metabolism ; *Oxidoreductases/genetics/metabolism ; *Ribonucleoproteins/metabolism/genetics ; Gene Knockout Techniques ; *Microalgae/genetics/metabolism ; *Chlamydomonas reinhardtii/genetics/metabolism ; Mutation/genetics ; Chloroplasts/metabolism/ultrastructure ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Phytoene desaturase (PDS; EC 1.3.5.5) is a key enzyme of the carotenoid biosynthetic pathway, catalyzing the desaturation of phytoene, precursor of all carotenoids. In this study, several PDS-knockout (PDS-KO) transformants of the chlorophyte microalga Chlamydomonas reinhardtii were generated using a reverse genetics strategy. Two single guide RNAs (sgRNA) were designed to target the first exon of the PDS gene, and pre-assembled Cas9 ribonucleoprotein (RNPs) complexes were delivered into microalgal nuclei by electroporation. Multiple white PDS-KO transformants were successfully obtained by this approach, and three independent transformant lines were subsequently characterized. By integrating ultrastructural, pigment and transcriptomic analyses of dark-grown cells of several PDS-KO carotenoid-deficient mutants in comparison with the parental strain, it was demonstrated that carotenoids are indispensable components of multiple cellular architectures. Chromatographic analysis confirmed that the only carotenoid accumulated in these transformants was phytoene, which lacks the critical structural and photoprotective functions of its colored derivatives. Transmission Electron Microscopy (TEM) observations revealed profound ultrastructure alterations, including poorly developed chloroplasts and effects on other cellular structures that were either absent or severely disorganized. Consistently, clustering differentially expressed genes into functional groups revealed downregulation of pathways associated with photosynthesis, chlorophyll and carotenoid biosynthesis, ribosome biogenesis, and vesicle and membrane trafficking in the PDS-KO lines. Conversely, upregulation of regulatory and retrotransposon-inducing genes was observed. These findings underscore the central metabolic role of colored carotenoids in plant cells, highlighting their essential contribution to cellular homeostasis and photosynthetic competence.},
}
@article {pmid41757335,
year = {2026},
author = {Shafiq, T and Khan, N and Kausar, T and Ahmed, W and Zhang, Z and Liang, Y and Duan, L},
title = {Red Blood Cell-Derived Extracellular Vesicles for Gene and RNA Therapeutics: Biological, Engineering, and Translational Challenges.},
journal = {International journal of nanomedicine},
volume = {21},
number = {},
pages = {579975},
pmid = {41757335},
issn = {1178-2013},
mesh = {Humans ; *Extracellular Vesicles/chemistry/metabolism ; *Genetic Therapy/methods ; *Erythrocytes/cytology/chemistry/metabolism ; CRISPR-Cas Systems ; Animals ; RNA, Messenger/genetics/administration &amp; dosage/therapeutic use ; *RNA/therapeutic use ; Gene Transfer Techniques ; },
abstract = {Gene therapy has great prospects of DNA/RNA manipulations and protein modulations. Its use in clinic is, however, stifled by risks of immunogenicity, low target specificity, and adverse effects. The red blood cell (RBC-EVs) extracellular vesicles can serve as a solution to this issue since they are biocompatible, long-term stable, and with low immunogenicity. RBC-EVs permit the accurate delivery of therapeutic cargo to space and time, thus minimizing systemic toxicity. This review presents the most recent developments on the expansion of the use of RBC-EVs to encapsulate the components of mRNA and CRISPR-Cas. Through the addition of the means to address these deficiencies, including stimulus-sensitive release mechanisms (eg, pH- or light-activated systems) and tissue-selective targeting approaches, RBC-EVs can be applied to enable the precise application in genetic diseases, inflammatory diseases, and cancer. Such innovations have the potential to overcome the clinical need and enable the biological complexity of mRNA- and CRISPR-Cas-based agents to provide a powerful delivery platform. Moreover, the review also demonstrates the unprecedented benefits of red blood cell EVs, which include immune evasion, scalability, and universal loading capacity, which can establish them as the next-generation delivery vehicles. Red blood cell EVs have the potential to increase the efficacy of precision medicine by increasing its feasibility. Lastly, we note the potential and translational issues in the provision of red blood cell EV-based mRNA and CRISPR-Cas therapeutic delivery of gene therapy.},
}
@article {pmid41755886,
year = {2026},
author = {Kaniganti, S and Saini, H and Chaitanya, AK and Hegde, N and Shah, P and Magar, ND and Rijal, R and Kaushik, JJ and Nanda, D and Sachan, S and Kumar, A and Bhoite, R and Jamedar, HR},
title = {CRISPR/Cas Genome Editing and Its Applications in Cereal Crop Improvement.},
journal = {Plant-environment interactions (Hoboken, N.J.)},
volume = {7},
number = {2},
pages = {e70133},
pmid = {41755886},
issn = {2575-6265},
abstract = {CRISPR/Cas-based genome editing has emerged as a transformative tool for precise genetic improvement of cereal crops. Recent advances in CRISPR technologies, including Cas9, Cas12, Cas13, base editing, and prime editing, have enabled targeted modification of genes and regulatory elements controlling yield, stress tolerance, and grain nutritional quality in major cereals such as rice, wheat, maize, and barley. This review summarizes current progress in CRISPR-mediated genome editing systems, delivery strategies, and representative applications in cereal crop improvement. Emphasis is placed on how genome editing reprograms enzymatic activities and biological pathways underlying complex agronomic traits rather than acting through single-gene effects. The review also discusses challenges related to trait complexity, regulatory considerations, and prospects for translating genome-edited cereal crops from laboratory research to field-level application. Collectively, this review highlights the potential of CRISPR/Cas genome editing as a powerful approach for developing high-yielding, resilient, and nutritionally improved cereal crops.},
}
@article {pmid41755634,
year = {2026},
author = {Hu, Z and Liu, Y and Han, Y and Li, M and Deng, K and Lu, X and Huang, Y and Liang, C and Wang, Y and Fu, Y and Xu, A},
title = {CRISPR/Cas9 screening with destabilized bicistronic fluorescent protein reporter revealed PABPN1 as a hub of regulators for alternative polyadenylation.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41755634},
issn = {1362-4962},
support = {2022YFA1103900//National Key Research and Development Program of China/ ; 32470586//National Natural Science Foundation of China/ ; 91942301//National Natural Science Foundation of China/ ; 81430099//National Natural Science Foundation of China/ ; 31930084//National Natural Science Foundation of China/ ; 32500472//National Natural Science Foundation of China/ ; 2023B1212060028//Guangdong Science and Technology Department/ ; 2022YFA1103900//National Key Research and Development Program of China/ ; },
mesh = {*Polyadenylation/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; Genes, Reporter ; *Poly(A)-Binding Protein I/genetics/metabolism ; HEK293 Cells ; DEAD-box RNA Helicases/metabolism/genetics ; *Luminescent Proteins/genetics/metabolism ; Cell Proliferation/genetics ; Polypyrimidine Tract-Binding Protein/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; },
abstract = {Alternative polyadenylation (APA) is intricately intertwined with diverse biological processes. Efficient approaches for screening the regulatory factors of specific APA events are essential to elucidate their regulation mechanisms. Here, we first engineered a destabilized bicistronic fluorescent protein reporter (dBFPR) to enhance the sensitivity of APA detection. Then, we developed a robust high-throughput screening platform for APA regulators by integrating CRISPR/Cas9, dBFPR, and fluorescence-activated cell sorting. With this method, we successfully screened the library of RNA binding proteins and found that PTBP1, ELAVL1, and DDX3X play significant roles in regulating APA and promoting cell proliferation through interaction with PABPN1, suggesting that PABPN1 is an important hub for APA regulation.},
}
@article {pmid41755633,
year = {2026},
author = {Aguilar-González, A and Martos-Jamai, I and Ramos-Hernández, I and Molina-Estévez, FJ and Villao, NV and Puig-Serra, P and Rodríguez-Perales, S and Torres, R and Labun, K and Sánchez-Martín, RM and Díaz-Mochón, JJ and Martín, F},
title = {A novel Dual-guide CRISPR-Cas13 strategy improves specificity for single-nucleotide variant detection.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41755633},
issn = {1362-4962},
support = {MCIN)/AEI/10.13039/501100011033//Spanish Ministry of Science and Innovation/ ; PID2022-141065OB-I00//European Union Next Generation/ ; CV20-77741//Consejer&#xed;a de Econom&#xed;a y Conocimiento/Project/ ; PI21/00298//Instituto de Salud Carlos III/ ; PI24/00888//Instituto de Salud Carlos III/ ; RD21/0017/0004//Instituto de Salud Carlos III/ ; RD24/0014/0005//Instituto de Salud Carlos III/ ; PI-0236-2024//Consejer&#xed;a de Salud y Familias/ ; PIP-0004-2025//Consejer&#xed;a de Salud y Familias/ ; GeneHumdi-CA21113//European Cooperation in Science and Technology/ ; FPU22/03455//Spanish Ministry of Science, Innovation and Universities/ ; RHJ-0053-2025//Consejer&#xed;a de Salud y Familias, Junta de Andaluc&#xed;a/ ; //European Social Fund/ ; //Universidad de Granada/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *SARS-CoV-2/genetics/isolation &amp; purification ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *COVID-19/diagnosis/virology/genetics ; RNA, Viral/genetics ; *Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins p21(ras)/genetics ; Sensitivity and Specificity ; Leishmania/genetics ; CRISPR-Associated Proteins/genetics ; },
abstract = {The emergence of CRISPR-Cas systems has transformed nucleic acid detection and manipulation. Cas13, a type VI CRISPR effector, targets RNA with high sensitivity through both cis (target RNA) and trans (collateral RNA) cleavage. This property enables the use of fluorescent reporters for sensitive diagnostics. However, Cas13's heightened sensitivity also leads to reduced specificity due to its susceptibility to single-nucleotide mismatches, potentially causing off-target effects. To overcome this limitation, we developed the first Dual-guide RNA system for Cas13 that improves mismatch discrimination and enhances target specificity. This system employs two distinct RNAs-dcrRNA and dtracrRNA-which cooperatively recognize the target and reduce off-target activity. In vitro experiments demonstrated robust cis- and trans-RNase activity, indicating efficient and specific cleavage. The system accurately detected SARS-CoV-2 RNA, distinguished KRAS G12D and G12C mutations, and differentiated mucocutaneous from cutaneous Leishmania sequences in analytical assays, with clinical validation confirming accurate detection of positive and negative samples. These results highlight the Dual-guide Cas13 platform's potential for precise, rapid, and reliable RNA detection. Overall, this approach represents a substantial advance over conventional Cas13 systems, offering improved specificity while maintaining clinically relevant sensitivity, and provides a generalizable tool for next-generation molecular diagnostics and precision RNA targeting and regulation.},
}
@article {pmid41754561,
year = {2026},
author = {Wupori, K and Garnett, L and Bello, A and Strong, JE},
title = {CRISPR-Based Detection of Viral Hemorrhagic Fevers at the Point of Care.},
journal = {Viruses},
volume = {18},
number = {2},
pages = {},
pmid = {41754561},
issn = {1999-4915},
support = {8//Genomics Research and Development Initiative/ ; CSSP-2022-CP-2546//Canadian Safety and Security Program/ ; },
mesh = {Humans ; *Hemorrhagic Fevers, Viral/diagnosis/virology ; *Point-of-Care Systems ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Point-of-Care Testing ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {Viral hemorrhagic fevers (VHFs) are highly lethal diseases that often present non-specific, influenza-like symptoms in their early stages, making clinical recognition and differentiation from other febrile illnesses difficult. This overlap underscores the critical need for diagnostic tests that are both sensitive and specific. Point-of-care (POC) diagnostic tests are an invaluable tool for detecting and controlling the spread of pathogens that threaten public health, such as VHFs, as these require fast, accurate diagnostics to ensure biosafety and appropriate mobilization of resources during outbreaks. Current molecular and serological diagnostic tests, while efficient and effective, lack the characteristics required of a POC test (POCT) to quickly and easily respond to a VHF outbreak while maintaining a low cost. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tests have gained popularity as POCTs due to their inherent attractive qualities, including high sensitivity and specificity, adaptability, low cost, quick turnaround time, and ease of use. However, studies on the development of CRISPR-based POC diagnostic tests for VHFs are limited. This review summarizes the current CRISPR-based POCTs for VHFs, including Ebola virus (EBOV), Lassa virus (LASV), Dengue virus (DENV), and Crimean-Congo hemorrhagic fever virus (CCHF). The isothermal pre-amplification methods commonly paired with CRISPR-based tests, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), are also discussed.},
}
@article {pmid41740934,
year = {2026},
author = {Sun, W and Zhu, S and Dong, Q and Tang, S and Liu, Q and Sha, Y and Chen, T and Wang, R and Chen, Y and Ying, H},
title = {Engineering Escherichia coli cell Factories for continuous 5'-cytidine monophosphate production via biofilm-anchored dual-enzyme cascade catalysis.},
journal = {Bioresource technology},
volume = {447},
number = {},
pages = {134267},
doi = {10.1016/j.biortech.2026.134267},
pmid = {41740934},
issn = {1873-2976},
mesh = {*Biofilms/growth &amp; development ; *Escherichia coli/metabolism/genetics/enzymology ; *Cytidine Monophosphate/biosynthesis ; Biocatalysis ; CRISPR-Cas Systems/genetics ; Uridine Kinase/metabolism ; },
abstract = {5'-cytidine monophosphate (5'-CMP) serves as a crucial intermediate for diverse nucleotide derivatives and finds extensive applications in the food and pharmaceutical industries. However, existing enzymatic production processes suffer from low catalytic efficiency and poor economic feasibility. In this study, we developed a continuous 5'-CMP production system based on a cell-enzyme co-immobilized biocatalytic platform. First, the pgaABCD gene cluster was integrated into ClearColi BL21(DE3) using CRISPR-Cas9, enhancing its biofilm-forming capacity by 168.93% and enabling robust cell immobilization on the carrier. Second, a dual-anchoring strategy utilizing ice-nucleation protein (INP) and autotransporter (AIDA-I) enabled surface display of uridine kinase (UDK) and acetate kinase (AckA) on the cell surface. This approach successfully addressed the instability and recovery issues of free enzymes by using biofilm engineering to co-immobilize cells and enzymes. The modified strain achieved a 5'-CMP productivity of 1.77 mmol/L/h, 5.98-fold higher than free intracellular enzyme catalysis, and was reused for ten consecutive cycles under the tested conditions while maintaining a cytidine conversion rate above 73.79%, and a 5'-CMP yield above 59.26%. This work demonstrates the first successful realization of continuous 5'-CMP biosynthesis and establishes an efficient route for its industrial production.},
}
@article {pmid41719905,
year = {2026},
author = {Yu, F and Yue, D and Wang, F and Fu, B and Qin, G and Wei, S and Zang, W and Zhang, Q and Cui, L and Wang, T},
title = {Structure-initiated CHA variant coordinating SDA for cascade amplification in CRISPR/Cas12a-based miRNA analysis.},
journal = {Talanta},
volume = {304},
number = {},
pages = {129558},
doi = {10.1016/j.talanta.2026.129558},
pmid = {41719905},
issn = {1873-3573},
mesh = {*MicroRNAs/genetics/analysis/blood ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {MicroRNAs (miRNAs) are well-established biomarkers for tumor diagnosis and monitoring. Herein, we report a novel biosensing platform by engineering a structure-initiated variant of catalytic hairpin assembly (VCHA) that coordinates with strand displacement amplification (SDA) to drive cascade amplification for CRISPR/Cas12a-based detection. This system employs three hairpin probes which, upon recognizing the target miRNA, self-assemble into a key 5'-end dangling three-way conjugate (5'-DTC) structure. This structure serves as the exclusive trigger, simultaneously propagating the VCHA cycle and priming the SDA process through the coordinated action of polymerase and nicking enzyme. Consequently, VCHA and SDA operate synergistically within a unified circuit, generating abundant single-stranded activator DNA (acDNA) products. These acDNA molecules then activate the trans-cleavage activity of CRISPR/Cas12a, yielding a significantly amplified fluorescence readout. The VCHA-SDA/Cas12a platform demonstrated excellent performance for miRNA-155 detection, achieving a broad dynamic range from 1 pmol/L to 10 nmol/L with an ultra-low detection limit of 0.166 pmol/L. Furthermore, the platform successfully quantified miRNA levels in clinical plasma specimens and various cell lines, confirming its considerable potential as a robust tool for molecular diagnostics and clinical translation.},
}
@article {pmid41713574,
year = {2026},
author = {Hu, T and Hou, Z and Zhang, Y and Jing, P and Dai, X and Wang, H},
title = {Development of a one-pot integrated rapid detection method for white spot syndrome virus based on RAA-CRISPR/Cas12a technology.},
journal = {Journal of invertebrate pathology},
volume = {216},
number = {},
pages = {108569},
doi = {10.1016/j.jip.2026.108569},
pmid = {41713574},
issn = {1096-0805},
mesh = {*White spot syndrome virus 1/isolation &amp; purification ; Animals ; *Penaeidae/virology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Aquaculture ; Sensitivity and Specificity ; },
abstract = {Pathogenic microorganisms, particularly white spot syndrome virus (WSSV), pose a major threat to global shrimp aquaculture, causing mass mortalities and substantial economic losses. To address the urgent need for rapid, accurate, and field-deployable detection methods, this study developed an innovative one-pot RAA-CRISPR/Cas12a assay. The platform integrates recombinase-aided amplification (RAA) with CRISPR/Cas12a technology using sucrose-mediated density gradient phase separation in a closed-tube format: sucrose acts as a density modifier to form distinct layers, spatially isolating RAA amplification reagents from CRISPR/Cas12a detection components to avoid cross-interference and enable sequential reactions without manual intervention. Under isothermal conditions at 37℃ for 60 min, the optimized assay achieves a limit of detection as low as 1 copy/μL, validated by both fluorescence and lateral flow dipstick (LFD) readouts. High specificity was confirmed by the absence of cross-reactivity with four other prevalent shrimp pathogens: infectious hypodermal and hematopoietic necrosis virus (IHHNV), Decapod iridescent virus 1 (DIV1), Enterocytozoon hepatopenaei (EHP), and Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (VpAHPND). Clinical validation with 30 field samples showed concordant results with the chinese national detection standard (GB/T 28630.2-2012). This novel nucleic acid detection platform combines highly sensitive, excellent specificity, and user-friendly visual interpretation, making it highly suitable for point-of-care testing and large-scale disease surveillance in shrimp aquaculture.},
}
@article {pmid41690121,
year = {2026},
author = {Yin, B and Wu, X and Zhou, H and Meng, Y and Liu, J and Ding, L and Zhu, C and Tai, Z and Zhu, Q and Chen, Z},
title = {Topical ionic liquid-mediated GLUT1 gene editing ameliorates psoriasis and prevents recurrence.},
journal = {Biomaterials},
volume = {330},
number = {},
pages = {124058},
doi = {10.1016/j.biomaterials.2026.124058},
pmid = {41690121},
issn = {1878-5905},
mesh = {Animals ; *Gene Editing/methods ; *Psoriasis/therapy/genetics/pathology ; *Glucose Transporter Type 1/genetics ; Mice ; *Ionic Liquids/chemistry/administration &amp; dosage ; Humans ; CRISPR-Cas Systems/genetics ; Recurrence ; Mice, Inbred C57BL ; Keratinocytes/metabolism ; Disease Models, Animal ; Administration, Topical ; },
abstract = {Psoriasis is a chronic inflammatory skin disorder characterized by immune dysregulation and a high relapse rate. Current therapies seldom achieve lasting remission. Aberrant overexpression of glucose transporter 1 (GLUT1) in keratinocytes enhances glycolysis, fueling inflammation and immune imbalance, thus positioning GLUT1 as a promising therapeutic target. In this work, a composite ionic liquid-mediated transdermal platform was established for the delivery of CRISPR-Cas9 ribonucleoprotein (CIL-RNP), aiming to achieve efficient GLUT1 gene editing in keratinocytes. The CIL-RNP achieved 76.6% editing efficiency, downregulated pyruvate kinase M (PKM) expression, and reduced inflammatory cytokine secretion. In a psoriasis mouse model, topical administration of CIL-RNP decreased lesion severity by 50% PASI (Psoriasis Area and Severity Index) score, alleviating epidermal hyperplasia and immune infiltration. Furthermore, the treatment inhibited M1 macrophage polarization, reduced reactive oxygen species generation, rebalanced Th17/regulatory T cells (Tregs) responses, and diminished the accumulation of tissue-resident memory T cells (TRMs), thereby lowering the risk of relapse. This study establishes ionic liquid-based CRISPR-RNP transdermal editing of GLUT1 as a novel and effective strategy for restoring immune homeostasis in psoriasis, with potential for long-term remission and broader applications in cutaneous immunopathological conditions.},
}
@article {pmid41685943,
year = {2026},
author = {Wang, C and Li, D and Yu, R and Xue, J and Xie, W and Zhang, Q and Gui, X and Wang, L and Guo, S and Xie, Y and Jiang, Y and Liu, G and Wu, J},
title = {A CRISPR/Cas9-regulated dual-ring topological allosteric probe for detection of the EGFR L858R resistance mutation in CTCs.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {9},
pages = {1815-1825},
doi = {10.1039/d5ay02124c},
pmid = {41685943},
issn = {1759-9679},
mesh = {Humans ; ErbB Receptors/genetics ; *Neoplastic Cells, Circulating/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Lung Neoplasms/genetics/blood ; *Carcinoma, Non-Small-Cell Lung/genetics/blood ; Polymorphism, Single Nucleotide ; *Drug Resistance, Neoplasm/genetics ; Mutation ; Fluorescent Dyes/chemistry ; Cell Line, Tumor ; },
abstract = {A single-nucleotide polymorphism (SNP) is a point mutation occurring at a defined genomic locus, and its precise and rapid detection in circulating tumor cells (CTCs) is essential for early diagnosis and therapeutic monitoring of non-small cell lung cancer (NSCLC). In this study, a CRISPR/Cas9-regulated dual-ring topological allosteric probe was developed for ultrasensitive and specific detection of the EGFR L858R mutation. The recognition ring selectively hybridizes with the target sequence and is cleaved by the Cas9-sgRNA complex, triggering the release of the reporter ring. The released reporter ring then serves as a template for rolling circle amplification (RCA), generating products that hybridize with dual-labeled fluorescent probes to yield measurable signals. This assay clearly distinguished L858R from the wild-type sequence and detected mutation frequencies as low as 1.0% with high specificity against other common EGFR variants. Its robustness was further validated using clinical blood samples, enabling sensitive detection of low-abundance L858R mutations. These results demonstrate that the integration of programmable target recognition, efficient signal amplification, and fluorescence readout provides a promising platform for SNP analysis in liquid biopsy, supporting precision diagnosis and treatment monitoring in NSCLC.},
}
@article {pmid41666850,
year = {2026},
author = {Zhao, R and Chen, J and Li, Y and Jin, M and Liu, K and Liu, Y and Gao, L and Yang, G and Yuan, X and Chu, X and Wang, JG},
title = {CRISPR/Cas9-mediated knockout of the 22 kDa α-prolamin genes orchestrates the regulation of functional amino acid content in foxtail millet.},
journal = {Journal of plant physiology},
volume = {318},
number = {},
pages = {154723},
doi = {10.1016/j.jplph.2026.154723},
pmid = {41666850},
issn = {1618-1328},
mesh = {*CRISPR-Cas Systems/genetics ; *Setaria Plant/genetics/metabolism ; *Amino Acids/metabolism ; Gene Knockout Techniques ; *Prolamins/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Genes, Plant ; },
abstract = {Improving the nutritional quality of cereal crops remains a primary objective in modern breeding programs. The composition and content of prolamins directly affect the overall nutritional value. This study elucidates the role of two 22 kDa α-prolamin genes (Seita.9G301400 and Seita.9G406400) in foxtail millet using CRISPR/Cas9-mediated knockout. While simultaneous disruption of both genes in double mutants reduced prolamin content, amino acids, and soluble sugars, single-gene mutants exhibited the opposite effect. These single mutants displayed increased grain size alongside significantly enhanced levels of essential amino acids and sugars. Starch pasting properties were also improved in single mutants but compromised in double mutants. Our findings demonstrate that individual knockout of the two prolamin genes enhances nutritional and sensory quality, providing a potential strategy for developing improved foxtail millet varieties.},
}
@article {pmid41653514,
year = {2026},
author = {Li, M and He, L and Wang, Z and Wang, L and Pan, Q and Sun, P and Li, D and Zhang, C},
title = {Amplified ferroptosis and immunomodulation triggered by NIR-II photothermal-controllable CRISPR/Cas9 nanoplatform to treat osteosarcoma and prevent postsurgical implant-associated infections.},
journal = {Biomaterials},
volume = {330},
number = {},
pages = {124043},
doi = {10.1016/j.biomaterials.2026.124043},
pmid = {41653514},
issn = {1878-5905},
mesh = {*Ferroptosis/drug effects ; Humans ; Animals ; *CRISPR-Cas Systems ; *Osteosarcoma/therapy/immunology ; *Immunomodulation ; Mice ; Cell Line, Tumor ; Infrared Rays ; HSP70 Heat-Shock Proteins/genetics ; NF-E2-Related Factor 2/genetics/metabolism ; },
abstract = {Ferroptosis has been proven as a promising therapeutic approach with immunomodulatory effect; however, intracellular antioxidant system maintains redox balance and diminishes its efficacy. Nuclear factor erythroid 2-related factor 2 (Nrf2) is identified as a central transcription factor for regulating oxidative homeostasis. Herein, we have developed a thermal-controllable genome-editing nanoplatform BF/pHCN. Specifically, a CRISPR/Cas9 plasmid with an upstream HSP70 promoter sequence (HSP70-Cas9-sgNrf2, named pHCN) was constructed. Subsequent Fe(II) and pHCN were co-loaded into organic small-molecule BTP with near infrared II (NIR-II) absorption and coated with DSPE-mPEG2000. The generated BF/pHCN (BTP@Fe/pHCN) could be internalized within osteosarcoma cells. Subsequent NIR-II laser-triggered hyperthermia at 42 °C activated the HSP70 promoter and facilitated the precise inhibition targeting Nrf2 genomic sequences while promoting Fe(II) release, ultimately disrupting oxidative stress states. Moreover, the amplified ferroptosis fully triggered immunogenic cell death, thus reprogramming macrophages, promoting maturation of dendritic cells, and activating cellular antitumoral immunity. Additionally, BF/pHCN exhibited direct bactericidal activity against planktonic bacteria, and effectively eliminated intracellular bacteria through iron metabolic disorders strategy targeting macrophages, thereby initiating adaptive antimicrobial immunity. Overall, our NIR-II thermal-controllable genome-editing nanoplatform BF/pHCN exhibits remarkable antitumoral properties alongside robust antiinfection and immunomodulation, providing feasible strategies toward effective management of osteosarcoma, and preventing postsurgical implant-associated infections.},
}
@article {pmid41649281,
year = {2026},
author = {Zhu, Z-J and Cui, M-L and Liu, Y and Yao, X-Q and Lu, M-J and Wang, M-C and Liu, J-H and Li, J-F and Li, E-Z},
title = {CRISPR/Cas14a combined with RPA for visual detection of Marek's disease virus.},
journal = {Microbiology spectrum},
volume = {14},
number = {3},
pages = {e0262525},
pmid = {41649281},
issn = {2165-0497},
support = {22A310017//Key Scientific Research Projects of Universities in Henan/ ; },
mesh = {Animals ; *Marek Disease/diagnosis/virology ; *CRISPR-Cas Systems ; *Herpesvirus 2, Gallid/isolation &amp; purification/genetics ; *Poultry Diseases/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; Chickens/virology ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; Recombinases/metabolism/genetics ; *Mardivirus/isolation &amp; purification/genetics ; },
abstract = {Marek's disease, a highly contagious avian immunosuppressive disorder caused by the α-herpesvirus MDV-1, poses a significant threat to poultry health. The development of rapid visual detection methods capable of distinguishing epidemic MDV-1 strains from vaccine strains is crucial for early disease warning, vaccine efficacy evaluation, and precise disease control. We developed a novel isothermal detection system that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas14a technology for the visual identification of epidemic MDV-1 strains. This method operates at a constant temperature of 37°C and allows for either real-time analysis or endpoint visual readout without the need for complex instrumentation. Our results showed no cross-reactivity with Newcastle disease virus, infectious bursal disease virus, MDV-1 vaccine strains, or herpesvirus of turkeys. Plasmid DNA standards were used to determine the sensitivity of the assay, and the detection limit was 24.6 copies/μL. Clinical evaluation using 24 field samples confirmed that the method successfully identified all Marek's disease virus-positive cases, demonstrating its diagnostic reliability. In conclusion, we have developed a rapid, highly specific nucleic acid detection platform for MDV-1 that enables visual readout without complex instrumentation by combining the sensitivity of RPA with the specificity of CRISPR/Cas14a technology, offering promising potential for field-based diagnostics and disease surveillance.IMPORTANCEMarek's disease virus (MDV-1) is a highly contagious and economically important avian pathogen. Existing diagnostic methods are unable to reliably distinguish between epidemic and vaccine strains in field settings, which hampers effective surveillance and evaluation of vaccination programs. To address this challenge, we developed a portable isothermal detection assay that combines recombinase polymerase amplification with CRISPR/Cas14a technology. This approach enables highly sensitive (24.6 copies/μL) and specific visual detection of epidemic MDV-1 strains without cross-reactivity with vaccine strains or related viruses. The assay demonstrated 100% agreement with reference methods when evaluated using clinical samples. As a cost-effective method that avoids the need for complex detection instruments, it offers a practical solution for rapid on-site diagnosis, facilitating enhanced outbreak control and improved poultry health management globally.},
}
@article {pmid41638449,
year = {2026},
author = {Qu, Y and Li, Y and Shao, T and Kuang, J and Qi, Y and Yang, J and Liu, Y and Wang, J and Fu, X and Liu, J and Zhang, X and Peng, T and Yuan, Q and Zhu, L},
title = {Optimizing prime editing: Advances in efficiency enhancement.},
journal = {Biotechnology advances},
volume = {88},
number = {},
pages = {108815},
doi = {10.1016/j.biotechadv.2026.108815},
pmid = {41638449},
issn = {1873-1899},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Protein Engineering/methods ; },
abstract = {Prime editing (PE) enables the precise installation of targeted insertions, deletions, and all possible base-to-base conversions without introducing double-strand breaks or donor DNA templates. However, its efficiency remains highly variable across genomic contexts. To address this, multi-faceted optimization strategies have been developed: protein engineering has yielded editor variants with enhanced reverse transcriptase activity and stability; structural refinements to pegRNA design improve its functional integrity and resistance to degradation; regulation of the PE-Flap-mismatch repair (MMR) process favors the retention of desired edits; and the development of protospacer adjacent motif (PAM)-relaxed Cas variants dramatically expands targetable sites. This review systematically consolidates these advances, illustrating how the integration of structural, mechanistic and targeting enhancements is overcoming fundamental bottlenecks. Together, these developments establish PE as a versatile and efficient system for precision genome engineering, paving the way for its reliable application in diverse biological settings.},
}
@article {pmid41506374,
year = {2026},
author = {Marschhofer, M and Chen, S and Molbay, M and Winkeljann, B and Villano, E and Giancaspro, C and Kourou, A and Berninghausen, O and Rieder, S and Ungewickell, C and Beckmann, R and Popper, B and Torres, AM and Vidal, A and Merkel, OM and Carneiro, SP},
title = {Optimized lipid nanoparticles for pulmonary delivery of CRISPR/Cas9 targeting KRAS G12S in lung cancer.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {391},
number = {},
pages = {114607},
doi = {10.1016/j.jconrel.2026.114607},
pmid = {41506374},
issn = {1873-4995},
mesh = {Animals ; *Lung Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; Humans ; *Nanoparticles/administration &amp; dosage/chemistry ; *Proto-Oncogene Proteins p21(ras)/genetics ; *Lipids/chemistry/administration &amp; dosage ; Gene Editing ; A549 Cells ; *Carcinoma, Non-Small-Cell Lung/genetics/therapy ; Mice ; *CRISPR-Associated Protein 9/genetics ; Mice, Nude ; RNA, Guide, CRISPR-Cas Systems/administration &amp; dosage/genetics ; Female ; RNA, Messenger/administration &amp; dosage/genetics ; Apoptosis ; Cell Line, Tumor ; Liposomes ; },
abstract = {KRAS G12S mutations in non-small cell lung cancer (NSCLC) remain refractory to current targeted therapies, with few clinical options and frequent resistance. While CRISPR/Cas9 enables mutation-specific gene disruption, its pulmonary application is limited by systemic clearance, hepatic tropism, and airway mucus barriers. Here, we present lipid nanoparticles (LNPs) specifically engineered for pulmonary delivery of Cas9 mRNA and KRAS G12S-targeting sgRNA, optimized through mRNA surrogate screening and orthogonal mixture design to guide lipid composition and Cas9:sgRNA weight-to-weight ratios. Two lead LNP formulations, A6 3:1 and A8 1:1, exhibited robust critical quality attributes, including particle sizes below 120 nm, low polydispersity, near-neutral zeta potential, and over 80 % encapsulation efficiency. Cryo-TEM revealed distinct morphologies correlated with enhanced transfection. In vitro, A8 1:1 achieved up to 90 % on-target gene editing in A549 cells and a 3.6-fold increase in apoptosis, while A6 3:1 induced a 3.7-fold apoptotic response. Both formulations efficiently traversed airway mucus in air-liquid interface cultures and preserved over 80 % cell viability across doses. In vivo, repeated pulmonary administration was well tolerated, with no signs of systemic toxicity or cytokine elevation in healthy or tumor-bearing mice. In an orthotopic A549-luc lung tumor model, intratracheal delivery of A6 3:1 and A8 1:1 modestly suppressed tumor growth, with histological evidence of tumor cell apoptosis for A8 1:1. Quantification confirmed a statistically significant increase of apoptosis in the A8 1:1 group, consistent with effective KRAS disruption in vivo. Overall, lead LNPs, particularly A8 1:1, enabled efficient and localized RNA-based gene editing that induced downstream apoptotic signaling, demonstrating a preliminary, yet promising, proof-of-concept for CRISPR/Cas9 therapy in NSCLC.},
}
@article {pmid41489552,
year = {2026},
author = {Fan, R and Tong, Y and Luo, S and He, Y and Yang, C and Li, W and Liu, J and Pan, J and Zhu, Y and Zhang, X and Zhu, J and Zhu, Y and Guo, Y and Li, L and Situ, B and Yan, X and Ma, W and Chang, L and Zhang, Y},
title = {Integrated Electroporated-Lysis Electrochemical Platform Enables Sensitive and Rapid EV Protein and miRNA Profiling Based on Multiplex-Responsive CRISPR/Cas12a.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {13},
pages = {e13331},
doi = {10.1002/smll.202513331},
pmid = {41489552},
issn = {1613-6829},
support = {2024YFC2419100//National Key R&amp;D Program of China/ ; 82272424//National Natural Science Foundation of China/ ; 82572682//National Natural Science Foundation of China/ ; 82372343//National Natural Science Foundation of China/ ; 82402753//National Natural Science Foundation of China/ ; 22304006//National Natural Science Foundation of China/ ; 62471021//National Natural Science Foundation of China/ ; 2023A1515010909//Guang Dong Basic and Applied Basic Research Foundation of China/ ; JCYJ20230807142204008//Shenzhen Science and Technology Innovation Committee/ ; 2024J004//Southern Medical University/ ; 2024M750606//China Postdoctoral Science Foundation/ ; 2025T180607//China Postdoctoral Science Foundation/ ; T2425021//National Science Fund for Distinguished Young Scholars/ ; },
mesh = {*MicroRNAs/metabolism/genetics ; Humans ; *Extracellular Vesicles/metabolism ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; *Electroporation/methods ; },
abstract = {Proteins and miRNAs in extracellular vesicles (EVs) have emerged as crucial biomarkers for tumor diagnosis. While CRISPR/Cas12a-based platforms have shown great promise in nucleic acid and protein detection, their susceptibility to off-target activation and structural instability remains a significant limitation. Here, we have developed an electroporation-lysis electrochemical platform integrated with DNA cube-cage-locked CRISPR/Cas12a (DC-Cas12a), termed EL-DC-Cas12a. This platform utilizes an electric field to rapidly lyse EVs, releasing their internal proteins and miRNAs. These released molecules then activate the DC-Cas12a system, thereby triggering the displacement of two distinct crRNA/Cas12a complexes that correspond to EV proteins and miRNAs, respectively. These complexes then specifically recognize and cleave electrochemical probes, generating quantifiable electrochemical signals that enable synchronous and accurate analysis of the two biomarkers. The integrated workflow for EV lysis and detection can be completed within 40 min, greatly simplifying the overall operation. The detection limits (LOD) of this platform for EV PD-L1 protein and miR-1246 were 5.44 × 10[4] particles/mL and 3.59 × 10[3] particles/mL, respectively. Moreover, by applying machine learning algorithms to analyze the EV-associated proteins and miRNAs profiling, the platform demonstrated a diagnostic accuracy of 98.3% in distinguishing healthy donors from early-stage GC patients, and 99% in differentiating early-stage from advanced-stage GC patients in a clinical gastric cancer cohort. Therefore, the proposed platform offers a promising strategy for multiplexed detection of EV biomarkers and precise discrimination of GC.},
}
@article {pmid41477961,
year = {2026},
author = {Gong, P and Tao, D and Chen, Q and Yang, Y and Xie, S and Chen, X and Shi, S and Wang, Y and Wang, L and Qian, Y and Ye, S},
title = {A rapid, visual, ultrasensitive and highly specific method for detecting adeno-associated virus 2020 based on the LAMP-CRISPR/Cas12a system.},
journal = {Poultry science},
volume = {105},
number = {2},
pages = {106344},
pmid = {41477961},
issn = {1525-3171},
mesh = {Animals ; *Poultry Diseases/diagnosis/virology ; *Parvoviridae Infections/veterinary/diagnosis/virology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *CRISPR-Cas Systems ; *Dependovirus/isolation &amp; purification/genetics ; *Ducks ; *Molecular Diagnostic Techniques/veterinary/methods ; Sensitivity and Specificity ; Phylogeny ; },
abstract = {Avian parvovirus infection would lead to growth retardation, weight loss, physical deformities and increased mortality in poultry, causing substantial economic losses to the poultry industry. Therefore, the development of a rapid, visual, ultrasensitive and highly specific method is essential for timely diagnosis and effective control of the avian parvovirus infection. In this study, we developed a detection platform based on loop-mediated isothermal amplification (LAMP) combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system. Firstly, we have identified a novel avian parvovirus strain from diseased Muscovy ducks. Through genome sequencing, sequence assembly and phylogenetic tree analysis, we have identified this novel avian parvovirus as an adeno-associated virus (AAA) belonging to the family Parvoviridae, subfamily Parvovirinae and genus Dependoparvovirus. So, the novel virus strain was named AAV-2020. Next, specific sgRNAs and LAMP primers targeting the 3 capsid proteins (Cap) genes of AAV-2020 were designed and optimized. Moreover, the CRISPR/Cas12a-based system demonstrated a limit of detection as low as 2 copies/μL for AAV-2020. Importantly, the system could effectively distinguish AAV-2020 from 3 closely related AAV strains with high sequence similarity, indicating excellent specificity. In summary, we developed a novel, rapid, visual, ultrasensitive and highly specific detection system for AAV-2020, offering a reliable tool for early diagnosis and on-site detection of avian parvovirus infections, which would aid in the prevention and control of avian parvovirus infection in poultry industry.},
}
@article {pmid41423250,
year = {2026},
author = {Sharma, A and Pathangey, L and Chirackal, SS and Shim, KG and Fonseca, R and Swaminathan, S},
title = {Ferritin H Knockout Induces Differential Immunomodulatory Drug Responses in Multiple Myeloma Cell Lines.},
journal = {European journal of haematology},
volume = {116},
number = {4},
pages = {391-401},
doi = {10.1111/ejh.70084},
pmid = {41423250},
issn = {1600-0609},
support = {//Paula and Rodger Riney Foundation/ ; },
mesh = {Humans ; *Multiple Myeloma/genetics/metabolism/drug therapy/pathology ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Gene Knockout Techniques ; *Ferritins/genetics ; Reactive Oxygen Species/metabolism ; Iron/metabolism ; CRISPR-Cas Systems ; *Immunomodulating Agents/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; Oxidative Stress ; Oxidoreductases ; },
abstract = {BACKGROUND: Immunomodulatory agents (IMiDs) are a cornerstone in the successful management of multiple myeloma (MM). However, acquired IMiD resistance leading to disease relapses remains a major barrier. Hydrogen peroxide generation and oxidative stress are key mediators that determine IMiD's effectiveness in MM. Iron plays a key role in the generation of oxidative stress; therefore, cellular iron levels are tightly governed. FTH1 is the major iron storage protein that tightly regulates cellular iron availability. Hence, the present study is targeted to investigate the role of FTH1 in MM and IMiD resistance.<br><br>METHODS: IMiD-sensitive and IMiD-resistant MM cells were analyzed for expression of iron-metabolism genes. CRISPR-cas9-mediated knockout of FTH1 was performed and the after-effects were assessed through multiple experiments.<br><br>RESULTS: Initial analysis showed a positive correlation between FTH1 expression and IMiD resistance in MM cells. FTH1-KO reduced IMiD sensitivity in the KMS11 cell line but had no effect on the RPMI8226 cell line. RNA-seq data showed downregulation of ER-stress and calcium signaling genes after FTH1-KO. Further, KMS11-FTH1KO cells exhibited lower intracellular ROS, labile-iron, and mitochondrial superoxide levels along with increased CD63, suggesting activation of L-ferritin secretory pathways.<br><br>CONCLUSION: Data reveals a link between FTH1, labile iron, ROS, and IMiD resistance in MM cells.},
}
@article {pmid41376159,
year = {2026},
author = {Ha, AS and Kalter, N and Rosenberg, M and Acevedo, LA and Liang, B and Liu, W and Paruthiyil, S and Sinha, M and Vu, A and Nguyen, V and Qi, Z and Krishnappa, N and Shu, J and Yu, J and Catanzaro, J and Bluestone, JA and Tang, Q and Urnov, FD and Marson, A and Hendel, A and Herold, KC and Shy, BR and Esensten, JH},
title = {Gene-corrected regulatory T cell therapy for IL2RA deficiency.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1367-1381},
doi = {10.1016/j.ymthe.2025.12.004},
pmid = {41376159},
issn = {1525-0024},
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology/metabolism/transplantation ; *Interleukin-2 Receptor alpha Subunit/genetics/deficiency ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Gene Editing ; Male ; Female ; Interleukin-2 ; Mutation ; },
abstract = {Bi-allelic germline deficiency of IL2RA causes a rare autoimmune disease with impaired regulatory T cell (Treg) function and interleukin-2 (IL-2) signaling. Definitive treatment is currently limited to allogeneic hematopoietic stem cell transplantation, which carries significant morbidity and mortality risks. We previously identified a family with three siblings affected by compound heterozygous mutations in their IL2RA gene, resulting in dysfunctional Tregs. Here, we introduce a novel therapeutic approach involving ex vivo generation of gene-corrected autologous regulatory T cells (gcTregs). One of the two disease-causing mutations in patient-derived Tregs was corrected with CRISPR-Cas9-mediated homology-directed repair, restoring IL2RA expression. The resulting gcTregs demonstrated robust suppressive activity in vitro. Clinical-scale manufacturing from a patient with IL2RA deficiency showed efficient gene correction, restored IL2RA expression, and functional equivalence to healthy donor Tregs. This work establishes a Good Manufacturing Practice-compatible manufacturing process for personalized gcTreg therapies, potentially providing a safer treatment option for patients with IL2RA deficiency as well as a framework for treating other inborn errors of immunity.},
}
@article {pmid41376155,
year = {2026},
author = {Jin, X and Wu, X and Song, J and Luo, M and Ye, Q and Ren, C and Song, L and Li, M and Hu, M and An, Y and Su, J and Fu, J and Xu, Q and Luo, M and Liu, F and Liu, M and Li, Q and Yao, S and Chen, L and Yang, Y},
title = {Comparative evaluation of liver-directed knockin strategies with viral and nonviral vectors in mouse inherited disease models.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1775-1793},
doi = {10.1016/j.ymthe.2025.12.012},
pmid = {41376155},
issn = {1525-0024},
mesh = {Animals ; *Genetic Vectors/genetics/administration &amp; dosage ; Mice ; Disease Models, Animal ; Dependovirus/genetics ; *Liver/metabolism ; *Gene Knock-In Techniques/methods ; Genetic Therapy/methods ; Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Recombinational DNA Repair ; *Hemophilia B/genetics/therapy ; Transgenes ; },
abstract = {CRISPR-Cas9-mediated gene knockin has emerged as a promising strategy for early-onset genetic disease intervention. However, the therapeutic efficacy and editing outcomes of different knockin strategies remain incompletely understood. Here, we systematically evaluated three major liver-directed knockin strategies, namely homology-directed repair (HDR), homology-independent targeted integration (HITI), and homology-mediated end joining (HMEJ), using neonatal mouse models of mucopolysaccharidosis type I and hemophilia B. Although all three approaches effectively rescued disease phenotypes, we observed distinct editing outcomes. Notably, the HMEJ approach, delivered via a combined adeno-associated virus-lipid nanoparticle (AAV-LNP) system, exhibited superior integration efficiency (5.8%-5.9%) and fidelity (97%-98%) compared with HDR and HITI. In contrast, whole-genome sequencing indicated that HITI induced a higher risk of random AAV donor integration than HDR or HMEJ. Furthermore, long-read sequencing analyses revealed that the frequencies of inverted terminal repeat (ITR)-mediated transgene integration differed between the 5' and 3' genomic junctions among the three strategies. Specifically, in HDR- and HMEJ-treated mice, ITR-mediated integration events were 7.7- to 19.7-fold more common at the 3' junctions than at the 5' junctions. These findings highlight the comprehensive advantages of the AAV-LNP-mediated HMEJ approach for liver-directed knockin therapy and suggest its strong potential for clinical translation.},
}
@article {pmid41370232,
year = {2026},
author = {Alok, A and Raman, V and D'Agostino, L and Kshetry, AO and Rai, KM and Wang, C and Gunapati, S and Stupar, RM and Patil, GB and Zhang, F},
title = {Developmental regulators enable rapid and efficient soybean transformation and CRISPR-mediated genome editing.},
journal = {Plant physiology},
volume = {200},
number = {3},
pages = {},
doi = {10.1093/plphys/kiaf640},
pmid = {41370232},
issn = {1532-2548},
support = {IOS-2040218//National Science Foundation/ ; IOS-2206920//National Science Foundation/ ; #2021-67013-34565//USDA NIFA/ ; //Texas Governor's University Research/ ; },
mesh = {*Glycine max/genetics/growth &amp; development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *Plant Growth Regulators/metabolism ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; },
abstract = {Soybean (Glycine max) transformation remains challenging and has not kept pace with rapid advances in genetic engineering technologies due to low efficiency, lengthy timelines, and genotype dependency. Here, we developed a streamlined transformation method by leveraging developmental regulators (DRs) to promote de novo shoot regeneration directly from growing soybean plants. By evaluating multiple DR combinations, our results showed that co-expression of WUSCHEL2 (WUS2) and the gene encoding isopentenyltransferase (IPT) achieved higher transformation efficiencies (14.6% to 22.3%) in Williams 82 and Bert varieties than individual DRs without requiring exogenous hormones or selection agents. Moreover, this method produced heritable transgenic events within 9 to 11 weeks and successfully delivered CRISPR-Cas9 components, generating heritable mutations with 20% efficiency. The temporal transcriptomic and gene regulatory network analyses revealed that WUS2/IPT synergistically modulates stress responses and activates developmental pathways, orchestrating a transition from initial stress adaptation to regenerative programming. Our findings demonstrate that this DR-enabled approach significantly enhances soybean transformation frequency, reduces tissue culture requirements, and offers a promising genome-editing platform for soybean improvement.},
}
@article {pmid41332134,
year = {2026},
author = {Inuzuka, T and Mouzannar, K and Zhang, M and Umarova, R and Park, SB and Uchida, T and Ma, CD and Liang, TJ},
title = {A CRISPR-based genome-wide loss-of-function screen defines a role of host metabolism in regulating hepatitis B virus infection.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1616-1632},
pmid = {41332134},
issn = {1525-0024},
support = {Z01 DK054500/ImNIH/Intramural NIH HHS/United States ; Z01 DK054504/ImNIH/Intramural NIH HHS/United States ; Z99 DK999999/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Humans ; *Hepatitis B virus/genetics/physiology ; *Hepatitis B/metabolism/virology/genetics ; *CRISPR-Cas Systems ; Hep G2 Cells ; Virus Replication/genetics ; Hepatocytes/metabolism/virology ; *Host-Pathogen Interactions/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Hepatitis B virus (HBV) co-opts and interacts with an extensive array of host factors for productive infection. Herein, we develop an HBV reporter virus expressing red fluorescent protein (HBV-RFP) that is suitable for a CRISPR-based genome-wide screen for HBV host dependency factors. HepG2[NTCP/Cas9] cells were transduced with a pooled lentiviral library of single-guide RNA (sgRNA) targeting 19,114 human genes, edited and infected with HBV-RFP. RFP-low cells were sorted using fluorescence-activated cell sorting. The sorted cells were expanded and underwent two additional rounds of infection and sorting to enrich for sgRNA-targeted proviral host factors. By next-generation sequencing and bioinformatic analyses, we identified 63 genes as candidate host proviral factors, including known HBV proviral factors: RXRA, POLL, LDLR, and NTCP. Among the novel candidate genes, knockout of 12 genes significantly decreased HBV replication markers. Validation using siRNA knockdown in primary human hepatocytes confirmed several factors including the monoacylglycerol acyltransferase 2 (MOGAT2) gene as a bona fide HBV proviral factor. Further analysis with MGAT2 inhibitors demonstrated that inhibition of MOGAT2 activity impairs HBV transcription and replication. Our study demonstrates the value of the HBV reporter system in identifying previously unrecognized host metabolic factors important for HBV infection, offering a potential avenue for therapeutic development.},
}
@article {pmid41267399,
year = {2026},
author = {Dua, PH and Simon, BMJ and Marley, CBE and Feliciano, CM and Watry, HL and Cowan, QT and Steury, D and Abraham, A and Gilbertson, EN and Ramey, GD and Capra, JA and Conklin, BR and Judge, LM},
title = {Haplotype editing with CRISPR-Cas9 as a therapeutic approach for dominant-negative missense mutations in NEFL.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1633-1651},
doi = {10.1016/j.ymthe.2025.11.026},
pmid = {41267399},
issn = {1525-0024},
support = {R01 NS119678/NS/NINDS NIH HHS/United States ; U01 ES032673/ES/NIEHS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Mutation, Missense ; *Haplotypes ; *Charcot-Marie-Tooth Disease/genetics/therapy ; Motor Neurons/metabolism ; Polymorphism, Single Nucleotide ; Alleles ; Genetic Therapy/methods ; Phenotype ; },
abstract = {Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene is haplosufficient. We previously edited a dominant NEFL missense mutation causing Charcot-Marie-Tooth type 2E (CMT2E) with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, a multitude of different NEFL missense mutations cause CMT2E. Here, we addressed this challenge by targeting common single-nucleotide polymorphisms in cis with NEFL disease mutations for gene excision. We validated this haplotype editing approach in two iPSC lines with different missense mutations and demonstrated phenotypic rescue in iPSC-motor neurons. Surprisingly, our analysis revealed that gene inversion, a frequent by-product of excision editing, failed to reliably disrupt mutant allele expression. We deployed novel molecular assays to optimize our approach and achieve therapeutic levels of editing in immature iPSC-motor neurons. Finally, population genetics analysis demonstrated the power of haplotype editing to enable therapeutic development for the greatest number of patients. Our data serve as an important case study for many dominant genetic disorders amenable to this approach.},
}
@article {pmid41247434,
year = {2026},
author = {Liu, F and Ge, D and Lian, G and Han, Z and Chai, J},
title = {Unraveling plant immunity: from pathogen perception to resistance engineering.},
journal = {Science China. Life sciences},
volume = {69},
number = {3},
pages = {779-793},
pmid = {41247434},
issn = {1869-1889},
mesh = {*Plant Immunity/genetics ; *Disease Resistance/genetics/immunology ; *Plant Diseases/immunology/microbiology ; Receptors, Pattern Recognition/metabolism/immunology ; NLR Proteins/metabolism/genetics ; Signal Transduction ; *Plants/immunology/genetics ; Calcium Signaling ; Plant Proteins/genetics/metabolism ; Host-Pathogen Interactions/immunology ; CRISPR-Cas Systems ; },
abstract = {Unlike animals, which rely on circulatory systems and mobile immune cells, each plant cell must autonomously detect and respond to pathogenic threats. Plant immunity operates through two major layers: pattern-triggered immunity (PTI), initiated by cell-surface pattern recognition receptors (PRRs), and effector-triggered immunity (ETI), primarily mediated by intracellular nucleotide-binding leucine-rich repeat (NLR) receptors. Recent advances have substantially enhanced our understanding of PTI and ETI signaling. Notably, some NLRs following activation by specific recognition of pathogen effectors form higher-order oligomeric complexes termed resistosomes that act as Ca[2+]-permeable channels to trigger immune signaling. Increasing evidence points to extensive crosstalk and mutual potentiation between PTI and ETI, with Ca[2+] functioning as a pivotal second messenger in both pathways. Elucidating the molecular basis of these pathways, combined with emerging tools like CRISPR/Cas9, offers new strategies for engineering durable disease resistance in crops. This review highlights current insights into PTI and ETI, with an emphasis on the central role of Ca[2+] signaling and key challenges in engineering NLR receptors.},
}
@article {pmid41170937,
year = {2026},
author = {Soliman, H and Akram, N and Saleh, M},
title = {CRISPR-Enhanced RAA-SHERLOCK Assay for Point-of-Care Detection of Cyprinid Herpesvirus-3: Development, Validation and Clinical Application.},
journal = {Journal of fish diseases},
volume = {49},
number = {4},
pages = {e70079},
pmid = {41170937},
issn = {1365-2761},
support = {//Veterinary Medicine University/ ; //Academy of Scientific Research and Technology/ ; },
mesh = {*Fish Diseases/diagnosis/virology ; Animals ; *Herpesviridae/isolation &amp; purification/genetics ; *Herpesviridae Infections/veterinary/diagnosis/virology ; *Carps ; CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Point-of-Care Systems ; Recombinases/metabolism ; },
abstract = {Koi herpesvirus disease (KHVD), caused by Cyprinid herpesvirus-3 (CyHV-3), poses a significant threat to global aquaculture due to its high mortality rates and economic impact. Current diagnostic methods, such as PCR, are limited by equipment dependency and procedural complexity, hindering point-of-care (POC) applications. To address this, we developed an integrated assay combining recombinase-aided amplification (RAA) with CRISPR-Cas13a-mediated SHERLOCK technology and lateral flow detection (LFD) for rapid and visual detection of CyHV-3 in clinical samples. The KHV-SHERLOCK assay targets a conserved region of the CyHV-3 thymidine kinase (TK) gene, demonstrating exceptional specificity with no cross-reactivity to related pathogens or host DNA. Sensitivity evaluations revealed a detection limit of 100 ag/μL for CyHV-3 plasmid DNA, tenfold more sensitive than the conventional PCR (1 fg/μL) assay, even in the presence of 100 ng of carp genomic DNA as background interference. Clinical validation using 50 archived samples showed 100% concordance with reference PCR results, confirming diagnostic reliability. The assay's isothermal RAA step (37°C, 40 min) and CRISPR-Cas13a detection (37°C, 1 h) enable equipment-free operation, while LFD provides unambiguous visual results within minutes. This platform merges high sensitivity with POC practicality, offering a transformative tool for field-based KHVD surveillance.},
}
@article {pmid41078118,
year = {2026},
author = {Wang, Y and Yang, J and Hou, H and Song, L and Cheng, X and Liu, YX},
title = {Advancing Plant Microbiome Research Through Host DNA Depletion Techniques.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1189-1203},
pmid = {41078118},
issn = {1467-7652},
support = {32470055//National Natural Science Foundation of China/ ; U23A20148//National Natural Science Foundation of China/ ; CAAS-BRC-CB-2025-01//Basic Research Center for Crop Biosafety Sciences/ ; CAAS-ZDRW202308//Agricultural Science and Technology Innovation Program/ ; },
mesh = {*Microbiota/genetics ; *Plants/microbiology/genetics ; *Metagenomics/methods ; *DNA, Plant/genetics/isolation &amp; purification ; CRISPR-Cas Systems ; },
abstract = {Plants provide ecological habitats for diverse microorganisms, making accurate metagenomic sequencing essential for understanding the complex interactions that support plant growth, development and disease resistance. However, host DNA contamination poses a major challenge in plant microbiome studies, obscuring microbial genetic signatures and complicating the accurate analysis of microbial genomes. This review provides a comprehensive overview of current host DNA depletion strategies, including physical separation (e.g., filtration, gradient centrifugation), selective lysis and enzymatic treatments targeting plant cell walls. Advanced techniques such as targeted sequence capture with magnetic beads, methylation-based enrichment and nanopore selective sequencing offer additional options for host DNA removal. Despite these advances, current methods still face challenges in efficiency, specificity and applicability, emphasising the need for tailored strategies and the exploration of novel approaches for microbial enrichment. Innovations like CRISPR-Cas9 and chromatin immunoprecipitation-based host DNA depletion methods are proposed to provide novel directions for addressing current limitations. The development and refinement of host depletion techniques tailored to plant systems are crucial for enabling high-resolution, cost-effective metagenomic studies. These efforts promise to deepen our understanding of microbial diversity and functionality, ultimately accelerating microbiome-based innovations in crop improvement, sustainable agriculture and ecosystem resilience.},
}
@article {pmid40744834,
year = {2026},
author = {Qin, Z and Deng, Z and Li, C and Wang, D and Ji, X and Chen, Z},
title = {AI sheds new light on genome editing.},
journal = {Trends in biotechnology},
volume = {44},
number = {3},
pages = {696-709},
doi = {10.1016/j.tibtech.2025.07.011},
pmid = {40744834},
issn = {1879-3096},
mesh = {*Gene Editing/methods ; *Artificial Intelligence ; CRISPR-Cas Systems/genetics ; Humans ; Biotechnology ; },
abstract = {Artificial intelligence (AI) has revolutionized life sciences, driving transformative advances in engineering clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)-based genome editors for therapeutic and agricultural applications. Recent breakthroughs demonstrate how deep learning accelerates the discovery, engineering, and design of next-generation genome editing tools. In this review, we explore how AI-driven approaches are supercharging genome editing in three aspects: (i) structure-based methods for discovering novel genome editors neglected by conventional methods, (ii) engineering genome editors with enhanced properties, and (iii) the de novo design of entirely new genome editors endowed with bespoke functions. Finally, we discuss the current challenges and envision the future potential of data-driven AI to unlock new possibilities in genome editing, catalyzing innovations across biology and biotechnology.},
}
@article {pmid41754535,
year = {2026},
author = {Li, H and Wang, R and Li, J and Duan, W and Liang, Y and Sun, Q and Zhou, J and Zhang, Y},
title = {SHFL Post-Transcriptionally Restricts Coxsackievirus A16 In Vitro and In Vivo.},
journal = {Viruses},
volume = {18},
number = {2},
pages = {},
pmid = {41754535},
issn = {1999-4915},
support = {GJJKJ-2024-ZY//National Disease Control and Prevention Administration Public Health Talent Training Support Project/ ; ZDGWNLJS25-36//National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases (NITFID)/ ; 2021YFC2302003//National Key Research and Development Program of China/ ; },
mesh = {Animals ; Mice ; Virus Replication ; *Coxsackievirus Infections/virology ; *Enterovirus A, Human/genetics/physiology ; Humans ; *Enterovirus/genetics/physiology ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; Hand, Foot and Mouth Disease/virology ; Cell Line, Tumor ; Viral Load ; Interferon-beta/pharmacology ; },
abstract = {Coxsackievirus A16 (CVA16), a major etiological agent of hand, foot, and mouth disease, is increasingly contributing to neurological complications, with no vaccines or virus-specific antivirals currently available. To identify CVA16-restricting host factors, we investigated the role of the interferon-stimulated gene shiftless (SHFL), previously implicated in the control of other RNA viruses. Using CRISPR-Cas 9, we generated SHFL knockout rhabdomyosarcoma cells and assessed viral replication, cytopathic effects, and replication stage dynamics. We evaluated disease progression and tissue injury in neonatal mice infected with a mouse-adapted CVA16 strain. SHFL expression was strongly induced during CVA16 infection and was inducible by exogenous interferon-β treatment, and its loss markedly increased infectious virus production, accelerated early replication, and exerted severe cytopathic effects. In vivo, SHFL deficiency led to rapid weight loss, pronounced neurological signs, increased viral burden across multiple tissues, and uniform mortality, together with high viral loads and extensive pathological damage in the central nervous system, lungs, and skeletal muscle. Transcriptomic analyses revealed SHFL-dependent modulation of adhesion- and mitogen-activated protein kinase-related pathways. Overall, our results suggest SHFL as a key determinant of host resistance to CVA16, acting mainly at the post-transcriptional stage to limit viral spread and tissue injury, and highlight SHFL-linked pathways as promising host-directed antiviral targets.},
}
@article {pmid41753780,
year = {2026},
author = {Palanisamy, V and Bosilevac, JM and Barkhouse, DA and Velez, SE and Dass, SC},
title = {Unraveling the Coevolutionary Dynamics of Phage and Bacterial Protein Warfare Occurring in the Drains of Beef-Processing Plants.},
journal = {Microorganisms},
volume = {14},
number = {2},
pages = {},
pmid = {41753780},
issn = {2076-2607},
support = {2020-67017-30776//USDA-NIFA/ ; },
abstract = {Phages, the most abundant entities on Earth, exhibit a complex interplay with bacteria, especially within environmental biofilms, resulting in an ecological arms race. This study investigates the interaction between phages and bacteria in the drains of beef-processing plants using high-throughput sequencing and metagenomic analysis. Metagenomic data collected from 75 drain samples from beef-processing plants were analyzed to investigate phage-bacterial interactions. First, assembled contigs were screened to identify viral sequences, which were then taxonomically annotated to determine the viral composition, including phages. Functional annotation of these viral sequences provided information about the viral genes and their roles in bacterial interactions specifically associated with attack and counterattack of bacteria. In parallel, bacterial contigs were examined to identify genes associated with antiphage defense systems, providing insights into the strategies adapted by bacteria to resist phage infection. Taxonomic annotation of viral sequences from the bulk metagenomic data revealed the presence of phages targeting Pseudomonas, Klebsiella, and Enterococcus. The higher abundance of Pseudomonas phages aligns with our previous study, where Pseudomonas was identified as the dominant bacterial genus, suggesting potential copersistence of phages and their hosts. Functional annotation of phage contigs revealed infective and lysis-related genes, highlighting their potential role in bacterial attack. Conversely, bacterial contigs encoded antiphage defense systems, including CRISPR-Cas, restriction-modification, and other defense-related genes. The study also uncovered the presence of anti-CRISPR proteins in phages, suggesting a counterattack on the bacterial defense. These findings provide evidence for phage attack, bacterial defense, and phage counterattack and may showcase the ongoing coevolutionary arms race between phages and bacteria. While this evidence looks promising, these results remain preliminary and further studies are needed to validate these findings. Still, this study provides a foundational understanding of bacteria-phage coexistence in beef-processing plant drains and paves the way for further explorations of these intricate interactions and their possible applications in controlling pathogenic microorganisms within biofilms.},
}
@article {pmid41752145,
year = {2026},
author = {Fayed, S and Amer, S and Badawy, M and Bou Malhab, L and Omran, N and Khoder, G and Ghemrawi, R and Haider, M and Hamoudi, R and Harati, R},
title = {The Role of CRISPR and Its Therapeutic Applications in Glioblastoma.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41752145},
issn = {1422-0067},
support = {210111350//University of Sharjah/ ; 2201110368//University of Sharjah/ ; 23010902146//University of Sharjah/ ; VRI-20-10//ASPIRE Precision Medicine Research Institute Abu Dhabi/ ; },
mesh = {Humans ; *Glioblastoma/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Brain Neoplasms/therapy/genetics ; Animals ; *Genetic Therapy/methods ; },
abstract = {Glioblastoma multiforme (GBM) remains the most aggressive and treatment-refractory form of primary brain tumor in adults, characterized by rapid proliferation, intratumoral heterogeneity and resistance to current therapies. Despite therapeutic advancements in surgical resection, radiotherapy and chemotherapy, clinical outcomes remain poor, underscoring the need for innovative molecular strategies. This review examines the therapeutic potential of CRISPR/Cas9 genome-editing technologies in GBM, highlighting their ability to model, dissect and potentially correct the genetic alterations that drive GBM tumorigenesis. Key molecular targets, such as EGFR, PTEN, TP53, NF1 and PIK3CA, are discussed within the context of GBM's mutational and signaling landscape. We further outline emerging CRISPR applications in preclinical models, the current status of CRISPR-based clinical trials and the major barriers hindering translation, including off-target effects, immunogenicity and the challenge of delivering gene-editing systems across the blood-brain barrier. Particular emphasis is placed on delivery technologies, viral and non-viral vectors, including lipid nanoparticles, polymeric systems, inorganic nanocarriers and DNA nanostructures, which are rapidly evolving to improve precision, safety and CNS penetrance. Collectively, this review highlights CRISPR/Cas9 as a powerful tool whose integration with molecular neuro-oncology and precision medicine may ultimately shift GBM treatment toward more personalized and durable therapeutic interventions.},
}
@article {pmid41751979,
year = {2026},
author = {Eskildsen, J and Dong, M and Hanak, T and Madsen, CK and Holme, I and Plaszkó, T and Vestergård, M and Nicolaisen, M and Thordal-Christensen, H and Brinch-Pedersen, H},
title = {Novel CRISPR/Cas9-Derived mlo Alleles in Barley: Resistance to Powdery Mildew and Microbiome Implications.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751979},
issn = {1422-0067},
support = {NNF19OC0056580//Novo Nordisk Foundation/ ; BarleyMicroBreed, 101060057//EU Horizon research and innovation/ ; },
mesh = {*CRISPR-Cas Systems ; *Hordeum/genetics/microbiology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; *Ascomycota/pathogenicity ; *Microbiota/genetics ; Alleles ; Plant Roots/microbiology/genetics ; *Plant Proteins/genetics ; Mutation ; },
abstract = {Barley grown in temperate regions is often challenged by powdery mildew disease. An effective solution is mildew resistance locus o (mlo)-based resistance, which is monogenic, durable, and broad-spectrum. While the pleiotropic effects of mlo mutations on above-ground tissues are well documented, their impact on the root-associated microbiome remains underexplored. We utilized CRISPR/Cas9 to generate novel mlo mutant lines and evaluated their resistance to causal fungus Blumeria hordei. We further examined if mlo knockout has any impact on the overall root microbiome diversity and composition under field-like conditions and applied DESeq2 to compare the abundance of microbial taxa between mutants and wild type. We created five novel resistant mlo lines, including the first mutants with amino acid alterations in the protein's extracellular region. Mutant lines showed significantly reduced B. hordei colony formation (0.5-5%). While microbial alpha and beta diversity were not significantly altered, a few microbial taxa displayed time-dependent shifts in abundance. Overall, our study demonstrates the effectiveness of CRISPR/Cas9 in generating mlo-based resistance. Moreover, the study revealed functionally important residues in the protein's extracellular region. Finally, we present the first evidence of limited mlo-associated effects on root microbiome diversity and relative abundance of microbial taxa.},
}
@article {pmid41751840,
year = {2026},
author = {Begum, SN and Hasan, SK},
title = {Prime Editing Driven Functional Genomics: Bridging Genotype to Phenotype in the Post-Genomic Era.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751840},
issn = {1422-0067},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Genomics/methods ; Phenotype ; Genotype ; Animals ; },
abstract = {The post-genomic era, defined by large-scale sequencing initiatives, has generated an unprecedented catalogue of human genetic variation. Yet, the vast majority of genetic variants remain classified as variants of uncertain significance or are located within poorly characterized non-coding regions, thereby hindering the effective translation of genomic data into meaningful biological understanding and clinical application. Bridging this genotype-to-phenotype gap requires precise, high-throughput functional genomics. Early CRISPR-Cas9 knockout and CRISPR interference/activation (CRISPRi/a) screens mapped gene-level functions but could not assess single nucleotide variants (SNVs). Bridging this genotype-to-phenotype gap demands precise, high-throughput functional genomics. Multiplexed assays of variant effect (MAVEs), like saturation genome editing, systematically test all possible mutations using CRISPR-Cas9 and donor libraries. Base editors allow targeted single-base changes without double-strand breaks but are limited in scope, while prime editing can introduce any small substitution, insertion, or deletion without double-strand breaks (DSBs) or donor templates. This review traces the evolution of functional screens from gene-level knockouts to saturation genomic editing (SGE), and highlights how prime editing is driving a new paradigm for the systematic functional characterization of thousands of variants across disease-relevant genes. We also detail the architecture, mechanism, and progressive optimization of PE systems and their delivery methods. Collectively, prime editing stands as a transformative platform poised to accelerate precision functional genomics and advance the diagnosis and treatment of genetic diseases.},
}
@article {pmid41751799,
year = {2026},
author = {Cai, X and Liang, X and Zou, P and Xiao, R and Wang, Y},
title = {CRISPRi Screening Identifies Essential E. coli Virulence Factors for Placental Barrier Breach in a Maternal-Fetal Infection Model.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751799},
issn = {1422-0067},
support = {82574169//National Natural Science Foundation of China/ ; 7232009, 7244289//Natural Science Foundation of Beijing Municipality/ ; 20240484724//Cross-cooperation project of Beijing Science and Technology New Star Program/ ; Subject leaders-03-02//High Level Public Health Technical Personnel Construction Project/ ; //Research Foundation of Capital Institute of Pediatrics/ ; //Beijing Chaoyang District Postdoctoral Research Foundation 2024/ ; },
mesh = {Female ; Pregnancy ; Animals ; *Virulence Factors/genetics/metabolism ; *Escherichia coli Infections/microbiology/genetics ; *Escherichia coli/pathogenicity/genetics ; Rats ; *Placenta/microbiology/metabolism ; Humans ; Disease Models, Animal ; Escherichia coli Proteins/genetics/metabolism ; Host-Pathogen Interactions ; Trophoblasts/microbiology/metabolism ; CRISPR-Cas Systems ; *Pregnancy Complications, Infectious/microbiology ; Neonatal Sepsis/microbiology ; },
abstract = {Early-onset neonatal sepsis caused by Escherichia coli (E. coli) threatens neonates' lives due to the pathogen's high virulence and multidrug resistance. The mechanisms that enable its placental barrier breach are poorly understood. Using a clinically isolated ST95 ExPEC strain from a neonatal sepsis case, along with a pregnant rat model and an in vitro placental barrier model, we performed CRISPR interference screening. This screen targeted 264 virulence factor genes and identified virulence factors for motility, iron acquisition, hemolysin secretion, and adherence/invasion as critical. We demonstrated that hlyB is essential for uterine infection, and we elucidated a mechanism for ibeA that facilitates syncytial trophoblast cell layer penetration by interacting with the host receptor(s) PSF/VIM to enhance bacterial internalization. Host cells countered ibeA+ E. coli infection via a novel host defense pathway involving upregulation of ASPHD1. This study systematically mapped the virulence factors required for E. coli placental translocation and delineated key host-pathogen interactions.},
}
@article {pmid41751614,
year = {2026},
author = {Sun, Q and Guo, Y and Wang, L and Jia, L and Wei, P and Ma, S},
title = {CRISPR-Mediated Silkworm: The Oncoming Agricultural Revolutions and a Rising Model Organism.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751614},
issn = {2073-4425},
support = {32570591//National Natural Science Foundation of China/ ; },
mesh = {*Bombyx/genetics/growth &amp; development ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Agriculture/methods ; Silk/genetics ; Genome, Insect ; Genomics/methods ; },
abstract = {The silkworm (Bombyx mori) is essential to sericulture and is also becoming a key model organism in genomics and agriculture. For decades, genetic studies of the silkworm were limited by inefficient and inflexible genome tools. CRISPR genome editing allows precise and scalable alterations to genes regulating development, physiology, and industrial traits. This review summarizes silkworm genome-editing breakthroughs, highlighting CRISPR's evolution from simple gene knockouts to large-scale genome-wide screening. We highlight how these advancements contribute to disease resistance, higher yields, and the development of new silk-based materials, as well as how they influence the development and growth rate of the sericulture. The creation of high-quality reference genomes, pangenomes, and genome-wide screening systems has made the silkworm a major model for integrating multiple biological datasets and approaches, such as genomic, transcriptomic, and proteomic. By considering the unique biological characteristics of the silkworm, this provides new insights for research on silk biology, piRNA synthetic biology, and hormonal signaling regulation. Finally, we examine new areas at the intersection of CRISPR, pangenomics, and artificial intelligence (AI) and suggest future paths for molecular breeding, pest control, and synthetic biology. Moreover, AI-assisted prediction of CRISPR outcomes is utilized to inform the design of targeted trait modifications, representing an approach to enhancing biomanufacturing efficiency and eco-friendly silk production. Together, these advances have made the silkworm a flexible genetic platform and an important part of sustainable agriculture and biomanufacturing.},
}
@article {pmid41751560,
year = {2026},
author = {Lee, S and Park, S and Bang, H and Kim, SU and Park, YH and Wee, G and Chae, U and Kim, E},
title = {VPS35 Deficiency Markedly Reduces the Proliferation of HEK293 Cells.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751560},
issn = {2073-4425},
mesh = {Humans ; *Cell Proliferation/genetics ; *Vesicular Transport Proteins/genetics/deficiency/metabolism ; HEK293 Cells ; Apoptosis/genetics ; CRISPR-Cas Systems ; Mitochondrial Dynamics/genetics ; Mitochondria/metabolism/genetics ; Gene Knockout Techniques ; },
abstract = {Background/Objectives: The retromer protein complex is involved in various physiological processes, especially endosomal trafficking, and its dysregulation has been linked to Alzheimer's disease and Parkinson's disease, as well as VPS35 knockout (KO), causing early embryonic lethality. We aimed to investigate the cellular consequences of VPS35 deficiency. Methods: To investigate the effects of VPS35 loss, we used CRISPR/Cas9 to generate VPS35 KO human embryonic kidney 293 (HEK293) cells. We analyzed changes in retromer component expression, cell proliferation, apoptosis, and mitochondrial dynamics using Western blotting, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and confocal microscopy. Results: VPS35 KO led to a significant reduction in cell proliferation and decreased expression of VPS29 and VPS26, both essential for retromer complex assembly. Consequently, retromer formation was impaired. Compared to control cells, KO cells exhibited elevated levels of cleaved caspase-3, poly(ADP-ribose) polymerase, cytochrome C, and p21, while the expression of Ki-67, CDK4, and cyclin D was reduced. Additionally, VPS35 deletion also promoted mitochondrial fragmentation, associated with increased expression of mitochondrial fission-related proteins. Finally, the rescue experiment using the human VPS35 gene confirmed that the recovery of VPS35 not only led to the recovery of the essential elements constituting the retromer but also the recovery of molecules related to the cell cycle, restoring cell death to a normal level. Conclusions: These findings suggest that VPS35 plays a critical role in cell growth and survival by modulating apoptosis, mitochondrial dynamics, and cell cycle progression.},
}
@article {pmid41751536,
year = {2026},
author = {Hawkins, V and Rudiger, SR and McLaughlan, CJ and Kelly, JM and Lehnert, K and Jacobsen, JC and Handley, RR and Henare, K and Verma, PJ and Snell, RG},
title = {Foundations of an Ovine Model of Fragile X Syndrome.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751536},
issn = {2073-4425},
support = {3914//Curekids/ ; 20/259//Health Research Council of New Zealand/ ; },
mesh = {Animals ; *Fragile X Syndrome/genetics/pathology ; *Disease Models, Animal ; *Fragile X Messenger Ribonucleoprotein 1/genetics ; Sheep/genetics ; Male ; Female ; CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; Humans ; },
abstract = {BACKGROUND: Fragile X Syndrome (FXS) is an X-linked neurodevelopmental disorder characterised by intellectual disability, developmental delays, anxiety, and social and behavioural challenges. Currently, no effective treatments exist to address the root cause of FXS. Mouse models are the most widely used for studying molecular pathogenesis and conducting preclinical treatment testing. However, therapeutic interventions that show promise in rodent models have yet to succeed in clinical trials. After evaluating the current models, we have developed an ovine model to address this clinical translation gap. We expect this model to more accurately reflect the human condition in brain size, structure, and neurodevelopmental trajectory. We aim to establish this model as a valuable preclinical platform for testing therapies for FXS.<br><br>METHODS: To generate the sheep model, we used CRISPR-Cas9 dual-guide editing to knock out the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene in ovine embryos.<br><br>RESULTS: Two founder animals were created, one ram (male) and one ewe (female), both of which carried FMR1 gene knockouts. The ewe carries inactivating mutations on both alleles, with the edits in both animals resulting in no detectable Fragile X Messenger Ribonucleoprotein (FMRP) as expected. Both founders have undergone molecular characterisation and basic health checks, with the female founder showing increased joint flexibility, a characteristic of FXS. The ram has been used for breeding, with the successful transmission of the edited allele to his offspring. Importantly, specific lamb cohorts for postnatal treatment testing can be produced efficiently utilising accelerated breeding methods and preimplantation selection.},
}
@article {pmid41750315,
year = {2026},
author = {Schulze, A and Kainz, K and Bauer, MA and Carmona-Gutierrez, D},
title = {Editing Candida: Origins and Advances of CRISPR Tools.},
journal = {Biomolecules},
volume = {16},
number = {2},
pages = {},
pmid = {41750315},
issn = {2218-273X},
support = {10.55776/P37278//FWF Austrian Science Fund/ ; not applicable//University of Graz/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Candida/genetics/pathogenicity ; Humans ; Virulence/genetics ; Genome, Fungal ; Candidiasis/microbiology/genetics ; },
abstract = {Pathogens causing candidiasis encompass a diverse group of ascomycetous yeasts that have become essential models for studying fungal adaptability, pathogenicity, and host-pathogen interactions. Although many candidiasis-promoting species exist as commensals within host microbiota, several have acquired virulence traits that enable opportunistic infections, positioning them as a leading cause of invasive fungal disease in humans. Deciphering the molecular and genetic determinants that underpin the biology of organisms responsible for candidiasis has long been a central objective in medical and molecular mycology. However, research progress has been constrained by intrinsic biological challenges, including noncanonical codon usage and the absence of a complete sexual cycle in diploid species, which have complicated traditional genetic manipulation. CRISPR-Cas9 genome editing has overcome many of these limitations, providing a precise, efficient, and versatile framework for targeted genomic modification. This system has facilitated functional genomic studies ranging from single-gene deletions to high-throughput mutagenesis, yielding new insights into the mechanisms governing virulence, antifungal resistance, and stress adaptation. Since its initial application in Candida albicans, CRISPR-Cas9 technology has been refined and adapted for other clinically and industrially relevant species, including Nakaseomyces glabratus (formerly referred to as Candida glabrata), Candida parapsilosis, and Candida auris. The present work provides an overview of the evolution of genetic approaches employed in research directed against candidiasis-associated species, with a particular focus on the development and optimization of CRISPR-based systems. It highlights how recent advancements have improved the genetic tractability of these pathogens and outlines emerging opportunities for both fundamental and applied studies in fungal biology.},
}
@article {pmid41748831,
year = {2026},
author = {Moreno, DS and Carvalho, JP and Murray, E and Colombo, NSR and Lamas, A and Cobas, AC and Hill, C and Azeredo, J and Domingues, L},
title = {Evaluation of the delivery of an anti-Listeria endolysin via CRISPR-Cas9 engineered probiotic Saccharomyces boulardii.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {41748831},
issn = {1432-0614},
mesh = {*Listeria monocytogenes/drug effects ; *Probiotics ; *CRISPR-Cas Systems ; *Endopeptidases/genetics/pharmacology/metabolism ; *Saccharomyces boulardii/genetics/metabolism ; Humans ; Bacteriophages/enzymology ; *Anti-Bacterial Agents/pharmacology/metabolism ; Saccharomyces cerevisiae/genetics ; },
abstract = {Listeriosis is a foodborne infection caused by Listeria monocytogenes that causes febrile gastroenteritis and central nervous system infections and that can often lead to fatality. Upon consumption of contaminated food, Listeria is able to survive a number of gastrointestinal stressors, including competition with the host microbiota. The emergence of antibiotic-resistant clones of L. monocytogenes, together with the side effects of antibiotic treatment, highlights the need for alternatives or additives for its treatment and prevention. Saccharomyces boulardii is a probiotic yeast that is often used alongside antibiotics to minimize side effects since it is not affected by them as a result of its eukaryotic nature. Furthermore, it can be engineered to produce a wide range of molecules. We previously engineered Saccharomyces cerevisiae through CRISPR-Cas9 integration to produce Ply511, a bacteriophage endolysin active against L. monocytogenes, showing the potential of engineered yeast to produce endolysins for biocontrol. In this study, we extended this approach to the probiotic yeast S. boulardii and directly compared the two yeasts as secretion hosts for Ply511. Using a simulated human gastrointestinal environment, we evaluated their ability to retain endolysin activity and reduce L. monocytogenes levels. We then tested the cell extracts from both yeasts in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI), confirming a specificity for Listeria. Finally, we evaluated their activity in a simulated intestinal fermentation using fecal samples from human donors. Overall, this study demonstrates the potential of delivering endolysins to the gut via engineered probiotic S. boulardii. KEY POINTS: CRISPR-Cas9-engineered S. boulardii and S. cerevisiae were compared, both allowing the expression and activity of endolysin Ply511 against L. monocytogenes. Endolysin Ply511 retained its activity against L. monocytogenes in simulated gastrointestinal digestion and was specific against Listeria in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI). Using fecal samples from human donors, the anti-Listeria effect was reduced potentially due to the lower metabolic activity of S. boulardii and the higher competition with the intestinal microbiome.},
}
@article {pmid41719925,
year = {2026},
author = {Park, YJ and Song, DY and Jeon, HB and Kim, DM},
title = {Nucleic acid detection via protein readout through Cas-controlled gating of cell-free protein synthesis.},
journal = {Biosensors &amp; bioelectronics},
volume = {301},
number = {},
pages = {118514},
doi = {10.1016/j.bios.2026.118514},
pmid = {41719925},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Cell-Free System ; *Protein Biosynthesis/genetics ; *Escherichia coli O157/genetics ; Bacillus anthracis/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Ribosomal, 16S/genetics/analysis ; *Nucleic Acids/analysis/genetics ; Bacterial Proteins/genetics ; },
abstract = {We present a modular platform that converts CRISPR target recognition into programmable protein outputs for nucleic acid detection. This system couples Cas-mediated collateral cleavage with cell-free protein synthesis. In the presence of a target, Cas-mediated collateral cleavage releases an extension-competent trigger DNA that gates reporter expression. Although collateral cleavage is inherently indiscriminate, we achieve deterministic fragment generation by employing chemically programmed precursors bearing backbone modifications-exemplified here by phosphorothioate linkages-that constrain cleavage to predefined sites. Using Bacillus anthracis and E. coli O157:H7 as a model, the developed CRIVER assay enables concurrent readouts of 16S rRNA together with the species-specific capB or ecf1 loci by integrating Cas13a-mediated RNA recognition and Cas12a-mediated DNA recognition into a dual-channel workflow. Taken together, proposed method establishes addressable signaling at the protein layer, supports protein-based outputs in a modular workflow, and provides a general route to sensitive, dual-channel nucleic acid detection.},
}
@article {pmid41700332,
year = {2026},
author = {Huang, Y and Zhao, Z and Li, J and Wang, X and Qu, H and Zheng, Y},
title = {An aptamer-CRISPR/Cas12a biosensor for rapid and sensitive detection of florfenicol.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {9},
pages = {1910-1916},
doi = {10.1039/d5ay01903f},
pmid = {41700332},
issn = {1759-9679},
mesh = {*Biosensing Techniques/methods ; *Thiamphenicol/analogs &amp; derivatives/analysis ; *Aptamers, Nucleotide/chemistry/genetics ; *CRISPR-Cas Systems ; *Anti-Bacterial Agents/analysis ; Limit of Detection ; Animals ; Food Contamination/analysis ; },
abstract = {Florfenicol (FF), a broad-spectrum antibacterial agent widely used in livestock and poultry farming, has raised significant food safety concerns due to the accumulation of its residues in animal-derived products (e.g., eggs), posing potential threats to human health. Herein, we developed a novel aptamer-CRISPR/Cas12a biosensor for the rapid and sensitive detection of FF. The biosensor employs streptavidin-modified magnetic beads (SA-MBs) as a solid carrier to achieve efficient enrichment of FF-specific aptamers (APT), while integrating the dual advantages of the APT's high-specificity target recognition and the CRISPR/Cas12a system's powerful signal amplification capability. The detection mechanism is based on a competitive displacement: APT pre-hybridizes with its complementary strand (APT-c) to form stable duplexes. The presence of FF triggers the release of APT-c from APT, and the liberated APT-c then activates the trans-cleavage activity of the CRISPR/Cas12a system. This process converts the small-molecule FF into a CRISPR/Cas12a-detectable nucleic acid signal and enables quantitative FF detection. Under optimized conditions, the biosensor demonstrated a linear detection range of 10 nM to 100 µM for FF (R[2] = 0.9907) and a limit of detection (LOD) of 1.41 nM. The accuracy and practicality were confirmed through spiked recovery experiments in egg samples, yielding recoveries between 97.1% and 100.8%. Furthermore, the modular design of this platform allows its easy adaptation for detecting other antibiotics simply by replacing the specific APT and its corresponding APT-c, highlighting its considerable potential for broad applications in food safety monitoring.},
}
@article {pmid41693124,
year = {2026},
author = {Xin, W and Tang, Z and Wang, S and Song, ZL and Luo, X},
title = {Scaffold-Proximal DNA Extensions Enhance Cas12a Trans-cleavage for Direct and Broad-Scope Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {8},
pages = {6149-6162},
doi = {10.1021/acs.analchem.5c07125},
pmid = {41693124},
issn = {1520-6882},
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry ; CRISPR-Cas Systems ; *DNA/chemistry/metabolism/genetics ; *MicroRNAs/analysis/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; Polymorphism, Single Nucleotide ; *RNA, Messenger/analysis ; *Bacterial Proteins/metabolism ; },
abstract = {The CRISPR/Cas12a system has revolutionized nucleic acid diagnostics, but its direct application for RNA detection remains constrained by the requisite reverse transcription step, insufficient sensitivity, and poor compatibility across diverse RNA targets such as microRNAs (miRNAs), long mRNAs, and single nucleotide polymorphisms (SNPs). Here, we introduce a rationally engineered DNA activator architecture that markedly enhances Cas12a trans-cleavage activity and expands its analytical utility. A key mechanistic finding is the strong positional dependence of activator extensions: appending an overhang specifically at the scaffold-proximal end of the DNA activator (termed Proximal-Extended Activator, PEA) potently boosts Cas12a activation through ribonucleoprotein (RNP) stabilization, whereas distal extensions are inhibitory due to steric hindrance of essential interdomain motions. This optimized PEA system facilitates direct, amplification-free RNA detection, achieving exceptional sensitivity with detection limits of 1.3 fM for miRNA and 93 fM for mRNA, all without reverse transcription. Furthermore, a Split-PEA format confers exceptional discriminatory power for SNPs, enabling robust identification of the EGFR T790 M mutation at a 0.1% allelic frequency. This work establishes a facile and versatile platform where simple sequence modification enables highly sensitive and specific analysis of a broad range of nucleic acid targets, effectively overcoming a significant hurdle in CRISPR-based diagnostics.},
}
@article {pmid41671611,
year = {2026},
author = {Raban, R and James, AA and Akbari, OS},
title = {Advances in CRISPR gene drives for mosquito population control.},
journal = {Current opinion in microbiology},
volume = {90},
number = {},
pages = {102712},
doi = {10.1016/j.mib.2026.102712},
pmid = {41671611},
issn = {1879-0364},
mesh = {Animals ; *Gene Drive Technology/methods ; *Culicidae/genetics ; *Mosquito Control/methods ; *CRISPR-Cas Systems ; *Mosquito Vectors/genetics ; },
abstract = {CRISPR-based gene drive (GD) systems bias allele inheritance during meiosis, enabling transgenes to spread at rates exceeding Mendel's law of segregation. This capability underlies their potential as powerful tools for controlling mosquito-borne diseases. GDs can be engineered either to suppress mosquito populations or to modify them by introducing traits that block pathogen transmission. Recent advances have focused on improving evolutionary stability, with modeling studies providing insights into expected population dynamics. With a focus on the most current population modification GDs, we discuss advances in GD architectures - including integral and allelic drives, combined modification-suppression systems, and both homing and non-homing toxin-antidote designs - that expand the range of possible strategies and address limitations of early homing drives. Numerous antipathogen effectors with strong pathogen-blocking activity can now be coupled to these systems, with current efforts assessing their durability against genetically diverse pathogens. Key challenges remain, including resistance evolution, ecological impacts, and long-term stability. Nonetheless, GDs offer a promising approach for reducing disease transmission, especially in regions where conventional interventions are difficult to sustain.},
}
@article {pmid41671237,
year = {2026},
author = {Bagheri, N and Bertucci, A and Merlo, R and Porchetta, A},
title = {Synthetic DNA Transducers Integrate DNA Repair to CRISPR Signal Transduction.},
journal = {ACS sensors},
volume = {11},
number = {2},
pages = {1634-1644},
doi = {10.1021/acssensors.5c04118},
pmid = {41671237},
issn = {2379-3694},
mesh = {*DNA Repair ; Humans ; *CRISPR-Cas Systems/genetics ; DNA Glycosylases/metabolism ; Uracil-DNA Glycosidase/metabolism ; *Signal Transduction ; *DNA/genetics/metabolism/chemistry ; CRISPR-Associated Proteins/metabolism/genetics ; Endodeoxyribonucleases/metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/metabolism/genetics ; },
abstract = {CRISPR-based molecular diagnostics have revolutionized nucleic acid detection, yet the integration of upstream enzyme activity into programmable CRISPR output remains largely unexplored. Here, we present a synthetic transduction platform that directly couples endogenous DNA repair activity with CRISPR-Cas12a activation. By linking base excision repair (BER) events to the structural switching of a programmable DNA transducer, we convert the activity of DNA glycosylases, such as uracil DNA glycosylase (UDG) and human 8-oxoguanine glycosylase (hOGG1), into a robust fluorescence signal via Cas12a-mediated collateral (trans-) cleavage. This one-step assay allows rapid and sensitive lysate-based detection of repair activity with high specificity. In addition, it can also be easily adapted to achieve rapid throughput screening of small molecule inhibitors. The rational modular design supports the adaptation to various glycosylase activities, establishing a general framework for transducing DNA repair activity into programmable CRISPR output. Beyond bioanalytical applications, this approach paves the way for the development of synthetic gene circuits that respond to DNA repair activity and CRISPR-based drug screening platforms.},
}
@article {pmid41670012,
year = {2026},
author = {Miao, Y and Li, C and Su, Y and Peng, T and Wang, J and Liu, S and Ma, C and Li, L and Wang, Y},
title = {The application of CRISPR-Cas9 system in brain diseases.},
journal = {Nanomedicine (London, England)},
volume = {21},
number = {6},
pages = {899-920},
pmid = {41670012},
issn = {1748-6963},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Brain Diseases/therapy/genetics ; *Gene Editing/methods ; Blood-Brain Barrier/metabolism ; *Genetic Therapy/methods ; Animals ; Genetic Vectors/genetics ; },
abstract = {As an efficient genome-editing technology, Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-CRISPR-associated protein9 (Cas9) system is increasingly being recognized as a significant therapeutic strategy for brain diseases. In recent years, researchers have continuously tried to regulate the expression of genes related to the nervous system through CRISPR-Cas9 system, which provides a new and efficient strategy for the treatment of brain diseases. At the same time, various delivery vectors of CRISPR-Cas9 system have been reported. Although some delivery vectors have not been applied to the research of brain diseases, they still provide valuable ideas for the brain delivery of CRISPR-Cas9 system. In this review, we summarized the principle of CRISPR-Cas9 system and its application in the nervous system, discussed the barrier of blood-brain barrier (BBB) to the treatment of brain diseases, overviewed various delivery vectors of CRISPR-Cas9 system and their applications, and highlighted advanced of CRISPR-Cas9 system applied to various brain diseases. Furthermore, we also discussed the existing obstacles and promising avenues for future investigation regarding CRISPR-Cas9-based therapeutic approaches. This article, through retrieving keyword combinations[PubMed,from Jan. 2018 to Dec. 2025], aims to elucidate the CRISPR-Cas9 system's potential for extensive future research and application as a therapeutic strategy for brain disorders.},
}
@article {pmid41663776,
year = {2026},
author = {Pearce, JC and Campbell, JS and Prior, JL and Titball, RW and Wakefield, JG},
title = {PiggyBac-mediated transgenesis and CRISPR-Cas9 knockout in the greater wax moth, Galleria mellonella.},
journal = {Lab animal},
volume = {55},
number = {3},
pages = {95-102},
pmid = {41663776},
issn = {1548-4475},
support = {NC/W002388/1//National Centre for the Replacement Refinement and Reduction of Animals in Research (NC3Rs)/ ; NC/T001518/1//National Centre for the Replacement Refinement and Reduction of Animals in Research (NC3Rs)/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Moths/genetics ; *DNA Transposable Elements/genetics ; *Gene Knockout Techniques/methods ; Larva/genetics ; Animals, Genetically Modified/genetics ; *Gene Transfer Techniques ; },
abstract = {The larvae of the greater wax moth, Galleria mellonella, are gaining prominence as a versatile nonmammalian in vivo model to study host-pathogen interactions. Their ability to be maintained at 37 °C, coupled with a broad susceptibility to human pathogens and a distinct melanization response that serves as a visual indicator for larval health, positions G. mellonella as a powerful resource for infection research. Despite these advantages, the lack of genetic tools, such as those available for zebrafish and Drosophila melanogaster, has hindered development of the full potential of G. mellonella as a model organism. Here we describe a robust methodology for generating transgenic G. mellonella using the PiggyBac transposon system and for precise gene knockouts via CRISPR-Cas9 technology. These advances significantly enhance the utility of G. mellonella in molecular research, paving the way for its widespread use as an inexpensive and ethically compatible animal model in infection biology and beyond.},
}
@article {pmid41649900,
year = {2026},
author = {Zakiyyah, SN and Irkham, and Wulandari, DA and Zein, MIHL and Gaffar, S and Ozsoz, M and Ogata, G and Einaga, Y and Hartati, YW},
title = {CRISPR-Cas13a-Powered Electrochemical Biosensor for Label-Free Detection of Chikungunya Virus RNA Using a Ceria-Modified Screen-Printed Carbon Electrode.},
journal = {ACS sensors},
volume = {11},
number = {2},
pages = {923-938},
doi = {10.1021/acssensors.5c01924},
pmid = {41649900},
issn = {2379-3694},
mesh = {*Biosensing Techniques/methods/instrumentation ; *Chikungunya virus/genetics/isolation &amp; purification ; *RNA, Viral/analysis/genetics ; *Cerium/chemistry ; Electrodes ; *Electrochemical Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; Carbon/chemistry ; Limit of Detection ; Humans ; },
abstract = {This study aims to develop a label-free electrochemical biosensor for the detection of chikungunya virus (CHIKV) RNA, based on CRISPR-Cas13a integrated with a cerium oxide (ceria)-modified screen-printed carbon electrode (SPCE). The ceria film was deposited through cathodic electrodeposition, forming a uniform, needle-like film, as observed by SEM, and a crystalline fluorite structure was confirmed by XRD with characteristic (111), (200), and (220) reflections. The results showed that Raman spectroscopy demonstrated a dominant F2g band at ∼463 cm[-1], indicative of cubic ceria, while XPS analysis displayed the presence of 13.65% Ce[3+], contributing to favorable surface reactivity for biomolecule immobilization. This interface enhanced the attachment of a biotinylated RNA probe through streptavidin binding. Furthermore, a guide RNA (gRNA) was rationally designed to target the conserved region of the CHIKV E1 gene, with high specificity confirmed through in silico arrangement against related viruses. Upon target recognition, the activated Cas13a enzyme triggered collateral cleavage of the immobilized probe, leading to a measurable reduction in the guanine oxidation signal, detected by differential pulse voltammetry (DPV). This detection strategy was entirely label-free and amplification-free, simplifying both sensor fabrication and operation. The biosensor achieved a detection limit of 1.325 ppt, had a linear response in the range of 1-10,000 ppt, and showed excellent selectivity against DENV and SARS-CoV-2. It also retained signal stability over 45 days and yielded a recovery rate of 94.98% in spiked human serum. In conclusion, this study represents a modular and programmable sensing platform for direct RNA detection that integrates RNA-guided molecular recognition and signal transduction without the need for labeled substrates or amplification, simplifying CRISPR-based diagnostics supporting good health and well-being through field-deployable applications.},
}
@article {pmid41643803,
year = {2026},
author = {Kim, C and Zhu, Z and Tagmount, A and Barbazuk, WB and Bacher, R and Vulpe, CD},
title = {Physiologically relevant 3D CRISPR screening enhances mechanistic insight into chemical toxicity compared to 2D screening.},
journal = {Toxicology},
volume = {522},
number = {},
pages = {154422},
doi = {10.1016/j.tox.2026.154422},
pmid = {41643803},
issn = {1879-3185},
mesh = {Humans ; Hep G2 Cells ; *Toxicogenetics/methods ; *CRISPR-Cas Systems ; *Doxorubicin/toxicity ; *Toxicity Tests/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Spheroids, Cellular/drug effects ; Bioreactors ; *Cell Culture Techniques, Three Dimensional ; },
abstract = {CRISPR-based approaches can complement other genomics-based toxicology studies by enabling causal interrogation of gene function modulating chemical-induced toxicity. Moreover, CRISPR screens enable scalable and systematic identification of functional pathways involved in cellular response to chemical exposure. Cell-based functional toxicogenomics approaches using CRISPR provide a potential powerful tool for the development of mechanism-driven new approach methodologies (NAMs) for toxicodynamic and toxicokinetic hazard screening to enable more effective risk assessment. To improve the physiological relevance of in vitro functional toxicogenomics, we developed a three-dimensional (3D) CRISPR screening platform using HepG2/C3A spheroids cultured in a continuously rotating bioreactor (ClinoStar). We evaluated the potential utility of a 3D CRISPR screen as compared to conventional 2D screen using a custom CRISPR sgRNA library representing common loss-of-function genetic variants in the human population and exposure to the well characterized DNA damaging toxicant, doxorubicin. The 3D platform identified more genes and pathways in which variants have previously been associated with doxorubicin toxicity in clinical studies than the 2D system. These results support the utility of 3D CRISPR screening to identify physiologically relevant genetic determinants underlying chemical toxicity.},
}
@article {pmid41637835,
year = {2026},
author = {Xie 谢飞, F and Liu 刘晓宙, X and Wang 王露露, L and Zhang 张聪, C and Liu 刘传宏, C and Huo 霍振庆, Z and Zhao 赵正东, Z and Zhao 赵清远, Q and He 贺秋月, Q and Guo 郭科男, K and Sun 孙宇, Y and Wang 王勇, Y},
title = {A porcine congenital deafness model with unconditional knockout of GJB2 generated by CRISPR/Cas9 genomic editing.},
journal = {Hearing research},
volume = {472},
number = {},
pages = {109552},
doi = {10.1016/j.heares.2026.109552},
pmid = {41637835},
issn = {1878-5891},
mesh = {Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Connexin 26/genetics ; *Gene Editing ; *Deafness/genetics/physiopathology/congenital/pathology ; *Connexins/genetics/deficiency ; Phenotype ; *Hearing/genetics ; Gene Knockout Techniques ; Sus scrofa ; Homozygote ; Female ; Organ of Corti/physiopathology/pathology ; Mutation ; Animals, Genetically Modified ; Hair Cells, Auditory/pathology/metabolism ; Genetic Predisposition to Disease ; Male ; },
abstract = {GJB2, the primary gene responsible for DFNB1, the most prevalent non-syndromic hearing loss (NSHL), has variants that account for over 50% of all prelingual hearing loss (HL). Mice are the main model for congenital hearing loss (CHL) research, but they have delayed auditory maturation postnatally, and unconditional Gjb2 knockout in mice causes embryonic lethality. Pigs have similar inner-ear anatomy to humans and, like humans, have matured auditory function and fully differentiated cochlea at birth. Currently, there is no GJB2 unconditional knockout animal model for GJB2-related CHLs research, and whether unconditional GJB2 deletion causes embryonic lethality in pigs or if GJB2-deficient pigs can recapitulate typical clinical pathological characteristics remains unclear. In this study, we employed CRISPR/Cas9 to establish the first unconditional GJB2 knockout pig model. The mutant GJB2 alleles in the founder pig were stably germline-transmitted to subsequent generations. Homozygous GJB2 knockout pigs exhibited no embryonic lethality and showed profound hearing loss, cochlear hair cell depletion and impaired Organ of Corti's development. This GJB2 unconditional knockout pig model has not been reported before and demonstrates GJB2 mutation pathological characteristics consistent with clinical patients, validating its potential in investigating the pathogenic mechanisms and therapeutic interventions of GJB2-deficient CHLs.},
}
@article {pmid41636718,
year = {2026},
author = {Xu, ZH and Hu, X and Weng, X and Lin, RM and Xu, W and Yu, LS and Gao, H},
title = {An "off-on" electrochemiluminescence biosensor based on CRISPR-Cas12a for ultrasensitive determination of aflatoxin B1.},
journal = {The Analyst},
volume = {151},
number = {5},
pages = {1325-1331},
doi = {10.1039/d5an01369k},
pmid = {41636718},
issn = {1364-5528},
mesh = {*Aflatoxin B1/analysis ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; *CRISPR-Cas Systems ; Aptamers, Nucleotide/chemistry/genetics ; Limit of Detection ; Quantum Dots/chemistry ; *CRISPR-Associated Proteins/genetics ; Food Contamination/analysis ; Electrodes ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Accurate and sensitive determination of aflatoxin B1 (AFB1) is of vital importance for food safety. Herein, an electrochemiluminescence (ECL) "off-on" switch sensor combined with CRISPR-Cas12a is fabricated for the quantitative analysis of AFB1. Initially, the DNA activator is effectively locked by two AFB1 aptamers. Once the target AFB1 is introduced, the activator is released in an open state and thus is recognized by the Cas12a-crRNA duplex. Black hole quencher (BHQ)-marked DNA strands are digested by the activated CRISPR-Cas12a system. Owing to the resonance energy transfer (RET) between aggregation-induced ECL active (AIECL-active) polymer dots (Pdots) and BHQ, the ECL signal of Pdots switches from signal "off" to "on" along with the detachment of BHQ from the electrode surface. The proposed ECL sensor thus achieves sensitive quantification of AFB1 with a detection limit of 0.06 pg mL[-1]. This work provides an effective strategy for mycotoxin determination.},
}
@article {pmid41620000,
year = {2026},
author = {Shah, W and Hussain, M and Serwat, A and Bilal, M and Raza, Y and Mansoor, A and Faraz, A},
title = {CRISPR/Cas9 and reproductive failure: applications, ethical challenges, and future perspectives in human germline genome editing.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {584},
number = {},
pages = {120875},
doi = {10.1016/j.cca.2026.120875},
pmid = {41620000},
issn = {1873-3492},
mesh = {Humans ; *Gene Editing/ethics ; *CRISPR-Cas Systems/genetics ; *Germ Cells/metabolism ; Animals ; *Infertility/genetics/therapy ; },
abstract = {Reproductive failure affects millions of couples worldwide and frequently arises from genetic defects that impair gametogenesis, fertilization, or early embryonic development. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome-editing technology has emerged as a powerful experimental platform for dissecting infertility-associated genes and, in principle, correcting pathogenic variants in germline cells or preimplantation embryos. This review critically examines current applications of CRISPR/Cas9 in reproductive biology, including disease modeling in animal systems, editing of spermatogonial stem cells (SSCs), manipulation of oocytes and zygotes, and proof-of-concept studies in human embryos. Particular emphasis is placed on the major technical barriers that currently preclude clinical translation, including off-target mutagenesis, embryo mosaicism, and the low efficiency of homology-directed repair relative to non-homologous end joining. Limitations related to delivery strategies, DNA damage responses, chromosomal rearrangements, and the genetic heterogeneity of infertility are also evaluated. Comparative discussion highlights how germline editing differs fundamentally from somatic CRISPR therapies that have already reached clinical application in hematologic disorders. The review further analyzes ethical and regulatory challenges associated with heritable genome modification, including long-term safety, consent across generations, international governance disparities, and the continued reliance on assisted reproductive technologies combined with preimplantation genetic testing as safer clinical alternatives. Collectively, current evidence indicates that CRISPR/Cas9 remains primarily a research tool for elucidating reproductive biology rather than an imminent therapeutic option for human infertility. Continued technological refinement, rigorous preclinical validation, and globally harmonized oversight will be essential before germline applications can be ethically or clinically justified.},
}
@article {pmid41616988,
year = {2026},
author = {Yao, B and Öktem, M and Yang, G and Wang, Q and Daniels, MA and Dokter, I and Lefferts, JW and Gonçalves, MAFV and Doevendans, PA and van Mil, A and Sluijter, JPG and Schiffelers, R and Mastrobattista, E and Lei, Z},
title = {LAH5-mediated delivery of prime editor ribonucleoprotein complexes for genome editing.},
journal = {International journal of pharmaceutics},
volume = {692},
number = {},
pages = {126622},
doi = {10.1016/j.ijpharm.2026.126622},
pmid = {41616988},
issn = {1873-3476},
mesh = {Humans ; *Gene Editing/methods ; *Ribonucleoproteins/administration &amp; dosage/genetics ; HEK293 Cells ; *Cell-Penetrating Peptides/administration &amp; dosage/chemistry ; Induced Pluripotent Stem Cells/metabolism ; Myocytes, Cardiac/metabolism ; CRISPR-Cas Systems ; },
abstract = {Prime editing (PE) is a precise gene-editing technology with potential for treating genetic disorders, but efficient delivery systems remain a challenge. Viral vectors offer high efficiency but pose safety concerns related with their immunogenicity, while non-viral methods struggle with stability and scalability. Cell-penetrating peptides (CPPs) present a promising alternative due to their low immunogenicity. In this study, we explored LAH5, a histidine-rich CPP, for delivering PE ribonucleoproteins (RNPs) into PLN R14del mutant cell lines. We purified engineered SpGPEmax protein, evaluating its intracellular uptake and editing frequency in HEK293T.PLN R14del reporter cells and human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Our results demonstrate that LAH5 effectively delivers intracellularly SpGPEmax RNP components, resulting in correction of the R14del mutation, thereby offering a viable non-viral strategy for direct cellular precise genome editing.},
}
@article {pmid41615337,
year = {2026},
author = {Liu, X and Wang, X and He, S and Li, M and Lu, C and Xing, C},
title = {An oxidative cleavage-based HCR-CRISPR/Cas12a biosensor for highly sensitive detection of hypochlorous acid.},
journal = {The Analyst},
volume = {151},
number = {5},
pages = {1317-1324},
doi = {10.1039/d5an01299f},
pmid = {41615337},
issn = {1364-5528},
mesh = {*Biosensing Techniques/methods ; *Hypochlorous Acid/analysis ; *CRISPR-Cas Systems ; Oxidation-Reduction ; Limit of Detection ; DNA/chemistry/genetics ; Nucleic Acid Hybridization ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; *Water Pollutants, Chemical/analysis ; Bacterial Proteins ; },
abstract = {Hypochlorous acid (HClO) is a critical disinfectant in public health and water treatment, yet its excessive presence in environmental matrices poses significant risks to human health (e.g., respiratory irritation, tissue damage) and aquatic ecosystems (e.g., biodiversity loss). Herein, we developed an oxidative cleavage-based hybridization chain reaction (HCR)-CRISPR/Cas12a biosensor for highly sensitive detection of HClO. The sensor utilizes a dual-lock switch mechanism: HClO selectively cleaves a phosphorothioated (PS) DNA hairpin (Lock 1), releasing an initiator strand to trigger the HCR and generate PAM-containing long dsDNA. Simultaneously, the locked crRNA (L-crRNA), which is caged by a PS-modified silent chain (Lock 2), can be released when the PS-modified silent chain is degraded under HClO incubation, allowing the activated crRNA to dynamically regulate Cas12a-crRNA complex formation. Then, HCR-dsDNA activates Cas12a's trans-cleavage activity, enabling fluorescence signal readout via reporter DNA cleavage. The dual-lock strategy minimizes nonspecific activation in CRISPR/Cas12a systems, significantly enhancing sensitivity and specificity. Our work establishes a robust platform for environmental pollutant monitoring, with applications in water safety assessment and food quality control.},
}
@article {pmid41605928,
year = {2026},
author = {Ganguly, C and Aribam, SD and Dos Santos, AM and Martin, L and Thomas, LM and Shao, Y and Rajan, R},
title = {Bridge helix of Cas12a is an allosteric regulator of R-loop formation and RuvC activation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41605928},
issn = {2041-1723},
support = {MCB-1716423 and MCB-2424888//National Science Foundation (NSF)/ ; R35GM153297 and R44GM133270//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics/chemistry ; CRISPR-Cas Systems/genetics ; Allosteric Regulation ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; *Bacterial Proteins/metabolism/genetics/chemistry ; *Francisella/genetics/enzymology ; *R-Loop Structures ; DNA Cleavage ; Gene Editing ; DNA/metabolism ; },
abstract = {CRISPR-Cas12a, an RNA-based DNA targeting system, is widely used for genome editing and biomarker detection. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Francisella novicida Cas12a variant (FnoCas12a[KD2P]) by introducing double proline substitutions (K969P/D970P) in a conserved arginine-rich helix called the bridge helix (BH). In this work, we use a combinatorial approach to understand the molecular mechanisms of BH-mediated activation of Cas12a for DNA cleavage. We report five structures of FnoCas12a[KD2P] that are at different states of conformational activation. Comparison of the variant and wild-type (FnoCas12a[WT]) structures, along with activity assays and computational simulations, establishes the loop-to-helical transition and bending of the BH as an allosteric trigger for RNA-DNA hybrid propagation. These changes track with the previously reported coupled remodeling of BH and helix 1 of RuvC motif-II as well as the REC lobe movements needed to accommodate the growing hybrid. The transition of the BH is essential for the loop-to-helical transition of the "lid", which in turn opens the RuvC active site pocket for DNA entry and cleavage. Pairwise 3D structural comparison of the BH and RuvC of Cas12 and Cas9 families provides insight into the diversity of BH's structural organization in these mechanistically similar enzymes.},
}
@article {pmid41603430,
year = {2026},
author = {Li, W and Yang, Y and Xu, W and Zhu, Y and Li, Y and Cao, L and Lyu, S and Liu, J and Xie, Y and Li, X and Hu, X and Huang, L},
title = {An Extraction-free One-Pot Assay for Rapid Field Discrimination of African Swine Fever Virus Variants by a Single-Step RPA-CRISPR/Cas12a Strategy.},
journal = {ACS sensors},
volume = {11},
number = {2},
pages = {1201-1213},
doi = {10.1021/acssensors.5c03287},
pmid = {41603430},
issn = {2379-3694},
mesh = {*African Swine Fever Virus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Animals ; Swine ; *African Swine Fever/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; },
abstract = {African swine fever virus (ASFV) causes devastating outbreaks in swine populations worldwide. The co-existence of wild-type and emerging gene-deleted variants (e.g., ASFV-ΔI177L) necessitates rapid on-site diagnostic tools for strain identification, which is critical for timely disease control and tailored farm management. Here, we developed a field-deployable, extraction-free one-pot assay (CORDSv2) that combines multiplex RPA and CRISPR/Cas12a to target conserved ASFV sequences and specific fluorescent markers (eGFP/mCherry) of gene-deleted variants. The assay achieved ultrasensitive detection (LOD: 6 copies/μL) and demonstrated 97.9% accuracy with 96 field samples. To streamline field operations, we integrated an extraction-free protocol (for serum/saliva) with freeze-dried reagent microspheres, enabling stable storage and direct use with minimal manual handling. When paired with a portable fluorometer (hippoCORDS), the system completes the entire sample-to-answer workflow within 1 h with a single step: addition of lysate to the microspheres. This robust, portable system addresses the urgent need for simple, on-site ASFV variant surveillance and is adaptable for other high-threat pathogens.},
}
@article {pmid41591278,
year = {2026},
author = {Li, L and Wang, Y and Liu, L and Lou, Y and Lin, K and Li, T and Yu, C and Han, Y and Wei, H and Wang, D and Wang, S and Rong, Z},
title = {Miniaturized Single-Step Duplex CRISPR Diagnostic Platform for At-Home Molecular Testing of HPV16 and HPV18.},
journal = {ACS sensors},
volume = {11},
number = {2},
pages = {1117-1128},
doi = {10.1021/acssensors.5c03020},
pmid = {41591278},
issn = {2379-3694},
mesh = {*Human papillomavirus 16/genetics/isolation &amp; purification ; *Human papillomavirus 18/genetics/isolation &amp; purification ; Humans ; Female ; *Papillomavirus Infections/diagnosis/virology ; *CRISPR-Cas Systems ; Uterine Cervical Neoplasms/diagnosis/virology ; Nucleic Acid Amplification Techniques ; Miniaturization ; Limit of Detection ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The early screening, precise diagnosis, and effective treatment of invasive cervical cancer necessitate at-home molecular testing of human papillomaviruses (HPVs). However, current HPV DNA tests cannot meet the need for an affordable, rapid, and accurate diagnosis using a streamlined workflow. Here, we present a miniaturized single-step duplex CRISPR diagnostic platform, termed SCOPEv2 (Streamlined CRISPR On Pod Evaluation platform, version 2), for rapid and highly sensitive at-home molecular testing of high-risk HPV16 and HPV18 for population screening of cervical cancer. Dual-target recombinase polymerase amplification (RPA) was initially incorporated with Cas12a/Cas13a cleavage reactions in a single-step reaction system. A miniaturized and low-cost dual-color wireless analysis device was further developed to execute the analysis workflow. SCOPEv2 can detect HPV16 and HPV18 with limits of detection of 2.5 copies/μL (5 copies/reaction) and 5 copies/μL (10 copies/reaction) in 30 min, respectively. The analysis results for 128 clinical cervicovaginal swab samples revealed 94.7% sensitivity and 100% specificity. SCOPEv2 demonstrates an easy-to-use workflow, low cost, high analytical performance, and superior clinical feasibility, which enable accurate and simultaneous point-of-care testing of HPV16 and HPV18.},
}
@article {pmid41582136,
year = {2026},
author = {Guo, S and Zhao, S and Tang, S and Leng, H and Wu, Y and Li, W and Xing, S and Feng, Y and Zhang, Y},
title = {Establishment of a CRISPR/Cas12a/13a-driven dual-detection platform for rapid diagnosis of swine influenza virus and porcine reproductive and respiratory syndrome virus infection.},
journal = {Virology journal},
volume = {23},
number = {1},
pages = {},
pmid = {41582136},
issn = {1743-422X},
support = {2021YFD1800200//National Key Research and Development Program of China/ ; 32170539//National Natural Science Foundation of China/ ; },
mesh = {Animals ; Swine ; *Porcine respiratory and reproductive syndrome virus/isolation &amp; purification/genetics ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Porcine Reproductive and Respiratory Syndrome/diagnosis/virology ; Sensitivity and Specificity ; *Orthomyxoviridae Infections/diagnosis/veterinary/virology ; *Swine Diseases/diagnosis/virology ; Coinfection/diagnosis/virology/veterinary ; },
abstract = {BACKGROUND: Swine influenza virus (SIV) and porcine reproductive and respiratory syndrome virus (PRRSV) are leading pathogens in pigs, whose co-infections exacerbate disease severity. Current diagnostics like RT-PCR lack suitability for rapid, on-site use, while CRISPR-based systems face challenges in convenient multiplex detection.<br><br>RESULTS: We developed an RT-LAMP-CRISPR-Cas12a/13a-LFD dual-detection platform that integrates reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the orthogonal trans-cleavage activities of CRISPR-Cas12a and Cas13a, followed by lateral flow dipstick (LFD) visualization. This assay achieved detection limits of 5 copies/&#xb5;L for SIV and 2 copies/&#xb5;L for PRRSV, and exhibited high specificity against other common swine pathogens. The entire process, including a 20-minute amplification at 40&#xa0;&#xb0;C and 5-minute LFD readout, enables rapid and visual diagnosis. A preliminary validation was conducted using respiratory infection samples, demonstrating high concordance with reference methods and specificity against non-target pathogens.<br><br>CONCLUSIONS: The RT-LAMP-CRISPR-Cas12a/13a-LFD assay provides a sensitive, specific, and potentially field-adaptable tool for the simultaneous detection of SIV and PRRSV. It is ideally suited for early screening and precise control of these pathogens in resource-limited settings.},
}
@article {pmid41577687,
year = {2026},
author = {Montagud-Martínez, R and Ruiz, R and Baldanta, S and Delicado-Mateo, R and Rodrigo, G},
title = {CRISPR-Cas9 trans-cleavage is hindered by a flanked R-loop, an elongated spacer, and an inactive HNH domain.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41577687},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *R-Loop Structures ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; Protein Domains ; DNA, Single-Stranded/metabolism/genetics ; Models, Molecular ; DNA/metabolism ; },
abstract = {Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key determinants underlying this function. First, we show that unflanked R-loops in the RNA 5' side favor trans-cleavage activity, which occur when targeting short double-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impair this collateral activity. Third, although trans-cleavage is mediated by the RuvC domain, we show that a catalytically active HNH domain contributes to an efficient process. Analysis of structural models provides tentative mechanistic insights. Together, these findings illustrate that fine modulation of Cas9 function can be achieved.},
}
@article {pmid41572361,
year = {2026},
author = {Mulero-Sánchez, A and Bosma, A and Visuvasam, B and Pouliopoulou, N and van de Ven, M and Proost, N and Boeije, M and Lieftink, C and Beijersbergen, R and Bernards, R and Mainardi, S},
title = {CRISPR knockout screens reveal JUN as the master mediator of resistance to MAPK inhibition in KRAS-mutant pancreatic cancer.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {45},
number = {1},
pages = {},
pmid = {41572361},
issn = {1756-9966},
support = {SU2C-AACR-PCC-01-18//Stand Up To Cancer/ ; KWF 12539//KWF Kankerbestrijding/ ; },
mesh = {Humans ; *Proto-Oncogene Proteins p21(ras)/genetics ; *Pancreatic Neoplasms/genetics/drug therapy/pathology/metabolism ; Animals ; Mice ; *Drug Resistance, Neoplasm/genetics ; Mutation ; Cell Line, Tumor ; *Protein Kinase Inhibitors/pharmacology ; CRISPR-Cas Systems ; *Carcinoma, Pancreatic Ductal/genetics/drug therapy/pathology ; Xenograft Model Antitumor Assays ; Gene Knockout Techniques ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is often driven by KRAS mutations, but inhibitors targeting the most frequent KRAS substitutions in PDAC are not yet approved in the clinic. We previously discovered that KRAS-mutant PDAC is sensitive to the combination of SHP2 and ERK inhibitors, recently investigated in the Phase I/Ib clinical trial NCT04916236. Lately, RAS(ON) multi-selective inhibitors have entered clinical development, representing a promise for mono or combination therapies in PDAC. However, resistance may arise even for combination therapies. Here, we aimed at anticipating mechanisms of resistance to SHP2 plus ERK or RAS(ON) multi-selective inhibitors.<br><br>METHODS: We performed a genome-wide CRISPR-KO screening, followed by four follow-up focused screenings, leading to the identification of resistance mediators, which were further validated through functional genetic and pharmacological experiments, both in vitro and in vivo.<br><br>RESULTS: Through unbiased CRISPR-based screenings, we identified mTOR and JUN hyperactivation as interconnected mechanisms that overcome MAPK suppression. Further investigation pointed at JUN as the most downstream resistance mediator, and indirect therapeutic target, using MAP2K4 inhibitors.<br><br>CONCLUSIONS: Alterations in the PI3K/AKT/mTOR and JUN pathways can induce resistance to multiple combinations of MAPK pathway inhibitors, and may serve as biomarkers for sensitivity/resistance in clinical trials exploring such combinations in KRAS-mutant PDAC.},
}
@article {pmid41512567,
year = {2026},
author = {Sushmita, and Srivastava, A and Jain, G and Singh, M and Verma, PC},
title = {Targeted disruption of a cell wall-modifying gene α-Mannosidase using CRISPR-Cas9 enhances post-harvest shelf life in tomato through ABA accumulation.},
journal = {Plant physiology and biochemistry : PPB},
volume = {231},
number = {},
pages = {111017},
doi = {10.1016/j.plaphy.2026.111017},
pmid = {41512567},
issn = {1873-2690},
mesh = {*Solanum lycopersicum/genetics/metabolism/enzymology ; *Abscisic Acid/metabolism ; *Cell Wall/metabolism/genetics ; *CRISPR-Cas Systems ; *alpha-Mannosidase/genetics/metabolism ; Plant Proteins/genetics/metabolism ; Fruit/genetics/metabolism ; Gene Expression Regulation, Plant ; Reactive Oxygen Species/metabolism ; },
abstract = {Tomato ripening is a complex process regulated by transcription factors, hormones, and physiological changes. While this intricate regulation ensures desirable traits such as color, texture, and flavor, it is often accompanied by rapid post-harvest deterioration and poor shelf life, leading to significant economic losses and limiting market potential. Various cell wall-modifying enzymes, including N-glycan-processing enzyme α-Mannosidase, play crucial roles in the softening and senescence of tomato fruits. Our study shows that α-Mannosidase knockout through CRISPR/Cas9 results in fruits with enhanced firmness, longer shelf life, and improved moisture retention. Additionally, the expression of SlRIN (Ripening Inhibitor), a key regulator of ripening and several downstream genes, including those involved in cell wall degradation, ethylene biosynthesis, and signaling, was downregulated. Interestingly, in the later stages of storage, they also exhibited higher accumulation of abscisic acid (ABA) and lower accumulation of reactive oxygen species, along with better antioxidant capacity, compared to the control fruits, which may confer delayed softening and increased shelf life. Our findings highlight α-Mannosidase as a ripening-specific regulator and a promising genetic target for extending tomato shelf life, offering a sustainable strategy to minimize post-harvest losses without compromising plant development.},
}
@article {pmid41505207,
year = {2026},
author = {Gao, JL and Li, Z and Calderon-Perez, R and Pavek, A and Kim, L and McDermott, DH and Murphy, PM},
title = {Gene therapy via CRISPR/Cas9-mediated Cxcr4 disease allele inactivation reverses leukopenia in WHIM mice.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {5},
pages = {},
pmid = {41505207},
issn = {1558-8238},
mesh = {Animals ; *Receptors, CXCR4/genetics ; Mice ; *CRISPR-Cas Systems ; *Genetic Therapy ; *Leukopenia/therapy/genetics/pathology ; *Warts/therapy/genetics/pathology ; *Alleles ; *Immunologic Deficiency Syndromes/therapy/genetics/pathology ; Primary Immunodeficiency Diseases/therapy/genetics ; Hematopoietic Stem Cell Transplantation ; Humans ; Gene Editing ; },
abstract = {Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is an immunodeficiency caused by autosomal dominant hyperfunctional mutations in chemokine receptor CXCR4 that promote panleukopenia due to BM retention. We previously reported a preclinical gene therapy protocol involving allele-nonspecific Cxcr4 CRISPR/Cas9 inactivation, leveraging the known in vivo dominance of Cxcr4+/o (+, WT; o, inactivated) hematopoietic stem cells (HSCs) for autologous BM engraftment and leukocyte reconstitution over HSCs with other Cxcr4 genotypes. Here, we show that without BM conditioning, this approach is not able to correct leukopenia in WHIM mice. We therefore modified the protocol by adding conditioning with a nongenotoxic CD117-targeted immunotoxin, CD117-antibody-saporin-conjugate. With this change, donor-derived blood cells rapidly reached ~95% chimerism after transplantation, which was stable without adverse events. Mice receiving edited HSCs showed rapid normalization of absolute myeloid cell counts, the key blood subset responsible for WHIM syndrome. In competitive transplants using equal numbers of edited and unedited donor HSCs, over 80% of blood cells originated from the edited population, predominantly with the Cxcr4+/o genotype. These results provide proof of principle that CRISPR/Cas9-mediated inactivation of the Cxcr4 disease allele, combined with nongenotoxic HSC-targeted conditioning, may offer a safe and effective gene therapy strategy generalizable to all WHIM-causing mutations.},
}
@article {pmid41485979,
year = {2026},
author = {Shimizu, T and Okamoto, M and Kawamoto, K},
title = {Evaluation of a d-octaarginine-linked polymer for CRISPR-Cas9 ribonucleoprotein (RNP) delivery and genome editing in murine dendritic cells.},
journal = {The Journal of veterinary medical science},
volume = {88},
number = {3},
pages = {374-383},
doi = {10.1292/jvms.25-0334},
pmid = {41485979},
issn = {1347-7439},
mesh = {Animals ; *Dendritic Cells/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Mice ; *Ribonucleoproteins/genetics ; *Oligopeptides/chemistry ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; Transfection ; *Polymers/chemistry ; },
abstract = {We previously reported that poly (N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) efficiently introduces proteins and nucleic acids into cells. Based on these results, we hypothesized that VP-R8 can introduce a complex composed of guide RNA and Cas9 (RNP complex) into cells and induce genome editing mediated by the CRISPR-Cas9 system. We tested this hypothesis using a mouse dendritic cell line and mouse primary dendritic cells. The RNP complexes formed by guide RNA consisting of CRISPR RNA (crRNA), fluorescently labeled trans-activating crRNA (tracrRNA), and GFP-fused Cas9 were introduced into a mouse dendritic cell line using VP-R8 or control transfection reagents. Cells transfected using VP-R8 exhibited higher fluorescence than those transfected with other transfection reagents, indicating that VP-R8 efficiently introduced the RNP complex into the mouse dendritic cell line. Genome editing of the target DNA was detected in cells transfected with the RNP complex using VP-R8 and dominant relative to other transfection reagents. We also observed that VP-R8 effectively delivered RNP complexes consisting of single-guide RNA and Cas9 and induced genome editing in the dendritic cell line. Additionally, VP-R8 efficiently delivered RNP complexes into mouse primary dendritic cells and induced genome editing of the functional gene without producing early inflammatory cytokines. Thus, VP-R8 shows potential as a transfection tool to generate dendritic cells with specific gene regions deleted by genome editing via the CRISPR-Cas9 system. This approach aims to elucidate the detailed molecular mechanisms of dendritic cell function for its application to vaccines.},
}
@article {pmid41432359,
year = {2026},
author = {Fruitet, E and de Fouchier, A and Heckel, DG and Groot, AT},
title = {Multiple CRISPR/Cas9 modifications of an esterase reveal its role in influencing acetate esters in the pheromone blend of a moth.},
journal = {Insect molecular biology},
volume = {35},
number = {2},
pages = {166-176},
doi = {10.1111/imb.70016},
pmid = {41432359},
issn = {1365-2583},
support = {//Instituut voor Biodiversiteit en Ecosysteem Dynamica, Universiteit van Amsterdam/ ; //Universiteit van Amsterdam/ ; //International Max Plank Research School, Max-Planck-Instituts f&#xfc;r chemische &#xd6;kologie/ ; //Max-Planck-Gesellschaft/ ; },
mesh = {*Moths/genetics/metabolism/enzymology ; Animals ; CRISPR-Cas Systems ; Female ; Male ; *Esterases/genetics/metabolism ; *Acetates/metabolism ; Esters/metabolism ; *Insect Proteins/genetics/metabolism ; *Sex Attractants/metabolism ; Quantitative Trait Loci ; },
abstract = {Sexual signalling by pheromones is essential for mate finding and mate choice in moths and plays an important role in reproductive isolation. Acetates (i.e., acetate esters) produced by females of Heliothis (Chloridea) subflexa Fabricius, 1777 (Lepidoptera: Noctuidae) attract conspecific males but repel Heliothis virescens Fabricius, 1777 (Lepidoptera: Noctuidae) males. A QTL (quantitative trait locus) harbouring carboxylesterases and lipases was previously shown to affect acetates, and CRISPR/Cas9-induced knockouts increased acetate amounts by blocking hydrolysis of the esters as expected. A second, unlinked QTL, containing a cluster of three different carboxylesterases (CXEs), unexpectedly yielded decreased acetate amounts. In one of these genes, esterase CXE24, we found a naturally occurring transposable element insertion in exon 8. A CRISPR/Cas9-induced frameshift at the same position yielded the same results. The paradox was resolved by a CRISPR/Cas9-induced frameshift in exon 2 of CXE24 which increased acetate amounts. The frameshift in exon 2 produced a truncated protein lacking the substrate binding site and the catalytic triad, while the frameshift in exon 8 removed only the third residue of the catalytic triad. In silico modelling showed that the exon-8-truncated protein could not hydrolyse the esters by itself, which likely explains the decreased acetate amounts. To place our findings in an evolutionary context, we explored variation in the esterase cluster in 16 species of Lepidoptera with completely sequenced genomes. Geographic and temporal variation in acetates has been observed in H. subflexa, and variation in the frequency of the transposable element could be a possible explanation.},
}
@article {pmid41411488,
year = {2026},
author = {Bär, I and Groten, SA and Barraclough, A and Bürgisser, PE and van Kwawegen, C and Lenting, PJ and van Moort, I and Eikenboom, JCJ and Leebeek, FWG and Voorberg, J and van den Biggelaar, M and Bierings, R},
title = {Allele-selective disruption of pathogenic VWF variants in type 2 von Willebrand disease using CRISPR/Cas9.},
journal = {Blood advances},
volume = {10},
number = {5},
pages = {1429-1443},
pmid = {41411488},
issn = {2473-9537},
mesh = {Humans ; *von Willebrand Factor/genetics ; *Alleles ; *CRISPR-Cas Systems ; *von Willebrand Disease, Type 2/genetics/therapy ; Polymorphism, Single Nucleotide ; *Gene Editing/methods ; Genetic Therapy/methods ; Mutation ; },
abstract = {In contrast to major innovations in treating severe hemophilia, the treatment of severe von Willebrand disease (VWD) remains limited to intravenous infusion of von Willebrand factor (VWF) concentrates. To date, no gene therapy-based approaches for the treatment of VWD have been developed, largely owing to the disease's heterogeneous mutational landscape and the challenge of specifically targeting VWF production in endothelial cells. In this study, we developed a novel gene therapy strategy for patients with VWD caused by heterozygous dominant-negative VWF variants. Our strategy permanently inactivates VWF variants by selectively disrupting the pathogenic allele's open reading frame via the introduction of indels by Cas9. To circumvent the challenge of designing variant-specific strategies, we targeted the common single nucleotide polymorphism (SNP) rs1800378 in VWF. We used endothelial colony-forming cells (ECFCs) from patients with VWD2A and VWD2B with heterozygous p.C1190R and p.R1306W variants, respectively, to demonstrate ex vivo proof of principle. Using next-generation sequencing analysis, we show efficient and allele-selective knockout of VWF, while maintaining VWF expression of the nontargeted allele. Variant mapping mass spectrometry that discriminates between wild-type and variant VWF proteoforms confirmed selective reduction of variant allele expression, which was accompanied by reversal of cellular disease phenotypes in ECFCs. This study shows the feasibility of a novel gene editing strategy for VWD that, by virtue of its targeting of a common SNP, can be broadly applicable and can be used to design treatments for VWD without being constrained by the disease-causing variant, pathogenic mechanism, or VWD subtype.},
}
@article {pmid41370881,
year = {2026},
author = {Li, SR and Li, Y and Yang, KB and Wang, SW and Sun, ML and Liu, Z and Zhang, XP and Zhong, Y and Yao, J},
title = {CRISPR/Cas12a coupled with MIRA: A specific and rapid assay for human DNA in challenging forensic matrices.},
journal = {Forensic science international. Genetics},
volume = {82},
number = {},
pages = {103393},
doi = {10.1016/j.fsigen.2025.103393},
pmid = {41370881},
issn = {1878-0326},
mesh = {Humans ; *CRISPR-Cas Systems ; Animals ; *Nucleic Acid Amplification Techniques/methods ; Cytochromes b/genetics ; Mice ; Swine ; Chickens ; *DNA Fingerprinting/methods ; DNA ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Human DNA detection is crucial in forensic medicine, particularly for trace, degraded, or mixed samples, which demand high sensitivity, specificity, and rapid processing. Traditional methods, such as immunological assays and PCR-based techniques, often suffer from operational complexity, limited sensitivity, or high equipment dependency. To address these challenges, we developed a novel detection system combining multienzyme isothermal rapid amplification (MIRA) with CRISPR-Cas12a for the rapid, specific, and portable human DNA identification. By targeting the human mitochondrial cytochrome b (CYTB) gene and sex-determining Region Y(SRY) gene, we designed MIRA primers and CRISPR-Cas12a crRNA to enable dual recognition and signal amplification. The method involves isothermal amplification at 37°C followed by CRISPR-Cas12a-mediated cleavage, producing detectable fluorescence or lateral flow chromatographic signals. Our system achieves ultra-sensitive detection and high specificity, distinguishing human DNA from non-human sources (e.g., pig, chicken, mouse), and also enables accurate gender identification, further enhancing its utility in forensic and genetic studies. Compared to traditional qPCR, this approach demonstrates superior sensitivity, faster turnaround (≤ 45 min), and minimal equipment requirements, making it ideal for forensic applications. Moreover, the blood, mixed, and degraded samples were used to confirm its robustness, with results interpretable via blue-light fluorescence or colloidal gold test strips. In summary, the MIRA-CRISPR/Cas12a system overcomes the limitations of conventional techniques, offering a rapid, cost-effective, and reliable solution for forensic human DNA identification, with potential extensions to wildlife conservation and food safety testing.},
}
@article {pmid41211856,
year = {2026},
author = {Kelly, AA and Fulda, M and Aden, M and Abreu, IN and Feussner, K and Feussner, I},
title = {Reducing the Sinapine Levels of Camelina sativa Seeds Through Targeted Genome Editing of REF1.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1839-1865},
pmid = {41211856},
issn = {1467-7652},
support = {INST 186/1434-1//Deutsche Forschungsgemeinschaft/ ; ZUK 45/2010//Deutsche Forschungsgemeinschaft/ ; 031B0343A//Bundesministerium f&#xfc;r Bildung und Forschung/ ; },
mesh = {*Seeds/metabolism/genetics ; *Gene Editing/methods ; *Choline/analogs &amp; derivatives/metabolism ; *Brassicaceae/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; CRISPR-Cas Systems ; },
abstract = {Sinapine (O-sinapoyl choline) is the major phenolic metabolite typically found in the oil-rich seeds of Brassicaceae such as Camelina sativa and Brassica napus. It imparts a bitter taste to the seeds as a defence mechanism against herbivores, but it also renders them less palatable to livestock. To improve Camelina flour for human consumption or as animal feed, we reduced sinapine content through CRISPR/Cas9-based genome editing of REF1 (REDUCED EPIDERMAL FLUORESCENCE1), which encodes the NADP[+]-dependent coniferaldehyde/sinapaldehyde dehydrogenase (CALDH/SALDH), a key enzyme for sinapine biosynthesis in Arabidopsis thaliana and B. napus. Inactivation of all three homoeologues found in C. sativa lowered the sinapine content in seeds by an overall 56% in two cultivars indicating the presence of a REF1-independent pathway for sinapine biosynthesis. Most importantly however, crucial agronomic seed traits such as total lipid or protein content of the seeds, seed weight or germination were not affected. Hence, the ref1 mutant lines produced here provide a valuable trait, that can be combined with other traits through gene stacking to obtain crops with significantly improved product quality. Furthermore, metabolite fingerprinting by ultra-performance liquid chromatography-electrospray ionisation-quadrupole time-of-flight mass spectrometry of ref1 mutant lines revealed a contrasting phenylpropanoid profile in seeds and leaves, indicating that REF1 oxidises sinapaldehyde to sinapate in seeds and coniferyl aldehyde to ferulate in leaves. In contrast to Arabidopsis however, Camelina accumulates no comparable levels of sinapoyl malate, but substantial amounts of chlorogenic acid, of which an additional chlorogenic acid isomer distinguishes the two different Camelina cultivars as a metabolite marker.},
}
@article {pmid41195477,
year = {2026},
author = {Lee, SK and Hong, WJ and Kim, EJ and Lee, C and Moon, S and Ha, SH and Jung, KH},
title = {Developing an Efficient System for Hybrid Rice Seed Production Using Partial Male Sterility.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1740-1749},
pmid = {41195477},
issn = {1467-7652},
support = {RS-2024-00322278//Rural Development Administration/ ; RS-2021-NR059380//National Research Foundation of Korea/ ; RS-2021-NR060084//National Research Foundation of Korea/ ; },
mesh = {*Oryza/genetics/physiology/growth &amp; development ; *Seeds/genetics/growth &amp; development ; *Plant Infertility/genetics ; *Plant Breeding/methods ; Pollination/genetics ; Hybridization, Genetic ; CRISPR-Cas Systems ; Pollen/genetics ; Mutation ; },
abstract = {Efficient production of hybrid rice seeds requires male sterility systems to overcome the challenges of self-pollination. In this study, we identified male gametic transfer defect (GTrD)5 and GTrD9 as essential genes for the process of male gamete transmission in rice. Mutations in these genes cause partial male sterility by impairing pollen tube elongation, thereby reducing the fertilisation rate. Using CRISPR/Cas9 technology, we developed gtrd5flo5 and gtrd9flo5 double mutants that combined the gtrd5 and gtrd9 genes responsible for partial male sterility with the floury endosperm (FLO)5 gene, whose defects result in seeds with an opaque endosperm, allowing us to easily differentiate between hybrid and self-pollinated seeds. This two-line hybrid system demonstrated a high rate of hybrid seed production with significantly reduced self-pollination ratios. The hybrid seeds resulted in plants with increased height, panicle size and grain yield compared with those obtained from the parental lines and also displayed heterosis. Unlike the current two-line hybrid systems based on photoperiod- and thermosensitive genic male-sterile lines, our approach is independent of environmental factors, ensuring a stable and reliable hybrid seed production. This novel method simplifies seed production, enhances efficiency and offers a cost-effective and environmentally sustainable solution for hybrid rice breeding.},
}
@article {pmid41190715,
year = {2026},
author = {Simon, MK and Yuan, L and Che, P and Day, K and Jones, T and Godwin, ID and Koltunow, AMG and Albertsen, MC},
title = {Induction of Synthetic Apomixis in Two Sorghum Hybrids Enables Seed Yield and Genotype Preservation Over Multiple Generations.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1712-1724},
pmid = {41190715},
issn = {1467-7652},
support = {INV-002955/GATES/Gates Foundation/United States ; OPP1076280//Bill and Melinda Gates Foundation/ ; //Corteva Agriscience/ ; INV-002955/GATES/Gates Foundation/United States ; },
mesh = {*Sorghum/genetics/physiology ; *Apomixis/genetics ; *Seeds/genetics/growth &amp; development/physiology ; Genotype ; Hybridization, Genetic ; CRISPR-Cas Systems ; Plant Breeding/methods ; Meiosis/genetics ; Parthenogenesis/genetics ; Plant Proteins/genetics/metabolism ; },
abstract = {Induction of apomixis, or clonal reproduction through seed, could economise commercial hybrid seed production and enable smallholder farmers to save and sow hybrid seed. Here, we demonstrate the synthetic induction of apomixis in two sorghum hybrids and show that the clonal hybrid seed can be maintained across multiple seed generations. This was achieved through the combination of avoidance of meiosis and induced parthenogenesis. Meiotic avoidance was generated by CRISPR/Cas9 knockout of the sorghum meiosis genes Spo11, Rec8, OsdL1 and OsdL3. Parthenogenesis was induced in the resultant diploid egg cell by the expression of the Cenchrus ASGR-BBML2 gene coding sequence. Two different strategies were used to combine these components to induce synthetic apomixis in two sorghum hybrids. Each hybrid used Tx623 as a female parent and either Tx430 or the African landrace Macia as a male parent. Seed yields in both apomictic hybrids were consistent and stable for multiple generations following self-pollination but reduced relative to the sexual hybrids. Sorghum contains two copies of the Osd1 gene that function in meiotic non-reduction. CRISPR/Cas9 knockout of both OsdL1 and OsdL3 loci was sufficient to produce clonal hybrid progeny in conjunction with the other components, but this led to a reduction in seed set. By contrast, a single in-frame edited allele of either OsdL1 or OsdL3 significantly improved seed set of clonal hybrid progeny. Fine-tuning OsdL activity appears to be essential to optimising fertility; however, additional improvements are required to unlock the agronomic potential of synthetically induced apomictic sorghum in the field.},
}
@article {pmid41186962,
year = {2026},
author = {Misceo, D and Terkelsen, T and Bøen Keim, SM and Bjørnstad, PM and Strand, ME and Orszagh, VC and Jensen, UB and Frengen, E},
title = {CRISPR Activation Reveals the Spliceogenicity of an Intronic NEB Variant in Fetuses With Arthrogryposis Multiplex Congenita 6.},
journal = {Clinical genetics},
volume = {109},
number = {4},
pages = {772-777},
pmid = {41186962},
issn = {1399-0004},
support = {//Norwegian National Advisory Unit on Rare Disorders/ ; //Legatet til Henrik Homans Minde/ ; },
mesh = {Humans ; *Arthrogryposis/genetics/diagnosis/pathology ; *Introns/genetics ; Female ; Fetus/pathology ; Pregnancy ; *RNA Splicing/genetics ; *CRISPR-Cas Systems/genetics ; Pedigree ; Male ; Frameshift Mutation ; RNA Splice Sites/genetics ; },
abstract = {Whole-genome sequencing identifies intronic variants whose pathogenicity can be predicted with tools like SpliceAI. However, an actionable classification of such variants may require RNA-based validation, which can be limited by low expression in clinically accessible tissues. We report two fetuses from one family with Arthrogryposis multiplex congenita 6 (AMC6 [OMIM # 619334]) and biallelic NEB variants: a paternally inherited likely pathogenic frameshift variant, Chr2(GRCh38):g.151579391del; NM_001164508.2:c.16653del; NP_001157980.2:p.(Asp5552ThrfsTer5), and a maternally inherited intronic variant of uncertain clinical significance, Chr2(GRCh38):g.151496267G>A; NM_001164508.2:c.24486+9C>T; NP_001157980.2:p.(?). Because NEB is poorly expressed in fibroblasts, we used CRISPR activation to induce its expression in fibroblasts from the heterozygous mother. RNA-sequencing subsequently confirmed that the intronic variant generated a novel splice donor site associated with inferred loss of splicing at the canonical donor site. After NMD-inhibition, we could thus identify 45.5% of NEB transcripts with a 7 bp exon extension, predicted to result in a protein-coding frameshift. The intronic variant was classified as likely pathogenic, allowing a genetic diagnosis.},
}
@article {pmid41159426,
year = {2026},
author = {Kato, M and Kleidon, J and Phillips, A and Tabrett, A and Harding, R and Dale, J and Paul, JY},
title = {Transgene-Free, Gene-Edited Cavendish Bananas (Musa acuminata, AAA).},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1620-1634},
pmid = {41159426},
issn = {1467-7652},
support = {//Cooperative Research Centre Projects/ ; //Australian Banana Research Pty Ltd/ ; },
mesh = {*Gene Editing/methods ; *Musa/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Transgenes/genetics ; DNA, Bacterial/genetics ; Oxidoreductases/genetics ; Intramolecular Lyases/genetics ; Carotenoids/metabolism ; },
abstract = {Global consumer acceptance of gene-edited food crops is increasing with new breeding technologies that can modify the genome without foreign DNA integration. Here, we report an Agrobacterium-based system for transgene-free, gene editing of the banana cultivar, Cavendish. The protocol uses a three-tiered approach whereby cells containing T-DNA are enriched by positive antibiotic selection over a 48-h period, CRISPR/Cas9-mediated gene editing occurs over a short transient window and negative selection of cells containing T-DNA is achieved by the addition of 5-FC, which is converted to cytotoxic 5-FU by the co-expressed CODA enzyme. Two key enzymes in the carotenoid biosynthesis pathway were targeted as visual markers of editing: phytoene desaturase (pds) and lycopene β-cyclase (LCYb). Disruptive edits of the LCYb gene were easily identifiable at the calli stage of the regeneration process with cells appearing pink due to lycopene accumulation. Eight of 32 plants for pds and 34 of 125 plants for LCYb contained edits and were likely free of integrated vector DNA as determined by targeted genome sequencing and T-DNA-specific PCR, respectively. Plants containing disruptive tri-allelic edits in either gene displayed an albino phenotype. A subset of potentially transgene-free, edited plants was verified by whole genome sequencing. The transient editing protocol has an estimated 17.6% to 21.9% efficiency in generating plants containing disruptive tri-allelic modifications that are free of novel DNA. The protocol overcomes the regulatory limitations associated with the release of gene-edited, vegetatively propagated crops and provides an effective means of creating new disease-resistant and agronomically superior Cavendish cultivars.},
}
@article {pmid41123354,
year = {2026},
author = {Choi, NY and Kim, MH and Jang, HA and Pyo, SW and Park, KY and Lee, H and Bae, EK and Ko, JH},
title = {Elucidating the Genetic Basis of Columnar Upright Architecture in Populus Through CRISPR Disruption of TILLER ANGLE CONTROL1.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1377-1397},
pmid = {41123354},
issn = {1467-7652},
support = {RS-2023-NR076519//National Research Foundation of Korea/ ; FG0702-2023-01-2025//Korea Forest Service/ ; 2023489B10-2325-AA01//R&amp;D Program for Forest Science Technology/ ; },
mesh = {*Populus/genetics/growth &amp; development/anatomy &amp; histology ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Gene Editing ; Plant Leaves/genetics/growth &amp; development/anatomy &amp; histology ; Gene Expression Regulation, Plant ; },
abstract = {Narrow or upright branch angles in shoots and leaves lead to columnar, upright-growing tree architectures, as observed in various tree species such as Lombardy poplar (Populus nigra var. italica). However, the genetic mechanism underlying this unique growth habit in Lombardy poplar has not yet been elucidated. In this study, we identified a nonsense mutation in the PnTAC1-1 gene of Lombardy poplar, an ortholog of the rice TILLER ANGLE CONTROL 1 (TAC1) gene known to regulate branch angles. To confirm the functional role of TAC1 in regulating tree architecture, we generated transgenic hybrid poplar (Populus alba × Populus glandulosa, clone BH) with targeted mutations in TAC1 homologues using CRISPR/Cas9 gene editing. The resulting TAC1-CRISPR hybrid poplars exhibited a stable upright branching phenotype closely resembling that of Lombardy poplar, as confirmed by two consecutive years of living modified organism (LMO) field trials. Anatomical analysis revealed increased cell elongation specifically in the lower petiole region and significantly enhanced gravitropic responses in TAC1-CRISPR hybrid poplars compared to wild-type BH clones. RNA sequencing analysis further demonstrated that TAC1 disruption triggered extensive transcriptomic reprogramming of axillary meristem, notably altering hormonal and photomorphogenic signalling pathways, which redirected auxin accumulation toward the abaxial region and increased gibberellin biosynthesis, ultimately promoting upright growth. This research uncovers the genetic and molecular mechanisms behind columnar growth in poplar and provides a promising approach for engineering tree architecture to enhance planting density, harvest efficiency and woody biomass productivity.},
}
@article {pmid41108596,
year = {2026},
author = {Liu, Y and Merino, I and Gutensohn, M and Johansson, AI and Johansson, K and Andersson, M and Hofvander, P and Sitbon, F},
title = {Glycoalkaloid-Free Starch Potatoes Generated by CRISPR/Cas9-Mediated Mutations of Genes in the Glycoalkaloid Biosynthesis Pathway Enable More Sustainable Uses of By-Products From Starch Production.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1344-1358},
pmid = {41108596},
issn = {1467-7652},
support = {//SLU Grogrund/ ; },
mesh = {*Solanum tuberosum/genetics/metabolism ; *Starch/metabolism/biosynthesis ; *CRISPR-Cas Systems/genetics ; Plant Tubers/metabolism/genetics ; Mutation ; Gene Editing ; Plants, Genetically Modified ; Biosynthetic Pathways/genetics ; },
abstract = {Steroidal glycoalkaloids (SGAs) are toxic cholesterol-derived secondary metabolites present in several Solanaceae species. In potato, tuber SGA levels are for reasons of toxicity of concern in both table and starch cultivars. In the latter, SGAs bind to proteins and fibres in starch production side-streams and prevent their further uses as food and feed. To enable more sustainable uses of starch by-products, we have here reduced SGA biosynthesis in a starch potato cultivar using DNA-free CRISPR/Cas9. Six SGA genes were targeted, encoding enzymes acting either before cholesterol (SMO1-L, DWF1-L, DWF7-L), or after (16DOX, CYP88B1, TAMiso2). Editing efficiencies varied between 20% and 49%, and generated mutants were investigated under greenhouse and field conditions. Target mass-spectrometric analyses confirmed reduced SGA levels and alterations of sterol metabolism in mutated events. Plant height and tuber yield were reduced in several events, although this was not correlated to low SGA levels. Several knockout mutants had almost SGA-free leaves and tubers, the latter also under two SGA-inducing conditions. Similarly, both fibre and protein fractions isolated from side-streams in the starch production process from mutant tubers had very low SGA levels. By contrast, the corresponding wild-type SGA levels were almost 10-fold and, respectively, 40-fold higher than the recommended upper safe limit. The results demonstrate that glycoalkaloid-free mutants can be generated and grown with moderate yield reductions under both greenhouse and field conditions. This suggests a potential for sustainable production of high-value products, e.g., food-grade protein and fibre, from starch production side-streams of SGA knockout tubers.},
}
@article {pmid41063698,
year = {2026},
author = {Roman, G and Lauritzen, KH and Harrison, SP and Bhattacharya, A and Andresen, MS and Mowinckel, MC and Smolkova, B and Henriksson, CE and Glosli, H and Iversen, N and Thiede, B and Sullivan, GJ and Almaas, R and Sandset, PM and Stavik, B and Chollet, ME},
title = {Ex vivo correction of severe coagulation Factor VII deficiency in patient-derived 3D liver organoids.},
journal = {Haematologica},
volume = {111},
number = {3},
pages = {941-954},
pmid = {41063698},
issn = {1592-8721},
mesh = {Humans ; *Factor VII Deficiency/therapy/genetics/pathology/metabolism ; *Organoids/metabolism/cytology ; *Liver/metabolism/pathology/cytology ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Factor VII/genetics/metabolism ; Mutation, Missense ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Coagulation factor (F) VII deficiency is the most frequent among the rare, inherited bleeding disorders and is predominantly caused by missense mutations in the F7 gene. The disease phenotype ranges from asymptomatic cases to extremely severe hemorrhagic forms, requiring prophylactic injections with plasma-derived or recombinant FVII concentrates. In response, we have developed an autologous cell-based approach that corrects the disease-causing mutation in patient- derived induced pluripotent stem cells (iPSC) and generates therapeutic, three-dimensional hepatic organoids (HO). We report the CRISPR-mediated correction of homozygous c.718G>C (p.G240R), a missense mutation associated with a severe, life-threatening bleeding phenotype. The HO contain all liver cell types and exhibit key liver functions, including coagulation factor production. After correction, our data indicate that the patient-derived HO secrete consistent amounts of functional FVII protein, resulting in improved thrombin generation times. These results represent a significant milestone toward the establishment of an autologous cell-based therapy for patients with FVII- and other coagulation factor deficiencies.},
}
@article {pmid40954224,
year = {2026},
author = {Hu, X and Atwal, RS and Xiao, S and Ahmed, SU and Wang, Z and Zhao, C and Wang, H and Kelley, SO},
title = {ELOVL6 activity attenuation induces mutant KRAS degradation.},
journal = {Nature chemical biology},
volume = {22},
number = {3},
pages = {424-434},
pmid = {40954224},
issn = {1552-4469},
mesh = {Humans ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; Mutation ; Fatty Acid Elongases/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Proteolysis/drug effects ; *Acetyltransferases/metabolism/genetics/antagonists &amp; inhibitors ; },
abstract = {KRAS is one of the most frequently mutated oncogenes in cancer. Targeting mutant KRAS directly has been challenging because of minor structural changes caused by mutations. Despite recent success in targeting KRAS-G12C, targeted therapy for another hotspot mutant, KRAS-G12V, has not been described. We used CRISPR-Cas9 genome-wide knockout screens to identify genes that specifically modulate mutant KRAS harboring the G12V substitution. Our top hit, a fatty acid elongase (ELOVL6), showed remarkable selectivity in diminishing KRAS-G12V protein expression and aberrant oncogenic signaling associated with mutant KRAS. Our studies reveal that ELOVL6 can be targeted to control the production of phospholipids exploited by KRAS mutants for function-targeted and trigger-targeted degradation of the protein. Our results demonstrate the basis for a first-in-class small-molecule inhibitor to selectively clear KRAS-G12V from cancer cells.},
}
@article {pmid40789664,
year = {2026},
author = {Li, H and Lu, Q and Yu, Z and Wu, Z and Zhu, Z and Li, J and Zhang, Z and Wang, Z and Yang, N and Chen, Y and Lu, H and Niu, T and Tong, A},
title = {CRISPR/Cas9-engineered universal CD123/B7-H3 tandem CAR-T cell for the treatment of acute myeloid leukemia.},
journal = {Chinese medical journal},
volume = {139},
number = {5},
pages = {728-740},
pmid = {40789664},
issn = {2542-5641},
mesh = {*Leukemia, Myeloid, Acute/therapy/immunology/genetics ; Animals ; Humans ; *CRISPR-Cas Systems/genetics ; *Interleukin-3 Receptor alpha Subunit/metabolism/genetics ; Mice ; *B7 Antigens/metabolism/genetics ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/metabolism ; T-Lymphocytes/metabolism ; Cell Line, Tumor ; Gene Editing/methods ; },
abstract = {BACKGROUND: Autologous chimeric antigen receptor T (CAR-T) cell therapy has demonstrated efficacy in the treatment of acute myeloid leukemia (AML). Nevertheless, the intrinsic characteristics of autologous therapy, such as extended manufacturing timelines and patient-specific limitations, contribute to delays in treatment availability. More critically, relapse due to antigen escape following single-targeted CAR-T therapy constitutes a significant clinical obstacle. To address the dual challenges of delayed treatment accessibility and antigen escape relapse, this study proposes the development of universal tandem CAR-T cells. These cells, engineered to target CD123 and B7-H3 through clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology, represent an innovative therapeutic strategy for AML.<br><br>METHODS: In this study, an immune phage display nanobody library was developed for the purpose of screening CD123-specific nanobodies. The CRISPR/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system was utilized to disrupt the T-cell receptor alpha chain (TRAC) and B2M genes present in T cells, resulting in the generation of universal CD123/B7-H3 bispecific universal CAR-T (UCAR-T) cells. The efficacy of these dual-specific UCAR-T cells in combating tumors was subsequently assessed through in vitro and in vivo experiments.<br><br>RESULTS: Through four rounds of panning against CD123 from an immunized camelid VHH library, we identified 21 antigen-specific nanobodies. Tandem bispecific UCAR-T engineered with these binders demonstrated CAR transduction efficiencies ranging from 82% to 87%. In vitro functional profiling revealed a significantly enhanced cytotoxicity of bispecific UCAR-Ts against CD123 + /B7-H3 + AML cell lines when compared to single-target constructs, while effectively regulating the secretion of effector cytokines (IL-2, IFN-&#x3b3;, TNF-&#x3b1;). In AML xenograft models, treatment with bispecific UCAR-T notably inhibited tumor progression, extended the survival of tumor-bearing mice with recurrence-free persistence throughout the observation period, and did not result in significant body weight loss or cytokine release syndrome.<br><br>CONCLUSIONS: The findings of the study address the issue of tumor antigen evasion in the treatment of AML, circumvent certain constraints associated with autologous CAR-T cell therapy, and offer novel insights and strategies for managing AML.},
}
@article {pmid41752921,
year = {2026},
author = {Panov, J and Elbert, A and Rosenthal, DS and Levi, M and Chumakov, K and Andino, R and Brodsky, L and Kaphzan, H},
title = {Spacio-Linear Screening for Ligand-Docking Cavities in Protein Structures: SLAM Algorithm.},
journal = {Life (Basel, Switzerland)},
volume = {16},
number = {2},
pages = {},
pmid = {41752921},
issn = {2075-1729},
support = {N/A//Tauber Foundation/ ; },
abstract = {Identifying structurally similar ligand-binding sites in unrelated proteins can facilitate drug repurposing, reveal off-target effects, and deepen our understanding of protein function. A number of tools were developed for structural screening, but many of them suffer from limited sensitivity and scalability. Using a data bank of crystallized protein structures, we aimed to discover novel protein targets for a ligand by leveraging a known ligand-binding query protein with a resolved structure. Here, we present SLAM (Spacio-Linear Alignment of Macromolecules), a novel alignment-based algorithm that detects local 3D similarities between ligand-binding cavities or protein-exposed surfaces of query and target proteins. SLAM encodes spatial substructure neighborhoods into short linear sequences of physicochemically annotated atoms, then applies pairwise sequence alignment combined with distance-correlation scoring to identify high-fidelity structural matches. Benchmarking using the Kahraman-36 dataset demonstrated that SLAM outperforms the state-of-the-art ProBiS algorithm in true-positive rate for predicting ligand-docking compatibility. Furthermore, SLAM identifies candidate ligands that may inhibit functionally critical domains of CRISPR-Cas proteins and predicts novel binding partners of toxic per- and polyfluoroalkyl Substance (PFAS) compounds (PFOA, PFOS) with plausible mechanistic links to toxicity. In conclusion, SLAM is a robust computationally efficient and flexible structural screening tool capable of detecting subtle physicochemical compatibilities between protein surfaces, promising to accelerate target discovery in pharmacology and elucidate protein-ligand interactions in environmental toxicology.},
}
@article {pmid40954296,
year = {2025},
author = {Zhang, R and Mirdita, M and Söding, J},
title = {De novo discovery of conserved gene clusters in microbial genomes with Spacedust.},
journal = {Nature methods},
volume = {22},
number = {10},
pages = {2065-2073},
pmid = {40954296},
issn = {1548-7105},
support = {CompLifeSci project horizontal4meta//Bundesministerium f&#xfc;r Bildung und Forschung (Federal Ministry of Education and Research)/ ; RS-2023- 00250470//National Research Foundation of Korea (NRF)/ ; },
mesh = {*Metagenomics/methods ; Metagenome/genetics/physiology ; *Multigene Family ; *Genome, Microbial/genetics ; Conserved Sequence ; *Software ; *Computational Biology/methods ; Algorithms ; Genome, Bacterial ; CRISPR-Cas Systems ; },
abstract = {Metagenomics has revolutionized environmental and human-associated microbiome studies. However, the limited fraction of proteins with known biological processes and molecular functions presents a major bottleneck. In prokaryotes and viruses, evolution favors keeping genes participating in the same biological processes colocalized as conserved gene clusters. Conversely, conservation of gene neighborhood indicates functional association. Here we present Spacedust, a tool for systematic, de novo discovery of conserved gene clusters. To find homologous protein matches, Spacedust uses fast and sensitive structure comparison with Foldseek. Partially conserved clusters are detected using novel clustering and order conservation P values. We demonstrate Spacedust's sensitivity with an all-versus-all analysis of 1,308 bacterial genomes, identifying 72,843 conserved gene clusters containing 58% of the 4.2 million genes. It recovered 95% of antiviral defense system clusters annotated by the specialized tool PADLOC. Spacedust's high sensitivity and speed will facilitate the annotation of large numbers of sequenced bacterial, archaeal and viral genomes.},
}
@article {pmid41751548,
year = {2026},
author = {Machel Gica, NG and Gica, WT and La, H and Mi, Y and Zhou, Y},
title = {Precision Breeding for a Global Staple Food: A Systematic Review with a Strategic Framework for CRISPR-Cas Applications in Rice (Oryza sativa L.).},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751548},
issn = {2073-4425},
support = {2025GBJ002388//Chinese Government Scholarship Council/ ; },
abstract = {Background: Rice is one of the world's main staple crops, and improving its productivity and resilience is important to achieving food security under varying climatic conditions. Objectives: This systematic review synthesizes the existing evidence on the application, technical limitations, and potential of the development of genome editing technologies (CRISPR-Cas) in rice (Oryza sativa L.), as well as presents a novel approach called the CRISPR Trait Prioritization and Readiness Framework (CTPRF). Methods: Peer-reviewed articles that reported applications of genome editing based on the CRISPR-Cas system in the genome of rice for trait improvement or functional genomics were identified through searches fromPubMed, Scopus, Web of Science, and Google Scholar with studies published between 2012 and 2025. Studies were screened on predefined inclusion criteria related to experimental validation, reporting of editing efficiency, and clear phenotypic results. Data on CRISPR systems, target genes, methods of delivery, traits modified, and phenotypic results were extracted and synthesized by comparative analysis. Results: A wide variety of different CRISPR systems have been used in rice, and our results indicate that NHEJ-mediated knockouts are effective in average genotypes with editing efficiencies in the range of 70-90%, but HDR and prime editing are still under 10%. The CTPRF is being introduced as a strategic decision support tool to evaluate traits from four dimensions: technical feasibility, phenotypic predictability, impact potential, and regulatory pathway. We use this framework for case studies in pioneering countries (USA, Japan, China) and show how it can be useful for guiding research investment and policy. Conclusions: CRISPR-Cas technologies have transformed rice breeding, but their introduction requires overcoming genotype-dependent barriers to transformation and negotiating patchwork regulatory environments. The CTPRF offers a roadmap for the acceleration of the development of climate-resilient and nutritious rice varieties for the action plan.},
}
@article {pmid41751014,
year = {2026},
author = {Wang, Q and Zheng, L and You, G and Dong, H and Chen, S and Wang, S and Chen, S},
title = {Navigating the Complexity: Advancing Diagnostic Strategies for Avian Reovirus in Chinese Poultry.},
journal = {Animals : an open access journal from MDPI},
volume = {16},
number = {4},
pages = {},
pmid = {41751014},
issn = {2076-2615},
support = {XTCXGC2021018, XTCXGC2021012//the '5511' Collaborative Innovation Project of Fujian Academy of Agricultural Sciences, China/ ; },
abstract = {Avian reovirus (ARV) infections pose a significant and evolving threat to China's poultry industry, the world's largest. Diverse farming systems-ranging from modern intensive operations to traditional waterfowl-poultry polyculture-foster a unique ecological niche for ARV, defined by complex serotypic and genotypic diversity, marked regional variations, potential interspecies transmission between chickens and waterfowl, and recurrent co-infections. Collectively, these factors undermine the efficacy of conventional diagnostic approaches. This review systematically outlines the current epidemic landscape of ARV in China, highlighting the molecular characteristics of prevailing strains (particularly those from waterfowl) and their roles in diagnostic evasion. We critically assess the performance and limitations of existing diagnostic techniques (virus isolation, ELISA, PCR/qPCR) within the Chinese epidemiological setting. Furthermore, we discuss innovative technologies-including multiplex qPCR, CRISPR-Cas systems, and next-generation sequencing (NGS)-that offer potential for developing next-generation diagnostics tailored to China's specific challenges. Finally, we propose future directions, with an emphasis on standardization, data sharing, and interdisciplinary collaboration to bridge the gap between cutting-edge innovation and on-farm application for precise ARV control.},
}
@article {pmid41744735,
year = {2026},
author = {Hu, X and Su, J and Song, S},
title = {CRISPR/Cas System-Based Biosensors.},
journal = {Biosensors},
volume = {16},
number = {2},
pages = {},
pmid = {41744735},
issn = {2079-6374},
abstract = {Over the past decade, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, originally identified as adaptive immune systems in bacteria and archaea that defend against invading nucleic acids, have revolutionized biological research [...].},
}
@article {pmid41741764,
year = {2026},
author = {Shivgotra, R and Soni, B and Bakrey, H and Kaur, P and Jain, SK},
title = {Bridging Gaps in Fungal Keratitis Management: Novel Diagnostics, Drug Delivery Systems, and Gene Therapies.},
journal = {AAPS PharmSciTech},
volume = {27},
number = {3},
pages = {},
pmid = {41741764},
issn = {1530-9932},
abstract = {Globally, fungal keratitis has become a major health concern, particularly in tropical and humid regions. The disease burden is exacerbated because of the underlying complexity of fungal pathogens, delayed or insufficient diagnosis, and limitations in existing therapeutic strategies, often leading to progressive corneal damage and impaired vision. Additional challenges include insufficient therapy optimization and variable efficacy of topical and systemic antifungal treatments, which may be influenced by factors such as rapid ocular drug clearance, ineffective ocular penetration, and the increasing prevalence of antifungal resistance. Conventional diagnostic techniques, such as microscopy and culture, continue to serve as reference standards but have limitations due to comparatively modest sensitivity and the prolonged turnaround times. In comparison, recent advances in diagnostic techniques, including CRISPR-based assays, PCR, MALDI-ToF MS, and in vivo confocal microscopy, as well as the novel drug-delivery nanocarriers, have been reported to show noticeable improvements in diagnostic accuracy and therapeutic outcomes. Furthermore, emerging DNA-based gene therapies and RNA-based therapeutics, along with advanced ocular drug carriers, have shown promising outcomes in preclinical research and early-phase clinical trials, suggesting potential advantages in enhanced tissue targeting and reduced therapeutic resistance. However, before widespread clinical acceptability, extensive clinical validation, long-term safety evaluations, and cost assessments are required, as existing findings are mainly limited to short-term and experimental research. This review focuses on the pathophysiology of fungal keratitis while underscoring the unmet diagnostic and therapeutic needs. It further explores the potential for developing translatable technologies aimed at predictive diagnosis and the effective management of this sight-threatening condition.},
}
@article {pmid41741653,
year = {2026},
author = {Saxton, DS and DeWeirdt, PC and Doering, CR and Roney, IJ and Laub, MT},
title = {A membrane-bound nuclease directly cleaves phage DNA during genome injection.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41741653},
issn = {1476-4687},
abstract = {From mammals to bacteria, the direct recognition and cleavage of viral nucleic acids is a potent defence strategy against viral infection, but it requires mechanisms for distinguishing self from non-self[1,2]. In bacteria, CRISPR-Cas and restriction-modification systems achieve this discrimination by recognizing specific DNA sequences or DNA modifications, respectively. Alternative mechanisms probably remain to be discovered. Here, we characterize SNIPE, an anti-bacteriophage defence system that constitutively localizes to the bacterial cell membrane in Escherichia coli to block phage λ infection. Using radiolabelled phage DNA and time-lapse microscopy to track phage genomes, we demonstrate that SNIPE directly cleaves phage DNA during genome injection. Based on proximity labelling, we find that SNIPE associates with host proteins essential for λ genome entry and with the λ tape measure protein, which facilitates λ genome injection across the inner membrane. SNIPE also defends against diverse siphoviruses, probably through direct interactions with their tape measure proteins. Our findings establish SNIPE as a widespread bacterial defence system that exploits the spatial organization of phage genome injection to specifically target viral DNA, representing a previously unknown strategy for distinguishing self from non-self in prokaryotic immune systems.},
}
@article {pmid41740122,
year = {2026},
author = {Yıldırım, K and Kavas, M},
title = {The Role of CRISPR in Modern Plant Breeding: Overcoming Breeding Barriers and Legislative Challenges Through Transgene-Free Genome Editing.},
journal = {Genome},
volume = {},
number = {},
pages = {},
doi = {10.1139/gen-2025-0063},
pmid = {41740122},
issn = {1480-3321},
abstract = {Traditional plant breeding techniques-such as crossbreeding, mutation breeding, and marker-assisted selection-have significantly contributed to crop improvement over the past century. However, these methods are often limited by long breeding cycles, low precision, and the unintended transfer of undesirable traits. To address these challenges, transgenic breeding emerged as a powerful tool, enabling the introduction of specific foreign genes to confer desirable traits such as pest resistance or herbicide tolerance. While highly effective and precise, transgenic approaches face considerable regulatory and public acceptance barriers, particularly in regions with strict GMO legislation.The advent of CRISPR/Cas genome editing has revolutionized plant breeding by enabling precise, efficient, and targeted modification of native genes, significantly accelerating the development of improved crops. Among CRISPR-based methods, transgene-free genome editing has gained prominence for its ability to produce enhanced plant varieties without integrating foreign DNA, thus avoiding many regulatory constraints associated with GMOs. This review aims to provide a comprehensive overview of transgenefree CRISPR-mediated genome editing technologies, emphasizing their use in modern plant breeding and their transformative potential to overcome the limitations of conventional methods while providing a regulation-friendly pathway for crop improvement.},
}
@article {pmid41740027,
year = {2026},
author = {Duarte, DF and Lucena, LP and Gonçalves, MHO and Benko-Iseppon, AM and Aburjaile, F and Azevedo, V and Brenig, B and Gama, MAS and Souza, EB},
title = {Phylogenomic analysis of Paracidovorax citrulli strains reveals the presence of two lineages in Brazil.},
journal = {Genetics and molecular biology},
volume = {48},
number = {4},
pages = {e20250046},
doi = {10.1590/1678-4685-GMB-2025-0046},
pmid = {41740027},
issn = {1415-4757},
abstract = {Paracidovorax citrulli is the causative agent of bacterial fruit blotch in melons and watermelons. This study used comparative genomic approaches of 17 Brazilian P. citrulli strains obtained from melons and watermelons to classify them into groups I and II and try to understand their genomic differences. The genomes of P. citrulli presented general characteristics similar to those shown for the genomes of the type strain of P. citrulli and reference strains of groups I and II. A phylogenomic analysis revealed two distinct groups of P. citrulli, in which most Brazilian P. citrulli strains were grouped with the strain representing group I. CRISPR-Cas analysis revealed the presence of two proteins, Cas3 and Cas10, in all Brazilian P. citrulli genomes. In addition, we observed the presence of two plasmids (pAMC6 and pAC53) in three Brazilian P. citrulli strains, all closely related to group I. The prediction of effector proteins revealed the XopE/AvrPphe protein as a differential between the strains of groups I and II. The present study will contribute to a more detailed understanding of aspects of host-pathogen interactions and will help improve the detection of strains from these groups, thus elucidating the population dynamics of Brazilian strains of P. citrulli.},
}
@article {pmid41739553,
year = {2026},
author = {Valinsky, WC and Ray, RP and Schaefer, KS and Grimm, JB and Nicolini, C and Lavis, LD and Clapham, DE},
title = {Phenotypic CRISPR screens identify NLRX1 as an essential activator of the human mitochondrial permeability transition.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {9},
pages = {e2535298123},
doi = {10.1073/pnas.2535298123},
pmid = {41739553},
issn = {1091-6490},
support = {NA//Janelia Research Campus (Janelia)/ ; },
mesh = {Humans ; Calcium/metabolism ; *Mitochondria/metabolism/genetics ; *Mitochondrial Proteins/metabolism/genetics ; Membrane Potential, Mitochondrial ; CRISPR-Cas Systems ; Mitochondrial Membrane Transport Proteins/metabolism/genetics ; Mitochondrial Membranes/metabolism ; Phenotype ; Clustered Regularly Interspaced Short Palindromic Repeats ; Permeability ; Calcium Channels/metabolism/genetics ; },
abstract = {The mitochondrial permeability transition (mPT) is an evolutionarily conserved destructive process that permeabilizes the inner mitochondrial membrane in response to calcium overload. The molecular mechanism underlying the mPT is not established. To unambiguously identify essential proteins, we designed two phenotypic assays for mitochondrial calcium overload and applied them to FACS-based CRISPR screening in human cells, ultimately evaluating 19,113 genes. The first screen studied mitochondrial membrane potential (MMP) collapse in response to calcium overload. Top-ranked genes were the essential proteins of the mitochondrial calcium uniporter complex, MCU and EMRE, reflecting that the calcium-induced MMP collapse results from mitochondrial calcium entry and not the mPT. The second screen measured the permeability of the inner mitochondrial membrane. Here, the fluorescent interaction of a membrane impermeant ~600 Da dye and a mitochondrial-targeted HaloTag protein was studied under mPT activating conditions; calcium overload and the thiol-reactive molecule phenylarsine oxide. With secondary validation, we identified four protein-encoding genes that delayed or prevented the mPT under knockout: NF2, REST, BPTF, and NRLX1. Knockout of the nonmitochondrial proteins BPTF, NF2, or REST increased mitochondrial calcium retention capacity (CRC). However, calcium release or sensitivity to cyclosporin A (CsA) persisted, indicative of mPT sensitizers. Only knockout of the mitochondrial matrix protein, NLRX1, increased CRC, abolished calcium release, and was CsA-insensitive. This top-ranked hit of the mitochondrial permeability screen meets the definition of an essential mPT activator. Integral membrane proteins, including all previously proposed mPT candidates, were not essential activators.},
}
@article {pmid41738762,
year = {2026},
author = {Irfan, M and Duran-Pinedo, A and Solbiati, J and Rocha, FG and Gibson, FC and Frias-Lopez, J},
title = {A CRISPR array orchestrates virulence and host response in Porphyromonas gingivalis.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0283425},
doi = {10.1128/spectrum.02834-25},
pmid = {41738762},
issn = {2165-0497},
abstract = {CRISPR-Cas systems are primarily recognized for their defensive role against foreign DNA. Recent studies, however, indicate involvement in regulatory functions. The persistence of a large spacer array without apparent phage targets in Porphyromonas gingivalis ATCC 33277 remains unexplained. This study demonstrates that deletion of the non-coding CRISPR array 30.1 in P. gingivalis ATCC 33277 results in increased biofilm formation, elevated virulence in a Galleria mellonella infection model, and significant alterations in the macrophage transcriptomic response. The ΔCRISPR-30.1 mutant forms twice as much biofilm as the wild type, induces 50% mortality in Galleria larvae within 130 h compared to 200 h for the wild type (P < 0.0001), and elicits a cytokine profile characterized by increased IL-6, CXCL1, CXCL2, and CXCL9 secretion. Dual RNA sequencing of THP-1 macrophages infected with wild-type and ΔCRISPR 30.1 strains reveals that the loss of the array activates bacterial metabolic and secretion pathways while suppressing host innate and adaptive immune signaling. Single-primer amplification (SPA) identifies numerous self-genome loci bound by individual 30.1 spacers, supporting a direct, spacer-mediated regulatory mechanism. These findings establish CRISPR array 30.1 as a previously unrecognized regulator of P. gingivalis physiology and host-pathogen interactions.IMPORTANCECRISPR-Cas systems are established as adaptive immune elements, yet spacer arrays without known targets are frequently observed in bacteria and often lack a defined function. In P. gingivalis, a keystone periodontal pathogen, a non-coding CRISPR array has been shown to regulate biofilm formation, virulence in an invertebrate model, and the macrophage transcriptional response. This expands the recognized functions of CRISPR arrays to include the direct regulation of bacterial physiology and the modulation of host immune responses, identifying CRISPR spacers as potential targets for antimicrobial interventions. Furthermore, elucidating the role of CRISPR arrays in P. gingivalis may have broader clinical implications, given the established associations between periodontal health and systemic inflammatory diseases. Targeting spacer arrays to modulate bacterial virulence could influence the management of these conditions and enhance the translational relevance of such therapeutic strategies.},
}
@article {pmid41738557,
year = {2026},
author = {Girard, V and Sorge, S and Kurth, J and Alexandre, C and Gould, A},
title = {ShineGAL4 drivers for tissue and cell-type specific optogenetics in Drosophila.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {4},
pages = {},
doi = {10.1242/dev.204981},
pmid = {41738557},
issn = {1477-9129},
support = {FC001088/CRUK_/Cancer Research UK/United Kingdom ; FC001088/MRC_/Medical Research Council/United Kingdom ; FC001088/WT_/Wellcome Trust/United Kingdom ; 223760/WT_/Wellcome Trust/United Kingdom ; 104566/WT_/Wellcome Trust/United Kingdom ; 543-2022//EMBO/ ; //Francis Crick Institute/ ; },
mesh = {Animals ; *Optogenetics/methods ; *Drosophila Proteins/genetics/metabolism ; Transcription Factors/genetics/metabolism ; *Drosophila melanogaster/genetics ; Organ Specificity/genetics ; CRISPR-Cas Systems/genetics ; Neurons/metabolism ; *Drosophila/genetics ; },
abstract = {An optogenetic split-GAL4 system, ShineGAL4, allows genes to be manipulated with unprecedented spatiotemporal precision. Here, we convert a panel of 14 GAL4 drivers widely used in Drosophila research into their ShineGAL4 counterparts. Homology assisted CRISPR knock-in (HACK) is used to replace GAL4 with the GAL4 DNA binding domain fused to a Magnet photoswitch. We show that the resulting ShineGAL4 drivers enable gene expression to be rapidly induced by light specifically in fat body, muscles, enterocytes, oenocytes, Malpighian tubules, neurons, neuroblast lineages, glial subtypes or in all glia. We also develop an optogenetic cassette for photoactivation of GAL4 in 'silent' FLP-out clones. This panel of optogenetic tools will enable precise spatiotemporal control of gene expression in a wide range of different Drosophila tissues and cell-types.},
}
@article {pmid41736546,
year = {2026},
author = {Zhang, C and Ye, K and Shang, Y and Song, Y and Li, P and Jiang, X and Yang, C and Liang, A and Zhang, J and Meng, F and Zhang, M},
title = {Spatially concentrated adenine base editors efficiently correct PLP1 mutations in oligodendrocytes.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
doi = {10.1093/nar/gkag156},
pmid = {41736546},
issn = {1362-4962},
support = {32370853//National Natural Science Foundation of China/ ; },
mesh = {*Oligodendroglia/metabolism ; *Gene Editing/methods ; Animals ; *Myelin Proteolipid Protein/genetics/metabolism ; *Adenine/metabolism ; Mutation ; Mice ; *Pelizaeus-Merzbacher Disease/genetics/therapy/pathology ; Humans ; CRISPR-Cas Systems ; Adenosine Deaminase/metabolism/genetics ; },
abstract = {Oligodendrocytes (OLs), the myelinating cells of the central nervous system, are particularly prone to pathogenic G-to-A mutations, such as PLP1A243V, which causes Pelizaeus-Merzbacher disease (PMD), a lethal hypomyelinating disorder lacking effective therapy. Although adenine base editors (ABEs) can in principle correct such mutations, their application in OLs is limited by inefficient on-target editing. Here, we develop a spatially concentrated ABE (cABE) strategy that enhances editing by promoting nuclear translocation of tRNA adenosine deaminase (TadA*) from the cytoplasm. Using a SunTag-based multivalent recruitment system, TadA* is locally enriched at genomic targets (cABE-1.0), achieving robust editing in vitro. To enable in vivo delivery while preserving high efficiency and fidelity, SpCas9 is replaced with compact eNme2-C Cas9, generating an AAV-compatible variant (cABE-2.0). Notably, cABE-2.0 forms dynamic nuclear puncta with properties of liquid-liquid phase separation, enhancing on-target editing while substantially reducing transcriptome-wide RNA off-target effects. Functionally, cABE-2.0 efficiently corrects the PLP1A243V mutation in OLs, restores Plp subcellular localization, and rescues myelination-related phenotypes. These findings demonstrate that spatial reorganization, rather than increasing intrinsic catalytic activity of TadA*, provides a distinct principle for improving base editing in difficult-to-edit cell types, such as OLs, offering a mechanistic and technical framework for gene therapy of PMD and related myelin disorders.},
}
@article {pmid40561247,
year = {2025},
author = {Damaskou, A and Wilson, R and Gozdecka, M and Giotopoulos, G and Asby, R and Eleftheriou, M and Gu, M and Récher, C and Mansat-De Mas, V and Vergez, F and Sahal, A and Vick, B and Papachristou, EK and Sawle, A and Yankova, E and Dudek, M and Liu, X and Russell, J and Rak, J and Hilcenko, C and D'Santos, C and Jeremias, I and Sarry, JE and Tzelepis, K and Huntly, BJP and Warren, AJ and Tavana, O and Vassiliou, GS},
title = {Posttranscriptional depletion of ribosome biogenesis factors engenders therapeutic vulnerabilities in NPM1-mutant AML.},
journal = {Blood},
volume = {146},
number = {10},
pages = {1239-1252},
doi = {10.1182/blood.2024026113},
pmid = {40561247},
issn = {1528-0020},
mesh = {*RNA Processing, Post-Transcriptional/drug effects/genetics ; *Ribosomes/genetics/metabolism ; *Leukemia, Myeloid, Acute/drug therapy/genetics/pathology ; Cytoplasm ; Dactinomycin/pharmacology ; Nuclear Proteins/metabolism ; Mice, Knockout ; Hematopoietic Stem Cells ; Ribosome Subunits, Small, Eukaryotic/chemistry ; CRISPR-Cas Systems ; Humans ; Animals ; Mice ; *Nucleophosmin/genetics/physiology ; Sulfonamides ; Tumor Suppressor Protein p53 ; Bridged Bicyclo Compounds, Heterocyclic ; },
abstract = {NPM1 is a multifunctional phosphoprotein with key roles in ribosome biogenesis among its many functions. NPM1 gene mutations drive 30% of acute myeloid leukemia (AML) cases. The mutations disrupt a nucleolar localization signal and create a novel nuclear export signal, leading to cytoplasmic displacement of the protein (NPM1c). NPM1c mutations prime hematopoietic progenitors to leukemic transformation, but their precise molecular consequences remain elusive. Here, we first evaluate the effects of isolated NPM1c mutations on the global proteome of preleukemic hematopoietic stem and progenitor cells (HSPCs) using conditional knockin Npm1cA/+ mice. We discover that many proteins involved in ribosome biogenesis are significantly depleted in these murine HSPCs, but also importantly in human NPM1-mutant AMLs. In line with this, we found that preleukemic Npm1cA/+ HSPCs display higher sensitivity to RNA polymerase I inhibitors, including actinomycin D (ActD), compared with Npm1+/+ cells. Combination treatment with ActD and venetoclax inhibited the growth and colony-forming ability of preleukemic and leukemic NPM1c+ cells, whereas low-dose ActD treatment was able to resensitize resistant NPM1c+ cells to venetoclax. Furthermore, using data from CRISPR dropout screens, we identified and validated TSR3, a 40S ribosomal maturation factor whose knockout preferentially inhibited the proliferation of NPM1c+ AML cells by activating a p53-dependent apoptotic response. Similarly, to low-dose ActD treatment, TSR3 depletion could partially restore sensitivity to venetoclax in therapy-resistant NPM1c+ AML models. Our findings propose that targeted disruption of ribosome biogenesis should be explored as a therapeutic strategy against NPM1-mutant AML.},
}
@article {pmid41735731,
year = {2026},
author = {Zhao, R and Chen, J and Li, Y and Chen, DY and Kang, X and Dong, S and Yuan, X and Li, X and Gao, L and Yang, G and Chu, X and Wang, JG},
title = {CRISPR/Cas9-mediated α-prolamin gene (Seita.8G190200) mutagenesis increases the content of functional amino acids in foxtail millet (Setaria italica).},
journal = {Plant cell reports},
volume = {45},
number = {3},
pages = {},
pmid = {41735731},
issn = {1432-203X},
support = {2023YFD1202702-6//National Key Research and Development Program of China/ ; 2025QNLJ203//"Youth Science and Technology Leadership Talent Training Program" of Shanxi Agricultural University/ ; },
mesh = {*Setaria Plant/genetics/metabolism ; *Amino Acids/metabolism ; *CRISPR-Cas Systems/genetics ; *Prolamins/genetics/metabolism ; Gene Editing ; *Mutagenesis/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Nutritive Value ; },
abstract = {The gene editing of α-prolamin gene (Seita.8G190200) exhibited significant increases in multiple functional aminoacids. The vast majority sugar components showed significant increases in the gene editing lines. Knockout of the prolamin gene not only improves the nutritional quality but also affects its eating quality. Proteins in foxtail millet exhibits a balanced amino acid composition that meets human dietary requirements.Prolamins are notably deficient in essential amino acids such as lysine, the structure, morphology, and functional characteristics of foxtail millet prolamin have been sufficiently elucidated, but the biological roles of prolamin genesstill need further exploration. This study demonstrates that the α-prolamin gene (Seita.8G190200) plays crucial roles in regulating prolamin content, functional amino acid and sugar components levels, potentially serving as an important target for improving both nutritional and eating quality of foxtail millet. These findings provide scientific foundations for developing novel nutritionally fortified functional foxtail millet products, breeding new foxtail millet varieties enriched with functional amino acids for human health benefits.},
}
@article {pmid41733973,
year = {2026},
author = {van der Meulen, SA and Roemhild, K and Driessen, M and van den Akker, E and Nethe, M},
title = {In vivo modeling of stress erythropoiesis through targeted gene editing of rat hematopoietic stem cells.},
journal = {Blood advances},
volume = {10},
number = {4},
pages = {1281-1292},
doi = {10.1182/bloodadvances.2025017433},
pmid = {41733973},
issn = {2473-9537},
mesh = {Animals ; *Erythropoiesis/genetics ; Rats ; *Gene Editing/methods ; *Hematopoietic Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Anemia/etiology/genetics/metabolism ; Cadherins/genetics/metabolism ; Disease Models, Animal ; *Stress, Physiological ; },
abstract = {In response to anemia, the erythroid lineage significantly expands. This growth is driven by extramedullary erythropoiesis in mice, but is additionally regulated within the bone marrow (BM) of rats, a process likely conserved in humans due to similar BM architecture. This process is, however, mostly elusive. We identified E-cadherin to mark the expansion of the erythroid lineage in BM from anemic rats. To explore the regulation of erythropoiesis in the BM in response to anemia, we studied the role of E-cadherin in the erythroid lineage of rats. As genetic methods to model erythropoiesis in rats are limited, we established a rat BM transplant model that, combined with CRISPR/Cas9 genome editing, enabled us to examine the control of E-cadherin in BM in response to anemia. We identified CD90+CD44+CD45R- cells to contain hematopoietic stem and progenitor cells (HSPCs) in rats. CD90+CD44+CD45R--enriched HSPCs can be efficiently edited using CRISPR/Cas9, which, upon transplant, induce high BM chimerism. Importantly, we identified that recovery from irradiation-induced anemia involves 2 phases. Phase 1 is marked by expansion of erythroid precursors in the BM, supported by extramedullary erythropoiesis in the spleen. This phase is followed by a second phase, characterized by accelerated terminal differentiation, which is selectively controlled in the BM. Finally, we discovered that genetic inactivation of hematopoietic-expressed E-cadherin delays recovery from radiation-induced anemia. Our work provides novel means to expand our knowledge on hematology, and the opportunity to dissect the molecular regulation underlying the erythroid response(s) to anemia in BM, using rat models.},
}
@article {pmid41732389,
year = {2026},
author = {Fanarraga, ML and García Hevia, L},
title = {Silica nanoparticles as advanced platforms for nucleic acid delivery.},
journal = {Materials today. Bio},
volume = {37},
number = {},
pages = {102921},
pmid = {41732389},
issn = {2590-0064},
abstract = {Nucleic acid therapeutics, including siRNA, mRNA, plasmid DNA, and CRISPR/Cas systems, have demonstrated remarkable potential but continue to face translational barriers related to systemic instability, immune activation, and inefficient intracellular delivery. Conventional lipid and polymeric carriers, although clinically validated, often lack the structural resilience and versatility required for large or complex cargos. Silica-based nanoparticles, particularly mesoporous silica nanoparticles, provide a distinctive combination of mechanical rigidity, tunable porosity, and abundant surface chemistry that enables robust encapsulation, protection, and controlled release of diverse nucleic acids. This review adopts a problem-driven perspective, analyzing how specific nanoarchitectural designs, surface functionalizations, and ligand-mediated targeting strategies address key limitations in nucleic acid delivery. Emphasis is placed on overcoming systemic barriers such as premature degradation, immune recognition, and restricted biodistribution, as well as intracellular challenges including endosomal escape and nuclear access. Hybrid and biomimetic silica platforms are highlighted for their capacity to integrate combinatorial and theranostic functionalities, expanding the therapeutic scope toward complex payloads and multifunctional formulations. By linking synthesis approaches with translational requirements, an integrated roadmap is proposed that positions silica nanocarriers as advanced platforms for next-generation gene therapy. The evidence underscores the potential of silica architectures to combine structural durability with versatile chemical adaptability, thereby enabling safe, efficient, and clinically relevant delivery of nucleic acids.},
}
@article {pmid41732321,
year = {2026},
author = {He, Y and Tu, X and Xue, Y and Chen, Y and Ye, B and Li, X and Li, D and Zhong, Z and Zhong, Q},
title = {CRISPR screening redefines therapeutic target identification and drug discovery with precision and scalability.},
journal = {Journal of pharmaceutical analysis},
volume = {16},
number = {2},
pages = {101357},
pmid = {41732321},
issn = {2214-0883},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening technology is redefining the landscape of drug discovery and therapeutic target identification by providing a precise and scalable platform for functional genomics. The development of extensive single-guide RNA (sgRNA) libraries enables high-throughput screening (HTS) that systematically investigates gene-drug interactions across the genome. This powerful approach has found broad applications in identifying drug targets for various diseases, including cancer, infectious diseases, metabolic disorders, and neurodegenerative conditions, playing a crucial role in elucidating drug mechanisms and facilitating drug screening. Despite challenges like off-target effects, data complexity, and ethical or regulatory concerns, ongoing advancements in CRISPR technology and bioinformatics are steadily overcoming these limitations. Additionally, by integrating with organoid models, artificial intelligence (AI), and big data technologies, CRISPR screening expands the scale, intelligence, and automation of drug discovery. This integration boosts data analysis efficiency and offers robust support for uncovering new therapeutic targets and mechanisms. This review outlines the fundamental principles and applications of CRISPR screening technology, delves into specific case studies and technical challenges, and highlights its expanding role in drug discovery and target identification. It also examines the potential for clinical translation and addresses the associated ethical and regulatory considerations.},
}
@article {pmid41732093,
year = {2026},
author = {Neupane, S and Pfrender, ME and Wang, L and Xu, S},
title = {Detection of CRISPR-Cas-induced mutations in Daphnia.},
journal = {G3 (Bethesda, Md.)},
volume = {},
number = {},
pages = {},
doi = {10.1093/g3journal/jkag050},
pmid = {41732093},
issn = {2160-1836},
abstract = {CRISPR-Cas9 has established itself as a robust tool for conducting loss of function gene research in emerging model species including the freshwater zooplankton Daphnia. However, sensitive detection of mutations, especially in genetic mosaic and pooled samples, remains a challenge. In this study we evaluate two of the most widely used mutation screening techniques, the T7 Endonuclease I (T7EI) assay and Fragment Analysis (FA) for their sensitivity, accuracy, and practical use in detecting CRISPR-induced indels in four targeted genes, DNMT3A, DNMT3B, PERIOD2, and DMRT1 in Daphnia magna. Here, we show that T7EI, although it offers a quick and cost-effective screening method, often produces false positives, especially when examining pooled samples. Conversely, FA facilitates detecting allele size differences at a fine resolution, reproducibility in detecting indels, and distinguishing zygosity and is more reliable as a method to detect mutation. Our comparative analyses convey the importance of carefully selecting the appropriate screening methods depending on research questions.},
}
@article {pmid41730595,
year = {2026},
author = {Chang, X and Han, C and Ji, H and Zeng, Z and Yang, J and Liu, Q and Jia, C and Zhao, L and Zhou, C and Chen, S and Knoll, W and Li, J and Wang, Z and Zhang, L},
title = {SPARC: A programmable molecular diagnostic platform based on a signal-triggered, self-supplied crRNA and tiered PER-transcription-CRISPR cascade for early detection of hepatocellular carcinoma.},
journal = {Analytica chimica acta},
volume = {1394},
number = {},
pages = {345209},
doi = {10.1016/j.aca.2026.345209},
pmid = {41730595},
issn = {1873-4324},
mesh = {Humans ; *Liver Neoplasms/diagnosis/genetics ; *Carcinoma, Hepatocellular/diagnosis/genetics ; *MicroRNAs/genetics/analysis ; *Early Detection of Cancer/methods ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Limit of Detection ; *Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Accurate quantification of microRNAs (miRNAs) is essential for early cancer detection, yet remains challenging due to their short length, low abundance, and high sequence similarity. Existing assays often struggle to achieve sufficient sensitivity, specificity, and robustness for reliable clinical deployment.<br><br>RESULTS: We introduce SPARC, a programmable molecular diagnostic platform that integrates a signal-triggered primer exchange reaction, self-supplied crRNA generation, and a tiered PER-transcription-CRISPR/Cas12a amplification cascade. Using miRNA-21 as a model, SPARC achieves an ultralow detection limit of 1.22&#xa0;fM and a broad quantitative range from 1&#xa0;fM to 100&#xa0;nM. The system exhibits high specificity, strong analytical stability, and modular adaptability to diverse targets, including miRNA-122. Notably, the dual-directional profiling of oncogenic and tumor-suppressive miRNAs enhances diagnostic resolution. When applied to HCC cell lines and clinical tissues, SPARC accurately distinguished malignant from normal samples and showed excellent agreement with qRT-PCR measurements and histopathological assessments.<br><br>SIGNIFICANCE: This streamlined and self-amplifying cascade system provides a scalable, robust, and clinically compatible platform for ultrasensitive miRNA detection. SPARC holds strong potential for early hepatocellular carcinoma screening, molecular subtyping, and broader precision oncology applications.},
}
@article {pmid41729380,
year = {2026},
author = {Liu, YY and Lv, YR and Jia, JT and Zhang, R and Yang, B and Xue, SY and Bayaer, H and Bagen, A and Chen, RB and Tunala, S and Wang, R and Ding, YL and Zhao, L and Liu, YH},
title = {Rapid and Simple Detection of Enterocytozoon Bieneusi Using Lateral Flow Assay Based on Recombinase Polymerase Amplification or Nested PCR Combined with CRISPR-Cas12a.},
journal = {Acta parasitologica},
volume = {71},
number = {2},
pages = {},
pmid = {41729380},
issn = {1896-1851},
support = {2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; },
mesh = {*Enterocytozoon/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Animals ; Feces/microbiology ; *Microsporidiosis/diagnosis/veterinary/microbiology ; *Polymerase Chain Reaction/methods ; Humans ; Recombinases/genetics ; DNA, Fungal/genetics ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Enterocytozoon bieneusi is an obligate intracellular microsporidian pathogen. It primarily causes diarrhea and weight loss in infected humans and animals, resulting in substantial economic losses to the livestock industry. Therefore, establishing a highly sensitive and specific detection method for E. bieneusi is critical for its prevention and control.<br><br>METHODS: crRNA and recombinase polymerase amplification (RPA) primers were designed based on partial sequences of the 18&#xa0;S ribosomal RNA gene and the internal transcribed spacer (ITS) of E. bieneusi. DNA extracted from fecal samples was amplified using RPA or nested polymerase chain reaction (PCR). PCR amplicons were treated with a Tris-saturated phenol-chloroform-isoamyl alcohol mixture to obtain purified target DNA, which was subsequently introduced into the CRISPR-Cas12a reaction system. Post-reaction detection was performed via qPCR instrumentation, fluorescence visualization, and lateral flow strip (LFS) assays. The operational parameters for E. bieneusi detection were subsequently optimized using RPA/CRISPR-Cas12a or nested PCR/CRISPR-Cas12a platforms. The aforementioned methodology was concurrently validated using 50 clinical specimens with known E. bieneusi infection status.<br><br>RESULTS: The limits of detection were 7.13 copies/&#xb5;L for RPA/CRISPR-Cas12a and 2.35&#x2009;&#xd7;&#x2009;10[-&#x2009;2] copies/&#xb5;L for nested PCR/CRISPR-Cas12a. When the concentration of unamplified DNA in the CRISPR-Cas12a reaction system reached&#x2009;&#x2265;&#x2009;1&#x2009;&#xd7;&#x2009;10[-&#x2009;4] &#xb5;g/&#xb5;L, the single-stranded DNA reporter was efficiently cleaved, resulting in a detectable fluorescence signal. The nested PCR/CRISPR-Cas12a technology was used to analyze 50 fecal samples with confirmed E. bieneusi-positive or -negative status. The results obtained from instrument-based detection, fluorescence observation, and lateral flow test strip detection were completely consistent.<br><br>CONCLUSIONS: We established the first integration of nested PCR with CRISPR-Cas12a for the detection of E. bieneusi. and were also the first to quantitatively explore the detection limit of Cas12a using non-amplified E. bieneusi DNA. This approach offers a rapid, specific, and highly sensitive diagnostic method. Furthermore, the wide selection of appropriate visualization methods facilitates adaptation to various laboratory conditions and sample template concentrations, enabling accurate result interpretation.},
}
@article {pmid41728952,
year = {2026},
author = {Feng, H and Li, Z and Zhang, H and Zheng, Y and Xu, B and Zhang, Y and Zou, L and Wu, L},
title = {Characterization of gRNA-dependent and gRNA-independent off-target binding sites of PspCas13b and RfxCas13d in mammalian cells.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41728952},
issn = {1362-4962},
support = {2021YFA1100201//National Key R&amp;D Program of China/ ; 2022YFA1303301//National Key R&amp;D Program of China/ ; XDB0570000//Chinese Academy of Sciences/ ; 82400181//National Natural Science Foundation of China/ ; 82270160//National Natural Science Foundation of China/ ; 2024M751998//China Postdoctoral Science Foundation/ ; 2023YFC2706401//Ministry of Science and Technology/ ; },
mesh = {Humans ; HEK293 Cells ; Binding Sites ; CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; Regulatory Factor X Transcription Factors ; Gene Editing ; Transcriptome ; },
abstract = {CRISPR-Cas13 systems, harnessed for RNA-guided transcriptome editing, hold significant promise for clinical and in vivo therapeutic applications. However, understanding their in vivo target specificity and recognition rules remains a challenge. In this study, we employed the uSpyCLIP method, which enhances sensitivity and specificity for identifying RNA-binding protein (RBP) binding sites, to map the transcriptome-wide binding sites of catalytically inactive PspCas13b (dPspCas13b) and RfxCas13d (dRfxCas13d) in HEK293T cells, using a variety of single guide RNAs (gRNAs). Surprisingly, we identified both gRNA-dependent and gRNA-independent off-target binding sites for both dCas13 complexes. These gRNA-independent off-target sites exhibited distinct RNA structural and sequence signatures: dPspCas13b's gRNA-independent binding was associated with specific RNA structural features, while dRfxCas13d's was linked to unique sequence motifs. Analysis of gRNA-dependent off-target sites revealed the crucial role of the DR-distal and middle regions of the gRNA in determining binding specificity. Further analysis demonstrated that some off-target binding events led to changes in gene expression at the messenger RNA and/or protein level. Collectively, our findings provide important insights into the characteristics of gRNA-dependent and gRNA-independent off-target binding for PspCas13b and RfxCas13d, offering valuable guidance for optimizing Cas13 and gRNA design in future applications.},
}
@article {pmid41728950,
year = {2026},
author = {King, HE and O'Connell, S and Kavanagh, D and Mason, S and McCool, C and Fernandez-Chamorro, J and Chaffer, CL and Clark, SJ and Vieira, HGS and Sterne-Weiler, T and Weatheritt, RJ},
title = {Isoform-specific single-cell perturb-seq reveals distinct functions of alternative promoters in drug response.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41728950},
issn = {1362-4962},
support = {DP250103133//Australian Research Council Discovery/ ; FT210100355//Future Fellowship/ ; //Scrimshaw Foundation/ ; },
mesh = {*Promoter Regions, Genetic ; Humans ; *Single-Cell Analysis/methods ; Tamoxifen/pharmacology/therapeutic use ; *Estrogen Receptor alpha/genetics ; *Breast Neoplasms/genetics/drug therapy/pathology ; CRISPR-Cas Systems ; Female ; Protein Isoforms/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic/drug effects ; },
abstract = {CRISPR interference (CRISPRi) screens have emerged as powerful tools for dissecting gene function, yet their application to genes with multiple promoters, which comprise over 60% of human genes, remains poorly understood. Here, we demonstrate that CRISPR-dCas9-based screens exhibit widespread promoter specificity, with untargeted promoters often showing compensatory upregulation to maintain gene expression. Leveraging this selective targeting of individual promoters within the same gene, we developed Isoform-Specific single-cell Perturb-Seq to systematically analyse alternative promoter function. Our analysis revealed that alternative promoters in 51.6% of targeted genes drive distinct transcriptional programs. This suggests that promoter selection represents a fundamental mechanism for generating cellular diversity rather than mere transcriptional redundancy. In breast cancer models, this promoter-specific targeting revealed differential effects on drug sensitivity, where distinct estrogen receptor (ESR1) promoters showed opposing influences on tamoxifen response and patient survival. These findings demonstrate the necessity of promoter-level analysis in functional genomics and suggest new strategies for therapeutic intervention through promoter-specific targeting.},
}
@article {pmid41728946,
year = {2026},
author = {Dang, QT and Chang, CW and Chen, PY and Truong, VA and Huang, PY and Nguyen, MTT and Hu, YC},
title = {CRISPR-associated transposon for programmable viral vector engineering and prime editing.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41728946},
issn = {1362-4962},
support = {NSTC 114-2223-E-007-013//National Science and Technology Council/ ; 113-2321-B-007-004//National Science and Technology Council/ ; 113-2223-E-007-010//National Science and Technology Council/ ; 113-2622-E-007-012//National Science and Technology Council/ ; 112-2622-E-007-030//National Science and Technology Council/ ; 112-2223-E-007-002//National Science and Technology Council/ ; //Veterans General Hospitals/ ; VGHUST115-G6-1-1//University System of Taiwan/ ; //National Science and Technology Council/ ; //Veterans General Hospitals/ ; VGHUST115-G6-1-1//University System of Taiwan Joint Research Program/ ; },
mesh = {*Gene Editing/methods ; Humans ; HEK293 Cells ; *Baculoviridae/genetics ; *Genetic Vectors/genetics ; *DNA Transposable Elements/genetics ; Animals ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; Transgenes ; *Genetic Engineering/methods ; },
abstract = {Baculovirus, an insect virus commonly used for recombinant protein expression in insect cells and gene delivery in mammalian systems, is often generated through bacmid-based engineering. To enable flexible and programmable bacmid engineering, we developed SHOT 2.0, an optimized CRISPR-associated transposon platform that mediates RNA-guided and customized bacmid editing in Escherichia coli. The edited bacmid can be transfected into insect cells to produce recombinant baculoviruses. SHOT 2.0 supported site-specific integration of large DNA cargos (at least 14 kb) into defined loci such as v-cath and ODVe56, with integration at ODVe56 markedly improving transgene stability during serial virus passaging. The system is fully compatible with the Bac-to-Bac® workflow, enabling dual-gene insertion into the bacmid and derived baculovirus. Leveraging this platform, we constructed an all-in-one baculovirus encoding the PE5max prime editor. This vector-mediated prime editing achieves efficiencies up to 85.6% in HEK293T cells and achieves robust prime editing in hard-to-transfect cell types, including iPSCs and liver cancer cells, with efficiencies up to 37.1%. These results demonstrate that SHOT 2.0 substantially expands the baculovirus engineering toolbox, providing a flexible platform for genome editing and future gene delivery.},
}
@article {pmid41728195,
year = {2025},
author = {Zhu, F and Liu, Z and Zheng, Z},
title = {An AI-driven framework for enhancing regulatory precision and efficiency in CRISPR-Cas gene-edited crops: challenges, opportunities, and global harmonization.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1693105},
pmid = {41728195},
issn = {1664-462X},
abstract = {INTRODUCTION: The rapid advancement and adoption of CRISPR-Cas technologies in crop improvement has significantly outpaced existing regulatory frameworks, leading to inconsistencies in the global oversight of gene-edited organisms. As governments and international bodies struggle to reconcile scientific innovation with policy governance, a pressing need has emerged for methodologies that can translate biological edits into regulatory-compliant representations across jurisdictions. Traditional approaches often compartmentalize genomic and legal domains, lacking the formalism to bridge biological intent and compliance precision. These methods are typically static, unable to adapt to jurisdictional policy drift or incorporate real-time exemption logic, thereby undermining both regulatory interpretability and technical fidelity.<br><br>METHODS: To address this gap, I propose a unified computational framework built around the novel GeneRegAlignNet model and the Constraint-Aware Policy Induction (CAPI) strategy. This framework embeds regulatory semantics directly into the learning architecture, enabling the alignment of gene-editing features with heterogeneous policy descriptors in a shared latent space. GeneRegAlignNet employs symbolic gating, contrastive manifold learning, and exemption-aware vectorization to predict alignment likelihoods between edits and legal categories with high precision. CAPI extends this model with a risk-calibrated policy optimization pipeline that accounts for policy evolution, regulatory variance, and jurisdictional priorities.<br><br>RESULTS AND DISCUSSION: Empirical validation demonstrates improved performance in regulatory alignment accuracy and resilience to policy drift across a diverse set of gene-editing scenarios. By tightly integrating formal representations of molecular edits with dynamic, multi-jurisdictional policy inference, our framework offers a scalable and interpretable path forward in enhancing regulatory precision and global harmonization in the oversight of CRISPR-Cas-edited crops.},
}
@article {pmid41727334,
year = {2025},
author = {Ramezani, R and Behbahani, M and Mohabatkar, H and Sarraf Mamouri, K and Hejazi, F},
title = {Comparison of CRISPR Sequences in Archaea and Bacteria with Eukaryotic microRNAs.},
journal = {Avicenna journal of medical biotechnology},
volume = {17},
number = {4},
pages = {258-276},
pmid = {41727334},
issn = {2008-2835},
abstract = {BACKGROUND: This study explores repetitive Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sequences from the archaea Acidianus sp. and Acidianus ambivalens (A. ambivalens), as well as from the bacterium Yersinia ruckeri (Y. ruckeri). These sequences are compared with human microRNA (miRNA) sequences to investigate potential genetic similarities and disease associations.<br><br>METHODS: CRISPR sequences were retrieved from the CRISPR/Cas[++] database, and human miRNA sequences were obtained from miRBase. Sequence alignments were performed using BLASTn with an E-value threshold of 1e-5 to identify significant similarities. Genes associated with matched human miRNAs were identified through the HGNC and GeneCards databases. Further analyses included comparison with disease-associated miRNAs reported in human and mouse datasets.<br><br>RESULTS: In Y. ruckeri, alignments revealed similarities to miRNAs linked with genes such as FOXO1, PTEN, PAX7, and DOCK3, which are associated with lung cancer and muscular dystrophies. In A. ambivalens, aligned miRNAs corresponded to loci including CHM13 and GRCh38, potentially linked to periembolic adenocarcinoma and mild pre-eclampsia. For Acidianus sp., matches were observed with miRNAs associated with genes like Irak2, NOS2, STAT1, and Numb, which have been implicated in Psoriatic arthritis, Alzheimer's disease, Hepatocellular carcinoma, and Coronary artery disease.<br><br>CONCLUSION: CRISPR sequences from these prokaryotes show notable similarities with human miRNAs, suggesting possible indirect links to genes involved in major diseases. These preliminary findings emphasize the need for further investigation into shared sequence motifs and their functional roles in host-pathogen interactions or evolutionary biology.},
}
@article {pmid41727253,
year = {2026},
author = {Meenakshi, and Komal, and Sharma, A and Prabhu, S and Awasthi, S},
title = {Modern plant stress adaptation: integrating defense, nanotechnology and genetics.},
journal = {3 Biotech},
volume = {16},
number = {3},
pages = {105},
pmid = {41727253},
issn = {2190-572X},
abstract = {This review critically analyses plant adaptive responses to biotic and abiotic stress, with a focus on recent advancements in molecular defense pathways, emerging nanotechnology approaches and CRISPR/Cas-based genome editing strategies. We critically reviewed structural, physiological, biochemical and genetic adaptations. Key regulatory processes include phytohormonal regulation, antioxidants, reactive oxygen species (ROS) signaling and stress-response gene networks are explored along with advances in nanotechnology-based strategies and CRISPR/Cas genome editing. A comparative evaluation of conventional breeding, molecular breeding, and genome-editing approaches highlights the advantages of CRISPR/Cas systems, particularly their precision, efficiency and ability to generate targeted phenotypic changes. In parallel, nanomaterials have shown promise in improved nutrient delivery, protecting cellular structures and enhancing genome-editing efficiency under stress conditions. By integrating nanotechnology and genome-editing approaches with traditional agricultural practices, it may be possible to enhance plant resilience, sustain crop productivity and reduce reliance on chemical inputs. Overall, this review provides a cohesive perspective on how these technologies can be combined to support future crop improvement efforts to tackle climate-induced agricultural challenges.},
}
@article {pmid41725298,
year = {2026},
author = {Chew, YP and Ferenczi, A and Dannay, M and Ponce-Lilly, C and Kovac, A and Tóth, D and Tóth, SZ and Molnar, A},
title = {Enhancing CRISPR/Cas-Mediated Gene Knockout With Short Non-Homologous Oligonucleotides.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70548},
pmid = {41725298},
issn = {1467-7652},
support = {BB/W003538/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //Darwin Trust of Edinburgh/ ; //UK Research and Innovation/ ; },
abstract = {Chlamydomonas reinhardtii is a model green microalga that has great industrial potential as a sustainable bio-factory for recombinant protein and high-value chemical production. Efficient genome editing tools are required to redesign this organism for synthetic biology applications. CRISPR-Cas editing technologies have already been adapted for gene knockout, transgene knock-in, and precise gene editing in C. reinhardtii. However, the efficacy of CRISPR/Cas-mediated gene knockout (KO) is low, which hampers pathway engineering and functional genomic studies. Here we report that co-delivery of CRISPR-Cas gene editing reagents with short double-stranded non-homologous oligodeoxynucleotides (dsNHO) increases gene knockout efficacy up to 100-fold in C. reinhardtii. This phenomenon, referred to as non-homologous oligonucleotide enhancement (NOE), is heavily affected by the length, structure, and chemical modifications of dsNHO, and is largely mediated by the DNA double-stranded break sensor KU70/80 (KU) heterodimer in a Cas nuclease-, locus-, and strain-independent manner. Our data suggest that dsNHOs disrupt the cell's double-stranded break (DSB) sensing pathways, consequently shifting the balance of DNA repair from canonical non-homologous end joining (c-NHEJ) towards the more error-prone, microhomology-mediated end joining (MMEJ), which could be harnessed as a strategy for improving gene KO efficiency in Chlamydomonas and beyond.},
}
@article {pmid41724842,
year = {2026},
author = {Jana, UK and Bawankar, P and Gupta, P and Kango, N},
title = {CRISPR/Cas systems in fungal biotechnology: advancing high-value metabolite synthesis for industrial and food security applications.},
journal = {Archives of microbiology},
volume = {208},
number = {5},
pages = {},
pmid = {41724842},
issn = {1432-072X},
}
@article {pmid41724841,
year = {2026},
author = {Ibrahim, R and Aranjani, JM},
title = {Bacterial defense mechanisms against bacteriophages: an evolutionary arms race.},
journal = {Archives of microbiology},
volume = {208},
number = {5},
pages = {},
pmid = {41724841},
issn = {1432-072X},
mesh = {*Bacteriophages/physiology/genetics ; *Bacteria/virology/genetics/immunology ; Biological Evolution ; CRISPR-Cas Systems ; Biofilms/growth &amp; development ; },
abstract = {Bacteria and bacteriophages are in a co-evolutionary arms race, developing intricate bacterial defense mechanisms that enable phage resistance and counterstrategies. Bacteria evolve diverse defense mechanisms to inhibit each stage of the phage infection cycle.Surface-based defenses prevent phage adsorption and infection, including receptor modifications, capsule production, and biofilm formation. Intracellular systems such as restriction-modification (R-M) and abortive infection (Abi) mechanisms degrade phage DNA or sacrifice infected cells to protect the population. Adaptive immunity, particularly through CRISPR-Cas systems, enables bacteria to recognize and neutralize recurring phage attacks. Phages counter these defenses through anti-CRISPR proteins, receptor mimicry, and depolymerization, which degrade capsules and biofilm matrices. These dynamic interactions shape microbial ecosystems, offering insights for the development of novel antimicrobial strategies. Emerging approaches, including engineered phages and combination therapies, hold promise for addressing bacterial resistance. Understanding these bacterial-phage dynamics is critical for advancing phage therapy as a powerful tool against multidrug-resistant bacterial infections. This review aims to systematically examine and integrate current knowledge on bacterial antiphage defense systems and the evolutionary adaptations employed by bacteriophages to overcome these barriers.},
}
@article {pmid41724570,
year = {2026},
author = {Vizoso, M},
title = {DiLiCre2.0 mouse model: An advanced genome-editing tool to induce mutagenesis in vivo with high spatio-temporal resolution.},
journal = {Methods in cell biology},
volume = {203},
number = {},
pages = {41-58},
doi = {10.1016/bs.mcb.2025.12.003},
pmid = {41724570},
issn = {0091-679X},
mesh = {Animals ; Mice ; *Mutagenesis/genetics ; Mice, Transgenic ; *Gene Editing/methods ; Disease Models, Animal ; Humans ; *Carcinogenesis/genetics ; Neoplasms/genetics/pathology ; CRISPR-Cas Systems/genetics ; },
abstract = {For decades, transgenic mouse models have been developed and utilized to study tumorigenesis in vivo, offering the ability to manipulate oncogene and tumor suppression gene expression systemically or within entire organs and tissue compartments. However, the induction of tumorigenesis in many of these experimental models contrasts sharply with the development of most human cancers, where mutations affecting gene expression occur in a spatially restricted manner and lesions generally originates from the clonal expansion of one single mutated cell. This discrepancy raises critical questions regarding the relevance of the existing transgenic mouse models in accurately replicating the mechanisms of tumor initiation observed in humans. To overcome this limitation and study tumor initiation in vivo, I developed an innovative mouse model to induce early tumorigenesis through light-targeted mutagenesis of single cells, achieving unprecedented spatio-temporal resolution. This model provides a more accurate representation of tumor initiation processes, thus enhancing our understanding of cancer mechanisms at its inception.},
}
@article {pmid41724454,
year = {2026},
author = {Ghasemian, A and Al-Marzoqi, AH and Ali, ZA and Nouruzi, F and Abdollahi, A and Montaseri, Z and Memariani, M and Zarenezhad, E},
title = {Engineered Bacteria as living detectors of tumor DNA: A new diagnostic frontier.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {},
number = {},
pages = {120914},
doi = {10.1016/j.cca.2026.120914},
pmid = {41724454},
issn = {1873-3492},
abstract = {The identification of tumor-generated DNA must be accurate, minimally invasive, and precise, as it forms a fundamental aspect of effective cancer diagnosis, prognosis, and customized treatment plans. Recent advances in synthetic biology have pioneered the creation of genetically engineered bacteria as innovative biosensors capable of detecting tumor-derived DNA directly in situ. This review explores key developments in designing these microbial sentinels to pinpoint oncogenic DNA alterations, particularly emphasizing KRAS mutations that drive many cancers. By leveraging natural competence and horizontal gene transfer, in combination with CRISPR-Cas tools for selective targeting and integration of mutant DNA sequences, engineered bacteria can distinguish between tumor and wild-type DNA and produce observable reporter outputs. We further elaborate on various molecular engineering strategies using unique genetic circuits, homologous recombination, multiplexed CRISPR systems and safety circuits to improve specificity, sensitivity and biosafety. An additional perspective in the discussion incorporates diverse bacterial species and various cancer types, with a specific emphasis on colorectal and gastrointestinal cancers, while also considering possible applications to other solid tumors. Detection modalities encompass in vitro assays, organoid models, in vivo mouse models, and non-invasive stool sampling, offering an impressive range of platforms for validating biosensors. The positive aspects of these approaches, such as real-time detection, affordability, programmability, and reduced invasiveness, need to be balanced with their negative aspects concerning biosafety, colonization efficiency, and detection sensitivity limitations. Looking forward, this review delves into the translational potential of engineered bacterial biosensors for clinical cancer diagnostics, their integration with therapeutic delivery systems, and future directions that involve multiplexed detection and the incorporation of digital health. Indubitably, engineered bacterial tumor DNA biosensors represent a key fusion of microbiology, synthetic biology, and oncology, aimed at revolutionizing the diagnosis and management of cancers.},
}
@article {pmid41723978,
year = {2026},
author = {Zhen, Z and Yu, L and Zhu, Y and Lu, Z and Huang, Z},
title = {Molecular mechanism of CRISPR-SpyCas9 inhibition by AcrIIA26.},
journal = {Biochemical and biophysical research communications},
volume = {809},
number = {},
pages = {153491},
doi = {10.1016/j.bbrc.2026.153491},
pmid = {41723978},
issn = {1090-2104},
abstract = {In the ongoing arms race between bacteria and phages, CRISPR-Cas systems and anti-CRISPR proteins (Acrs) have evolved to counteract one another. Recently, AcrIIA26 from Streptococcus sp. was identified as a modulator of SpyCas9 activity, although its molecular mechanism remains unclear. Here, we present the cryo-EM structure of the SpyCas9-sgRNA-AcrIIA26 complex, revealing a two-domain architecture. The 5A domain of AcrIIA26 binds the PI and WED domains of Cas9, while the 4A domain contacts the REC2 domain. This dual interaction sterically blocks target DNA binding and prevents the conformational changes required for cleavage. Notably, AcrIIA26's sgRNA-independent binding to Cas9 allows the modulation of gene editing across an extended temporal window. Our study elucidates the molecular mechanism of AcrIIA26 and provides novel strategies for precise SpyCas9 regulation.},
}
@article {pmid41721881,
year = {2026},
author = {Jin, Z and Liu, M and Liu, M and Qi, X and Zhao, L and Yu, X and Guo, Y and Wen, Y},
title = {CRISPR/Cas9-mediated dual editing of VviGAI and VviFLC generates a novel early-flowering grapevine germplasm.},
journal = {Planta},
volume = {263},
number = {3},
pages = {},
pmid = {41721881},
issn = {1432-2048},
support = {No.32272670//Natural Science Foundation for Young Scientists of Shanxi Province/ ; No.31972986//the National Natural Science Foundation of China/ ; },
mesh = {*Vitis/genetics/growth &amp; development/physiology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Flowers/genetics/growth &amp; development/physiology ; *Plant Proteins/genetics/metabolism ; Phenotype ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; Mutation ; },
abstract = {CRISPR/Cas9-mediated dual knockout of VviGAI1 and VviFLC in grapevine promotes early flowering and induces distinctive morphological changes, offering novel genetic resources for breeding. CRISPR/Cas9-mediated genome editing offers a transformative approach for grapevine improvement. In this study, we achieved simultaneous knockout of two central flowering regulators VviGAI1, a DELLA protein ortholog, and VviFLC, a floral repressor in Vitis vinifera 'Cabernet Sauvignon' using a dual-sgRNA vector system. Remarkably, all 15 independent edited lines exhibited biallelic mutations in both genes, primarily consisting of frameshifts that led to premature termination. The dual-mutant plants displayed a range of distinctive phenotypic alterations, including dwarfism, shortened internodes, modified leaf morphology, and disrupted tendril development. Notably, one line (EL-43) showed precocious flowering under greenhouse conditions, underscoring the synergistic role of VviGAI1 and VviFLC in repressing floral transition. Comparative analysis with previously reported gai mutants revealed both conserved and novel traits, suggesting that structural variation within the DELLA domain contributes to phenotypic diversity. Collectively, our findings establish that dual editing of VviGAI1 and VviFLC not only accelerates flowering but also introduces unique vegetative and reproductive characteristics, providing a valuable genetic resource for future grapevine domestication and precision breeding efforts.},
}
@article {pmid41686483,
year = {2026},
author = {Tanaka, PP and Cotta-Almeida, V and Donadi, EA and Westerberg, L and Passos, GA},
title = {Distinct mutations in the autoimmune regulator gene differentially affect transcriptional and functional properties of medullary thymic epithelial cells.},
journal = {Human molecular genetics},
volume = {},
number = {},
pages = {},
doi = {10.1093/hmg/ddag004},
pmid = {41686483},
issn = {1460-2083},
support = {17/10780-4//S&#xe3;o Paulo Research Foundation/ ; 311304/2021//National Council for Scientific and Technological Development/ ; 302060/2019-7//National Council for Scientific and Technological Development/ ; 88887.642780/2021-00//CAPES-STINT/ ; 001//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; },
abstract = {Autoimmune Polyendocrine Syndrome Type 1 (APS-1) is a rare monogenic disorder caused by mutations in the autoimmune regulator (AIRE) gene. Although AIRE is essential for central immune tolerance, how distinct APS-1-associated mutations differentially affect medullary thymic epithelial cell (mTEC) biology remains incompletely understood. Here, we investigated the molecular and functional consequences of three Aire/AIRE variants using complementary murine mTEC models. To define transcriptional effects, we performed single-cell RNA sequencing (scRNA-seq) on mTECs carrying a heterozygous genomic Aire c.735delG mutation generated by CRISPR-Cas9. This analysis revealed reduced transcriptional heterogeneity, decreased expression of tissue-restricted antigens (TRAs) mRNAs (including Col4a3, Col7a1, and Neto2), and downregulation of key mTEC lineage markers (Epcam, Cldn4, Krt14). Mutant cells also displayed altered expression of mRNAs involved in chemokine-mediated migration (Ccl25, Cxcl16), extracellular matrix and cell adhesion (Fn1, Lama5, Col4a1, Nectin1, Cdh1), and actin cytoskeleton organization (Gsn, Rac1, Wasl, Actn1), indicating broad disruption of pathways governing mTEC identity and cell-cell interactions. Guided by these findings, we assessed mutation-specific functional outcomes using a CRISPR-derived Aire functional knockout and lentiviral expression of the human AIRE missense variants p.G229W and p.C313Y in wild-type mTECs. Functional assays revealed mutation-dependent alterations in mTEC morphology, thymocyte migration, and adhesion, with the p.C313Y variant exerting the strongest effects. Together, these data demonstrate that heterozygous and missense AIRE mutations exert distinct yet convergent effects on mTEC transcriptional programs and cellular behavior, providing mechanistic insight into AIRE-dependent immune tolerance failure in APS-1.},
}
@article {pmid41720886,
year = {2026},
author = {Nagalakshmi, U and Rodriguez, JE and Nguyen, T and Weissman, RF and Thornton, BW and Terrace, CI and Savage, DF and Dinesh-Kumar, SP},
title = {High-efficiency, transgene-free plant genome editing by viral delivery of an engineered TnpB.},
journal = {Nature plants},
volume = {},
number = {},
pages = {},
pmid = {41720886},
issn = {2055-0278},
support = {IOS-2303522//National Science Foundation (NSF)/ ; DGE-2146752//National Science Foundation (NSF)/ ; DGE-2146752//National Science Foundation (NSF)/ ; },
abstract = {Genome editing has revolutionized plant biology research[1], yet the efficient delivery of editing reagents remains a challenge. Current methods are labour intensive, involving lengthy tissue culture and complex transformation and regeneration steps. Viral delivery can mitigate these issues[2] but CRISPR-Cas nucleases exceed viral cargo limits, restricting guide RNA (gRNA) delivery into Cas9-expressing transgenic plants[2-11]. This requires generating an initial Cas9 transgenic line. Furthermore, gRNAs delivered by plant viral vectors can induce somatic edits, although only a few produce heritable edits[3-7,9-12]. Some engineered plant negative-strand rhabdoviruses can deliver both Cas9 and gRNA, but they face other challenges, including the need for tissue regeneration or pruning infected plants, and some rhabdoviruses can be delivered only through vector transmission[13-16]. Recently, smaller editors such as TnpBs were discovered, but they are significantly less active than Cas9[17-19]. Here we optimized a tobacco rattle virus-based system to deliver recently engineered, highly active ISDra2 TnpB variants. The eTnpBc variant enables effective somatic editing in systemic leaves and achieves up to 90% editing efficiency at target loci. In addition, up to 89% of offspring exhibit a mutant phenotype, with editing efficiencies reaching 100%. The design principles outlined here could promote wider use of eTnpBc for efficient, transformation- and transgene-free plant genome editing.},
}
@article {pmid41720881,
year = {2026},
author = {Ong, JK and Bhunia, S and Hilbert, B and Kirschner, V and Duglosz, S and Zimmermann, F and Freichel, M and Cornean, A},
title = {ABE9 fused to SpRY Cas9 nickase enables precise generation of bystander free mouse models.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41720881},
issn = {2045-2322},
mesh = {Animals ; *Gene Editing/methods ; Mice ; *CRISPR-Cas Systems ; *Deoxyribonuclease I/genetics/metabolism ; Humans ; Disease Models, Animal ; *CRISPR-Associated Protein 9/genetics/metabolism ; Point Mutation ; },
abstract = {Point mutations cause many genetic disorders, but modelling them in organisms is technically challenging. Creating mouse models that mimic these mutations is crucial for establishing a causal relationship between mutations and disease phenotype, thereby supporting the development of therapeutic strategies. Adenine base editors (ABEs) can correct single-nucleotide variants (SNVs) in disease modelling without double-stranded breaks (DSBs) or donor DNA, achieving higher product purity than traditional Cas9 methods. Earlier ABE techniques faced issues like limited targetability, bystander editing, and off-target effects. By combining two editor advancements, we introduced and tested ABE9-SpRY, an improved ABE variant fused with a PAM-flexible SpRY-Cas9 nickase. Our results show that ABE9-SpRY effectively generates three out of four targeted A-to-G mutations in mouse embryos, achieving desired editing efficiencies of up to 96% in individual adult founder mice. Furthermore, we observe fewer off-target events at predicted DNA sites in mouse embryos and in an orthogonal R-loop assay compared with ABE8e-SpRY. ABE9-SpRY also enhances product purity in mouse embryos under pooled sgRNA injections and, as a proof-of-concept, at a single endogenous locus in human induced pluripotent stem cells (hiPSCs), relative to ABE8e-SpRY. Our findings support ABE9-SpRY's precision at the loci tested and PAM-flexible versatility. Although performance remains sequence-dependent, these data support ABE9-SpRY as a PAM-flexible tool for generating precise point-mutation models where bystander editing is a concern.},
}
@article {pmid41720778,
year = {2026},
author = {Lee, YJ and Zhang, D and Stolze, SC and Saridis, G and Ebert, MK and Nakagami, H and Doehlemann, G},
title = {Ustilago maydis disrupts carbohydrate signaling networks to induce hypertrophy in host cells.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41720778},
issn = {2041-1723},
support = {DO1421/3-3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Zea mays/microbiology/metabolism/genetics ; Signal Transduction ; Plant Proteins/metabolism/genetics ; *Plant Diseases/microbiology ; Starch/metabolism/biosynthesis ; Gene Expression Regulation, Plant ; Host-Pathogen Interactions ; Fungal Proteins/metabolism/genetics ; Plant Leaves/microbiology/metabolism ; Mesophyll Cells/metabolism/microbiology ; *Carbohydrate Metabolism ; Endoreduplication ; Virulence Factors/metabolism/genetics ; Cell Cycle/genetics ; CRISPR-Cas Systems ; Basidiomycota ; },
abstract = {Ustilago maydis infection in maize causes hypertrophic leaf tumors; however, the underlying mechanisms driving this excessive cell growth are unknown. In this study, we identify Hap1 (hypertrophy-associated protein 1) as an effector and virulence factor that regulates mesophyll cell hypertrophy. Using CRISPR-Cas9 mutagenesis, we demonstrate that Hap1 contributes to endoreduplication and starch accumulation in infected tissues. Transcriptomics revealed Hap1-dependent upregulation of starch biosynthesis and cell cycle genes, as well as suppression of plant defense. This links Hap1 to metabolic and cell cycle reprogramming, and immune suppression. To identify the target of Hap1 that drives metabolic reprogramming, we investigated its interaction with ZmSnRK1α in maize. We found that Hap1 interferes with the phosphorylation of SnRK1 substrates and that two Hap1-interacting effectors, Hip1 and Hip2, enhance its protein stability. We conclude that Hap1 contributes to the reprogramming of maize metabolism and cell cycle, as well as mesophyll cell hypertrophy, by modulating the SnRK1 signaling pathway to regulate starch biosynthesis and host defense responses.},
}
@article {pmid41720308,
year = {2026},
author = {Shi, Q and Huang, W and Hu, D and Zhang, P and Chen, X and Hu, H and Wang, Y and Zhou, J and Weng, R and Quan, J and Zhao, D and Du, X and Yu, Y and Jiang, Y},
title = {The nationwide genomic characteristics and phylogenetic evolution of ST23-K1 hypervirulent Klebsiella pneumoniae in relation to virulence and antimicrobial resistance acquisition.},
journal = {The Journal of infection},
volume = {},
number = {},
pages = {106709},
doi = {10.1016/j.jinf.2026.106709},
pmid = {41720308},
issn = {1532-2742},
abstract = {OBJECTIVES: Hypervirulent Klebsiella pneumoniae (hvKp) ST23-K1 poses a global health threat due to its high virulence and increasing antimicrobial resistance. This study aimed to characterize the genomic feature and phylogenetic evolution of ST23-K1 in China.<br><br>METHODS: K1 isolates from a nationwide epidemiological surveillance project underwent whole-genome sequencing. Virulence was assessed using hypermucoviscosity phenotyping and a murine infection model. For ST23-K1 carrying acquired antimicrobial resistance genes (ARGs), the CRISPR/Cas system, protospacers, anti-CRISPR (Acr) genes, and plasmidome were characterized. Time-resolved phylogenetic analysis was performed using integrated locally generated and publicly available data.<br><br>RESULTS: Among 400 K1 isolates, ST23 was the most prevalent sequence type, and its effective population size increased following CG23-I divergence. The CG23-I sub-lineage was widely distributed nationwide with limited evidence of clonal transmission. Isolates with an incomplete cps locus exhibited significantly reduced virulence compared with those carrying an intact locus. The prevalence of extended-spectrum &#x3b2;-lactamase-positive ST23-K1 isolates increased over time, whereas carbapenemase-producing isolates remained stable. Among acquired ARGs-positive ST23-K1 isolates, a conserved protospacer corresponding to a prevalent spacer was identified. This protospacer, together with AcrIE genes, were frequently co-located on IncFII-type plasmids.<br><br>CONCLUSION: ST23-K1 remains a hypervirulent lineage undergoing ongoing evolutionary expansion. The presence of acquired ARGs in ST23-K1 may be associated with AcrIE-harboring IncFII plasmids, and functional validation is required to clarify the underlying mechanisms. Continuous genomic surveillance is essential to monitor the evolution and antimicrobial resistance trends of ST23-K1.},
}
@article {pmid41717201,
year = {2026},
author = {Mittal, A and Manna, S and Nelson, V and Ladha, N},
title = {In Silico Design of gRNA for CRISPR System for Detection of Multidrug Resistant Tuberculosis Using Indian Mycobacterium tuberculosis Genomes: A Computational Study.},
journal = {Cureus},
volume = {18},
number = {1},
pages = {e101851},
pmid = {41717201},
issn = {2168-8184},
abstract = {Background Multidrug-resistant tuberculosis (MDR-TB) continues to pose a major challenge to TB elimination in India, where drug resistance and delayed diagnosis contribute significantly to ongoing transmission. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based diagnostics have emerged as versatile tools, compared to GeneXpert, capable of detecting resistance-associated mutations with rapid turnaround and high accuracy. This study aimed to design and in silico validate Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein (CRISPR-Cas)-based guide RNAs (gRNAs) targeting major drug-resistance mutations in Indian Mycobacterium tuberculosis (M. tuberculosis) isolates. Methods Whole-genome mutation profiles were analyzed using TBProfiler, and gRNAs were designed using CHOPCHOP. Off-target evaluation was performed using Cas-OFFinder and Basic Local Alignment Search Tool (BLAST). High-confidence mutations in gyrA, rpoB, katG, rpsL, embB, and ethA were selected based on prevalence in Indian isolates and WHO-defined resistance markers. Results Numerous drug resistance-associated mutations were identified in the drug-resistant tuberculosis genome isolates. The study identified six key genetic mutations identified in MTB isolates that are associated with phenotypic drug resistance, including gyrA (Asp94Gly), rpoB (Ser450Leu), and katG (Ser315Thr). For each of the six genes, the chromosome position, locus ID, mutation type, and affected amino acids were identified, and tailored guide RNAs were designed in silico. Top-ranked gRNAs demonstrated optimal GC content, high predicted cleavage efficiency, and zero off-target activity. Each resistance locus yielded multiple candidate gRNAs suitable for CRISPR-based assays. Conclusions This computational in silico analysis provides a robust panel of mutation-targeted gRNAs tailored to Indian MDR-TB genomic profiles. These findings lay a strong foundation for developing rapid, affordable CRISPR diagnostics for point-of-care detection of drug resistance. Future laboratory validation and clinical testing are essential for translation into diagnostic practice.},
}
@article {pmid41717108,
year = {2026},
author = {Balasubramani, S and Li, Z and Gayathri, E and Gurusamy, D and Singh, A},
title = {Editorial: Advancing plant defense: genome editing, RNAi, and synthetic biology for sustainable pest control.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1785705},
pmid = {41717108},
issn = {1664-462X},
}
@article {pmid41661055,
year = {2026},
author = {Bu, L and Yuan, A and Zhao, M and Pang, B and Li, J and Shang, Y and Xie, W and Peng, H},
title = {An Asymmetric Stem-Loop Translator Enhances Specificity of One-Pot Isothermal CRISPR/Cas12a Assay.},
journal = {Analytical chemistry},
volume = {98},
number = {7},
pages = {5513-5522},
doi = {10.1021/acs.analchem.5c07049},
pmid = {41661055},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *MicroRNAs/genetics/analysis ; Limit of Detection ; Humans ; *CRISPR-Associated Proteins/metabolism ; Aptamers, Nucleotide/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; },
abstract = {Sensitive and specific detection of molecular biomarkers is fundamental to clinical diagnostics and biomedical research, yet existing CRISPR-based assays often suffer from nonspecific activation and cross-reactivity. Here, we introduce an asymmetric stem-loop translator that markedly enhances the specificity of a one-pot isothermal strand displacement amplification (SDA)-CRISPR/Cas12a assay. The asymmetric probe enables precise molecular recognition and controlled signal transduction, converting diverse targets into amplifiable DNA intermediates. Within the integrated one-tube system, SDA-generated DNA products directly trigger Cas12a trans-cleavage, yielding rapid fluorescence responses without thermal cycling or manual intervention. Systematic optimization of reaction parameters effectively mitigated enzyme inhibition and aerosol contamination. The resulting assay achieves highly specific and sensitive detection of miRNA with a detection limit of 500 fM, accurately distinguishing single- and double-base mutations. Owing to its modular design, the asymmetric stem-loop translator is readily adaptable to other analytes. By coupling to an aptamer-based recognition element, the system enables sensitive aflatoxin B1 detection. This work establishes a generalizable framework for enhancing CRISPR/Cas12a specificity through asymmetric molecular translation, offering a versatile platform for rapid nucleic acid and small-molecule diagnostics in clinical and point-of-care settings.},
}
@article {pmid41643679,
year = {2026},
author = {Ferrández-Roldán, A and Piulachs, MD},
title = {Using DIPA-CRISPR for simple and efficient endogenous protein tagging in insects.},
journal = {Cell reports methods},
volume = {6},
number = {2},
pages = {101297},
doi = {10.1016/j.crmeth.2025.101297},
pmid = {41643679},
issn = {2667-2375},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Insect Proteins/genetics/metabolism ; Genes, Reporter ; *Insecta/genetics ; *Blattellidae/genetics/metabolism ; },
abstract = {CRISPR-Cas9 is rapidly expanding across diverse organisms. Among these advances, in-frame knockins of reporter genes have become essential for studying gene expression and protein localization. However, in hemimetabolan insects such as the German cockroach Blattella germanica, a phylogenetically basal and relevant pest species, functional fusion proteins have remained technically difficult to obtain. We present a streamlined gene-editing strategy to knock in a reporter gene in-frame with the distal-less gene, generating a functional fusion protein in B. germanica. By combining direct parental CRISPR with donor constructs designed for homology-directed repair carrying the mCherry gene, we successfully achieved targeted integration at the distal-less locus. The resulting fusion protein was functional and heritable and enabled live visualization of Distal-less protein distribution, showing fluorescence in developing appendages and the nervous system. This simple and robust methodology opens the door to generating fusion proteins in non-model insects, providing a valuable molecular tool for ecological, developmental, and pest-management research.},
}
@article {pmid41247804,
year = {2026},
author = {Li, A and Yang, C and Zhao, Z and Lu, T and Yin, C and Xu, C and Lv, C and Liu, Y and Zhan, Y and Li, S and Cao, C},
title = {Strategic engineering of DNA aptamers as precision modulators in CRISPR-driven oncogene control.},
journal = {International journal of surgery (London, England)},
volume = {112},
number = {2},
pages = {3015-3027},
doi = {10.1097/JS9.0000000000003837},
pmid = {41247804},
issn = {1743-9159},
mesh = {*Aptamers, Nucleotide/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; Mice ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Oncogenes/genetics ; Cell Line, Tumor ; *Neoplasms/genetics/therapy ; *CRISPR-Associated Protein 9/genetics ; Gene Editing/methods ; *Genetic Therapy/methods ; },
abstract = {OBJECTIVE: The goal of this study was to address the limited availability of intracellular protein-binding aptamers that restrict the potential of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based signal conductor systems. We aimed to develop a programmable CRISPR-SaCas9 platform using DNA aptamers to achieve precise and dynamic gene regulation for cancer therapy.<br><br>METHODS: We identified and characterized five DNA aptamers with high specificity for the SaCas9 protein. Functional assays were performed to evaluate their effects on the transcriptional regulatory activity of SaCas9. Some aptamers were engineered to associate with promoter elements, enabling transcription factor-responsive control of downstream gene expression. To test therapeutic efficacy, the system was delivered in vivo using In vivo-jetPEI reagents, and tumor progression and metastasis were monitored.<br><br>RESULTS: The identified aptamers exhibited distinct functionalities - some partially inhibited SaCas9 activity, while others enabled precise gene expression control in response to endogenous transcription factors. The aptamer-CRISPR complexes formed ternary assemblies with SaCas9 and single-guide RNAs, enabling logic-gated responses. In vivo , the system significantly suppressed tumor growth and metastasis.<br><br>CONCLUSION: We developed a modular CRISPR signal conductor platform that leverages SaCas9-binding DNA aptamers for transcription factor-mediated logic operations and precise gene control. This system expands the toolkit for genetic circuit design and offers a promising strategy for targeted cancer gene therapy.},
}
@article {pmid41224955,
year = {2026},
author = {Gil, JS and Lee, S and Koo, T},
title = {Therapeutic in vivo genome editing: innovations and challenges in rAAV vector-based CRISPR delivery.},
journal = {Gene therapy},
volume = {33},
number = {1},
pages = {97-106},
pmid = {41224955},
issn = {1476-5462},
mesh = {*Dependovirus/genetics ; *Gene Editing/methods ; *Genetic Vectors/genetics ; *Genetic Therapy/methods ; Humans ; *CRISPR-Cas Systems ; Animals ; Gene Transfer Techniques ; },
abstract = {The integration of CRISPR systems with recombinant adeno-associated virus (rAAV) vectors has opened new possibilities for therapeutic genome editing, offering potential treatments for both genetic and non-genetic disorders. rAAV vectors have emerged as promising vehicles for in vivo gene therapy due to their favorable safety profile, high tissue specificity, and ability to induce sustained transgene expression. However, their limited packaging capacity has been a significant challenge for delivering large CRISPR molecules. To overcome this limitation, innovative strategies have been developed, including the use of compact Cas orthologs, dual rAAV vector systems, and trans-splicing rAAV vectors. These approaches have significantly improved the efficiency of genome editing for therapeutic applications. This review presents recent advancements in rAAV-CRISPR-mediated in vivo gene therapy, highlighting key technological innovations, current challenges, and the therapeutic potential of these strategies in the development of next-generation gene therapies.},
}
@article {pmid41715936,
year = {2026},
author = {Shimizu, Y and Kataoka, K},
title = {DIPA-CRISPR Mediated Knockout of Vermilion Generates a Visible Eye Color Marker for The Band-Legged Ground Cricket Dianemobius nigrofasciatus.},
journal = {Archives of insect biochemistry and physiology},
volume = {121},
number = {2},
pages = {e70135},
doi = {10.1002/arch.70135},
pmid = {41715936},
issn = {1520-6327},
support = {JPJ009237//Bio-oriented Technology Research Advancement Institution, BRAIN/ ; 21K05614//JSPS KAKENHI Grant-in-Aid for Scientific Research (C)/ ; 21J23478/22KJ2609//Grant-in-Aid for JSPS Fellows/ ; },
mesh = {Animals ; Female ; *Gryllidae/genetics/growth &amp; development/physiology ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; *Eye Color/genetics ; Photoperiod ; Gene Knockout Techniques ; Gene Editing ; },
abstract = {The molecular basis of photoperiodism, by which insects use photoperiodic cues to anticipate seasonal changes and regulate key life-history events such as development, diapause, and reproduction, remains poorly understood. Studies on the molecular mechanisms of photoperiodism in hemimetabolous insects are limited compared with those in holometabolous insects, largely due to the lack of appropriate model organisms. The band-legged ground cricket Dianemobius nigrofasciatus represents a valuable model system because it exhibits clear photoperiodic responses in the maternal induction of embryonic diapause, the wing morph, and the rate of nymphal development. With the recent availability of the D. nigrofasciatus genome sequence, the establishment of effective genome-editing methods and reliable marker genes is expected to promote functional genomic analyses. In this study, we aimed to establish a direct parental (DIPA)-CRISPR genome-editing approach and evaluate the utility of vermilion (Dn-v), a gene involved in ommochrome synthesis, as a visible eye color marker for mutant screening. Cas9 ribonucleoprotein complexes were injected into females 3-5 days after adult emergence, during the vitellogenic stage, successfully yielding Dn-v knockout mutants. These mutants had white compound eyes throughout development, with pigmentation reaching a vermilion color about 20 days after adult emergence. We further examined the photoperiodic response associated with maternal diapause induction in knockout mutants. Similar to the wild-type, knockout mutants exhibited low and high diapause incidence under long-day and short-day conditions, respectively. Our results demonstrate that DIPA-CRISPR is an effective genome-editing method in D. nigrofasciatus and that Dn-v serves as a practical and reliable marker gene. The establishment of these genomic tools provides a foundation for future functional analyses aimed at elucidating the molecular basis of photoperiodism in hemimetabolous insects.},
}
@article {pmid41713039,
year = {2026},
author = {Zhang, H and Cui, C and Wang, X and Liu, S and Wang, X and Wang, Y and Ge, S and Cai, Y and Bao, J and Wang, Z},
title = {Development of a one-pot RT-RAA/CRISPR-Cas13a assay for rapid genotyping of Nipah virus in pigs.},
journal = {Diagnostic microbiology and infectious disease},
volume = {115},
number = {2},
pages = {117316},
doi = {10.1016/j.diagmicrobio.2026.117316},
pmid = {41713039},
issn = {1879-0070},
abstract = {INTRODUCTION: Nipah virus (NiV) is a highly pathogenic zoonotic virus transmitted from bats to humans through pigs as a key intermediate host. Given the existence of two distinct NiV genotypes, which differ in clinical manifestations and transmission patterns in both humans and pigs, rapid and sensitive method for detection and genotyping is crucial for effective disease control. Isothermal amplification combined with CRISPR/Cas-based assay provides a promising approach to meet this need.<br><br>METHODS: Conserved regions were identified by aligning the N gene sequences from 67 NiV strains. Specific primers and probes were designed for reverse transcription recombinase-aided amplification (RT-RAA) to detect NiV. Subsequently, single nucleotide polymorphisms within the conserved region were analyzed, and corresponding crRNAs were designed to establish a one-pot RT-RAA/CRISPR-Cas13a assay for NiV genotyping. The assays were evaluated using simulated pig serums spiked with NiV pseudovirus.<br><br>RESULTS: The RT-RAA assay exhibited a detection sensitivity of 10[-2] Infection Unit/mL (IU/mL) for NiV pseudovirus, outperforming conventional qRT-PCR in simulated pig serum samples. No cross-reactivity was observed with viral RNA or DNA of PCV2, PEDV, PRRSV, PRV and SVA, confirming high specificity. The entire one-pot RT-RAA/CRISPR-Cas13a assay could be completed within 1 hour and clearly discriminated between the two NiV genotypes without requiring sophisticated instruments. Evaluation with simulated samples showed a sensitivity of 100% (95% CI, 92.87-100%) and a specificity of 94% (95% CI, 83.78-98.36%), with a detection limit of 10[-1] IU/mL for NiV pseudovirus.<br><br>CONCLUSION: The one-pot RT-RAA/CRISPR-Cas13a assay provides a rapid and sensitive platform for NiV genotyping.},
}
@article {pmid41712626,
year = {2026},
author = {Sünderhauf, D and Ringger, JR and Payne, LJ and Pinilla-Redondo, R and Gaze, WH and Brown, SP and van Houte, S},
title = {CRISPR-Cas is beneficial in plasmid competition, but limited by competitor toxin-antitoxin activity when horizontally transferred.},
journal = {PLoS biology},
volume = {24},
number = {2},
pages = {e3003658},
doi = {10.1371/journal.pbio.3003658},
pmid = {41712626},
issn = {1545-7885},
abstract = {Bacteria can encode dozens of different immune systems that protect them from infection by mobile genetic elements (MGEs). MGEs themselves may also carry immune systems, such as CRISPR-Cas, to target competitor MGEs. It is unclear when this is favored by natural selection, and whether toxin-antitoxin (TA) systems-common competitive mechanisms carried by plasmids-can alter their efficacy. Here, we develop and test novel theory to analyze the outcome of competition between plasmids when one carries a CRISPR-Cas system that targets the other plasmid. Our mathematical model and experiments using Escherichia coli and competing IncP plasmids reveal that plasmid-borne CRISPR-Cas is beneficial to the plasmid carrying it when the plasmid has not recently transferred to a new host. However, CRISPR-Cas is selected against when the plasmid carrying it transfers horizontally, if a resident competitor plasmid encodes a TA system that elicits post-segregational killing. Consistent with a TA barrier to plasmid-borne CRISPR-Cas, a bioinformatic analysis reveals that naturally occurring CRISPR-Cas-bearing plasmids avoid targeting other plasmids with TA systems across bacterial genera. Our work shows how the benefit of plasmid-borne CRISPR-Cas is severely reduced against TA-encoding competitor plasmids, but only when plasmid-borne CRISPR-Cas is horizontally transferred. These findings have key implications for the distribution of prokaryotic defenses and our understanding of their role in competition between MGEs, and the utility of CRISPR-Cas as a tool to remove plasmids from pathogenic bacteria.},
}
@article {pmid41712102,
year = {2026},
author = {Lin, J and Hazaisi, H and Guan, Y and Bai, M},
title = {Multiplex gene editing drives revolution in crop breeding: overlaid editing of multiple genes and customization of complex traits.},
journal = {Advanced biotechnology},
volume = {4},
number = {1},
pages = {5},
pmid = {41712102},
issn = {2948-2801},
support = {2023ZD040360104//Chinese Academy of Agricultural Sciences from Institute of Crop Science/ ; },
abstract = {Modern agriculture currently demands higher standards for the simultaneous improvement of crop yield, quality and stress resistance. However, traditional crop breeding methods can no longer meet the needs of modern agricultural development. Improving a single trait is no longer sufficient to meet the multifaceted demands of modern agricultural production and consumer expectations. Multiple traits breeding has increasingly become a key objective in current crop breeding. Over the past decade, CRISPR/Cas9-based multiplex genome editing (MGE) has enabled efficient pyramiding and precise regulation of multiple traits via targeted editing of multiple gene loci, revolutionizing crop breeding. In this review, we briefly describe the core CRISPR/Cas-based MGE strategies and technical workflows, and thoroughly discuss the practical outcomes of MGE applications in various fields, such as enhancing crop stress resistance, increasing yield and improving quality. This review aims to provide a summary and theoretical reference for crop breeding, as well as open up new ideas for achieving different breeding goals.},
}
@article {pmid41711690,
year = {2026},
author = {Shi, H and Chi, H},
title = {Next-generation CRISPR screens enable causal systems immunology.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {3},
pages = {},
doi = {10.1084/jem.20241266},
pmid = {41711690},
issn = {1540-9538},
support = {//American Lebanese Syrian Associated Charities/ ; CA253188/NH/NIH HHS/United States ; CA281868/NH/NIH HHS/United States ; AI105887/NH/NIH HHS/United States ; AI131703/NH/NIH HHS/United States ; AI140761/NH/NIH HHS/United States ; AI150241/NH/NIH HHS/United States ; AI150514/NH/NIH HHS/United States ; //Lupus Research Alliance/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Systems Biology/methods ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Analysis ; Immunoinformatics ; },
abstract = {Mapping the causal circuits that shape the phenotypic and functional landscape of immune cells remains a formidable challenge. Recent advances in pooled CRISPR-based screens, coupled with multiplexed single-cell profiling and imaging-based spatial readouts, make this goal increasingly attainable. In this Perspective, we discuss how CRISPR-based genetic screens will fundamentally transform our understanding of immunobiology. We highlight the applications of state-of-the-art, high-throughput pooled perturbation approaches, including emerging methodologies for bulk, single-cell, and spatial CRISPR screens, to advance our understanding of immunity and in vivo biology. Additionally, we summarize new strategies to address the complexity of combinatorial perturbations to uncover genetic interactions and mechanistic drivers of immunity at unprecedented scale and resolution. By integrating CRISPR screening data with experimental insights, we advocate a new framework in immunology research that leverages perturbation-driven regulatory effects and networks to discover new therapeutic targets and establish causal systems biology and immunology for advancing immunological knowledge and therapeutic application.},
}
@article {pmid41710881,
year = {2026},
author = {Karaoglu, IC and Odabas, A and Önder, T and Kizilel, S},
title = {Single-gene knockout of RNLS or HIVEP2 are insufficient to protect β-cell spheroids from allo- and xeno-rejection.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1759835},
pmid = {41710881},
issn = {1664-3224},
mesh = {Animals ; Mice ; *Insulin-Secreting Cells/transplantation/immunology/metabolism ; Humans ; *Graft Rejection/genetics/immunology/prevention &amp; control ; Gene Knockout Techniques ; *Spheroids, Cellular/immunology/transplantation/metabolism ; CRISPR-Cas Systems ; *Islets of Langerhans Transplantation ; Cell Line ; Gene Editing ; Transplantation, Heterologous ; },
abstract = {INTRODUCTION: β-Cell replacement therapy offers a potential cure for type 1 diabetes, but its success is limited by rapid graft rejection. While genome-wide CRISPR screens have recently identified RNLS and HIVEP2 as candidate genes capable of protecting β-cells from autoimmune destruction, their efficacy against the distinct mechanisms of allogeneic and xenogeneic rejection remains unknown. This study aimed to test the hypothesis that single-gene ablation of RNLS or HIVEP2 protects β-cell spheroids from allo- and xenorejection in immunocompetent hosts.<br><br>METHODS: Murine &#x3b2;-TC-6 and human EndoC-&#x3b2;H1 &#x3b2;-cell lines were genetically edited using CRISPR-Cas9 to knockout RNLS or HIVEP2. Editing efficiencies were confirmed via T7 endonuclease I assay and Tracking of Indels by Decomposition (TIDE) analysis. Cells were aggregated into uniform, size-controlled spheroids using an optimized agarose suspension culture. Functional integrity was assessed via glucose-stimulated insulin secretion (GSIS). To evaluate immune evasion in vivo, luciferase-labeled spheroids were transplanted subcutaneously into immunocompetent CD-1 mice, modelling allogeneic (murine-to-murine) and xenogeneic (human-to-murine) rejection, with graft survival monitored longitudinally by bioluminescence imaging.<br><br>RESULTS: Robust editing efficiencies were achieved for both targets. Functional characterization indicated that Rnls deletion modestly impaired GSIS in murine cells, whereas HIVEP2 deletion showed no functional alterations in either cell line. In vivo assessment revealed no protective effects of RNLS or HIVEP2 deletion; grafts from both knockout groups displayed rejection kinetics indistinguishable from non-targeting controls. While allogeneic grafts survived longer than xenogeneic grafts, both were ultimately cleared by the host immune system regardless of genotype.<br><br>DISCUSSION: These data indicate that single-gene deletions of RNLS or HIVEP2 are insufficient to protect &#x3b2;-cell grafts from the barriers of allo- or xenorejection. By defining the limitations of these targets in isolation, our findings highlight the necessity for combinatorial genome editing strategies or complementary integration with immunomodulatory biomaterials to achieve effective and sustained &#x3b2;-cell graft survival.},
}
@article {pmid41709870,
year = {2026},
author = {Feng, L and Yu, P and He, N and Zhang, Q and Tang, Y and Geng, J and Lu, Q and Song, C and Chen, F},
title = {Label-Free MicroRNA Diagnostics: From CRISPR Nucleases to Nanomaterial-Enhanced Transducers.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00868},
pmid = {41709870},
issn = {2161-5063},
abstract = {Label-free detection of microRNAs (miRNAs) has emerged as a pivotal approach in molecular diagnostics, integrating the programmability of CRISPR systems with the high sensitivity of nanomaterial-based transduction. miRNAs are short, noncoding RNAs that play central roles in gene regulation and disease pathogenesis, serving as valuable biomarkers for early diagnosis and prognosis. Conventional miRNA detection methods rely on labeling and multistep amplification, which hinder their adaptability for rapid and point-of-care applications. In contrast, label-free biosensing translates molecular recognition into intrinsic optical, electrochemical, or mechanical signals, enabling real-time, amplification-free analysis. This review summarizes recent advances in label-free miRNA biosensing, with emphasis on CRISPR/Cas12a, Cas13a, and Cas14a systems that couple target recognition with signal transduction, and nanomaterial-assisted platforms including gold and silver nanoparticles, carbon nanotubes, quantum dots, silica nanostructures, and magnetic composites. Particular attention is given to innovations that achieve attomolar-level sensitivity, single-nucleotide discrimination, and multiplex detection. We also discuss integration into microfluidic and wearable platforms, addressing persistent challenges in repeatability and stability, antifouling performance, and clinical translation. Emerging trends in artificial intelligence-assisted data processing, molecular logic circuits, and digital single-molecule biosensing are highlighted. These advances collectively outline the pathway toward intelligent, amplification-free, and portable miRNA diagnostics, bridging molecular biology and synthetic bioengineering for next-generation healthcare applications.},
}
@article {pmid41709648,
year = {2026},
author = {Doghish, AS and Ghaiad, HR and Elfar, N and El Said, NH and Radwan, AF and Abd-Elmawla, MA and Mohamed, HH and Mohammed, OA and Rizk, HA},
title = {Unraveling the Function of lncRNAs in Gliomas: Interaction With Signaling Pathways and Therapeutic Opportunities.},
journal = {Journal of biochemical and molecular toxicology},
volume = {40},
number = {3},
pages = {e70756},
doi = {10.1002/jbt.70756},
pmid = {41709648},
issn = {1099-0461},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Glioma/genetics/metabolism/therapy/pathology ; *Signal Transduction ; *Brain Neoplasms/metabolism/genetics/therapy/pathology ; Animals ; *Gene Expression Regulation, Neoplastic ; *RNA, Neoplasm/metabolism/genetics ; },
abstract = {Brain tumors represent some of the most formidable challenges in neuro-oncology due to their aggressive clinical course, resistance to therapy, and profound molecular heterogeneity. Among the emerging regulatory elements reshaping our understanding of tumor biology are long non-coding RNAs (lncRNAs), a diverse class of RNA transcripts that modulate gene expression and cellular behavior without encoding proteins. This review provides an in-depth and integrative examination of the biogenesis, regulatory mechanisms, and functional roles of lncRNAs in brain tumor development and progression. We systematically explore both canonical and non-canonical pathways of lncRNA biogenesis, detailing how these influence structural specificity and molecular interactions. This review synthesized evidence retrieved from PubMed/MEDLINE, Scopus, and Web of Science, covering publications from January 2010 to June 2025. This analysis highlights key gaps, such as context-dependent therapeutic effects that limit translational applicability. A major focus is placed on the interplay between lncRNAs and core oncogenic signaling pathways, including Phosphoinositide 3-kinase (PI3K)/serine/threonine kinase (AKT), Signal Transducer and Activator of Transcription 3 (STAT3), Wingless/Int-1 (Wnt)/β-catenin, and Transforming Growth Factor-Beta (TGF-β), which drive malignant transformation, invasion, stemness, and therapeutic resistance in gliomas. Furthermore, we dissect the molecular functions of lncRNAs as epigenetic regulators, competitive endogenous RNAs (ceRNAs), and structural scaffolds, and discuss their contribution to the dynamic tumor microenvironment. By synthesizing the latest findings, this review underscores the academic and translational importance of targeting lncRNA-associated networks. It also highlights emerging therapeutic approaches, such as antisense oligonucleotides, RNA interference, CRISPR-Cas systems, and natural lncRNA-modulating compounds, which collectively represent a promising frontier in precision medicine for brain tumors. This work offers a critical framework for future research and therapeutic innovation in the lncRNA landscape of neuro-oncology.},
}
@article {pmid41707086,
year = {2026},
author = {Banerjee, S and Banerjee, A and Ray, S and Ray, A and Paul, D and Dastidar, SG and Willard, B and Biswas, K},
title = {dCas9 Targeted Proteome Profiling Reveals p300-Mediated Reciprocal Regulation of SMAD and SP1 as a Driver of GM2-synthase Transcription in Renal Cell Carcinoma.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {4},
pages = {e71597},
doi = {10.1096/fj.202502746R},
pmid = {41707086},
issn = {1530-6860},
support = {2019-0137-CMB/adhoc/BMS//MOHFW | DHR | Indian Council of Medical Research (ICMR)/ ; EMR/2016/001983//Department of Science and Technology, Ministry of Science and Technology, India (DST)/ ; CRG/2021/004623//Department of Science and Technology, Ministry of Science and Technology, India (DST)/ ; },
mesh = {Humans ; *Carcinoma, Renal Cell/genetics/metabolism/pathology ; *Sp1 Transcription Factor/metabolism/genetics ; *Kidney Neoplasms/genetics/metabolism/pathology ; *Smad Proteins/metabolism/genetics ; *Sialyltransferases/genetics/metabolism ; Cell Line, Tumor ; *E1A-Associated p300 Protein/metabolism/genetics ; *Proteome/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; Transcription, Genetic ; CRISPR-Cas Systems ; *p300-CBP Transcription Factors/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Glycolipids constitute an important component of the plasma membrane based on both abundance as well as function. Gangliosides, being a class of structurally diverse and functionally varied glycolipids, can act both as a receptor as well as a ligand and therefore are established as a crucial player in several normal cellular processes. In certain diseases, and in particular cancer, select gangliosides are over-expressed often leading to disease manifestation. GM2-synthase, the enzyme responsible for the formation of a pro-tumorigenic ganglioside, GM2, is well reported to be over-expressed across various cancer tissues and cell lines. This over-expression of GM2-synthase has been linked with increased migration, invasion, and epithelial to mesenchymal transition (1) as well as induction of a local and systemic host immune suppression in cancer. Despite only a handful of studies demonstrating an epigenetic regulation underlying the transcriptional regulation of the GM2-synthase (B4GalNT1) gene, the detailed mechanism still remains unclear. Here we identified the total proteome associated with the GM2-synthase promoter through a two-step CRISPR-dCas9 based proteome profiling approach by categorizing all the identified proteins leading to a detailed elucidation of the molecular drivers behind GM2-synthase transcription. While the previous study identified an acetylation-dependent de-repression of the transcription factor SP1 causing GM2-synthase activation, the underlying molecular mechanism driving its activation wasn't clear. This study demonstrated that the histone acetyl transferase p300, acts as a pivotal factor which on one hand causes acetylation-mediated degradation of SP1, and on the other hand activates SMAD2/4 to have a direct positive impact on GM2-synthase gene transcription. We identified p300 to have an activator role in GM2-synthase gene transcription through knock out, knock down, and over-expression experiments. Furthermore, SP1 degradation, SMAD activation, and their DNA binding patterns show the reciprocal role of p300 on SP1 and SMAD complexes. Altogether we have identified SMAD2/4 as an activator complex, p300 as a positive regulator, and uncovered a critical p300-SMAD-SP1 regulatory axis in GM2-synthase transcriptional regulation.},
}
@article {pmid41706678,
year = {2026},
author = {Verhezen, T and Van Den Eynde, A and Verstraelen, P and Gehrcken, L and Palmiotto, G and Lau, HW and De Vos, WH and Van Der Heijden, S and Brants, L and Melis, J and Van Audenaerde, J and Rodrigues Fortes, F and Le Compte, M and Roeyen, G and Prenen, H and Campillo-Davo, D and Lion, E and Argüello, RJ and Van Laere, S and Lardon, F and Deben, C and Wouters, A and Smits, E and De Waele, J},
title = {DRP1 depletion protects NK cells from hypoxia-induced dysfunction.},
journal = {Redox report : communications in free radical research},
volume = {31},
number = {1},
pages = {2626181},
pmid = {41706678},
issn = {1743-2928},
mesh = {*Killer Cells, Natural/metabolism ; Humans ; *Dynamins/metabolism/genetics ; Reactive Oxygen Species/metabolism ; Mitochondria/metabolism ; Cell Line, Tumor ; Cell Hypoxia ; Membrane Potential, Mitochondrial ; *Hypoxia/metabolism ; CRISPR-Cas Systems ; Tumor Microenvironment ; },
abstract = {OBJECTIVES: The efficacy of cellular therapies has been disappointing in solid tumors. A major barrier that contributes to the low success rate, is hypoxia within the tumor microenvironment. In this study, we investigated the influence of hypoxia on natural killer (NK) cell function and to evaluated a strategy to restore their activity in hypoxia.<br><br>METHODS: Unarmed or CAR NK cells were placed in normoxia (21% O2) or hypoxia (1% O2) prior to experimental readouts. Mitochondrial content and morphology were assessed by confocal microscopy, membrane potential and reactive oxygen species (ROS) by flow cytometry, and global transcriptional changes by RNA sequencing. Cytotoxicity was evaluated against tumor cell lines and patient-derived cancer organoids, which were characterized by RNA sequencing. DRP1 function was inhibited pharmacologically or through CRISPR-Cas9-mediated knockout.<br><br>RESULTS: Hypoxia reduced NK cell mitochondrial content and membrane potential, while increasing mitochondrial ROS and inducing broad transcriptional changes in stress response pathways. Their cytotoxic activity was drastically impaired, which could not be prevented by CD70-CAR-IL-15 engineering. Pharmacological inhibition of DRP1 restored mitochondrial content and cytotoxic function. To confirm the role of DRP1, CRISPR-Cas9-mediated DRP1 knockout (KO) NK cells preserved mitochondrial load and membrane potential under hypoxia, and DRP1[KO] CAR NK cells retained cytotoxic activity under hypoxic conditions against cancer cell lines. Patient microtumor models with distinct transcriptomic profiles exhibited divergent responses to DRP1[WT] and DRP1[KO] CAR NK cells.<br><br>CONCLUSION: These findings indicate that DRP1 inactivation supports NK cell function in hypoxia and metabolic engineering may enhance CAR-NK efficacy in solid tumors.},
}
@article {pmid41644992,
year = {2026},
author = {Oishi, S and Doi, S and Sekida, T and Yamashita, K and Yamada, Y and Muramoto, T},
title = {Genome editing across Dictyostelia species enables comparative functional genetics of social amoebas.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41644992},
issn = {2045-2322},
support = {23KJ1977//Japan Society for the Promotion of Science/ ; 23K05785//Japan Society for the Promotion of Science/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Dictyostelium/genetics ; *Genome, Protozoan ; Phylogeny ; },
abstract = {Gene manipulation is essential for understanding biological mechanisms, yet genetic modification in the social amoebas (Dictyostelia) has been largely limited to Dictyostelium discoideum. Here, we aimed to establish a CRISPR/Cas9-based genome-editing system applicable across the phylogenetic breadth of Dictyostelia, spanning Groups 1-4. Using an extrachromosomal CRISPR/Cas9 vector from D. discoideum, we disrupted stlA and pkaC in Polysphondylium violaceum and pkaC in two early-branching species, Heterostelium pallidum and Cavenderia fasciculata. In D. discoideum, co-introduction of donor oligos with the CRISPR vector enabled selection-free knockout generation of pkaC with 28.6% efficiency. In H. pallidum, where genome editing is typically inefficient, co-electroporation of donor oligos with the CRISPR/Cas9 vector followed by 4 days of drug selection increased the frequency of pkaC disruption from 0.9% to 8.3%. These results demonstrated that the D. discoideum CRISPR/Cas9 system can be extended across Dictyostelia, providing a versatile platform for comparative genetic and evolutionary developmental studies.},
}
@article {pmid41554885,
year = {2026},
author = {Zhang, L and Cai, C and Chen, Q and Tan, X and Chen, S and Zhang, K and Cheng, F},
title = {A CRISPR-based sequence proximity binding protein labelling system for scanning upstream regulatory proteins.},
journal = {Nature plants},
volume = {12},
number = {2},
pages = {277-283},
pmid = {41554885},
issn = {2055-0278},
support = {31972411//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; 32302570//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Arabidopsis/genetics/metabolism ; Oryza/genetics/metabolism ; Promoter Regions, Genetic ; *DNA-Binding Proteins/genetics/metabolism ; Brassica/genetics/metabolism ; Gene Expression Regulation, Plant ; *CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Arabidopsis Proteins/genetics/metabolism ; },
abstract = {Transcriptional regulation involves complex and dynamic protein-DNA interactions, which alter chromatin states and, consequently, regulate gene expression. In plants, current technologies face challenges in efficiently capturing dynamically DNA-binding proteins, especially transcription factors. Here, by leveraging the binding ability of dead Cas9 to specific DNA fragments and the labelling capacity of the TurboID protein for adjacent proteins, we have developed a CRISPR-based sequence proximity binding protein labelling system (CSPL) to detect promoter-binding proteins. Using this approach, we identified both known and novel upstream binding proteins on the PIF4 promoter in Arabidopsis, cabbage and rice. This demonstrates the powerful capabilities and broad potential applications of CSPL for detecting promoter-binding proteins in plants.},
}
@article {pmid41354630,
year = {2026},
author = {Wang, W and Chen, K and Wang, Z},
title = {Genome-Wide CRISPR Screen Reveals PIK3CA Inhibition Enhances Lipid Nanoparticle-Mediated siRNA Delivery.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {11},
pages = {e17617},
doi = {10.1002/advs.202517617},
pmid = {41354630},
issn = {2198-3844},
support = {//International Society for Advancement of Cytometry/ ; //Chan Zuckerberg Biohub/ ; //Chan Zuckerberg Initiative/ ; },
mesh = {Humans ; *RNA, Small Interfering/genetics/administration &amp; dosage ; *Nanoparticles/chemistry ; *Class I Phosphatidylinositol 3-Kinases/genetics/antagonists &amp; inhibitors/metabolism ; Animals ; Mice ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Lipids/chemistry ; CRISPR-Cas Systems/genetics ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are useful carriers for therapeutic siRNA delivery, yet their clinical efficacy remains constrained by insufficient cellular uptake. Here, using a genome-wide CRISPR knockout screen, multiple genetic modulators of LNP uptake is uncovered, with PIK3CA emerging as a top druggable target. Pharmacologic inhibition of PIK3CA with BAY1082439 - a clinically evaluated small molecule - significantly enhances LNP uptake, siRNA delivery, and gene silencing across diverse epithelial cancer cell lines in vitro. Co-administration of BAY1082439 with siRNA-loaded LNPs also better suppressed tumor growth and reduced liver inflammation in vivo, respectively. These findings establish PIK3CA inhibition as a broadly applicable strategy to boost LNP-mediated RNA interference and highlight the promise of combining functional genomics with nanomaterials to advance RNA-based therapeutics.},
}
@article {pmid41705810,
year = {2026},
author = {Shin, K and Kim, ET},
title = {Viral genome editing methods and applications in the CRISPR era.},
journal = {Journal of virology},
volume = {},
number = {},
pages = {e0204825},
doi = {10.1128/jvi.02048-25},
pmid = {41705810},
issn = {1098-5514},
abstract = {CRISPR-Cas systems have transformed viral genetics by enabling precise and efficient manipulation of large DNA virus genomes. This review provides a practical framework for applying CRISPR technology to herpesviruses and other large DNA viruses as an alternative and complement to traditional BAC recombination. Key considerations include nuclease choice; sgRNA design that minimizes cut-to-edit distance and prevents re-cutting; donor template configuration and homology arm length; and synchronized delivery of Cas complexes and donor DNA. Strategies to promote HDR efficiency, such as the use of small-molecule modulators, are also summarized. In addition, practical workflows for clone selection, genotypic validation, and phenotypic confirmation are summarized. Case studies in herpes simplex virus type 1 and human cytomegalovirus illustrate how optimized CRISPR designs achieve reproducible, scarless knock-ins and conditional gene manipulation at essential loci without complementing cell lines. Together, these approaches establish CRISPR as a flexible, scalable platform for functional genomics, antiviral target discovery, and translational virology, enabling direct editing of clinical isolates previously inaccessible with bacterial artificial chromosome-based methods.},
}
@article {pmid41705504,
year = {2026},
author = {Sun, K and Wu, H},
title = {A highly-efficient isothermal nano-detection platform coupling CRISPR/Cas technology for detection of circRNA.},
journal = {The Analyst},
volume = {},
number = {},
pages = {},
doi = {10.1039/d6an00107f},
pmid = {41705504},
issn = {1364-5528},
abstract = {Triple-negative breast cancer (TNBC), an aggressive molecular subtype of breast cancer with poor prognosis, is characterized by a high rate of metastasis and proliferation, which makes early detection particularly challenging. Early diagnosis of TNBC through biomarkers and prompt development of treatment methods can lower its mortality rate. This work has designed a nano-detection platform for TNBC biomarker circRNA based on the CRISPR/Cas system and isothermal amplification strategy. Specifically, this detection system uses functional nucleic acid molecules for recognition of circCD44, as well as dual signal amplification using Klenow(3'-5'exo-) and Cas9n. Furthermore, it combines Cas12a and immunomagnetic beads for an extra signal boost and output. After confirming its feasibility and optimizing the conditions, the detection system achieved a 1.73-fold enhancement in sensitivity, offering a linear detection range of 1 pM to 100 nM, with the limit of detection lowered to 95.1 fM. It also showed good specificity through testing against 5 biomarkers. Therefore, this detection system provides a novel strategy for the early diagnosis of TNBC and other diseases.},
}
@article {pmid41705235,
year = {2026},
author = {Li, Z and Yu, T and Ge, L and Lv, S and Fu, Q and Shi, H},
title = {Genome-wide CRISPR/Cas9 screening identifies host factors critical for antiviral defense against equine herpesvirus type 1.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1764863},
pmid = {41705235},
issn = {1664-3224},
mesh = {Animals ; *CRISPR-Cas Systems ; *Herpesvirus 1, Equid/physiology/immunology ; Virus Replication/genetics ; Cell Line ; *Host-Pathogen Interactions/genetics/immunology ; *Herpesviridae Infections/immunology/virology/genetics/veterinary ; Horses ; Gene Knockout Techniques ; *Horse Diseases/virology/immunology/genetics ; Genome-Wide Association Study ; },
abstract = {INTRODUCTION: Equine herpesvirus type 1 (EHV-1) is a major veterinary pathogen causing significant economic losses in the livestock industry. Despite its impact, effective vaccines and targeted antiviral strategies remain limited, largely due to an incomplete understanding of host factors regulating viral replication and pathogenesis.<br><br>METHODS: To systematically identify host genes essential for EHV-1 infection, we established a BHK-21 cell line stably expressing Cas9 and performed a genome-wide CRISPR/Cas9 knockout screen using a pooled lentiviral single-guide RNA library. Significantly enriched candidate genes from positive selection were validated by generating knockout cell lines. Viral replication and protein expression were assessed using quantitative polymerase chain reaction and Western blot analysis. Pathway enrichment and protein interaction network analyses were subsequently conducted.<br><br>RESULTS: Genome-wide CRISPR/Cas9 screening identified multiple host factors critical for EHV-1 replication. Pathway enrichment analysis revealed that these genes were involved in key cellular signaling and regulatory networks associated with viral infection. Functional validation demonstrated that knockout of selected host genes significantly suppressed EHV-1 replication and viral protein synthesis.<br><br>DISCUSSION: These findings highlight essential host determinants required for EHV-1 replication and suggest that targeting host factors may represent a promising strategy for antiviral intervention. This study provides a foundation for the development of host-directed immunotherapeutic and antiviral approaches against EHV-1 infection.},
}
@article {pmid41704956,
year = {2026},
author = {Wang, Y and Diao, Y and Zhang, T and Zhang, F and Wang, W},
title = {Sensitive, specific, and rapid on-site detection of calf diarrhea pathogens using the RPA-CRISPR/Cas 12a assay.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1734185},
pmid = {41704956},
issn = {2235-2988},
mesh = {Animals ; Cattle ; Sensitivity and Specificity ; *Diarrhea/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; *Cattle Diseases/diagnosis/virology/microbiology ; *Molecular Diagnostic Techniques/methods ; Diarrhea Viruses, Bovine Viral/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Enterotoxigenic Escherichia coli/isolation &amp; purification/genetics ; },
abstract = {Calf diarrhea is a common gastrointestinal disease that usually occurs within one month of birth. The disease causes the greatest economic losses to the cattle industry. Currently, a variety of diagnostic methods have been developed for calf diarrhea infections. However, existing methods are still unsatisfactory in terms of sensitivity, specificity, simplicity, cost, and speed.To provide a more sensitive, specific, simpler, and faster detection method, we recently developed an RPA-CRISPR/Cas12a assay that can detect BVDV, BCoV, BRV, and ETEC infections in cattle on-site. Testing for each pathogen is performed in a single test tube, without the need to open the tube in the middle, and can be completed in under 50 minutes.The RPA-CRISPR/Cas12a assay can detect BVDV, BCoV, BRV, and ETEC at concentrations of at least 10 copies/μL. The RPA-CRISPR/Cas12a assay does not produce false-positive results due to the presence of other pathogens. The sensitivity of BCoV, BRV, and ETEC in the RPA-CRISPR/Cas12a quadruple assay is equivalent to that of single qPCR. The sensitivity of BVDV in the quadruple assay is slightly lower than that of the single qPCR method.Due to its sensitivity, specificity, simplicity, and rapidity, the RPA-CRISPR/Cas12a assay is more practical for on-site detection of cattle diarrhea pathogens than any existing detection method.},
}
@article {pmid41704216,
year = {2026},
author = {Khweis, S and Blackburn, M and Perdigao, C and Pierce, M and Lewis, C and Dunkle, J},
title = {Cas10 residues lining the target RNA binding channel regulate interference by distinguishing cognate target RNA from mismatched targets.},
journal = {RNA biology},
volume = {},
number = {},
pages = {},
doi = {10.1080/15476286.2026.2633385},
pmid = {41704216},
issn = {1555-8584},
abstract = {Type III CRISPR systems are defined by the presence of the Cas10 protein and are among the most abundant CRISPR systems in nature. Cas10 forms a complex with crRNA and several Cas proteins that surveils prokaryotic cells for foreign RNA molecules and when they are detected it activates a cascade of interference activities. The synthesis of the cyclic oligoadenylate signalling molecule by Cas10 is a key aspect of the interference cascade. Despite structures of the Cas10 complex bound to target RNAs, the molecular mechanism by which Cas10 senses the bound state to licence interference is lacking. We identified five residues in S. epidermidis Cas10, two in the Cas10 Palm2 domain and three in domain 4, that line the target RNA binding channel. We assessed the contribution of these residues to interference in the context of a cognate or mismatched target RNA. We found that the residues regulate whether a mismatched crRNA-target RNA duplex is able to activate interference in vivo. We purified two site-directed mutants of Cas10-Csm and show with in vitro cOA synthesis assays that they demonstrate enhanced discrimination of cognate versus mismatched target RNAs.},
}
@article {pmid41679604,
year = {2026},
author = {Li, L and Wang, Y and Wang, B and Shen, L and Gao, Y and Lin, W and Li, Z},
title = {A dCas9-integrated iLight9O system enables dynamic regulation for enhanced patchoulol biosynthesis in Saccharomyces cerevisiae.},
journal = {Bioresource technology},
volume = {446},
number = {},
pages = {134195},
doi = {10.1016/j.biortech.2026.134195},
pmid = {41679604},
issn = {1873-2976},
mesh = {*Saccharomyces cerevisiae/metabolism/genetics ; Metabolic Engineering/methods ; *CRISPR-Cas Systems/genetics ; Optogenetics/methods ; Light ; },
abstract = {Numerous organisms have evolved the ability to utilize light through photoreceptor proteins that mediate diverse biological processes. Currently, several optogenetic sensor systems are widely used in yeast. However, when these systems are applied for gene repression to regulate endogenous yeast gene expression, they typically require the insertion of corresponding target sites near the native promoter of the gene of interest to achieve precise modulation. To address these constraints, a novel blue light-inducible optogenetic tool designated iLight9 was developed, a single-component optogenetic biosensor integrated with the CRISPR-dCas9 platform. The stability of the iLight9 system was further enhanced by employing a strategy involving the addition of a protein degradation tag. The resulting system was designated as iLight9O, which facilitated programmable regulation of distinct genes through the introduction of specific sgRNAs. Subsequently, systematic metabolic engineering strategies were employed to construct an efficient patchoulol-producing cell factory in Saccharomyces cerevisiae. Moreover, a two-step isoprenol utilization (IU) pathway was introduced into the recombinant strain to enhance its capacity for patchoulol biosynthesis. Crucially, the iLight9O system was adopted to dynamically downregulate squalene synthase, a key enzyme in the competing squalene biosynthetic pathway. This optogenetic flux control strategy increased patchoulol titers by 66 % in the IU-optimized strain and 24 % in the MVAIU2 strain, demonstrating significant improvements over static engineering approaches.},
}
@article {pmid41672287,
year = {2026},
author = {Chaumont, L and Peruzzi, M and Huetz, F and Raffy, C and Le Hir, J and Minke, J and Leong, JC and Boudinot, P and Collet, B},
title = {Knockout of the antiviral genes mx1 or mx3 modulates the expression of paralogous genes in a salmonid cell line.},
journal = {Fish &amp; shellfish immunology},
volume = {171},
number = {},
pages = {111204},
doi = {10.1016/j.fsi.2026.111204},
pmid = {41672287},
issn = {1095-9947},
mesh = {Animals ; *Myxovirus Resistance Proteins/genetics/immunology ; Cell Line ; *Fish Proteins/genetics/immunology ; Gene Knockout Techniques/veterinary ; *Fish Diseases/immunology ; *Immunity, Innate/genetics ; *Salmo salar/genetics/immunology ; *Gene Expression Regulation/immunology ; CRISPR-Cas Systems ; },
abstract = {Mx dynamin-like GTPases genes are Interferon Stimulated Genes (ISGs) encoding the Mx (myxovirus resistance) proteins that have an antiviral effect against a wide array of RNA viruses as well as some DNA viruses. In salmonids, the mx genes reside in four distinct chromosomal loci, that encode proteins clustering together into separate phylogenetic clades. To understand the contribution of Mx to the innate antiviral resistance, we knocked out the mx1 or mx3 gene by generating single mutant CHSE-derived cell lines by CRISPR/Cas9 genome editing. sgRNA were designed within the first coding exon of mx1 (LOC112247236), or mx3 (LOC112247235) genes, located 27 kb apart on the same chromosome LG02. Mx1, Mx2 and Mx3 proteins were quantified by western blotting. Only the Mx3 protein was found induced in the wild type EC cells after stimulation with recombinant Atlantic salmon interferon A2. mx1 gene knockout reduced the up-regulation of Mx3 protein expression, while mx3 gene knockout resulted in the induction of Mx1 and Mx2 proteins with and without additional stimulation. This was observed at the transcriptional level with the induction of mx1 gene was increased in mx3[-/-] cells compared to wild type cells. These results illustrate the complex interplay between mx1 and mx3 genes in salmonids.},
}
@article {pmid41671823,
year = {2026},
author = {Wang, Z and Yan, R and Lin, P and Yao, Y and He, H and Zhuang, W and Hu, J and Chen, Y and Xu, S and Hu, WW},
title = {CRISPR/Cas12a-nanozyme visual biosensor for detection of microRNA-21.},
journal = {Talanta},
volume = {303},
number = {},
pages = {129517},
doi = {10.1016/j.talanta.2026.129517},
pmid = {41671823},
issn = {1873-3573},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; DNA, Single-Stranded/chemistry ; Limit of Detection ; *Endodeoxyribonucleases/metabolism/chemistry ; *CRISPR-Associated Proteins/metabolism ; *Bacterial Proteins/metabolism ; Nucleic Acid Amplification Techniques ; Silver/chemistry ; },
abstract = {The precise diagnosis of aggressive tumors is crucial for guiding therapy, with microRNAs (miRNAs) increasingly recognized as pivotal biomarkers due to their regulatory roles in tumor development and dissemination. Herein, we report a CRISPR/Cas12a-nanozyme visual biosensor for direct and sensitive detection of tumor-associated miRNA-21. In this design, Ag/NiFe layered double hydroxide acts as a peroxidase-like nanozyme and is tethered to magnetic beads via single-stranded DNA (ssDNA) linkers. Upon recognition of miRNA-21, strand displacement amplification (SDA) can generate a DNA activator that activates CRISPR/Cas12a. The activated Cas12a then cleaves the ssDNA linkers, releasing the nanozyme to catalyze a chromogenic reaction that yields a visible color change. This approach enables sensitive detection of miRNA-21 with a detection limit of 420.0 fM, providing a direct visual readout and demonstrating strong potential for point-of-care nucleic acid diagnostics.},
}
@article {pmid41653624,
year = {2026},
author = {Sun, Y and Demachi-Okamura, A and Shinohara, S and Wang, Y and Guo, Z and Yamaguchi, R and Matsushita, H and Nabekura, T and Muraoka, D},
title = {Dual knockout of Fas and TCRα in Jurkat reporter cells enables highly sensitive identification of antigen-specific TCRs.},
journal = {Biochemical and biophysical research communications},
volume = {805},
number = {},
pages = {153400},
doi = {10.1016/j.bbrc.2026.153400},
pmid = {41653624},
issn = {1090-2104},
mesh = {Humans ; Jurkat Cells ; *fas Receptor/genetics/immunology ; CRISPR-Cas Systems ; *Gene Knockout Techniques ; CD8-Positive T-Lymphocytes/immunology ; Lung Neoplasms/immunology/genetics ; *Receptors, Antigen, T-Cell, alpha-beta/genetics/immunology ; *Receptors, Antigen, T-Cell/genetics ; },
abstract = {T-cell receptors (TCRs) that target tumor antigens are crucial for antitumor immunity; however, tumor-specific TCRs often exhibit low affinity for their cognate antigens, making the identification of functional TCRs challenging due to the limited sensitivity of current detection methods. In this study, we established a high-sensitivity TCR screening platform by generating Jurkat cell reporter clones with dual knockout (DKO) of endogenous Fas and TCRα via CRISPR-Cas9 system. In a viral antigen model system, these DKO Jurkat cells exhibited approximately 100-fold greater sensitivity to antigen stimulation compared with parental Jurkat cells. Notably, our DKO Jurkat-based platform enabled the identification of tumor-specific CD8[+] T cells from a lung cancer patient that could not be detected using parental Jurkat cells. Moreover, the identified tumor-specific T-cell clone exhibited a unique phenotype characterized by robust cytotoxic T lymphocyte (CTL) activity and natural killer-like properties. Together, these findings demonstrate that dual deletion of Fas and TCRα in Jurkat cells enables highly sensitive functional TCR screening. Integration of this platform with single-cell analysis facilitates the discovery of previously uncharacterized tumor-reactive TCRs and provides a powerful tool for advancing TCR-based cancer immunotherapy.},
}
@article {pmid41288537,
year = {2026},
author = {He, J and Papa, G and Azizi, F and Kirsche, L and Artola-Boran, M and Ferreira Cassio, R and Hotz, AL and Geiger, G and Francas, B and Weber, A and Tzankov, A and Kontarakis, Z and Leary, P and Müller, A},
title = {Gastric Organoid-Based Ectopic and Orthotopic In Vivo CRISPR Screening for Tumor Suppressors in Gastric Cancer.},
journal = {Gastroenterology},
volume = {170},
number = {3},
pages = {495-510},
doi = {10.1053/j.gastro.2025.09.009},
pmid = {41288537},
issn = {1528-0012},
mesh = {Animals ; *Stomach Neoplasms/genetics/pathology/microbiology/metabolism ; *Organoids/pathology ; Mice ; *CRISPR-Cas Systems ; Helicobacter pylori ; Humans ; *Genes, Tumor Suppressor ; Helicobacter Infections/microbiology ; Signal Transduction/genetics ; PTEN Phosphohydrolase/genetics ; Gene Knockout Techniques ; Disease Models, Animal ; },
abstract = {BACKGROUND &amp; AIMS: CRISPR-Cas9 screening is a powerful tool for the in vivo discovery of cancer dependencies. The aim of this study was to perform in vivo CRISPR knockout screening for gastric tumor suppressors using gastric murine organoids in a subcutaneous as well as a surgical model of orthotopic tumor growth.<br><br>METHODS: In vivo screening was performed using a custom library targeting 49 putative gastric tumor suppressor genes, as well as a "cancer genome-wide" library targeting 5000 genes, in immunocompetent and -deficient mice, and in the presence or absence of the gastric pathogen Helicobacter pylori. The top hits were selected for individual validation and mechanistic follow-up.<br><br>RESULTS: Our custom library knockout screens revealed single-guide RNAs targeting Pten, Fbxw7, and genes encoding several components of the transforming growth factor-&#xdf; signaling pathway (Smad4, Tgfbr1, Tgfbr2, and Acvr2a) to be recurrently enriched both in subcutaneously and orthotopically growing tumors. The same, and several additional genes were identified by cancer genome-wide CRISPR screening. Ten of our top hits could be validated individually in vivo. Pten inactivation resulted in large tumors characterized by increased neo-angiogenesis, neutrophil recruitment, and T-cell exclusion. Inactivation of Smad4, Tgfbr1, or Acvr2a all produced phenotypes that were reminiscent of early gastric cancer precursor lesions such as intestinal Alcian blue-positive metaplasia and compensatory hyperplasia. Helicobacter pylori infection failed to affect the mutational landscape of tumors; rather, we found that H pylori modulates the tumor microenvironment and recruits large numbers of tumor-promoting SiglecF[+] neutrophils.<br><br>CONCLUSIONS: In summary, we describe here a versatile model of gastric carcinogenesis that uncouples the genetics of the tumor and the host, and that faithfully recapitulates key risk factors of the malignancy.},
}
@article {pmid41703314,
year = {2026},
author = {Rocha, DC and Omoregbee, MO and Luo, W and Fang, H and Ye, Q and Liu, Y and Li, G and Mascoveto, J and de Souza, AA and Coleman, G and Culver, JN and Qi, Y},
title = {Transgene-free genome editing in citrus and poplar meristem tissues via biolistic ribonucleoprotein delivery of CRISPR-Cas9.},
journal = {Plant cell reports},
volume = {45},
number = {3},
pages = {58},
pmid = {41703314},
issn = {1432-203X},
support = {2020/07045-3, 2021/03466-7, and 2023/09068-9//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; DE-SC0023011//U.S. Department of Energy/ ; IOS-2132693 and IOS-2428015//Directorate for Biological Sciences/ ; 2020-70029-33161, 2021-67013-34554, 2024-33522-42755//National Institute of Food and Agriculture/ ; MD-PSLA-24014//McIntire Stennis Forest Research Program/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Meristem/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Populus/genetics ; *Citrus/genetics ; *Biolistics/methods ; Plants, Genetically Modified ; Transgenes/genetics ; },
abstract = {Biolistic particle bombardment was used to deliver CRISPR-Cas9 ribonucleoprotein complexes (RNP) into the shoot apical meristem tissue of citrus and axillary meristem tissue of poplar, generating directed mutations in target genes. The use of meristematic tissues offers a strategic approach to genome editing in woody species, especially those that are recalcitrant to conventional tissue culture, as these regions contain totipotent, highly regenerative cells capable of giving rise to whole plants. Here, we employed biolistic delivery of genome-editing reagents into theshoot apical meristem (SAM) of citrus and the axillary meristems (AXM) of poplar. The system was first validated using a GFP expression construct and subsequently applied for targeted genome editing. In citrus, edited plants were obtained at the CsNPR3 locus exclusively through the delivery of CRISPR/Cas9 ribonucleoproteins (RNPs), whereas plasmid-based vectors were unsuccessful. Similarly, genome editing in poplar was achieved using RNPs targeting the Pt4CL1 gene. Although chimeric events were detected, this approach provides a feasible and innovative framework for producing transgene-free edited perennial plants.},
}
@article {pmid41620608,
year = {2026},
author = {Pahlevan Kakhki, M and Rangani, F and Ewing, E and Starvaggi Cucuzza, C and Zheleznyakova, G and Kalomoiri, M and Kenny, L and Raghavan, A and Rao Prakash, C and van den Hoeven, G and Venkata S Badam, T and Covacu, R and Andreou, I and Needhamsen, M and Kular, L and Jagodic, M},
title = {Comprehensive profiling of CRISPR/dCas9 epigenome editors indicates a complex link between on and off target effects.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {41620608},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; DNA Methylation ; *Gene Editing/methods ; Humans ; *Epigenome ; DNA Methyltransferase 3A ; DNA (Cytosine-5-)-Methyltransferases/metabolism/genetics ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; Epigenesis, Genetic ; },
abstract = {BACKGROUND: CRISPR/dCas9-based epigenome editing systems, including DNA methylation epimodifiers, have greatly advanced molecular functional studies, revolutionizing their precision and applicability. Despite their promise, challenges such as the magnitude and stability of the on-target editing and unwanted off-target effects underscore the need for improved tool characterization and design.<br><br>RESULTS: We systematically compare specific targeting and genome-wide off-target effects of available and novel dCas9-based DNA methylation editing tools over time. We demonstrate that multimerization of the catalytic domain of DNA methyltransferase 3A enhances editing potency but also induces widespread, early methylation deposition at low-to-medium methylated promoter-related regions with specific gRNAs and also with non-targeting gRNAs. A small fraction of the methylation changes associated with transcriptional dysregulation and mapped predominantly to bivalent chromatin associating both with transcriptional repression and activation. Additionally, specific non-targeting control gRNAs cause pervasive and long-lasting methylation-independent transcriptional alterations particularly in genes linked to RNA and energy metabolism. CRISPRoff emerges as the most efficient tool for stable promoter targeting, with fewer and less stable off-target effects compared to other epimodifiers but with persistent transcriptome alterations.<br><br>CONCLUSIONS: Our findings highlight the delicate balance between potency and specificity of epigenome editing and provide critical insights into the design and application of future tools to improve their precision and minimize unintended consequences.},
}
@article {pmid41527385,
year = {2026},
author = {Pang, Y and Duan, Y and Sun, Y and Zhou, T and Li, A and Ran, R and Hou, H and Liu, SM},
title = {One copy in one-pot for rapid and accurate SFTSV testing by LAC12b-2M.},
journal = {Clinical chemistry and laboratory medicine},
volume = {64},
number = {4},
pages = {957-967},
pmid = {41527385},
issn = {1437-4331},
mesh = {Humans ; *Phlebovirus/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; RNA, Viral/genetics/blood ; CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Real-Time Polymerase Chain Reaction ; },
abstract = {OBJECTIVES: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne pathogen that can cause a fatality rate as high as 12-50 %, posing a significant threat to public health. SFTSV is prevalent in mountainous and hilly regions with relatively poor medical conditions. Therefore, there is an urgent need to develop a new convenient, rapid and sensitive method for SFTSV detection in low-resource environments.<br><br>METHODS: We developed a one-pot and visualized method for SFTSV detection using loop-mediated isothermal amplification assisted by CRISPR/Cas12b with G478A/K396A double mutations (LAC12b-2M). The specificity, sensitivity, accuracy and limit of detection (LOD) of LAC12b-2M were evaluated using clinical reverse transcription-quantitative PCR (RT-qPCR) as the reference method, with gradient dilutions of strong positive SFTSV RNA samples and 215 clinical serum samples from two cohorts.<br><br>RESULTS: LAC12b-2M is sensitive to detect SFTSV with a LOD of 1 copy/&#x3bc;L at 61&#x202f;&#xb0;C within 30&#x202f;min. Compared to clinical RT-qPCR, LAC12b-2M demonstrated a sensitivity of 98.8&#x202f;% (82/83), a specificity of 100.0&#x202f;% (96/96), and an accuracy of 99.4&#x202f;% (178/179) in cohort 1 (n=179), and an accuracy of 100.0&#x202f;% in cohort 2 (n=36).<br><br>CONCLUSIONS: Our LAC12b-2M method holds promise for point-of-care SFTSV testing in different healthcare settings, particularly in low-resource region where SFTSV is prevalent.},
}
@article {pmid41702797,
year = {2026},
author = {Li, C and Mei, D and Cheng, H and Pan, X and Zhang, B},
title = {CRISPR genome editing in plants without tissue culture.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.12.017},
pmid = {41702797},
issn = {1879-3096},
abstract = {Conventional plant genome editing relies on tissue culture-mediated somatic cell regeneration, a technically demanding process that limits its application across diverse species. Emerging strategies now circumvent this bottleneck by enabling direct genome editing of meristematic or germline cells. Key advances include (i) genome editing via de novo meristem induction or dormant meristem activation; (ii) germline editing facilitated by graft-mobile tRNA-like sequence systems and haploid induction technologies; and (iii) optimized viral delivery platforms that exploit mobile RNA elements and compact editors such as TnpB to achieve efficient, transgene-free, heritable modifications across a broad range of genotypes and species. The development of robust, tissue culture-free editing platforms promises to revolutionize crop improvement pipelines and accelerate trait development for sustainable agriculture.},
}
@article {pmid41702404,
year = {2026},
author = {Li, Z and Li, Y and Kong, J and Wu, Q and Huang, P and Zhang, Y and Wu, W and Chen, M and Liu, Y and Lin, H and Hou, L and Liu, G and Zeng, T and He, Y and Hu, C and Yang, Z and Lu, M and Luo, M and Xiao, Y},
title = {Structural basis for Cas9-directed spacer acquisition in type II-A CRISPR-Cas systems.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2026.01.024},
pmid = {41702404},
issn = {1097-4164},
abstract = {CRISPR-Cas systems confer prokaryotic immunity by integrating foreign DNA (prespacers) into host arrays. Type II-A systems employ Cas9 for protospacer-adjacent motif (PAM) recognition and coordinate with Csn2 and the Cas1-Cas2 integrase during spacer acquisition, yet their structural basis remains unresolved. Here, we report cryo-electron microscopy (cryo-EM) structures of the Enterococcus faecalis Cas9-Csn2-Cas1-Cas2 supercomplex in apo and DNA-bound states. The apo state (Cas92-Csn28-Cas18-Cas24) is a resting complex, while DNA binding forms a prespacer-catching complex threading DNA through Csn2's channel, enabling Cas9 to interrogate the PAM sequence while sliding along the DNA. Cas9 and Csn2 jointly define a 30-bp DNA segment matching the prespacer length. Cas9 dissociation triggers structural reconfiguration of the Csn2-Cas1-Cas2 assembly. This exposes the PAM-proximal DNA, allowing Cas1-Cas2 to bind the exposed site for subsequent prespacer processing and directional integration. These findings reveal how Cas9, Csn2, and Cas1-Cas2 couple PAM recognition with prespacer selection, ensuring fidelity during adaptation.},
}
@article {pmid41702403,
year = {2026},
author = {Gaizauskaite, U and Tamulaitiene, G and Silanskas, A and Gasiunas, G and Siksnys, V and Sasnauskas, G},
title = {Structural insights into Cas9-mediated prespacer selection in CRISPR-Cas adaptation.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2026.01.022},
pmid = {41702403},
issn = {1097-4164},
abstract = {During CRISPR-Cas adaptation, prokaryotic cells become immunized by the insertion of foreign DNA fragments, termed spacers, into the host genome to serve as templates for RNA-guided immunity. Spacer acquisition relies on the Cas1-Cas2 integrase and accessory proteins, which select DNA sequences flanked by the protospacer adjacent motif (PAM) and insert them into the CRISPR array. It has been shown that in type II-A systems, selection of PAM-proximal prespacers is mediated by the effector nuclease Cas9, which forms a "supercomplex" with the Cas1-Cas2 integrase and the Csn2 protein. Here, we present cryo-electron microscopy structures of the Streptococcus thermophilus type II-A prespacer selection supercomplex in the DNA-scanning and two distinct PAM-bound configurations, providing insights into the mechanism of Cas9-mediated prespacer selection in type II-A CRISPR-Cas systems. Repurposing Cas9 by the CRISPR adaptation machinery for prespacer selection, as characterized here, demonstrates Cas9 plasticity and expands our knowledge of Cas9 biology.},
}
@article {pmid41702027,
year = {2026},
author = {Galdikaite-Braziene, E and Krušnauskas, R and Henderson, E and Bujakowska, KM},
title = {CRISPR as a therapeutic tool for inherited retinal degenerations: Advances, challenges, and future directions.},
journal = {Molecular aspects of medicine},
volume = {108},
number = {},
pages = {101462},
doi = {10.1016/j.mam.2026.101462},
pmid = {41702027},
issn = {1872-9452},
abstract = {Inherited retinal diseases (IRDs) are a genetically diverse group of disorders characterized by progressive photoreceptor degeneration, leading to vision loss and blindness. With over 320 associated genes and significant phenotypic variability, effective treatment remains challenging. Recent advances in genome editing, particularly CRISPR/Cas-based technologies, have revolutionized therapeutic approaches by enabling precise and customizable DNA and RNA editing. This review explores the application of various CRISPR strategies-such as gene knockout via non-homologous end joining (NHEJ), exon skipping using dual-sgRNAs, homology-directed repair (HDR), base editing (BE), prime editing (PE), RNA editing with Cas13, and epigenetic modulation through CRISPRa/i-in preclinical models of IRDs. Emphasis is placed on allele-specific targeting, gene-agnostic approaches, and mutation-independent strategies to address dominant and recessive forms of disease. We also highlight recent clinical milestones, including the first human trial using CRISPR gene editing for CEP290-associated Leber congenital amaurosis. Finally, we discuss critical challenges, including delivery constraints, immune responses, and off-target effects, along with emerging solutions such as engineered Cas variants, split-intein systems, and advanced off-target detection methods. Together, these advances underscore the transformative potential of CRISPR technologies in treating IRDs and lay the foundation for future clinical translation.},
}
@article {pmid41699368,
year = {2026},
author = {Khalid, M and Ishaq, A and Arshad, M and Kaul, H and Majeed, M},
title = {Multiplex CRISPR/Cas9 editing of gliotoxin biosynthesis genes in Aspergillus fumigatus reduces pathogenicity in broilers.},
journal = {Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]},
volume = {57},
number = {1},
pages = {55},
pmid = {41699368},
issn = {1678-4405},
support = {13224/2020//Higher Education Commision, Pakistan/ ; },
mesh = {Animals ; *Aspergillus fumigatus/genetics/pathogenicity/metabolism ; *Gliotoxin/biosynthesis ; Chickens ; *CRISPR-Cas Systems ; *Gene Editing ; *Aspergillosis/veterinary/microbiology/pathology ; *Poultry Diseases/microbiology/pathology ; Virulence ; Fungal Proteins/genetics/metabolism ; Spores, Fungal/genetics ; },
abstract = {Gliotoxin of Aspergillus fumigatus has been extensively studied for its role in pathogenesis in animals and humans. It triggers pathogenesis by its immunosuppressive and cytotoxic effects. Biosynthetic gene cluster (BGC) consisting of 13 genes regulates its biosynthesis. We targeted gliZ, gliP and gliA genes of this BGC using CRISPR/Cas9 system in a multigene editing approach to check the pathogenesis in broilers. crRNAs were designed using EuPaGDT and 3 single guide RNAs (sgRNA) were commercially synthesized. Each sgRNA was combined with Cas9 to form ribonucleoprotein complexes which were then used for simultaneously transfecting fungal protoplasts. Thin-layer chromatography showed the absence of gliotoxin on silica plate and DNA sequencing showed various indels in target genes. These indels caused amino acid substitutions in all three gene products but, the gliP mutation, since it was synonymous, was likely not functionally relevant. Regenerated protoplasts were matured to form fungal hyphae and spore production was induced. These spores were inoculated intra-air sac in broiler chicks. During one-week infection trial, birds infected with the wild-type spores (group 1) showed morbidity and their mortality rate was 30%. Birds inoculated with RNP-treated spores (group 2) showed mild clinical signs and no mortality. No morbidity or mortality was recorded in birds in negative control group (group 3). Histopathological analysis of lungs showed necrosis and congestion, and presence of mixed population of inflammatory cells in wild-type infected birds, while no such lesions were seen in birds infected with RNP-treated spores. These results show that multigene editing approach was successful in creating indels simultaneously in 3 gliotoxin genes which resulted in amino acid substitution which negatively impacted gliotoxin biosynthesis and export. In vivo experiment results show that RNP-treated fungal spores were unable to cause A. fumigatus pathogenicity in broiler. Targeting gliotoxin biosynthesis could thus be a promising approach to develop antifungal therapy.},
}
@article {pmid41669836,
year = {2026},
author = {Dai, YM and Zhao, L and Xu, T and Duan, JQ and Wang, YM and Yan, YX and Ge, LP and Liu, ZH and Sun, J and Zeng, X and Lai, SY and Ai, YR and Huang, JB and Zhu, L and Xu, ZW},
title = {Fast and Simplified CRISPR-Cas13d Assay for Sensitive Detection of Porcine Deltacoronavirus.},
journal = {ACS synthetic biology},
volume = {15},
number = {2},
pages = {834-845},
doi = {10.1021/acssynbio.5c00909},
pmid = {41669836},
issn = {2161-5063},
mesh = {Animals ; Swine ; *CRISPR-Cas Systems/genetics ; *Deltacoronavirus/genetics/isolation &amp; purification ; RNA, Viral/genetics/analysis ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; *Swine Diseases/diagnosis/virology ; },
abstract = {Rapid and portable diagnostic technologies are essential for controlling infectious diseases. Here, we describe RAPID (Rapid Automated Portable Integrated Detection), a single-step, extraction-free CRISPR-Cas13d-based assay for sensitive and specific detection of porcine deltacoronavirus (PDCoV) RNA. RAPID integrates isothermal recombinase polymerase amplification with EsCas13d-mediated collateral cleavage in a one-pot reaction, enabling sample-to-answer detection within 30 min. A brief room-temperature lysis step allows direct RNA release from unextracted samples, simplifying sample preparation and reducing equipment requirements. Lyophilized reagents enhance stability during refrigerated storage (≤4 °C) and facilitate simplified transportation using conventional cooling measures, thereby reducing reliance on strict cold-chain logistics. The assay operates optimally at 37 °C and remains functional under ambient (∼25 °C) conditions with reduced sensitivity, permitting instrument-free operation when temperature control is unavailable. Detection is achieved via in-tube fluorescence or lateral-flow readouts. Clinical validation using porcine samples showed complete concordance with RT-qPCR, achieving 100% sensitivity and specificity. RAPID provides a practical point-of-care diagnostic platform for on-farm surveillance and deployment in resource-limited settings.},
}
@article {pmid41645934,
year = {2026},
author = {Wang, Z and Wang, Y and Ji, Q},
title = {Protocol for Discovery and Characterization of Miniature Cas12 Systems.},
journal = {ACS chemical biology},
volume = {21},
number = {2},
pages = {401-409},
doi = {10.1021/acschembio.6c00016},
pmid = {41645934},
issn = {1554-8937},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Dependovirus/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {Efficient delivery remains a major challenge for therapeutic genome editing because many widely used CRISPR nucleases are large and leave limited space for regulatory elements or additional payloads in a single adeno-associated virus (AAV) vector. Miniature Cas12 nucleases are particularly appealing, as their reduced size alleviates packaging constraints while preserving RNA-guided DNA cleavage. Here, we outline a workflow that links large-scale sequence mining with phylogenetic and structural filtering, followed by PAM profiling, in vitro cleavage, bacterial genome interference, and genome-editing assays in human cells to confirm activity. This protocol is intended to distill broad sequence collections into a small set of compact Cas12 nucleases with demonstrated functions that can serve as starting points for further engineering in delivery-limited settings.},
}
@article {pmid41639367,
year = {2026},
author = {Barreiro-Docío, E and Guerrero-Peña, L and Soni, P and Méndez-Martínez, L and Costas-Prado, C and Alvarado, MV and Vázquez, JA and Tort, L and Cerdá-Reverter, JM and Rotllant, J},
title = {Loss-of-function mutations in the melanocortin-2-receptor (mc2r) lead to skin hyperpigmentation in teleost fish.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41639367},
issn = {2045-2322},
mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Receptor, Melanocortin, Type 2/genetics/metabolism ; *Hyperpigmentation/genetics/metabolism ; *Loss of Function Mutation ; *Skin Pigmentation/genetics ; *Zebrafish Proteins/genetics/metabolism ; Melanophores/metabolism ; Melanins/biosynthesis ; CRISPR-Cas Systems ; },
abstract = {Melanocortins regulate pigmentation via melanocortin receptors (MCRs), which are highly conserved across vertebrates. Unlike other MCRs, the melanocortin 2 receptor (MC2R) is exclusively activated by ACTH; however, its role in pigmentation remains unclear. Using CRISPR/Cas9-generated mc2r knockout zebrafish, we demonstrated that the loss of mc2r in zebrafish results in impaired interrenal steroidogenesis and pronounced hyperpigmentation characterized by an increased number of melanophores and xanthophores while preserving normal patterning. Transcriptomic analyses revealed the upregulation of genes involved in melanosome formation, melanin synthesis, lipid metabolism, and carotenoid accumulation. These findings demonstrate that, in addition to controlling steroidogenesis, mc2r plays a key role in pigment cell development and metabolic regulation.},
}
@article {pmid41571527,
year = {2026},
author = {Kaneko, Y and Kawabe, Y and Nishijima, KI and Kamihira, M},
title = {Evaluation of cellular characteristics and genome editing responses in chicken primordial germ cell lines.},
journal = {Journal of bioscience and bioengineering},
volume = {141},
number = {4},
pages = {290-299},
doi = {10.1016/j.jbiosc.2025.12.010},
pmid = {41571527},
issn = {1347-4421},
mesh = {Animals ; *Chickens/genetics ; *Germ Cells/cytology/metabolism ; *Gene Editing/methods ; Male ; CRISPR-Cas Systems ; Female ; Cell Proliferation ; Cell Line ; },
abstract = {Chicken primordial germ cells (cPGCs) hold great potential for genetic modification and germ cell research in chickens. In this study, we evaluated the cellular characteristics of three cPGC lines: cPGC-1, cPGC-2, and cPGC-3. cPGC-1 and cPGC-2 were derived from male chickens, whereas cPGC-3 was derived from a female chicken. We analyzed and compared cell proliferation rates, marker gene expression, and gonadal colonization abilities. Three different cell culture temperatures were assessed (37 °C, 39 °C, and 41 °C) and proliferation rates were highest for all cPGC lines at 39 °C. Additionally, cPGC-1 demonstrated a higher proliferation rate than cPGC-2. No significant differences were observed between cPGC-1 and cPGC-2 with regard to the expression of germ cell and pluripotency marker genes (Cvh, Dazl, Pou5f3, and Nanog). To assess changes in cellular characteristics before and after genetic modification, we performed a green fluorescent protein (GFP) gene knock-in using the CRISPR/Cas9 system, followed by site-specific integration of the scFv-Fc gene using the Cre-loxP system. Transplantation experiments revealed that cPGC-2/GFP exhibited higher gonadal colonization efficiency than cPGC-1/GFP. This study demonstrates differences in cellular characteristics among established cPGC lines and highlights the impact of genetic modification on cPGC function. Our findings emphasize the importance of selecting appropriate cell lines and optimizing culture conditions based on cPGC traits to achieve efficient and reproducible production of transgenic chickens. These insights will aid in the conservation of poultry genetic resources and the advancement of transgenic chicken production for both research and industrial applications.},
}
@article {pmid41554741,
year = {2026},
author = {Li, S and Xu, K and Li, G and Jiang, H and Wu, Z and Gao, P and Yue, Y and Chen, Y and Liu, Z and Zhou, B and Zhou, M and Chen, Y and Liu, X and Wang, X and Wu, Z and Wei, Y},
title = {Engineering the MmeFz2-ωRNA system for efficient genome editing through an integrated computational-experimental framework.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41554741},
issn = {2041-1723},
support = {32441080, 32301251//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22207074//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; Animals ; Humans ; Mice ; Muscular Dystrophy, Duchenne/genetics/therapy ; Male ; Dystrophin/genetics/metabolism ; HEK293 Cells ; Protein Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Dependovirus/genetics ; CRISPR-Cas Systems ; },
abstract = {Eukaryotic Fanzor proteins are compact, programmable RNA-guided nucleases with substantial potential for genome editing, although their efficiency in mammalian cells remains suboptimal. Here, we present a combinatorial engineering strategy to optimize a representative Fanzor system, MmeFz2-ωRNA. AlphaFold3-powered rational redesign produced a minimized ωRNA scaffold that is 30% smaller while maintaining up to 82.2% efficiency. Synergistic structure-guided and AI-augmented protein engineering generated two variants, enMmeFz2 and evoMmeFz2, which exhibited an average ~32-fold increase in activity across 38 genomic loci. Moreover, fusion of the non-specific DNA-binding domain HMG-D further enhanced editing performance (enMmeFz2-HMG-D and evoMmeFz2-HMG-D). Notably, evoMmeFz2-HMG-D demonstrated robust in vivo genome editing activity, enabling dystrophin restoration in humanized male Duchenne muscular dystrophy mouse models via single adeno-associated virus (AAV) delivery. This study establishes Fanzor2 as a gene editing platform for genome engineering and therapeutic applications, and underscores the power of AI-guided engineering to accelerate genome editor development while reducing experimental burden.},
}
@article {pmid41538882,
year = {2026},
author = {Loubat, A and Wolfender, C and Calabre, M and Beaude, N and Tavares, P and Planson, AG and Jules, M},
title = {Advancing Fast-Track Genome Engineering in Bacillus subtilis Phages.},
journal = {ACS synthetic biology},
volume = {15},
number = {2},
pages = {687-700},
doi = {10.1021/acssynbio.5c00727},
pmid = {41538882},
issn = {2161-5063},
mesh = {*Bacillus subtilis/virology/genetics ; *Genome, Viral/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Bacillus Phages/genetics ; *Genetic Engineering/methods ; },
abstract = {Phage genome engineering methods accelerate the study of phage biology, the discovery of new functions, and the development of innovative genetic engineering tools. Here, we present QuickPhage, a rapid, technically accessible, precise, and cost-effective method for engineering Bacillus subtilis phages. Our approach uses CRISPR-Cas9 as a counter-selection system to isolate mutants of the model lytic siphovirus phage, SPP1. Efficient genome editing was achieved using homologous repair patches as short as 40 nucleotides, enabling streamlined patch construction and parallel engineering, resulting in highly accurate genome edits within a day. We applied QuickPhage to delete both essential and nonessential phage genes and to insert reporter genes. Protein production, such as GFP, was synthetically regulated using inducible systems without significantly affecting phage fitness, achieving induction levels of up to 400-fold. Time-series coinfection experiments with fluorescent protein expressing phages also revealed a highly efficient superinfection arrest mechanism that prevents reinfection as early as 13 min after initial infection. These findings highlight the potential of phages for protein production, opening new opportunities for metabolic engineering. This work also lays the foundation for systematic phage genome refactoring workflows and the development of phage-based tools for efficient DNA delivery, thereby expanding the synthetic biology toolbox for B. subtilis.},
}
@article {pmid41529994,
year = {2026},
author = {Ebe, S and Nakamura, H and Matsuda, M and Terauchi, Y and Akada, R and Hoshida, H},
title = {Identification and overexpression of genes encoding sugar alcohol transporter and metabolic enzymes for accelerated utilization in the yeast Kluyveromyces marxianus.},
journal = {Journal of bioscience and bioengineering},
volume = {141},
number = {4},
pages = {221-229},
doi = {10.1016/j.jbiosc.2025.12.003},
pmid = {41529994},
issn = {1347-4421},
mesh = {*Kluyveromyces/genetics/metabolism/enzymology/growth &amp; development ; Mannitol/metabolism ; Sorbitol/metabolism ; CRISPR-Cas Systems ; *Fungal Proteins/genetics/metabolism ; L-Iditol 2-Dehydrogenase/genetics/metabolism ; },
abstract = {The yeast Kluyveromyces marxianus assimilates various sugars, including sorbitol and mannitol. However, the metabolic pathways for sugar utilization, including sugar transporters, remain to be elucidated. To identify these genes in this study, first 13 candidate transporter genes were disrupted using a newly developed non-homologous end joining (NHEJ)-mediated gene disruption method, combined with targeted digestion using the CRISPR-Cas9 system. While most disruptants exhibited no clear growth defects in various sugar media, a disruptant of the KmMLEV2025 gene (named KmSAT1) failed to grow in either sorbitol or mannitol media, suggesting that it encodes a sugar alcohol transporter. Next, we investigated the candidate dehydrogenase genes crucial for sugar alcohol metabolism, as they are converted to fructose by dehydrogenases. KmXyl2p, a known xylitol dehydrogenase, is a candidate sorbitol dehydrogenase. Disruption of KmXYL2 caused growth defects in sorbitol medium, but not in mannitol medium. We disrupted several genes to identify the mannitol dehydrogenase, revealing that the disruption of KmSOU2, annotated as a sorbose reductase, resulted in a growth defect in the mannitol medium. The identified genes were overexpressed for the efficient utilization of sugar alcohols. The strain overexpressing KmSAT1, but not the dehydrogenase genes, started growing immediately, whereas the wild-type strain exhibited a lag time of several days. Furthermore, the final cell optical densities in both the sorbitol and mannitol media were higher than those observed in the glucose medium. These results indicated that overexpression of a sugar alcohol transporter is a highly effective strategy for biotechnological applications.},
}
@article {pmid41526975,
year = {2026},
author = {Hu, Q and Zhang, R and Liu, J and Zhang, W and Liao, X and Guo, Y and Lu, Q and Yang, B and Zhang, T and Zhai, X and Luo, Q},
title = {A rapid and field-deployable RAA-CRISPR/Cas12a platform for detection of Mycoplasma gallisepticum in poultry.},
journal = {BMC veterinary research},
volume = {22},
number = {1},
pages = {117},
pmid = {41526975},
issn = {1746-6148},
support = {2025BEB053//the Hubei Province Technology Innovation Plan Project/ ; 2024BBA004//the Hubei Province Technology Innovation Plan Project/ ; NYWSWZX2025-3432027-04//the Major Special Project for the Development of Agricultural Microbial Industry in Hubei Province/ ; 2023HBSTX4-04//the Hubei Province Modern Agricultural Industry Technology System/ ; CARS-41//the China Agriculture Research System/ ; },
mesh = {Animals ; *Mycoplasma gallisepticum/isolation &amp; purification/genetics ; *Poultry Diseases/diagnosis/microbiology ; *Mycoplasma Infections/veterinary/diagnosis/microbiology ; *CRISPR-Cas Systems ; Chickens ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; Recombinases ; Ducks ; Columbidae ; },
abstract = {BACKGROUND: Mycoplasma gallisepticum (MG) is a major pathogen that causes respiratory diseases 14in poultry, resulting in reduced production and severe economic losses. Current MG detection methods are time-consuming, labor-intensive, and expensive. Hence, the rapid and accurate detection of MG is critical for effective disease control. Therefore, this study aimed to develop a dual-mode diagnostic assay for sensitive and specific detection of MG by combining recombinase-aided amplification (RAA) with CRISPR/Cas12a technology. Conserved regions of the mgc2 gene were used for primer and CRISPR RNA design, and the reaction conditions were optimized to maximize detection efficiency.<br><br>RESULTS: The assay achieved a detection limit of 2 copies/&#xb5;L and demonstrated high specificity against seven other common avian pathogens. Detection was visualized within 1&#xa0;h using either fluorescence or lateral flow dipstick. Moreover, clinical validation of chicken samples showed complete concordance with quantitative real-time polymerase chain reaction results. Furthermore, an epidemiological investigation revealed that chickens had the highest positivity rate for MG among chickens, ducks, and pigeons in Hubei Province.<br><br>CONCLUSIONS: This simple, rapid, field-deployable method is valuable for timely MG surveillance and effective disease management in poultry production.},
}
@article {pmid41486850,
year = {2026},
author = {Shi, M and Yu, P and Liu, L and Cheng, J and Shao, R and Sun, Y and Lv, J and Li, Y and Zheng, Z and Yu, J and Xu, B and Gan, L and Liang, Y and Zhang, Y and Fang, Y and Shen, W and Huang, J and Zhu, X and Hong, J and Ma, R and Wu, L and Cheng, Y and Zhao, C},
title = {Fluoropolymer-Mediated Delivery of a Dual TSHR/IGF1R-Targeting CRISPR-Cas9 System for Localized Therapy in Thyroid-Associated Ophthalmopathy.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {11},
pages = {e11078},
doi = {10.1002/adma.202511078},
pmid = {41486850},
issn = {1521-4095},
support = {DGF828030-3/005//Clinical Scientist Foundation of Fudan University/ ; pp25014//Clinical Scientist Foundation of Fudan University/ ; U25A20258//National Natural Science Foundation of China/ ; 82271126//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Receptor, IGF Type 1/genetics/metabolism ; *Graves Ophthalmopathy/therapy/genetics/pathology ; *CRISPR-Cas Systems/genetics ; Humans ; Mice ; *Receptors, Thyrotropin/genetics/metabolism ; Gene Editing ; *Polymers/chemistry ; Fibroblasts/metabolism ; Disease Models, Animal ; Adipocytes/metabolism ; Genetic Therapy ; Organoids/metabolism ; Antibodies, Monoclonal, Humanized ; },
abstract = {Thyroid-associated ophthalmopathy (TAO), a vision-threatening and disfiguring autoimmune orbital disorder, remains a therapeutic challenge due to the lack of therapies with orbital specificity, sustained efficacy, and minimal side effects. Herein, we present G4F7-CRISPR, a fluoropolymer-based CRISPR-Cas9 delivery platform engineered for localized and efficient disruption of thyroid-stimulating hormone receptor (TSHR) and insulin-like growth factor 1 receptor (IGF1R), two key mediators of TAO pathogenesis. G4F7-CRISPR achieved high insertion/deletion frequencies in primary orbital fibroblasts (Tshr: 37.2%; Igf1r: 42.8%) and mature adipocytes (Tshr: 22.4%; Igf1r: 24.3%), and maintained robust editing efficiency in orbital adipose tissue of TAO mouse models (Tshr: 30.7%; Igf1r: 32.4%). In both TAO mouse models and 3D human orbital organoids, dual-gene editing of Tshr and Igf1r via G4F7-CRISPR significantly suppressed orbital adipogenesis, inflammation, and fibrosis, demonstrating superior therapeutic efficacy over either single-gene approaches. Comprehensive off-target analyses in both TAO mouse models and orbital organoids revealed minimal off-target activity. Furthermore, G4F7-CRISPR exhibited excellent short- and long-term ocular and systemic safety in TAO mouse models. Notably, it outperformed teprotumumab-the FDA-approved therapy for TAO-in both therapeutic efficacy and safety, highlighting its potential clinical advantages. Collectively, these findings highlight the translational promise of G4F7-CRISPR as a safe, precise, and clinically viable gene therapy for TAO.},
}
@article {pmid41254174,
year = {2026},
author = {Thomson, T and Li, G and Strilchuk, A and Cui, H and Wang, B and Li, B},
title = {Harnessing artificial intelligence to advance CRISPR-based genome editing technologies.},
journal = {Nature reviews. Genetics},
volume = {27},
number = {3},
pages = {212-230},
pmid = {41254174},
issn = {1471-0064},
mesh = {*Gene Editing/methods ; Humans ; *Artificial Intelligence ; *CRISPR-Cas Systems/genetics ; Animals ; Machine Learning ; Deep Learning ; },
abstract = {CRISPR-based genome editing technologies, including nuclease-based editing, base editing and prime editing, have revolutionized biological research and modern medicine by enabling precise, programmable modification of the genome and offering new therapeutic strategies for a wide range of genetic diseases. Artificial intelligence (AI), including machine learning and deep learning models, is now further advancing the field by accelerating the optimization of gene editors for diverse targets, guiding the engineering of existing tools and supporting the discovery of novel genome-editing enzymes. In this Review, we summarize key AI methodologies underlying these advances and discuss their recent noteworthy applications to genome editing technologies. We also discuss emerging opportunities, such as AI-powered virtual cell models, which can guide genome editing through target selection or prediction of functional outcomes. Finally, we identify key directions where the integration of AI methods is poised to have a substantial impact going forward.},
}
@article {pmid40738974,
year = {2026},
author = {Qu, Y and Huang, K and Yin, M and Zhan, K and Liu, D and Yin, D and Cousins, HC and Johnson, WA and Wang, X and Shah, M and Altman, RB and Zhou, D and Wang, M and Cong, L},
title = {CRISPR-GPT for agentic automation of gene-editing experiments.},
journal = {Nature biomedical engineering},
volume = {10},
number = {2},
pages = {245-258},
pmid = {40738974},
issn = {2157-846X},
support = {R35HG011316, 1R01GM141627//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; 1653435//National Science Foundation (NSF)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Artificial Intelligence ; Automation ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Performing effective gene-editing experiments requires a deep understanding of both the CRISPR technology and the biological system involved. Meanwhile, despite their versatility and promise, large language models (LLMs) often lack domain-specific knowledge and struggle to accurately solve biological design problems. We present CRISPR-GPT, an LLM agent system to automate and enhance CRISPR-based gene-editing design and data analysis. CRISPR-GPT leverages the reasoning capabilities of LLMs for complex task decomposition, decision-making and interactive human-artificial intelligence (AI) collaboration. This system incorporates domain expertise, retrieval techniques, external tools and a specialized LLM fine tuned with open-forum discussions among scientists. CRISPR-GPT assists users in selecting CRISPR systems, experiment planning, designing guide RNAs, choosing delivery methods, drafting protocols, designing assays and analysing data. We showcase the potential of CRISPR-GPT by knocking out four genes with CRISPR-Cas12a in a human lung adenocarcinoma cell line and epigenetically activating two genes using CRISPR-dCas9 in a human melanoma cell line. CRISPR-GPT enables fully AI-guided gene-editing experiment design and analysis across different modalities, validating its effectiveness as an AI co-pilot in genome engineering.},
}
@article {pmid41699287,
year = {2026},
author = {Pindi, C and Palermo, G},
title = {Computation and deep-learning-driven advances in CRISPR genome editing.},
journal = {Nature structural &amp; molecular biology},
volume = {33},
number = {2},
pages = {203-214},
pmid = {41699287},
issn = {1545-9985},
mesh = {*Deep Learning ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; Neural Networks, Computer ; Algorithms ; },
abstract = {Genome editing with CRISPR-Cas systems is revolutionizing medicine, molecular biology and biotechnology. In this Review, we discuss the contributions of deep learning-based structure prediction algorithms, physics-based simulations, neural networks, graph neural networks and generative models, including diffusion and large language models, in engineering and optimizing CRISPR systems and in understanding their mechanistic basis. We highlight the challenges and limitations to the transformative effects of computational modeling and tools in the context of the development of programmable genome editors for biomedicine and biotechnology.},
}
@article {pmid41696049,
year = {2026},
author = {Ahmadzadeh, M and Akbarian, F and Sanati, MH and Motaharirad, H and Farrokhi, F},
title = {Computational Optimization of CRISPR-Cas13a sgRNAs Targeting the SARS-CoV-2 Spike Gene for SHERLOCK-Based Diagnostics.},
journal = {Evolutionary bioinformatics online},
volume = {22},
number = {},
pages = {11769343251414318},
pmid = {41696049},
issn = {1176-9343},
abstract = {BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global health crisis, emphasizing the urgent need for accurate and rapid diagnostic tools. Modern molecular biology technologies, including CRISPR-Cas systems, provide highly efficient strategies for viral detection. Bioinformatic pipelines are essential for identifying conserved genomic regions and enabling rational single-guide RNA (sgRNA) design.<br><br>METHODS: This study aimed to design specific sgRNAs targeting the spike gene of SARS-CoV-2 isolates from Iranian patients using the SHERLOCK diagnostic platform. Complete genomes of the RefSeq virus and 470 SARS-CoV-2 isolates, representing all variants of concern (VOCs) detected in Iran, were retrieved from the NCBI and GISAID databases. Multiple sequence alignment with ClustalW identified conserved sequences within the receptor-binding domain (RBD) that differ from the RBD of SARS-CoV and MERS-CoV RefSeq genomes. Based on these regions, sgRNAs and isothermal amplification primers were designed using ADAPT, OLIGO7, and the UCSC Genome Browser to maximize diagnostic sensitivity and specificity. Secondary and tertiary structures of sgRNA-target complexes were analyzed via RNAfold and RNAup to select the most efficient sgRNA-amplicon combination.<br><br>RESULTS: Twenty-two-nucleotide sgRNA candidates were initially selected based on sequence alignment, showing high similarity to the SARS-CoV-2 RefSeq and low homology to SARS-CoV and MERS-CoV genomes. Analyses of secondary structures, RNA-RNA interactions, and free energy identified 6 sgRNAs with favorable 2-dimensional conformations and strong interaction profiles. Among these, the sgRNA1-Amplicon2 sequence exhibited the most stable 3-dimensional structure and a molecular docking score of -309.67, indicating high sensitivity and specificity for viral detection.<br><br>CONCLUSION: This study successfully designed an sgRNA with high sensitivity and specificity for rapid SARS-CoV-2 detection using the CRISPR-Cas13a system, informed by genomic analysis of Iranian isolates. The proposed approach provides an efficient framework for the rapid design and deployment of CRISPR-based diagnostic tools applicable to diverse viral pathogens.},
}
@article {pmid41695711,
year = {2026},
author = {Browne, TS and Edgell, DR and Gloor, GB},
title = {Better data for better predictions: data curation improves deep learning for sgRNA/Cas9 prediction.},
journal = {PeerJ},
volume = {14},
number = {},
pages = {e20706},
pmid = {41695711},
issn = {2167-8359},
mesh = {*Deep Learning ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Gene Editing/methods ; },
abstract = {The Cas9 enzyme along with a single guide RNA molecule is a modular tool for genetic engineering and has shown effectiveness as a species-specific antimicrobial. The ability to accurately predict on-target cleavage is critical as activity varies by target. Using the sgRNA nucleotide sequence and an activity score, predictive models have been developed with the best performance resulting from deep learning architectures. Prior work has emphasized robust and novel architectures to improve predictive performance. Here, we explore the impact of a data-centric approach through optimization of the input target site adjacent nucleotide sequence length and the use of data filtering for read counts in the control conditions to improve input data utility. Using the existing crisprHAL architecture, we develop crisprHAL Tev, a bacterial SpCas9 prediction model with performance that generalizes across related species and across data types. During this process, we also rebuilt two prior Escherichia coli Cas9 datasets, demonstrating the importance of data quality, and resulting in the production of an improved bacterial eSpCas9 prediction model. The crisprHAL models are available through GitHub: https://github.com/tbrowne5/crisprHAL.},
}
@article {pmid41693565,
year = {2026},
author = {Sinnott, RW and Solanki, A and Govind, AP and Green, WN and Dickinson, BC},
title = {Engineering a human-based translational activator for targeted protein expression restoration.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41693565},
issn = {1362-4962},
support = {FP106237//G. Harold and Leila Y. Mathers Charitable Foundation/ ; //Dr. Ralph and Marian Falk Medical Research Trust/ ; //Bank of America/ ; /EB/NIBIB NIH HHS/United States ; R01-EB035016/NH/NIH HHS/United States ; DGE-2022294368//National Science Foundation/ ; },
mesh = {Animals ; Humans ; Mice ; *Protein Biosynthesis ; *Epilepsies, Myoclonic/genetics/therapy ; Disease Models, Animal ; RNA, Messenger/genetics/metabolism ; *NAV1.1 Voltage-Gated Sodium Channel/genetics/metabolism ; CRISPR-Cas Systems ; *Protein Engineering/methods ; Haploinsufficiency ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Therapeutic modalities to programmably increase protein production are in critical need to address diseases caused by deficient gene expression via haploinsufficiency. Restoring physiological protein levels by increasing translation of their cognate messenger RNA (mRNA) would be an advantageous approach to correct gene expression but has not been evaluated in an in vivo disease model. Here, we investigated whether a translational activator could improve phenotype in a Dravet syndrome mouse model, a severe developmental and epileptic encephalopathy caused by SCN1a haploinsufficiency, by increasing translation of the SCN1a mRNA. We identify and engineer human proteins capable of increasing mRNA translation using the CRISPR-Cas-inspired RNA-targeting system (CIRTS) platform to enable programmable, guide RNA-directed translational activation with entirely engineered human proteins. We identify a compact (601 amino acid) CIRTS translational activator (CIRTS-4GT3) that can drive targeted, sustained translation increases up to 100% from three endogenous transcripts relevant to epilepsy and neurodevelopmental disorders. AAV-delivery of CIRTS-4GT3 targeting SCN1a mRNA to a Dravet syndrome mouse model led to increased SCN1a translation and improved survivability and seizure threshold-key phenotypic indicators of Dravet syndrome. This work validates a strategy to address SCN1a haploinsufficiency and emphasizes the preclinical potential of targeted translational activation to address neurological haploinsufficiency.},
}
@article {pmid41691452,
year = {2026},
author = {Li, T and Zeng, F and Zhang, J and Zhang, Y and Yin, W},
title = {Reversing Antibiotic Resistance: Strategies From Adjuvants to Innovative Therapeutics.},
journal = {MicrobiologyOpen},
volume = {15},
number = {1},
pages = {e70233},
pmid = {41691452},
issn = {2045-8827},
support = {H2024201044//Natural Science Foundation of Hebei Province/ ; 20231560//The Medical Scientific Research of Hebei Health Commission/ ; },
mesh = {*Anti-Bacterial Agents/pharmacology/therapeutic use ; Humans ; *Bacteria/drug effects/genetics ; *Drug Resistance, Bacterial/drug effects/genetics ; Gene Editing ; *Bacterial Infections/drug therapy/microbiology ; CRISPR-Cas Systems ; Photochemotherapy ; Nanotechnology ; },
abstract = {The escalating prevalence of antibiotic resistance has become a major threat to the effectiveness of conventional antibiotics. Meanwhile, the development of novel antibiotics faces substantial challenges, including lengthy research cycles, high costs, and the rapid emergence of bacterial tolerance, making it difficult for new drugs to keep pace with bacterial evolution. In this context, molecular reversal strategies targeting antibiotic resistance genes have emerged as a promising avenue to overcome this impasse. Among them, the use of antibiotic adjuvants, agents that enhance the efficacy of existing antibiotics by inhibiting resistance gene function, preventing their horizontal transfer or modulating host defense has gained considerable attention. Furthermore, innovative approaches such as CRISPR-Cas gene editing, photodynamic therapy, nanotechnology, and ecological competition strategies have shown great potential in reversing antimicrobial resistance. Collectively, these strategies offer novel insights into addressing the global crisis of antibiotic resistance, paving the way for more effective clinical interventions and ensuring the sustained efficacy of current antibiotic therapies.},
}
@article {pmid41691442,
year = {2026},
author = {Thevendran, R and Maheswaran, S},
title = {Molecular Genetics as the Leading-Edge Approach in Driving the Development of Live Attenuated Vaccines.},
journal = {Biotechnology journal},
volume = {21},
number = {2},
pages = {e70185},
doi = {10.1002/biot.70185},
pmid = {41691442},
issn = {1860-7314},
support = {//MOHE/ ; },
mesh = {*Vaccines, Attenuated/genetics/immunology ; Humans ; *Vaccine Development/methods ; Gene Editing ; *Molecular Biology/methods ; Animals ; CRISPR-Cas Systems ; Biotechnology ; },
abstract = {Molecular genetics has propelled advancements in scientific instrumentation, yielding transformative discoveries from CRISPR-mediated gene editing and detailed protein identification to sophisticated biosensor fabrications. This profound shift has also reshaped the landscape of live attenuated vaccine (LAV) development in contrast to conventional methods. Here we explore how modern molecular strategies have superseded previous empirical approaches, moving toward deliberate genetic modifications that both enhance and balance the aspects of LAV safety, stability, and potent immunogenicity. By detailing the transition from classical approaches to targeted, molecular-driven attenuation, our work reviews how advanced genetic methods address historical limitations and expand the potential for vaccine design. Hence, the current paper bridges the gap between foundational vaccine practices and cutting-edge biotechnology, offering a comprehensive perspective on the progression of molecular genetic strategies on LAV development and its future trajectory. The paper also elaborates on the key challenges of raising LAVs to clinical standards while describing in tandem the genetic approaches to overcome the limitations. Critical performance factors governing the LAV market and clinical deployment, alongside the pivotal role of artificial intelligence in refining LAV rational design, are also further discussed.},
}
@article {pmid41690735,
year = {2026},
author = {Li, G and Su, Z and Li, F and Liu, J and Shi, Y and Chen, J and Wang, P and Wang, R},
title = {Advances in rapid on-site detection techniques for food safety and authenticity.},
journal = {Advances in food and nutrition research},
volume = {118},
number = {},
pages = {43-87},
doi = {10.1016/bs.afnr.2025.08.004},
pmid = {41690735},
issn = {1043-4526},
mesh = {*Food Safety/methods ; *Food Contamination/analysis ; Biosensing Techniques/methods ; *Food Analysis/methods ; Humans ; Food Microbiology ; },
abstract = {Ensuring food safety and authenticity is a pressing global concern. This chapter provides a comprehensive overview of advanced rapid, on-site detection technologies targeting foodborne hazards, including pathogens, toxins, chemical contaminants, and authenticity issues such as adulteration and species fraud. Key aspects of these technologies include signal amplification strategies like catalytic hairpin assembly (CHA) and nanozyme-enhanced systems, as well as signal output platforms encompassing colorimetric, electrochemical, and fluorescence-based methods. The chapter also explores the emerging role of nanopore-based sensing as a novel signal output platform, emphasizing its single-molecule precision and broad applicability. By integrating laboratory innovations with practical field applications, this chapter underscores the potential of these technologies to address challenges in food safety and authenticity monitoring effectively.},
}
@article {pmid41679990,
year = {2026},
author = {Carota, AG and Spiaggia, F and Poma, N and Palladino, P and Cuffaro, D and Vivaldi, F and Ravelet, C and Di Francesco, F and Minunni, M},
title = {Highly fluorescent copper nanoclusters as programmable reporters for CRISPR/Cas12a-based detection of bacterial DNA.},
journal = {Biosensors &amp; bioelectronics},
volume = {300},
number = {},
pages = {118492},
doi = {10.1016/j.bios.2026.118492},
pmid = {41679990},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Copper/chemistry ; *Biosensing Techniques/methods ; *DNA, Bacterial/isolation &amp; purification/genetics ; Fluorescent Dyes/chemistry ; *Escherichia coli/genetics/isolation &amp; purification ; *Metal Nanoparticles/chemistry ; Humans ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Early and accessible pathogen detection is crucial for global health security and demands diagnostic assays that are rapid, affordable, and suitable for Point-of-Care use. This study presents a cost-effective, rapid, one-pot fluorescence assay for bacterial DNA detection that exploits the unique optical properties of DNA-templated copper nanoclusters (CuNCs). These nanoclusters offer a sustainable alternative to conventional fluorophores, thanks to their eco-friendly synthesis, high photostability, and large Stokes shift. The assay integrates CuNCs with the CRISPR/Cas12a system to achieve programmable and highly specific target recognition. Upon target binding, activation of the Cas12a/gRNA complex triggers collateral cleavage of rationally designed DNA templates that normally support CuNCs formation, resulting in a marked fluorescence decrease. A panel of hairpin and poly-thymine DNA structures was systematically evaluated to maximize both CuNCs fluorescence and responsiveness to Cas12a/gRNA trans-cleavage, ultimately identifying an AT-rich stem-loop reporter that provided strong signal intensity and complete signal shutdown upon target recognition. The final CRISPR-CuNCs assay achieved picomolar sensitivity, accurately detected E. coli DNA from reference strains, clinical isolates, and serum-spiked samples, and required no fluorophore-quencher probes or multistep procedures. Overall, this work demonstrated that combining the programmability of CRISPR/Cas12a with the versatility and low-cost of DNA-templated CuNCs enables a robust and accessible platform for molecular diagnostics, with strong potential for Point-of-Care deployment.},
}
@article {pmid41679028,
year = {2026},
author = {Yao, F and Qi, X and Yongli, S and Xiaofen, Z},
title = {Generation of a SV2A knockout human embryonic stem cell line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103924},
doi = {10.1016/j.scr.2026.103924},
pmid = {41679028},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Membrane Glycoproteins/genetics/metabolism/deficiency ; *Nerve Tissue Proteins/genetics/metabolism/deficiency ; *Gene Knockout Techniques ; Cell Line ; },
abstract = {Synaptic Vesicle Glycoprotein 2A (SV2A) is a ubiquitously expressed brain glycoprotein, localized to synaptic terminals. It regulates vesicle exocytosis, maintains neurotransmitter release, and serves as a receptor for both botulinum neurotoxins (e.g., BoNT/A) and tetanus neurotoxin (TeNT). It is a target for antiseizure drugs and implicated in epilepsy, Alzheimer's, and Parkinson's diseases. We generated a homozygous SV2A-knockout human embryonic stem cell (hESC) line WAe001-A-3F (H1-SV2A[-/-]), using CRISPR/Cas9 genome editing technology. The SV2A-knockout embryonic stem cell lines provide a precise in vitro model to dissect its roles in synaptic function and disease mechanisms.},
}
@article {pmid41655494,
year = {2026},
author = {Kantor, I and Wright, JL and Amor, DJ and Lockhart, PJ},
title = {Generation of two tetracycline-inducible NGN2 iN iPSC lines carrying a heterozygous floating-Harbor syndrome SRCAP truncating mutation.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103922},
doi = {10.1016/j.scr.2026.103922},
pmid = {41655494},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology/drug effects ; Humans ; *Nerve Tissue Proteins/genetics/metabolism ; *Mutation/genetics ; *Tetracycline/pharmacology ; Heterozygote ; Cell Line ; Cell Differentiation ; CRISPR-Cas Systems ; },
abstract = {Floating-Harbor syndrome (FHS) is a rare neurodevelopmental disorder caused by truncating variants in the last two exons of the gene encoding the chromatin remodeler SRCAP. We used CRISPR-Cas9 genome editing to introduce a monoallelic c.7330C > T (p.Arg2444*) truncating mutation into a published WTC11 iPSC line containing a tetracycline-inducible NGN2 transgene. We characterised two independent lines that maintained a normal karyotype, pluripotency and the ability to differentiate in vitro into all three embryonic germ layers. These lines can be rapidly differentiated into cortical neurons through the addition of doxycycline, making them a useful model for understanding the pathogenic mechanisms underlying FHS.},
}
@article {pmid41653914,
year = {2026},
author = {Chandrasekaran, SS and Tau, C and Fu, BXH and Nemeth, M and Bartie, L and Pawluk, A and Konermann, S and Hsu, PD},
title = {Rewriting endogenous human transcripts with dual CRISPR-guided 3' trans-splicing.},
journal = {Cell systems},
volume = {17},
number = {2},
pages = {101487},
doi = {10.1016/j.cels.2025.101487},
pmid = {41653914},
issn = {2405-4720},
mesh = {Humans ; *Trans-Splicing/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Messenger/genetics ; RNA Editing/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; RNA Precursors/genetics ; Exons/genetics ; },
abstract = {Unlike genome editing, RNA editing offers the ability to transiently alter cells with minimal risk from off-target effects. While exon-skipping technologies can influence splice site selection, many desired perturbations to the transcriptome require replacement or addition of exogenous exons to target mRNAs, such as replacing disease-causing exons, repairing truncated proteins, or engineering protein fusions. Here, we report the development of RNA-guided trans-splicing with Cas editor (RESPLICE). RESPLICE uses two orthogonal RNA-targeting CRISPR effectors to co-localize a trans-splicing pre-mRNA and to inhibit the cis-splicing reaction, respectively. We demonstrate efficient, specific, and programmable trans-splicing of RNA cargo (up to 2.1 kb) into 11 endogenous transcripts across 3 cell types, achieving up to 45% trans-splicing efficiency in bulk or 90% when sorting for high effector expression. Our results present RESPLICE as a mode of RNA editing that could provide fine-tuned and transient control of cellular programs.},
}
@article {pmid41649621,
year = {2026},
author = {Alavian, F and Ghasemi, S},
title = {CRISPR-Based Therapy for Ischemic Stroke: A Narrative Review.},
journal = {Cellular and molecular neurobiology},
volume = {46},
number = {1},
pages = {45},
pmid = {41649621},
issn = {1573-6830},
mesh = {Humans ; Animals ; *Ischemic Stroke/therapy/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods ; },
abstract = {Ischemic stroke (IS) is one of the most common neurological diseases worldwide and is caused by the blockage of cerebral blood vessels, leading to reduced blood flow and neuronal damage. Given the limitations of existing treatments, CRISPR gene-editing technology has emerged as a promising strategy to precisely target the molecular pathways underlying IS pathophysiology. By enabling intervention in genes regulating inflammation, apoptosis, and repair, CRISPR enables more precise and effective therapies. Various CRISPR delivery systems, including viral vectors, nanocarriers, and extracellular vesicles, play crucial roles in the effective access of this tool to neural cells. Studies have shown that the use of CRISPR-Cas9 to modulate key pathogenic pathways, including those governing inflammation, oxidative stress, and cell death, can prevent neuronal damage and improve neurological function. Additionally, targeting ncRNAs and RNA methylation with CRISPR-based systems plays a role in regulating oxidative stress and stress granule formation. The use of CRISPR to modulate cell communication and organelle transfer and correct mitochondrial mutations has also been considered a neuroprotective mechanism. Despite persistent challenges in targeted and safe delivery, substantial preclinical advances, primarily in rodent models, underscore the potential for CRISPR-based therapies to transform future stroke treatment. These findings suggest that CRISPR-based strategies could evolve into precision neurotherapeutics that address root molecular pathologies, potentially complementing or surpassing current stroke interventions.},
}
@article {pmid41645777,
year = {2026},
author = {Wu, H and Lin, S and Zuo, X and Hua, W and Sun, T and Shao, G and Li, F and Zhao, D and Feng, Z and Zhu, D and Wang, L},
title = {RAA-CRISPR-HCR cascade amplification for ultrasensitive visual detection of African swine fever virus DNA.},
journal = {Chemical communications (Cambridge, England)},
volume = {62},
number = {14},
pages = {4329-4332},
doi = {10.1039/d5cc06815k},
pmid = {41645777},
issn = {1364-548X},
mesh = {*African Swine Fever Virus/genetics/isolation &amp; purification ; *DNA, Viral/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods ; Animals ; Colorimetry/methods ; Limit of Detection ; Swine ; *CRISPR-Cas Systems ; African Swine Fever/diagnosis/virology ; },
abstract = {An RAA-CRISPR-HCR (RCH) cascade amplification system was developed, which generates an ultrasensitive colorimetric response to low concentrations of African swine fever virus (ASFV) DNA with a detection limit of 1 copy µL[-1]. It demonstrates excellent accuracy and specificity in real samples, facilitating efficient on-site visual early detection of ASFV.},
}
@article {pmid41643424,
year = {2026},
author = {Wang, S and Li, C and Zhu, L and Liu, K and Jing, L and Xie, L and Guo, H and Ge, S and Yu, J},
title = {Engineered DNA hydrogel paper chip biosensor by cascaded hybridization chain reaction-assisted CRISPR/Cas12a system for sensitive detection of miRNA 622.},
journal = {Biosensors &amp; bioelectronics},
volume = {300},
number = {},
pages = {118482},
doi = {10.1016/j.bios.2026.118482},
pmid = {41643424},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation &amp; purification/analysis ; *Biosensing Techniques/instrumentation ; CRISPR-Cas Systems/genetics ; Hydrogels/chemistry ; Limit of Detection ; Nucleic Acid Hybridization ; Humans ; Paper ; *DNA/chemistry/genetics ; Endodeoxyribonucleases/chemistry/genetics ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Electrochemical Techniques ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {The integration of DNA hydrogel with electrochemiluminescence (ECL) technology represents a synergistic enhancement through molecular-level precision design and nanoscale coordination. This strategic integration confers biosensors novel functionalities including intelligent responsiveness and environmental adaptability. In this study, a cascaded hybridization chain reaction (HCR) and CRISPR/Cas12a-integrated DNA hydrogel paper chip was engineered for ultrasensitive microRNA 622 (miRNA 622) detection. Target miRNA 622 triggered HCR amplification via hairpin DNA assembly, while Cas12a recognized protospacer adjacent motif (PAM) sequences within the HCR-generated double-stranded products to activate its trans-cleavage ability. The DNA hydrogel was constructed through copolymerization of acrylamide-modified DNA single strands (SA and SB) with Ru (II) complex-functionalized linker DNA. Activated Cas12a cleaved single-stranded DNA within the DNA hydrogel network, thereby releasing Ru (II) complexes. AuPd nanoparticles (AuPd NPs) served as the co-reactant accelerator, amplifying the cathodic ECL signals of the liberated Ru (II) complexes. The developed platform demonstrated a dynamic detection range from 0.001 to 500 pM with a detection limit of 0.33 fM, establishing a groundbreaking approach for detecting miRNA 622 in clinical diagnostics.},
}
@article {pmid41642051,
year = {2026},
author = {Yang, Y and Yang, L and Ma, H and Zhang, S and Zhu, Y and Zhang, S and Lin, X and La, H and Gu, X and Ma, J and Zhao, S and Yang, Y and Lei, H and Yang, Y},
title = {Precision detection of rifampicin-resistant rpoB_L378R mutation in Mycobacterium tuberculosis with CRISPR-Cas12a.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {7},
pages = {1442-1453},
doi = {10.1039/d5ay01718a},
pmid = {41642051},
issn = {1759-9679},
mesh = {*Rifampin/pharmacology ; *Mycobacterium tuberculosis/genetics/drug effects ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Bacterial/genetics ; *Bacterial Proteins/genetics ; *DNA-Directed RNA Polymerases/genetics ; Mutation ; Humans ; Antitubercular Agents/pharmacology ; Nucleic Acid Amplification Techniques/methods ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rifampicin is one of the most effective anti-tuberculosis drugs. However, certain strains of Mycobacterium tuberculosis (MTB) have developed resistance to rifampicin, making it crucial to identify alternative drugs for treating rifampicin-resistant MTB infections. Mutations in the rpoB gene play a pivotal role in MTB's resistance to rifampicin. Therefore, identifying these mutations is essential for effectively managing rifampicin-resistant MTB strains. Here, we developed a CRISPR-Cas12a platform integrated with recombinase polymerase amplification (RPA) and fluorescence detection, which was specifically designed to identify the rpoB_L378R mutation associated with rifampicin resistance in MTB. Our findings indicated that this detection technique exhibited high specificity and did not cross-react with reference samples constructed from the genomes of MTB H37Rv, Mycobacterium smegmatis, Mycobacterium aurum, and Escherichia coli. The RPA-CRISPR-Cas12a-based platform established in this research was simple, sensitive, and specific for detecting the rifampicin-resistant MTB strain with the rpoB_L378R mutation. These results suggest its potential applicability in clinical diagnosis for identifying the MTB rpoB_L378R mutation.},
}
@article {pmid41638184,
year = {2026},
author = {Li, Z and Zhang, W and Feng, Z and Liu, Z and Feng, Z and Shi, Y and Zhan, J and Zhang, J},
title = {A turn-on CRISPR/Cas12a strategy featuring a sterically-hindered activator for in situ fluorescence imaging of H2O2 in vivo.},
journal = {Biosensors &amp; bioelectronics},
volume = {300},
number = {},
pages = {118449},
doi = {10.1016/j.bios.2026.118449},
pmid = {41638184},
issn = {1873-4235},
mesh = {*Hydrogen Peroxide/isolation &amp; purification/analysis/chemistry ; *Biosensing Techniques/methods ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Humans ; *Optical Imaging/methods ; Limit of Detection ; *Endodeoxyribonucleases/chemistry/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Hydrogen peroxide (H2O2) serves as a key biomarker of oxidative stress in pathological processes such as cancer and inflammation. However, its in vivo visualization remains challenging due to the lack of sensitive, rapid, and bioorthogonal imaging methods. Here, we present a H2O2-activatable CRISPR/Cas12a strategy, termed A-BO-CRISPR, for real-time fluorescence imaging in living systems. This biosensing strategy employs a 4-bromomethylphenylboronic acid pinacol ester-caged DNA activator whose binding to crRNA is initially blocked by steric hindrance, effectively suppressing Cas12a trans-cleavage activity. Upon encountering endogenous H2O2, the boronate ester is selectively hydrolyzed, restoring activator/crRNA hybridization and triggering amplified fluorescent signal generation via Cas12a-mediated collateral cleavage of a ssDNA reporter. The system achieves a detection limit of 0.64 μM and responds within minutes, enabling real-time monitoring of H2O2 fluxes in living cells and tumor-bearing mice. It exhibits high selectivity and robust stability in complex biological environments. By integrating a chemical gating mechanism with CRISPR-based signal amplification, this work paves the way for potential applications in probing redox biology, imaging-guided diagnostics and therapeutic monitoring.},
}
@article {pmid41628878,
year = {2026},
author = {Gao, S and Wang, L and Hou, M and Zhang, M and Zhu, X and Luo, H and Yu, X and Lv, H and Chen, S and Huang, Y and Zhang, K and Wu, J},
title = {Gene insertion and transcriptional regulation of Escherichia coli based on CRISPR-associated transposases.},
journal = {International journal of biological macromolecules},
volume = {348},
number = {},
pages = {149850},
doi = {10.1016/j.ijbiomac.2025.149850},
pmid = {41628878},
issn = {1879-0003},
mesh = {*Escherichia coli/genetics ; *Gene Expression Regulation, Bacterial ; *Transposases/genetics/metabolism ; *CRISPR-Cas Systems ; *Transcription, Genetic ; Plasmids/genetics ; },
abstract = {Synthetic biology aims to construct robust microbial cell factories for sustainable biomanufacturing. A major obstacle lies in the difficulty of efficiently integrating large polycistronic expression cassettes into the genome and flexibly regulating gene expression. Here, a programmable tool MUSCULAR-CAST was developed based on type IF CRISPR-associated transposases (CAST) system Tn6677. Using MUSCULAR-CAST, we have achieved efficient genomic integration of various size polycistronic expression cassettes (1-10K). Among these, a human milk oligosaccharide 3-fucosyllactose (3-FL) chassis strain was successfully constructed, with similar yields and better growth compared with that of plasmid expression strain, and a plasmid-free cutinase recombinant expression strain was constructed, with enzyme activity higher than that of plasmid containing strain. Meanwhile, we developed a gene repression tool Tn-CRISPRi based on the targeting module of MUSCULAR-CAST, which achieved high single-gene repression across diverse PAM sequences and near-complete dual-gene suppression (98.6-99.8%). Applying Tn-CRISPRi to repress 17 genes competing with 3-FL biosynthesis or nonessential for growth revealed that knockdown of osmoregulated periplasmic glucans biosynthesis protein H (mdoH) and motility protein A (motA) increased 3-FL titers by 2.79- and 4.4-fold, respectively. This study establishes MUSCULAR-CAST and Tn-CRISPRi as efficient tools for genomic integration and transcriptional regulation, providing a scalable framework for advanced chassis strain engineering in synthetic biology.},
}
@article {pmid41622828,
year = {2026},
author = {Sen, MK and Roy, A and Varshney, RK and Chakraborty, A},
title = {Engineering next-generation crops through CRISPR-mediated horizontal gene transfer.},
journal = {The New phytologist},
volume = {249},
number = {6},
pages = {2683-2689},
doi = {10.1111/nph.70951},
pmid = {41622828},
issn = {1469-8137},
mesh = {*Crops, Agricultural/genetics ; *Gene Transfer, Horizontal/genetics ; Gene Editing ; *CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Crops increasingly face overlapping stresses such as heat, drought, salinity, and pathogens that conventional breeding or genome editing rarely overcome in combination. To address this, we propose CRISPR-enabled horizontal gene transfer (CRISPR-HGT) as a programmable framework that recreates the evolutionary process by which plants historically acquired adaptive microbial genes. Microbial genes, refined under extreme environments, provide a naturally preadapted resource for multi-trait resilience. By integrating tools such as Cas12a, CasΦ, RNA-targeting, and dCas-based epigenome editors with AI-guided microbial gene discovery, CRISPR-HGT enables modular and inducible stress regulation. This approach shifts genome editing from allelic modification to evolution-guided design. We outline a conceptual pipeline spanning microbial gene mining to adaptive field deployment, highlighting the ecological, biosafety, and regulatory dimensions, from the European Union's cautious oversight to the UK's product-based framework. CRISPR-HGT thus introduces an evolution-informed paradigm for engineering crops that anticipate stress and sustain yield under climate uncertainty.},
}
@article {pmid41620031,
year = {2026},
author = {Jiang, Y and Yang, Z and Yang, J and Li, Y and Liu, J and Zhao, L and Ge, J},
title = {Development of a rapid and portable detection method for canine distemper virus based on CRISPR-Cas13a.},
journal = {Journal of virological methods},
volume = {342},
number = {},
pages = {115355},
doi = {10.1016/j.jviromet.2026.115355},
pmid = {41620031},
issn = {1879-0984},
mesh = {Animals ; *Distemper Virus, Canine/isolation &amp; purification/genetics ; Sensitivity and Specificity ; Dogs ; *CRISPR-Cas Systems ; *Distemper/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; RNA, Viral/genetics ; },
abstract = {Canine distemper virus (CDV) is a pathogenic microorganism that severely affects the respiratory, digestive, and nervous systems, causing multi-systemic symptoms. It infects nearly all terrestrial carnivores worldwide, particularly the Canidae and Mustelidae families, posing a serious threat to global socio-economic and public health security. Given the importance of etiological treatment and early diagnosis, developing novel detection methods with improved accuracy, rapidity, and user-friendliness is necessary for effective prevention and control of CDV infection. In this study, we established a novel testing method using recombinase-aid amplification (RAA) coupled with CRISPR-Cas13a and optimized the working concentration of CRISPR RNA (crRNA) and Cas13a for the lateral flow detection (LFD) of CDV. The RAA-CRISPR-Cas13a-LFD for CDV did not cross-react against other prevalent canine pathogens and the sensitivity can detect as little as 10[2] copies/μL of CDV cDNA plasmids. Additionally, combined with HUDSON this RAA-CRISPR-Cas13a-LFD method could be used to detect clinical samples within 1.5 h, with performance comparable to that of RT-PCR. The results for the RAA-CRISPR-Cas13a detection could be visualized using either fluorescence or lateral flow strips for in field-deployable viral diagnosis. Overall, our developed method showed good potential in point-of-care testing (POCT) to control and reduce the losses by CDV infection.},
}
@article {pmid41593699,
year = {2026},
author = {Zhu, J and Li, Y and Yu, C and Huang, W and Chen, J and Liu, X and Qin, R and Li, J and Xu, R and Wei, P},
title = {Engineering hypercompact IscB nucleases for efficient and versatile genome editing in rice.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {49},
pmid = {41593699},
issn = {1474-760X},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Genome, Plant ; *Endonucleases/genetics/metabolism ; },
abstract = {BACKGROUND: IscB (Insertion sequences Cas9-like OrfB) represents a novel class of RNA-guided nucleases, approximately one-third the size of Cas9 proteins. Despite the limited natural efficiency in eukaryotic cells, recent advances have led to the engineering of several IscBs for mammalian genome editing.<br><br>RESULTS: In this study, we screen and identify high-activity IscB variants for rice. A version of pIscB-v3, combining enOgeuIscB and &#x3c9;RNA-v13, demonstrated superior mutagenesis efficiency compared to other systems. The average editing efficiency of pIscB-v3 is 17.61% from ten endogenous targets, and we obtain edited lines in up to 83.33% of T0 generation with 33.33% of homozygous and bi-allelic mutations. Further analysis reveals that pIscB-v3 exhibits high editing specificity and relaxed target-adjacent motif (TAM) compatibility in rice. Beyond gene knockout systems, we develop cytosine base editors (CBEs) and adenine base editors (ABEs) from pIscB-v3. We find that the ssDNA-targeting SCP1.201 family deaminase Sdd7 outperformed human APOBEC3A in IscB-CBEs for C-to-T conversions in rice. The Sdd7-nIscB achieves precise edits in 22.92% of lines on average, with a maximum frequency of 47.92%. Additionally, TadA8e-nIscB exhibits limited activity. However, fusing an extra copy of TadA-8e to either terminus of TadA8e-nIsc significantly enhances A-to-G conversions.<br><br>CONCLUSIONS: Collectively, our results demonstrate the robust capabilities of IscB to develop an efficient and versatile miniature plant genome editing toolkit to substantially facilitate crop breeding.},
}
@article {pmid41579593,
year = {2026},
author = {Jovanovic, VM and Rausch, R and DeRosa, MC and Castellano, D and McKee, C and Sen, C and Daly, F and Doege, CA and Tristan, CA},
title = {Generation and characterization of POMC-tdTomato reporter human pluripotent stem cell lines.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103905},
doi = {10.1016/j.scr.2026.103905},
pmid = {41579593},
issn = {1876-7753},
mesh = {Humans ; *Pro-Opiomelanocortin/metabolism/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; Neurons/metabolism/cytology ; Cell Line ; Cell Differentiation ; Genes, Reporter ; CRISPR-Cas Systems ; Red Fluorescent Protein ; },
abstract = {Proopiomelanocortin (POMC) is a precursor polypeptide that undergoes extensive, tissue-specific post-translational processing. It is expressed in several tissues, including pituitary gland, hypothalamus, brain stem, and skin. The hypothalamic POMC neurons in the arcuate nucleus are major neuronal populations involved in the regulation of body weight. In these neurons, POMC is processed into several peptides, among them the anorexigenic alpha-melanocyte stimulating hormone. Thus, the POMC neurons in the ARC have been named "satiety" neurons and are highly desirable drug targets. Here, we performed CRISPR/Cas9-mediated insertion of tdTomato reporter at the endogenous POMC locus, enabling direct visualization of POMC expression through tdTomato fluorescence in human pluripotent stem cell (hPSC)-derived hypothalamic neurons. This reporter line enables real-time visualization of POMC neuron differentiation, and selective enrichment of these populations for molecular, functional, and pharmacological studies. This line is readily available as new alternative method (NAM) platform, to support disease modeling and drug discovery in metabolic and neuroendocrine disorders within a human context.},
}
@article {pmid41546923,
year = {2026},
author = {Patil, S and Das, A and Inamdar, MS},
title = {Generation of a Brachyury reporter cell line (BJNhem20 Brachyury (TBXT)-2A-EGFP) in human embryonic stem cells using CRISPR-Cas9 gene targeting.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103907},
doi = {10.1016/j.scr.2026.103907},
pmid = {41546923},
issn = {1876-7753},
mesh = {Humans ; Brachyury Protein ; *T-Box Domain Proteins/genetics/metabolism ; *Fetal Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Line ; *Green Fluorescent Proteins/metabolism/genetics ; Genes, Reporter ; Cell Differentiation ; },
abstract = {Brachyury is a key transcription factor, which is required for mesoderm lineage development. Here, we have generated a Brachyury/TBXT-2A-EGFP knock-in Reporter line in the BJNhem20 human embryonic stem cell line, using CRISPR/Cas9-based gene editing. Successful gene editing was verified by DNA sequencing and comparing endogenous gene expression to reporter gene expression. This reporter line represents an important tool for tracking and assessing mesoderm differentiation.},
}
@article {pmid41539085,
year = {2026},
author = {Dong, T and Zhao, Y and Jin, HF and Pan, HM and Yue, LL and Lin, Y and Shen, L},
title = {The establishment of a GPD1L knockout human embryonic stem cell line (WAe009-A-80) using the CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103910},
doi = {10.1016/j.scr.2026.103910},
pmid = {41539085},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Line ; *Glycerolphosphate Dehydrogenase/genetics/metabolism/deficiency ; *Gene Knockout Techniques ; Cell Differentiation ; },
abstract = {The GPD1L gene is located on 3p22.3. It encodes the glycerol phosphate dehydrogenase 1-like protein with homology to glycerol phosphate dehydrogenase (GPD1L), but the function of this enzyme is unclear. Mutations in GPD1L have been associated with BrS (Brugada syndrome) and SIDS (sudden infant death syndrome) and reduce Na[+] inward current through an unknown mechanism in human cardiomyocytes. Here, a GPD1L knockout human embryonic stem cell line was generated using CRISPR/Cas9 system. The GPD1L knockout human embryonic stem cell maintains the pluripotency, differentiation into three germ layers, forming normal EBs.},
}
@article {pmid41532790,
year = {2026},
author = {Cutts, WD and Flanagan, AW and Gorman, BK and Sweten, A and Estrada, BJ and Subash, VN and Klemp, BT and Seely, KN and Sandobal, AD and Stilen, KR and Vaghela, T and Mehvish, A and Wood, JF and Govert, AM and Hobson, KE and Hillebrand, GH and Hooven, TA and Kim, BJ},
title = {CRISPR interference in a Streptococcus agalactiae multi-locus sequence type 17 strain.},
journal = {Journal of bacteriology},
volume = {208},
number = {2},
pages = {e0037625},
doi = {10.1128/jb.00376-25},
pmid = {41532790},
issn = {1098-5530},
support = {R03AI185593//National Institute of Allergy and Infectious Diseases/ ; R15NS131921/NS/NINDS NIH HHS/United States ; R01AI177991//National Institute of Allergy and Infectious Diseases/ ; R01AI182835//National Institute of Allergy and Infectious Diseases/ ; R21AI178067//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Streptococcus agalactiae/genetics/pathogenicity ; Humans ; *CRISPR-Cas Systems ; Streptococcal Infections/microbiology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; Virulence Factors/genetics ; Gene Knockdown Techniques ; Gene Expression Regulation, Bacterial ; Endothelial Cells/microbiology ; },
abstract = {UNLABELLED: Group B Streptococcus (GBS), a common colonizer of the human genital and gastrointestinal tracts, is a leading cause of neonatal bacterial meningitis, which can lead to severe neurological complications. The hypervirulent serotype III, sequence type 17 (ST-17) strain COH1 is strongly associated with late-onset disease due to its unique set of virulence factors. However, genetic manipulation of ST-17 strains remains challenging, limiting the ability to study key pathogenic genes. In this study, we developed a CRISPR interference (CRISPRi) system utilizing an endogenous catalytically inactivated Cas9 (dCas9) in the COH1 strain, enabling targeted and tunable gene expression knockdown. We confirmed the efficacy of this system through hemolysis assays, qPCR transcriptional analysis, and in vitro infection models using human brain endothelial cells. The CRISPRi system successfully produced phenotypic knockdowns of key virulence genes, including PI-2b, srr2, and iagA, reducing adhesion, invasion, and inflammatory responses at the blood-brain barrier (BBB). This platform enables rapid gene knockdowns for functional genomics in ST-17 GBS, enabling high-throughput screening and pathogenesis research.<br><br>IMPORTANCE: Group B Streptococcus (GBS) remains the world's leading cause of neonatal meningitis. GBS-host interactions at the blood-brain barrier (BBB) are dependent on bacterial factors, including surface factors and two-component systems. Multi-locus sequence type 17 (ST-17) GBS strains are highly associated with neonatal meningitis, and these strains harbor many virulence factors for infection at the BBB. Historically, these factors have been studied using traditional knockout mutagenesis, which has been challenging in the most common ST-17 lab strain, COH1. This study utilizes CRISPR interference (CRISPRi) to generate rapid expression knockdown. This study validates a CRISPRi-enabled COH1 dCas9 strain as a versatile tool for probing GBS pathogenesis at the BBB.},
}
@article {pmid41690159,
year = {2026},
author = {Ha, E and Shin, D and Ryu, S and Kong, M},
title = {Deciphering the role of endolysin LysCPD7 harboring C. perfringens spore binding domain.},
journal = {Microbiological research},
volume = {307},
number = {},
pages = {128474},
doi = {10.1016/j.micres.2026.128474},
pmid = {41690159},
issn = {1618-0623},
abstract = {Due to their potent bactericidal activity, phage-derived endolysins are considered promising alternatives to conventional antibiotics. Although some endolysins from phages infecting spore-forming bacteria contain a spore binding domain (SBD), their biological function remains unclear. LysCPD7, an endolysin from the Clostridium perfringens phage CPD7, showed high antimicrobial activity, effectively reducing C. perfringens contamination in milk and beef broth. Fluorescence assays and immunogold electron microscopy showed that LysCPD7 lacks a C-terminal cell wall binding domain, but contains a SBD that localizes to the spore cortex layer. We found that an E187K mutation in the SBD resulted in reduced spore binding capacity while retaining lytic activity. Infection with the wild-type CPD7 led to a decrease in sporulation efficiency in C. perfringens, whereas the mutant CPD7 carrying the E187K substitution in the SBD had no impact on sporulation, suggesting that the SBD may play a role in the inhibition of sporulation in C. perfringens. Our findings could contribute to the rational design of effective antimicrobials or diagnostic tools for controlling C. perfringens and provide new insights into the interactions between phages and their spore-forming hosts.},
}
@article {pmid41689014,
year = {2026},
author = {Lou, C and Wang, J and Dai, C and Wang, J and Yang, J and Fang, Y and Jiang, H and Pan, X and Li, H and Lan, C and Xu, G and Iqbal, S and Bao, J and Cai, L and Zheng, W},
title = {Engineered Cas9 exosome vesicles as a novel gene editing tool for targeted ASPN editing in osteoarthritis.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {165},
pmid = {41689014},
issn = {1477-3155},
support = {LQ24H060008//Basic Public Welfare Research Program of Zhejiang Province/ ; 2025HY0585//Zhejiang Medicine Health Science and Technology Program/ ; GY20250280//Wenzhou Science and Technology Plan Project/ ; },
mesh = {*Osteoarthritis/genetics/therapy ; *Gene Editing/methods ; *Exosomes/metabolism/genetics/chemistry ; Animals ; *CRISPR-Cas Systems/genetics ; Chondrocytes/metabolism ; Humans ; *CRISPR-Associated Protein 9/metabolism/genetics ; Mice ; Male ; Mesenchymal Stem Cells/metabolism ; },
abstract = {CRISPR-Cas9, an innovative genome-editing technique, holds immense promise in therapeutic applications; nevertheless, the lack of effective delivery methods for in vivo gene editing limits its utility in osteoarthritis (OA) treatment. Recently, exosomes, naturally derived nanosized vesicles secreted by cells, have attracted significant attention as potential vehicles for therapeutic cargo delivery. This study proposes a bioinspired engineered exosome-mediated CRISPR/Cas9 delivery platform for targeted editing of the Asporin (ASPN) gene as a potential precision therapy for OA. Specifically, chondrocyte affinity peptide (Cap)-modified MSC-derived exosomes were employed as natural, biocompatible carriers to deliver CRISPR/Cas9 components specifically to OA-affected chondrocytes, thereby achieving precise and efficient ASPN knockout. Flow cytometry analysis confirmed a modification efficiency of 79.1% for Cap, while the encapsulation efficiency of the ASPN-Cas9 plasmid into exosomes reached 9.5% ± 0.6%. Both in vivo and in vitro investigations revealed that this delivery approach markedly improved cellular uptake and gene-editing efficacy, achieving a substantial reduction of ASPN expression by 61.7%. This, in turn, alleviated ferroptosis, improved mitochondrial function, reduced chondrocyte senescence, inhibited inflammation, and enhanced the cartilage microenvironment. Altogether, these findings strongly suggest the promising therapeutic efficacy of this method in OA models, emphasizing its potential as a precise gene-targeting therapeutic intervention for OA.},
}
@article {pmid41689002,
year = {2026},
author = {Zhong, X and Gong, X and Zeng, N and Xie, T and Wang, S and Xia, Q},
title = {Programmable hooded DNA switches for conditional control of CRISPR/Cas12a in multiplexed biosensing.},
journal = {Journal of nanobiotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12951-026-04122-w},
pmid = {41689002},
issn = {1477-3155},
abstract = {The CRISPR/Cas system has become an indispensable tool for programmable and accurate biosensing, with its performance critically dependent on precise activity control. While most regulatory strategies have focused on engineering Cas proteins or modifying CRISPR RNAs, relatively little attention has been given to the design of substrate probes. Here, we systematically characterize the trans-cleavage activity of split CRISPR/Cas12a on structured substrates and leverage this insight to engineer a tunable "Hooded" probe with switchable properties. This probe architecture confers protection against trans-cleavage, and its activity can be progressively modulated by varying the probe length. Utilizing this design, we constructed a multiplexed logic-gated detection platform for direct and simultaneous analysis of miRNA and PSA, which demonstrated high sensitivity and specificity. Furthermore, we validated the robust performance of this system for logic-operated imaging in diverse cellular models, confirming its reliability in complex biological settings. Overall, our Hooded probe strategy not only broadens the applicability of CRISPR/Cas12a in molecular diagnostics, but also provides a novel design principle for the multiplexed biosensing.},
}
@article {pmid41688767,
year = {2026},
author = {Mansi, M and Danai, P},
title = {The emerging impact of CRISPR and gene editing on global crop improvement.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {8},
pmid = {41688767},
issn = {1573-9368},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics/growth &amp; development ; Genome, Plant ; },
abstract = {The advent of CRISPR-based genome editing has revolutionized crop improvement, offering unprecedented precision and efficiency in modifying key agronomic traits. This review comprehensively examines the mechanisms, applications, and future potential of CRISPR technology in enhancing global crop production. CRISPR-Cas systems, originally identified as adaptive immune mechanisms in bacteria and archaea, have been repurposed for targeted genome editing in plants. The CRISPR-Cas9 system, in particular, has emerged as a powerful tool for introducing site-specific double-strand breaks, enabling precise genetic modifications. The three-stage process of adaptation, expression, and interference underlies the CRISPR mechanism, with guide RNAs directing Cas endonucleases to specific genomic loci. Advances in CRISPR technology have expanded its applications beyond gene knockouts, encompassing base editing, prime editing, and epigenome editing. These innovations have facilitated the development of crops with enhanced yield, stress tolerance, disease resistance, nutritional content, and post-harvest quality. However, challenges related to off-target effects, regulatory hurdles, ethical concerns, and public acceptance must be addressed to fully harness the potential of CRISPR in agriculture. Integration of CRISPR with other cutting-edge technologies, such as synthetic biology, artificial intelligence, and high-throughput phenotyping, holds immense promise for accelerating crop improvement efforts. As research continues to refine CRISPR tools and expand their applicability across diverse plant species, this transformative technology is poised to play a pivotal role in shaping a sustainable, resilient, and productive global food system for future generations.},
}
@article {pmid41688147,
year = {2026},
author = {Mu, M and Melms, JC and Ho, P and Izar, B},
title = {Large-scale CRISPR-Cas9 screens to define regulators of immune checkpoints.},
journal = {Methods in cell biology},
volume = {202},
number = {},
pages = {117-132},
doi = {10.1016/bs.mcb.2025.10.011},
pmid = {41688147},
issn = {0091-679X},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Flow Cytometry/methods ; *Melanoma/genetics/immunology/pathology ; *Immune Checkpoint Proteins/genetics ; Animals ; Cell Line, Tumor ; },
abstract = {Immune checkpoints, which have emerged as potent target for the treatment of a variety of cancers, are central to tumor immunobiology and deciphering their dynamic regulation will continue to enable therapeutic development. CRISPR-Cas9 screening has recently been leveraged as a powerful tool to systematically interrogate regulators of immune checkpoints. Here, we describe a framework for such screens coupled with fluorescence-activated cell sorting (FACS) as a reliable and direct method of isolating and comparing how specific CRISPR perturbations impact the expression and maintenance of immune checkpoints. This approach has provided critical insights into immune checkpoint regulation and interactions in melanoma models and can feasibly be expanded to other systems.},
}
@article {pmid41688137,
year = {2026},
author = {Mathew, AE and Arivarasan, VK},
title = {Cell-free systems for nanobiomaterials assembly.},
journal = {Progress in molecular biology and translational science},
volume = {219},
number = {},
pages = {189-210},
doi = {10.1016/bs.pmbts.2025.11.002},
pmid = {41688137},
issn = {1878-0814},
mesh = {Cell-Free System ; Humans ; Animals ; *Nanostructures/chemistry ; *Biocompatible Materials/chemistry ; },
abstract = {Nanobiomaterials-engineered constructs operating at the 1-100 nm scale-integrate biological macromolecules such as nucleic acids and proteins with synthetic polymers or inorganic nanocomponents to achieve programmable functionality in biomedical and industrial contexts. Representative systems, including DNA origami-based drug carriers, CRISPR-Cas delivery scaffolds, and artificial protein vesicles, demonstrate subcellular targeting precision exceeding 90 % and up to a fivefold enhancement in intratumoral drug accumulation relative to conventional nanocarriers. Despite these advances, in vivo nanomanufacturing remains constrained by cytotoxicity, intracellular metabolic load, and limited spatiotemporal control over synthetic parameters. Cell-free systems (CFS) mitigate these limitations by utilizing crude lysates from prokaryotic or eukaryotic cells that retain functional transcription-translation machinery while eliminating the constraints of cellular viability. Platforms such as the reconstituted PURE system enable high-throughput, template-directed synthesis of nanoscale architectures incorporating noncanonical elements, including synthetic polymers, fluorinated analogs, and unnatural amino acids. This open, tunable environment permits over fivefold increases in yields of cytotoxic or aggregation-prone peptides, accelerates DNA origami prototype fabrication to under 24 h, and supports assembly of hybrid enzyme-polymer conjugates with retained catalytic activity. Distinct CFS sources impart complementary advantages: bacterial extracts offer rapid, cost-effective protein expression; yeast lysates facilitate eukaryote-specific glycosylation for ligand-specific nanocapsules; and mammalian systems enable near-physiological post-translational modification essential for therapeutic nanobiomaterials. Coupling CFS with artificial intelligence-based design optimization and microfluidic automation now underpins a new paradigm of programmable, scalable nanobiomanufacturing. By decoupling molecular construction from living systems, cell-free biofabrication establishes a controllable, high-fidelity platform for the rational engineering of nano-bio hybrid systems in precision medicine, biosensing, and tissue regeneration.},
}
@article {pmid41687992,
year = {2026},
author = {Bulle, M and Rahman, MM and Kota, S and Islam, MR and Keya, SS and Abbagani, S and Kirti, PB},
title = {Advancing chloroplast bioengineering: Innovations, regulatory challenges, and translational pathways for sustainable agriculture.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {150873},
doi = {10.1016/j.ijbiomac.2026.150873},
pmid = {41687992},
issn = {1879-0003},
abstract = {Escalating climate instability and rising global food demand necessitate the development of resilient crop systems underpinned by precise, predictable, and rapidly deployable genetic innovations. Chloroplast bioengineering has emerged as a vanguard strategy, offering a uniquely tractable platform characterized by the organelle's distinct plastome, discrete copy number, and predominantly maternal inheritance. Recent advances in plastid transformation and base editing now enable the high-fidelity, multiplex introduction of photosynthetic, osmoprotective, and redox-regulating pathways across diverse plant lineages. Controlled-environment and field-proximal trials demonstrate that chloroplast-engineered metabolic modules enhance CO2 assimilation, stabilize photochemistry under heat and drought stress, and improve osmotic buffering capacity. Beyond trait improvement, chloroplasts function as high-capacity production organelles capable of accumulating oral or mucosal biologics at levels that reduce reliance on cold-chain logistics. However, challenges such as species-level recalcitrance, prolonged timelines for achieving homoplasmy, and limited scalability of current plastid biomanufacturing pipelines continue to constrain broad agricultural and biopharmaceutical deployment. To overcome these barriers, we propose a standardized, empirically testable framework integrating optimized transformation workflows, quantitative trait benchmarking, multi-location field validation, and techno-economic analyses. This framework embeds long-term stewardship principles, including marker-free selection, proactive resistance management, and FAIR-aligned data transparency, while emphasizing equitable access pathways for resource-limited regions. Collectively, this work positions chloroplast engineering as a mechanistically grounded, field-ready platform poised to reshape crop resilience, sustainable bioproduction, and global biologic accessibility amid accelerating climate stress.},
}
@article {pmid41630603,
year = {2026},
author = {Rodriguez-Parks, A and Beezley, EG and Manna, S and Silaban, I and Almutawa, SI and Cao, S and Ahmed, H and Guyer, M and Baker, S and Richards, MP and Kang, J},
title = {Advancing knock-in approaches for robust genome editing in zebrafish.},
journal = {Biology open},
volume = {15},
number = {2},
pages = {},
doi = {10.1242/bio.062472},
pmid = {41630603},
issn = {2046-6390},
support = {R35GM137878/NH/NIH HHS/United States ; GR000042507/NH/NIH HHS/United States ; P30CA014520/NH/NIH HHS/United States ; R21OD037634/NH/NIH HHS/United States ; R01HL151522/NH/NIH HHS/United States ; 7000320//National Institute of Food and Agriculture/ ; 2019-67017-29179//National Institute of Food and Agriculture/ ; 2137434//National Science Foundation/ ; //NIH/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Gene Editing/methods ; *Gene Knock-In Techniques/methods ; Genome ; CRISPR-Cas Systems ; Genetic Vectors ; Exons ; },
abstract = {Precise genome editing remains a major challenge in functional genomics, particularly for generating knock-in (KI) alleles in model organisms. Here, we introduce the mini-golden system, a versatile Golden Gate-based subcloning platform that enables rapid assembly of donor constructs containing homology arms and a gene of interest. This system offers a library of middle entry vectors including diverse genes, enhancing the preparation of donor minicircles for KI applications. Using the mini-golden system, we efficiently generated a foxd3CreER KI zebrafish line, allowing conditional recombination in neural crest cells. To further improve genome editing precision, we developed a synthetic exon-based donor template strategy combined with fluorescence screening. Using this approach, we successfully engineered a targeted isoleucine-to-valine substitution (Ile-to-Val) in hbaa1.2, one of the two adult hemoglobin alpha genes in zebrafish. Importantly, despite the high sequence similarity between hbaa1.2 and its paralog hbaa1.1, our strategy specifically edited hbaa1.2, demonstrating the effectiveness of the synthetic exon approach. This method minimized undesired recombination and significantly improved the identification of lines carrying the edited genome. Together, we provide a robust toolkit for efficient and precise genome engineering in zebrafish, with broad applicability to other model systems.},
}
@article {pmid41620519,
year = {2026},
author = {Popović, J and Hahut, A and Torres, GE and Vincent, A and Soto-Echevarria, N and Wray, B and Bartom, ET and Paunesku, T and Goodman, CR and Woloschak, GE},
title = {Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {6797},
pmid = {41620519},
issn = {2045-2322},
support = {CA221848-07/NH/NIH HHS/United States ; CA221848-07/NH/NIH HHS/United States ; },
mesh = {Humans ; *ErbB Receptors/genetics/metabolism/chemistry ; *CRISPR-Cas Systems ; *Epidermal Growth Factor/metabolism ; *Gene Editing ; Protein Binding ; Cell Line, Tumor ; Protein Domains ; Uterine Cervical Neoplasms/genetics/metabolism ; Mutation ; Amino Acid Substitution ; Female ; Binding Sites ; },
abstract = {Elevated Epidermal Growth Factor Receptor (EGFR) expression is observed in most cervical cancers, and it is frequently associated with poor clinical outcomes. The limited efficacy of existing EGFR-targeted therapies in cervical cancer highlights the need for a deeper understanding of EGFR role in this cancer type. To investigate EGFR separately from its interaction with Epidermal Growth Factor (EGF), we removed the key amino acids from the ligand bindings site. We used CRISPR/Cas9 genome editing to generate a panel of EGFR mutant cell lines and then sequenced and characterized them in detail. Studying the phenotypes of mutant cell clones, we show that a pair of amino acid substitutions L14R and Y45M within Domain I of EGFR protein completely disrupts EGF binding and changes EGFR subcellular distribution. A single substitution Y45M significantly reduced EGF binding but did not lead to subcellular redistribution of EGFR. Upon editing, EGFR mRNA and protein expression were decreased in mutant clones compared to wild type cells. Genome wide profiling of different CRISPR/Cas9 clones confirmed correct editing of EGFR with no off target CRISPR/Cas9 generated mutations. At the same time, spontaneous mutations that could impact cell phenotypes were detected in mutant clones. Disruption of ligand binding domain of EGFR by sequential knock in CRISPR/Cas9 genome editing altered subcellular localization and phosphorylation of EGFR in cervical cancer cells. The results presented here provide insights that may accelerate the development of CRISPR/Cas9-based therapies for EGFR-dependent cancers and reinforce the importance of thorough evaluation of CRISPR/Cas9-generated phenotypes.},
}
@article {pmid41547662,
year = {2026},
author = {Mu, S and Li, Q and Chen, M and Li, Z and Ma, Y and Li, Y and Song, Y and Hou, S and Ding, Y and Ju, J and Lin, Y and Zhang, J and Yang, Y and Ren, X and Li, N and Jin, Q and Lai, L and Wang, K and Shi, H},
title = {Coiled-coil heterodimer-mediated split base editing systems enable flexible and robust nucleotide substitutions.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1765},
pmid = {41547662},
issn = {2041-1723},
support = {32570625//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; Humans ; Animals ; Mice ; Dependovirus/genetics ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; Proprotein Convertase 9/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Cytidine/metabolism ; Genetic Vectors/genetics ; *Nucleotides/genetics ; },
abstract = {Base editors (BEs) enable precise base substitutions, but their size exceeds the packaging capacity of adeno-associated virus (AAV), impeding in vivo applications. Here we design a split BE system that recruits deaminases to Cas9 nickase via coiled-coil heterodimers, resulting in various coiled-coil heterodimers-mediated base editors (CC-BEs), including cytidine base editor (CC-CBE), adenine base editor (CC-ABE), and their derivatives. We reveal that CC-BEs maintain and even improve the editing efficiency of the original unsplit BEs across various cell types and editing scopes, achieving maximum enhancements of 9.6-fold in human immortalized cells and 12.4-fold in primary somatic cells for CC-CBE. Using CC-ABE, we validate in vivo editing efficiency and successfully achieve A-to-G conversion in the Pcsk9 and Dmd genes via dual-AAV vectors in mice. Altogether, we develop a simple and universal strategy to address the challenges posed by the large size of BEs without compromising editing efficiency for base substitutions in vivo.},
}
@article {pmid41545355,
year = {2026},
author = {Tian, T and Zhang, T and Zhang, W and Qiu, Z and Guo, X and Chen, Y and Lin, M and Qi, W and Shen, Y and Hao, M and Xiao, H and Xiang, B and Pang, F and Song, J and Sun, B and Cheng, M and Zhou, X},
title = {Identification of thermotolerant non-canonical PAMs for robust one-pot CRISPR-Cas12a detection.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1771},
pmid = {41545355},
issn = {2041-1723},
support = {32150019//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82502830//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023M741238//China Postdoctoral Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Humans ; *Thermotolerance ; Nucleotide Motifs/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The canonical PAM site TTTV (where V = A, G, or C) is widely used in the design of CRISPR-Cas12a systems for both genome editing and diagnostic applications. Although several non-canonical protospacer-adjacent motifs (PAM) have been identified, they generally exhibit weak Cas12a cleavage activity. In this study, we find that increasing the reaction temperature to 45 °C or higher allows the identification of numerous non-canonical PAMs with trans-cleavage activity comparable to that of canonical PAMs, while displaying only weak cis-cleavage activity. Moreover, we observe that combining these non-canonical PAMs with elevated temperatures significantly enhances the Cas12a system's ability to discriminate highly similar sequences. Based on these findings, we develop a non-canonical PAM-mediated, poikilothermal, one-pot CRISPR-Cas12a detection platform (POP-CRISPR), which demonstrates substantial improvements in sensitivity, specificity, speed, and target adaptability for nucleic acid detection compared to existing methods. These advantages are validated through the reliable detection of clinical samples, including those of Human papillomavirus (HPV), Mycoplasma pneumoniae (MP), and its drug-resistant strains. Additionally, we show that POP-CRISPR enables rapid, on-site pathogen detection within 20 min, using a fast sample processing protocol and a miniaturized detection device.},
}
@article {pmid40905941,
year = {2026},
author = {Chaiyadet, S and Ittiprasert, W and Smout, MJ and Khowawisetsut, L and Ruangsuwast, A and Brindley, PJ and Loukas, A and Laha, T},
title = {Gene Editing of a Carcinogenic Liver Fluke Tetraspanin Impairs Parasite Surface Biogenesis and Extracellular Vesicle Uptake by Human Host Cells.},
journal = {The Journal of infectious diseases},
volume = {233},
number = {2},
pages = {e510-e520},
doi = {10.1093/infdis/jiaf466},
pmid = {40905941},
issn = {1537-6613},
support = {/CA/NCI NIH HHS/United States ; /NH/NIH HHS/United States ; //National Research Council of Thailand/ ; //Fundamental Fund of Khon Kaen University/ ; //the National Science, Research and Innovation Fund/ ; //National Health and Medical Research Council/ ; },
mesh = {*Extracellular Vesicles/metabolism ; Animals ; *Opisthorchis/genetics/metabolism ; Humans ; *Tetraspanins/genetics/metabolism ; *Gene Editing ; *Opisthorchiasis/parasitology ; *Helminth Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Host-Parasite Interactions ; },
abstract = {Opisthorchiasis remains a significant public health concern throughout Southeast Asia. The liver fluke Opisthorchis viverrini resides within the biliary tract, and chronic infection leads to bile duct cancer, or cholangiocarcinoma. Here, we examined the functions of liver fluke tetraspanins, 4-transmembrane domain proteins expressed on the surface of the fluke tegument, and extracellular vesicles (EVs) derived from this syncytial surface. We undertook CRISPR-Cas9 gene knockout (KO) of the O viverrini tetraspanin 2 (Ov-tsp-2) gene and found that Ov-tsp-2-KO flukes had abnormal tegument biogenesis. The tegument of Ov-tsp-2-KO flukes was increasingly vacuolated, and fewer EVs were secreted. EVs that were secreted were deficient in Ov-TSP-2, and their uptake by cholangiocytes was diminished. The findings indicate a critical role for Ov-TSP-2 in maintenance of the tegument, EV production, and uptake by host target cells; they also support the development of this parasite antigen as an anti-infection and anticancer vaccine for opisthorchiasis and opisthorchiasis-associated cholangiocarcinoma.},
}
@article {pmid41686883,
year = {2026},
author = {Rathod, BU and Rajyaguru, R and Dhawale, RN and Tomar, RS and Sharma, S and Chaskar, MG and Alsaidan, OA and Hajare, ST},
title = {CRISPR/Cas9-Mediated Editing in FAD2 Gene to Enhance Oil Quality in Soybean [Glycine max (L.) Merrill].},
journal = {PloS one},
volume = {21},
number = {2},
pages = {e0342660},
pmid = {41686883},
issn = {1932-6203},
mesh = {*Glycine max/genetics/metabolism ; *Gene Editing/methods ; *Fatty Acid Desaturases/genetics ; *CRISPR-Cas Systems/genetics ; *Soybean Oil/metabolism ; Plants, Genetically Modified/genetics ; Linoleic Acid/metabolism ; *Plant Proteins/genetics ; },
abstract = {Conventional soybean oil contains high levels of linoleic acid, which reduces oxidative stability and necessitates hydrogenation, leading to trans-fat formation. In this study, 40 Indian soybean genotypes were screened for fatty acid composition, and Gujarat Junagadh Soybean-3 (GJS-3) was selected for CRISPR/Cas9-mediated editing of the fatty acid desaturase-2 (FAD2) gene. Agrobacterium-mediated transformation produced 22 regenerated plants, of which 57.1% were PCR-positive for Cas9/sgRNA. Targeted single-nucleotide substitutions were confirmed by Sanger sequencing in three edited lines (T3, T7, and T15), corresponding to an editing efficiency of 13.63%. These lines exhibited a marked increase in oleic acid content (42-45%) compared with the wild type (22%) and a concomitant reduction in linoleic acid (30-32% vs. 54%), resulting in nearly a two-fold improvement in the oleic/linoleic acid ratio. PCR analysis confirmed the absence of Cas9 and U3 sequences, indicating transgene-free edited plants. This study provides the first evidence of CRISPR/Cas9-mediated FAD2 editing in an Indian soybean cultivar and demonstrates its effectiveness in improving oil quality, oxidative stability, and processing efficiency.},
}
@article {pmid41686849,
year = {2026},
author = {Dueñas, E and Tirado, I and Huaihua, P and Del Riego, AP and Cabrera-Sosa, L and Nakamoto, JA and Cruz, M and Restrepo, CM and Arévalo, J and Adaui, V},
title = {LAMP-coupled CRISPR-Cas12a assays: A promising new tool for molecular diagnosis of leishmaniasis.},
journal = {PLoS neglected tropical diseases},
volume = {20},
number = {2},
pages = {e0013456},
doi = {10.1371/journal.pntd.0013456},
pmid = {41686849},
issn = {1935-2735},
abstract = {BACKGROUND: Tegumentary leishmaniasis is a parasitic disease endemic in the Americas. Its clinical management and control rely on early and accurate diagnosis and adequate treatment. PCR-based molecular diagnostics offer high sensitivity and specificity over microscopy or culture but are less accessible in low-resource settings. New molecular tools for detecting Leishmania infections are needed in rural endemic regions. A promising tool harnessing CRISPR-Cas technology enables highly specific and sensitive detection of nucleic acid targets, offering an exciting potential for portable molecular diagnostics. Previously, we developed CRISPR-Cas12a-based assays coupled to PCR preamplification for Leishmania detection. Here, we adapted our assays, which target the multicopy 18S rDNA and kinetoplast DNA (kDNA) minicircles, by replacing PCR with loop-mediated isothermal amplification (LAMP).<br><br>LAMP-coupled CRISPR assays were optimized for fluorescence-based and lateral flow readouts. The assays could detect as low as 0.2 genome equivalents per reaction using L. braziliensis M2904 strain genomic DNA. The kDNA assay reliably detected all tested species of the L. (Viannia) subgenus, while the 18S assay showed pan-Leishmania detection capability. There was no cross-reactivity with other protozoan (Trypanosoma cruzi and Plasmodium falciparum) and bacterial (Mycobacterium tuberculosis) pathogen DNA, nor with human DNA. When applied to 90 clinical samples (skin lesions) from the Cusco region of Peru and compared to kDNA real-time PCR, LAMP-CRISPR assays with a fluorescence readout achieved a sensitivity of 90.9% (95% CI: 80.1-97.0%) for kDNA and 72.7% (95% CI: 59.0-83.9%) for 18S rDNA, both with 100% (95% CI: 90-100%) specificity. Overall, lateral flow strip results agreed with fluorescence-based detection in 18 tested samples, with one discrepancy observed in the 18S assay associated with low parasite load.<br><br>CONCLUSIONS/SIGNIFICANCE: These new proof-of-concept LAMP-CRISPR assays, combining high sensitivity, multiple Leishmania species detection capability, and a portable lateral flow readout, hold promise as next-generation molecular tools to improve leishmaniasis diagnosis and surveillance, supporting One Health strategies for disease control.},
}
@article {pmid41686830,
year = {2026},
author = {Caluianu, M and Owen, KA},
title = {A VPS33B CRISPR knockout study: In vitro evidence of an adhesion defect.},
journal = {PloS one},
volume = {21},
number = {2},
pages = {e0343240},
pmid = {41686830},
issn = {1932-6203},
mesh = {*Vesicular Transport Proteins/genetics/metabolism ; Humans ; *Cell Adhesion/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Epithelial Cells/metabolism ; Cell Line ; *Kidney Tubules, Proximal/metabolism/cytology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {VPS33B is a ubiquitously expressed regulator of vesicular membrane fusion and protein sorting involved in a broad range of cellular functions from organelle biogenesis to the establishment of apicobasal polarity. Loss-of-function mutations in VPS33B cause arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, a rare autosomal recessive disorder with multi-organ involvement, including a characteristic proximal tubular dysfunction in the kidney. While VPS33B has been studied in several cell types, its role in proximal tubular epithelial cells remains poorly understood. To investigate its function, a proximal tubular cell line (RPTEC-TERT1) was CRISPR-edited to generate VPS33B knockout (KO) cells. These cells were characterised using brightfield imaging, immunostaining, RNA sequencing, and cell detachment assays, revealing a distinct 'peeling' phenotype and altered adhesion properties. Transcriptional profiling indicated changes in genes linked to cell adhesion. Together, these findings offer preliminary evidence that loss of VPS33B impairs cell-matrix attachment and reveal the first insights into the role of VPS33B within proximal tubular epithelial cells.},
}
@article {pmid41685373,
year = {2025},
author = {Hanafiah, A and Sukri, A and Asmawi, MA and Yusoff, H and Mohd Puzi, S and Neoh, HM and Lopes, BS},
title = {Recent Advancements in Development and Characterization of Phages Targeting Helicobacter pylori.},
journal = {PHAGE (New Rochelle, N.Y.)},
volume = {6},
number = {4},
pages = {282-291},
pmid = {41685373},
issn = {2641-6549},
abstract = {Helicobacter pylori remains a significant global health concern, with rising antibiotic resistance posing challenges for conventional treatments. Bacteriophages, viruses that specifically target and lyse bacterial cells, present a promising alternative therapeutic approach. This review explores the advancements in phage research related to H. pylori, including the isolation, genomic and proteomic characterization, and therapeutic potential of lytic and lysogenic phages. Novel isolation techniques have identified diverse phages from clinical and environmental sources, such as sewage and wastewater, revealing unique genetic and structural adaptations that enhance their effectiveness in targeting H. pylori. Genomic analysis has highlighted the role of prophages in H. pylori evolution, while proteomic studies have uncovered structural features that enable phages to survive the acidic gastric environment. High-throughput technologies, such as next-generation sequencing, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems, and microfluidic platforms, have revolutionized phage discovery and characterization. Furthermore, the potential for phage-antibiotic synergy offers new avenues for combating antibiotic resistance. Despite these advancements, challenges such as H. pylori's genetic diversity, its fastidious growth requirements, and the development of robust delivery mechanisms for gastric application persist. This review highlights the need for further research to optimize phage-based therapies as a viable alternative or adjunct to current treatments for H. pylori infections.},
}
@article {pmid41683990,
year = {2026},
author = {Psaras, AM and McKay, SJ and Vasquez Vilela, J and Ospina Sanchez, E and Cintrón, MG and Elder, KK and Brooks, TA},
title = {Ovarian Cancer Susceptibility and Chemosensitivity to KRAS Modulation.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683990},
issn = {1422-0067},
mesh = {Humans ; Female ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism/antagonists &amp; inhibitors ; *Ovarian Neoplasms/genetics/drug therapy/pathology/metabolism ; *Drug Resistance, Neoplasm/genetics/drug effects ; Cell Line, Tumor ; Paclitaxel/pharmacology ; Cisplatin/pharmacology ; Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems ; },
abstract = {KRAS is frequently amplified or overexpressed in ovarian cancer and represents a potential therapeutic target for overcoming chemoresistance. We employed complementary approaches-CRISPR/Cas9 gene editing, Tet-ON inducible knockdown, polypurine reverse Hoogsteen hairpin (PPRH) oligonucleotides, and the pan-KRAS inhibitor BI2865-to investigate whether KRAS modulation enhances chemotherapeutic efficacy in ovarian cancer models. CRISPR-mediated KRAS knockdown in SKOV-3 cells dramatically altered three-dimensional spheroid morphology, reducing the average area six-fold, and significantly enhanced sensitivity to both cisplatin and paclitaxel in 3D cultures, where paclitaxel resistance was completely reversed. The Tet-ON system demonstrated dose-dependent chemosensitization with optimal effects at intermediate KRAS knockdown levels (~50-60%). PPRH oligonucleotides at sub-cytotoxic concentrations (50 nM) reduced cisplatin and paclitaxel IC50 values by approximately 50% in 2D cultures. Pharmacological KRAS inhibition with BI2865 produced striking synergy with paclitaxel (several hundred-fold sensitizations in 2D; complete reversal of 3D resistance), and additive effects with cisplatin. In KRAS-amplified Kuramochi cells (representing high-grade serous ovarian carcinoma), BI2865 enhanced paclitaxel efficacy, despite greater baseline chemoresistance. These findings establish KRAS as a promising chemosensitization target in ovarian cancer, with particular potential for taxane-based combination therapies.},
}
@article {pmid41683941,
year = {2026},
author = {Skaliter, O and Gura, A and Livneh, Y and Cohen, R and Shklarman, E and Edelbaum, O and Masci, T and Vainstein, A},
title = {Targeted Gene Modification of HMGR Enhances Biosynthesis of Terpenoid and Phenylpropanoid Volatiles in Petunia and Lettuce.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683941},
issn = {1422-0067},
support = {1368/23//Israel Science Foundation/ ; 20-01-0209//Office of the Chief Scientist/ ; },
mesh = {*Petunia/genetics/metabolism ; *Terpenes/metabolism ; *Lactuca/genetics/metabolism ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics/metabolism ; *Volatile Organic Compounds/metabolism ; },
abstract = {Terpenoids constitute the largest class of plant-specialized metabolites, playing essential roles throughout the plants' life cycle and having diverse applications for humans in nutrition, medicine, and flavor. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a rate-limiting enzyme of the mevalonate (MVA) pathway, producing sesquiterpenes, saponins, and other terpenoids. HMGR is post-translationally regulated by downstream MVA products through its N-terminal regulatory domain, limiting terpenoid production. To overcome this bottleneck, we employed a virus-based CRISPR/Cas9 system to genetically modify the N-terminal regulatory domain of HMGR in petunia (Petunia × hybrida) and lettuce (Lactuca sativa L.). In petunia, HMGR1-edited lines exhibited vigorous growth, larger flowers, and increased production of sesquiterpenes. Interestingly, they also showed enhanced production of phenylpropanoid volatiles, revealing a connection between these pathways. Transcript analysis revealed altered expression of genes involved in terpenoid biosynthesis, pyruvate metabolism, phenylpropanoid biosynthesis, and gibberellin- and auxin-related pathways, indicating enhanced carbon flux through these metabolic networks. In lettuce, HMGR7-edited plants displayed elevated emission of sesquiterpenes, apocarotenoids, and the phenylpropanoid benzaldehyde. Together, these results establish a transgene-free strategy to enhance the production of terpenoid and phenylpropanoid volatiles, and provide a framework for developing resilient, nutrient-enriched crops.},
}
@article {pmid41683905,
year = {2026},
author = {Ortiz-Bueno, M and Zinghirino, F and Serra, PP and Paschoudi, K and Montoliu, L and Atilla, E and Luo, Y and Cavazza, A and Lederer, CW and Benabdellah, K},
title = {From Bench to Bedside: Ethical and Clinical Best Practices for Genome Editing Applications.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683905},
issn = {1422-0067},
support = {CA21113//COST (European Cooperation in Science and Technology)./ ; ProyExcel_00875//Consejer&#xed;a de Universidad, Investigaci&#xf3;n e Innovaci&#xf3;n/ ; },
mesh = {Humans ; *Gene Editing/ethics/legislation &amp; jurisprudence/methods ; *Genetic Therapy/ethics/methods ; Animals ; CRISPR-Cas Systems ; *Translational Research, Biomedical/ethics ; },
abstract = {Genome editing (GE) has transformed medicine by allowing precise changes to DNA, offering potential treatments for a range of inherited and acquired disorders. Several technologies support these advances, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-based systems, of which the latter has emerged as the most accessible, versatile, and popular. While GE holds great promise, its clinical use requires careful attention to safety, ethics and regulatory standards. Inadvertent on- and off-target DNA alterations and unintended modification of non-target cells pose major technical challenges, while bioethical considerations and the need for harmonized safety standards create regulatory challenges. The Food and Drug Administration (FDA) and European Medicines Agency (EMA), as regulatory agencies for key advanced therapy markets, provide detailed guidance on these aspects, emphasizing rigorous preclinical testing, patient monitoring, ethical consent, and compliance with legal frameworks. This concise review summarizes what is currently published in the scientific literature and recommended by regulatory agencies, providing an overview of the responsible clinical application of GE, with emphasis on patient safety, adherence to regulatory guidance, and ethical practice.},
}
@article {pmid41645678,
year = {2026},
author = {Ren, Y and Xu, J and Luo, Q and Li, P and Jiang, M and Cheng, Y and Zhang, H and Wei, D and Bai, W and Liu, S and Wang, G and Xie, Y and Li, J and Lu, W},
title = {A Recombinant Targeted Bionanoparticle In Vitro Expressing a Gene-Editing Protein for Epidermal Growth Factor Receptor Mutant Lung Cancer.},
journal = {ACS nano},
volume = {20},
number = {6},
pages = {4920-4941},
doi = {10.1021/acsnano.5c17709},
pmid = {41645678},
issn = {1936-086X},
mesh = {*Lung Neoplasms/genetics/therapy/pathology ; *ErbB Receptors/genetics ; Humans ; *Gene Editing/methods ; Animals ; *Nanoparticles/chemistry ; Mice ; Mutation ; HEK293 Cells ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Recombinant Proteins/genetics ; Mice, Nude ; },
abstract = {Epidermal growth factor receptor (EGFR) mutation is a significant driving factor in the occurrence and progression of lung cancer. How to effectively block the abnormal expression of EGFR remains a key issue that urgently needs to be addressed, as it is of vital importance for the effective treatment of this cancer. Here, we report a recombinant targeted bionanoparticle that in vitro expresses Cas9 protein (tBioNP vitro-Cas9) for gene editing of EGFR mutant lung cancer. The nanoparticle was developed by transfection of four plasmids (Gag-Cas9, Gag, sgRNA, VSV-G Azi) into 293T cells to form a type of bionanoparticle and modifying it with a targeted polymer material (DBCO-PEG-FA), and it showed a cancer-targeted property, faster cancer cellular uptake, higher gene editing efficiency with lower off-target effects, and therapy efficacy in mice, indicating a translational prospect. In conclusion, the study provides a recombinant bionanoparticle in vitro expressing a Cas9 gene editing system and offers a potential strategy for gene therapy of EGFR mutant lung cancer.},
}
@article {pmid41640077,
year = {2026},
author = {Xu, YC and Liu, WJ and Li, CC and Zhang, D and Ma, F and Zhang, CY},
title = {CRISPR/Cas13a-Engineered RNA-Based Fluorogenic Biosensor for Label-Free Quantification of RNA in Colorectal Tissues.},
journal = {Analytical chemistry},
volume = {98},
number = {6},
pages = {5076-5084},
doi = {10.1021/acs.analchem.5c07694},
pmid = {41640077},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Colorectal Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems ; *Fluorescent Dyes/chemistry ; *RNA/analysis/genetics ; Aptamers, Nucleotide/genetics/chemistry ; },
abstract = {Colorectal cancer (CRC) continues to represent a serious threat to global health, and its diagnosis faces significant challenges, especially in the early phases of malignant tumors. Herein, we demonstrate that BRD2 RNA can serve as a potent noninvasive CRC biomarker and construct a CRISPR/Cas13a-engineered RNA-based fluorogenic biosensor for label-free detection of BRD2 RNA in colorectal tissues. In this assay, the specific recognition of BRD2 RNA by the substrate probe activates Cas13a/crRNA, leading to the trans-cleavage of the substrate probe and the generation of the T7 promoter sequence. The resulting T7 promoter subsequently induces efficient transcription amplification to synthesize abundant Pepper RNA aptamers that can light up HBC620. Leveraging the synergistic advantages of Cas13a precision, efficient transcription amplification, and superior signal-to-noise ratio of RNA aptamer-fluorophore complex, this fluorogenic biosensor enables sensitive detection of BRD2 RNA down to 0.39 fM and accurate quantification of its expression at the single-cell level. In addition, this fluorogenic biosensor can successfully distinguish CRC patient tissues from adjacent normal tissues based on distinct BRD2 RNA expression profiles. Importantly, the programmability of crRNA makes this fluorogenic biosensor readily adapted for detecting a broad range of RNA targets (e.g., noncoding RNAs and viral RNAs) by simply modifying the spacer sequence of crRNA, providing a new paradigm for early clinical diagnostics.},
}
@article {pmid41634962,
year = {2026},
author = {Li, W and Duan, M and Sun, S and Li, J and Wang, M and Zhao, H},
title = {A CRISPR Switch Integrated with Strand Displacement Amplification for Binary Channel Detection of SARS-CoV-2 Gene Fragments and Infectious Diagnosis.},
journal = {Langmuir : the ACS journal of surfaces and colloids},
volume = {42},
number = {6},
pages = {5070-5078},
doi = {10.1021/acs.langmuir.5c06262},
pmid = {41634962},
issn = {1520-5827},
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *COVID-19/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The development of sensitive, accurate, and multimodal approaches for the detection of viral gene fragments and the diagnosis of infections is essential for effective pandemic management across various contexts. This study introduces a CRISPR switch integrated with strand displacement amplification (SDA) for the binary channel detection of SARS-CoV-2 gene fragments and the diagnosis of SARS-CoV-2 infections. In the conventional channel, a specific single gene fragment can directly facilitate the formation of a three-way junction, thereby initiating the SDA process and resulting in the production of a substantial amount of single-stranded DNA. In the logical channel, two gene fragments can first induce the release of a substitute, which subsequently leads to the formation of the three-way junction and the ensuing SDA process. The single-stranded SDA product acts as the target sequence that activates the CRISPR switch, which performs reporter cleavage functions, thereby generating enhanced and detectable fluorescence signals. This method achieves sensitive and selective detection of SARS-CoV-2 gene fragments, with limits of detection (LODs) of 1.0 aM for the ORF1ab gene and 0.9 aM for the N gene in the conventional channel and 3.7 aM for simultaneous detection of both ORF1ab and N in the logical channel. Furthermore, accurate detection of these gene fragments in real samples obtained from patients exhibiting upper respiratory symptoms was successfully conducted, along with the corresponding diagnosis of SARS-CoV-2 infections. Consequently, this method represents a novel binary channel approach for viral gene detection and holds significant promise for clinical diagnosis and potential future epidemic control.},
}
@article {pmid41632863,
year = {2026},
author = {Shinoda, H and Makino, A and Yoshimura, M and Minagawa, N and Iida, T and Nakano, M and Noda, T and Toyoda, M and Watanabe, R},
title = {Multicolor Amplification-Free RNA Detection with Cas13a and Cas13b.},
journal = {Analytical chemistry},
volume = {98},
number = {6},
pages = {4705-4714},
doi = {10.1021/acs.analchem.5c06305},
pmid = {41632863},
issn = {1520-6882},
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Viral/analysis/genetics ; *COVID-19/diagnosis ; Influenza A virus/genetics/isolation &amp; purification ; Limit of Detection ; *CRISPR-Cas Systems ; Influenza, Human/diagnosis ; *CRISPR-Associated Proteins/genetics ; },
abstract = {The COVID-19 pandemic and recurring outbreaks of infectious diseases underscore the urgent demand for multiplex diagnostics capable of rapid and accurate pathogen identification. Although multiplex nucleic acid amplification tests (NAATs) are widely used for diagnosing diverse infectious diseases, their inherent amplification bias and long turnaround times highlight the demand for faster and reliable alternatives. Here, we present multicolor SATORI (mSATORI), an amplification-free single-molecule genetic test that leverages the complementary activities of CRISPR-Cas13a and Cas13b to achieve simultaneous detection of dual RNA targets. mSATORI identified Influenza A and SARS-CoV-2 RNAs within ∼10 min, with analytical limits of detection (LoD) of 86 aM and 52 aM, respectively. Validation using clinical specimens demonstrated robust diagnostic performance, achieving femtomolar limits of detection (550 aM for Influenza A and 640 aM for SARS-CoV-2), along with sensitivities exceeding 80% and specificities of 100%. Collectively, these results establish mSATORI as a platform for next-generation molecular diagnostics, with broad implications for clinical implementation, outbreak preparedness, and global infectious disease surveillance.},
}
@article {pmid41626798,
year = {2026},
author = {Schuster, I and Shlipak, KK and Qin, PZ},
title = {Impacts of DNA Supercoiling on the Sequence-Dependent Nuclease Activity of CRISPR-Cas9 with Truncated Guides.},
journal = {Biochemistry},
volume = {65},
number = {4},
pages = {371-384},
pmid = {41626798},
issn = {1520-4995},
support = {R35 GM145341/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; *DNA, Superhelical/chemistry/metabolism/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; Kinetics ; DNA Cleavage ; Nucleic Acid Conformation ; Base Sequence ; DNA/chemistry/metabolism ; Gene Editing ; },
abstract = {CRISPR-Cas9 is natively present in the adaptive immune systems of a multitude of bacteria and has been adapted as an effective genome engineering tool. The Cas9 effector enzyme, which is composed of a single Cas9 protein and a single-guide RNA (sgRNA), identifies and cleaves double-stranded DNA targets through a series of conformational changes that require DNA distortion and unwinding. While most studies of Cas9 specificity have focused on the DNA sequence, the role of intrinsic DNA physical properties ("DNA shape") in modulating Cas9 activity remains insufficiently defined. We previously showed that with a 16-nucleotide (-nt) truncated guide, the intrinsic DNA duplex dissociation energy at the PAM+(17-20) segment beyond the RNA-DNA hybrid tunes Cas9 cleavage rates of linear substrates. Here, we examined the impact of DNA supercoiling on Cas9 cleavage with the 16-nt truncated guide. Enzyme kinetic analysis revealed that PAM+(17-20) DNA sequences beyond the RNA/DNA hybrid preserve their effects on Cas9 cleavage in the supercoiled state. Furthermore, combining a novel asymmetric hairpin construct with a parallel-sequential kinetics model, rates for first-step nicking and second-step cleavage by Cas9 were obtained for both supercoiled and linear substrates. With both topologies, it was found that first-step nicking is clearly impacted by PAM+(17-20) DNA sequences, and the effects can be correlated with DNA unwinding, which dictates R-loop dynamics. This work expands our understanding of DNA target recognition by Cas9, and the methods developed, in particular those for analyzing the progression of Cas9-induced nicks, will aid in further in-depth mechanistic investigation.},
}
@article {pmid41603277,
year = {2026},
author = {Li, J and Shao, T and Cao, XJ and Wang, YX and Kong, DM},
title = {An allosteric key strand controlled adaptable CRISPR/Cas12a biosensing platform for point-of-care testing of multiple types of targets.},
journal = {Lab on a chip},
volume = {26},
number = {4},
pages = {917-929},
doi = {10.1039/d5lc01029b},
pmid = {41603277},
issn = {1473-0189},
mesh = {*Biosensing Techniques/instrumentation ; *CRISPR-Cas Systems ; *Point-of-Care Testing ; DNA/genetics/chemistry/analysis ; Humans ; Allosteric Regulation ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Currently, the CRISPR/Cas12a based sensor has become a powerful tool for gene editing and molecular diagnostics. However, most CRISPR/Cas12a sensors are primarily limited to the detection of a single target type, due to their strict dependence on the specific recognition of the PAM sequence within a precisely designed double-stranded DNA (dsDNA) and crRNA for cleavage activity regulation. Herein, we designed an allosteric key strand (KS) controlled CRISPR/Cas12a biosensor via toehold-based strand displacement reaction (TSDR). By simply reconfiguring KS into different conformations with functional nucleic acid structures, this sensor could selectively respond to various target molecules from nucleic acids to non-nucleic acid molecules without changing the sequence of crRNA and targeted PAM-dsDNA. The trans-cleavage activity of CRISPR/Cas12a could be triggered through leveraging proximity-based TSDR in response to target binding. The proposed sensor achieved sensitive and specific detection of various targets, including nucleic acids (HPV-16), small molecules (kanamycin), and enzymes (uracil-DNA glycosylase). Furthermore, by integrating lateral flow assay technology, this CRISPR/Cas12a-based system enabled point-of-care testing (POCT) for the detection of multiple target types. This approach can overcome the sequence-specific limitations, thereby improving the versatility of CRISPR/Cas12a sensors for extending more target types detection. We anticipate this innovative technology will serve as a flexible and accessible sensing platform, facilitating rapid diagnosis in the field of POCT and enabling its broader application across diverse biotechnological domains.},
}
@article {pmid41578398,
year = {2026},
author = {Ferreira de Vasconcellos, J and Friedman, L and Satapathy, I and Cubbage, N and Palmer, J and Majumder, S and Kono, M},
title = {A genome-wide CRISPR/Cas9 screen reveals novel positive regulators of FTY720 sensitivity in acute lymphoblastic leukemia cells.},
journal = {BMC research notes},
volume = {19},
number = {1},
pages = {75},
pmid = {41578398},
issn = {1756-0500},
mesh = {Humans ; *Fingolimod Hydrochloride/pharmacology ; *CRISPR-Cas Systems/genetics ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics/drug therapy ; Cell Line, Tumor ; },
abstract = {OBJECTIVE: Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. Fingolimod (FTY720) is a sphingosine-1-phosphate (S1P) receptor agonist that prevents lymphocytes from egressing from lymphoid tissues and has shown a cytotoxic effect on T-cell ALL (T-ALL) cells. However, the mechanism of action of FTY720 cytotoxicity in hematological malignancies is still unclear, and cell-specific effects have been reported. Here, we investigated the mechanism of cytotoxicity of FTY720 in T-ALL cells using a CRISPR-Cas9 genomic screening. Our goal was to identify novel positive regulators for the cytotoxic effect of FTY720 in T-ALL.<br><br>RESULTS: Cells treated with FTY720 were enriched for single-guide RNAs (sgRNAs) such as ZNF575, GPX3, FBXL15, DNAJB5, UBE2D1, ATXN7, C6orf201, RIC8A, RAB13, and C10orf12 when compared to the DMSO (vehicle control) samples. Altogether, our study identified novel genes that, when silenced, were positively correlated with the survival of T-ALL cells treated with FTY720.},
}
@article {pmid41578383,
year = {2026},
author = {Tao, T and Lin, L and Tang, Y and Liu, Z and Liu, Y and Xie, Y and Hu, X and Wang, J and Wang, T and Zhang, GF and Wang, Y and Zhu, S},
title = {Generation of an isogenic human induced pluripotent stem cell line with a mutant propionyl-CoA carboxylase α subunit.},
journal = {Orphanet journal of rare diseases},
volume = {21},
number = {1},
pages = {61},
pmid = {41578383},
issn = {1750-1172},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Methylmalonyl-CoA Decarboxylase/genetics/metabolism ; Myocytes, Cardiac/metabolism ; Cell Differentiation ; Propionic Acidemia/genetics ; Mutation/genetics ; Cell Line ; CRISPR-Cas Systems ; Gene Editing ; Propionyl-Coenzyme A Carboxylase ; },
abstract = {BACKGROUND: Propionic acidemia (PA) is a rare autosomal recessive metabolic disorder caused by defects in propionyl-CoA carboxylase (PCC), a mitochondrial enzyme composed of six alpha (PCCA) and six beta (PCCB) subunits. Mutations in PCCA/PCCB genes disrupt PCC function, leading to toxic metabolite accumulation and clinical manifestations. Current research is limited by inadequate patient-derived cellular models and ethical constraints in sample acquisition.<br><br>METHOD: Using CRISPR/Cas9-mediated gene editing, we established an isogenic human induced pluripotent stem cell (iPSC) line carrying the PCCA c.2002G>&#x2009;A mutation. The mutant iPSCs were further subjected to directed cardiac differentiation. Characteristic metabolites in the iPSC-derived cardiomyocytes (iPSC-CMs) culture medium were analyzed via untargeted metabolomics, and contractile function was assessed by video-based motion analysis under propionate challenge.<br><br>RESULTS: The mutant iPSCs showed sustained expression of pluripotency markers (OCT4, NANOG, SOX-2), maintained normal karyotype (46, XY), and retained trilineage differentiation capacity. Functional characterization demonstrated significantly reduced PCC enzyme activity, accurately modeling PA metabolic pathology. Furthermore, the mutant iPSCs successfully differentiated into cardiomyocytes and exhibited a PA-specific metabolic profile, including significantly elevated propionylcarnitine levels. Upon propionate treatment (2.5&#x2009;mM), the contractile function of mutant iPSC-CMs was significantly impaired, whereas wild-type iPSC-CMs showed the opposite response with enhanced contraction.<br><br>CONCLUSIONS: This isogenic iPSC line provides an ethically unconstrained platform to investigate PA molecular mechanisms and genotype-phenotype relationships. The model enables systematic drug screening and therapeutic development while overcoming patient sample limitations.},
}
@article {pmid41577693,
year = {2026},
author = {Shankar, A and Olender, L and Hsu, I and Miyauchi, M and Pálovics, R and Meaker, GA and Kaito, S and Rizq, O and Khoo, HM and Bozhilov, Y and Igarashi, KJ and Bhadury, J and Munson, C and Mack, PK and Tan, TK and Rehwinkel, J and Iwama, A and Wyss-Coray, T and Nakauchi, H and Haney, MS and Wilkinson, AC},
title = {In vivo CRISPR screening identifies SAGA complex members as key regulators of hematopoiesis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1756},
pmid = {41577693},
issn = {2041-1723},
support = {KKL1378//Kay Kendall Leukaemia Fund (KKLF)/ ; 302479Z23Z//Wellcome Trust (Wellcome)/ ; K99HL150218//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; 3385-19//Leukemia and Lymphoma Society (Leukemia &amp; Lymphoma Society)/ ; RG-202211-02958//European Hematology Association (EHA)/ ; },
mesh = {*Hematopoiesis/genetics ; Humans ; Hematopoietic Stem Cells/metabolism/cytology ; Animals ; Mice ; Histones/metabolism ; Acetylation ; Ubiquitination ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Mice, Inbred C57BL ; },
abstract = {The biological mechanisms that sustain the vast blood production required for healthy life remain incompletely understood. To search for cell intrinsic regulators of hematopoiesis, we perform a genome-wide in vivo hematopoietic stem and progenitor cell (HSPC)-based CRISPR knockout screen. We discover SAGA complex members, including Tada2b and Taf5l, as key regulators of hematopoiesis. Loss of Tada2b or Taf5l strongly inhibits hematopoiesis in vivo, causing a buildup of immature hematopoietic cells in the bone marrow. The SAGA complex deposits histone H3 lysine 9 acetylation (H3K9ac) and removes histone H2B ubiquitination (H2Bub). Loss of Tada2b leads to a reduction in H3K9ac levels and altered H2Bub enrichment in HSPCs, implicating disruption of SAGA complex activity. This is associated with upregulation of interferon pathway genes, reduced mitochondrial activity, and increased megakaryocyte progenitor cell commitment. Loss of these factors also enhances the cell outgrowth and the interferon pathway in an in vivo human myelodysplastic syndrome cell line model. In summary, this study identifies the SAGA complex as an important regulator of hematopoiesis.},
}
@article {pmid41566394,
year = {2026},
author = {Xin, C and Xiang, G and Cao, S and Wang, Y and Yuan, S and Liu, X and Huo, Y and Sun, J and Wan, X and Liu, D and Hong, J and Hu, J and Wang, H},
title = {Comprehensive assessment of activity, specificity, and safety of hypercompact TnpB systems for gene editing.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {39},
pmid = {41566394},
issn = {1474-760X},
support = {24YF2703900//the "Rising-Star Program" of "Shanghai 2024 Science and Technology Innovation Action Plan/ ; 32101204//NSFC grant/ ; 31771485//NSFC grant/ ; 202423110050063//Science and Technology Major Project of Anhui Province/ ; 2024YFA0917301//the National Key R&amp;D Program of China/ ; 2022YFC3400201//National Key Research and Development Program of China/ ; 2023ZD04074//Biological Breeding-Major Projects of the ministry of Agriculture and Rural Affairs of China/ ; 2022FH122//Program of Beijing Institute for Stem Cell and Regenerative Medicine/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Animals ; Genetic Therapy ; HEK293 Cells ; },
abstract = {BACKGROUND: As the ancestor of CRISPR-Cas12 nucleases, TnpB represents the most compact gene editing tool currently available. Recent studies have identified multiple TnpB systems with gene editing activity in mammalian cells, and the potential of TnpB in treating diseases has been demonstrated in animal models. However, the editing characteristics of various TnpB systems, comparable to CRISPR tools, require more extensive investigation.<br><br>RESULTS: Using a standardized evaluation framework, we conduct a thorough analysis of the editing properties of four TnpB variants alongside representative Cas12 and Cas9 tools applications. Overall, TnpBs exhibit intermediate editing activity and safety profiles among all tested systems, with ISYmu1 TnpB demonstrating a good performance in both editing activity and specificity. Considering its compact size, potent editing efficiency and high specificity, ISYmu1 TnpB represents a promising candidate for gene therapy.<br><br>CONCLUSIONS: By comprehensively analyzing genome editing outcomes, we characterize TnpB systems for genome editing and identify ISYmu1 TnpB as an optimal miniature RNA-guided genome editors with balanced performance, highlighting its potential for therapeutic applications.},
}
@article {pmid41547832,
year = {2026},
author = {Wang, X and Deng, X and Qiu, L and Liu, J and Shen, H and Du, H and Li, W and Song, L and Deng, W and Dong, X and Han, Y and Liu, B and Huang, J and Li, Z and Zhang, Y},
title = {Af-CUT&amp;Tag: a sensitive and antibody-free chromatin profiling method using genetically encoded tags and high-affinity binders fused to Tn5.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1746},
pmid = {41547832},
issn = {2041-1723},
mesh = {*Chromatin/metabolism/genetics ; Humans ; Animals ; YAP-Signaling Proteins ; Mice ; MicroRNAs/metabolism/genetics ; *Transposases/metabolism/genetics ; Transcription Factors/metabolism/genetics ; Liver Regeneration/genetics ; Adaptor Proteins, Signal Transducing/metabolism/genetics ; Chromatin Assembly and Disassembly ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Conventional chromatin profiling techniques are often limited by antibody availability and performance. Here, we introduce Af-CUT&amp;Tag, a target antibody-free method that overcomes these limitations by using CRISPR-integrated peptide tags (HiBiT/ALFA-tag) recognized by engineered binders (LgBiT/NbALFA) fused to a Tn5 transposase. Af-CUT&amp;Tag eliminates dependence on traditional target antibodies, achieving robust specificity and sensitivity with as few as 500 cells. It provides high-quality chromatin profiles, with improved signal-to-noise ratios and library quality compared with conventional antibody-based counterparts, while also enabling single-cell resolution (scAf-CUT&amp;Tag). Applying Af-CUT&amp;Tag to Hippo effectors (YAP1/TAZ) during liver regeneration reveals dynamic chromatin remodeling, including YAP1/TAZ-mediated control of lipid metabolism (e.g., Lpin1, Fasn) and heme clearance (Hpx, Trf). We further identify miR-122 as a critical regulator of these processes, impacting liver regeneration. The versatility of Af-CUT&amp;Tag in cell lines, bulk tissues, and single nuclei establishes it as a powerful tool for studying gene regulation in development, disease, and regeneration.},
}
@article {pmid41474015,
year = {2026},
author = {Cheng, X and Dong, J and Jain, P and Qin, S and Miao, Y and Liu, K and Theja, MLV and Butch, CJ and Wang, Y and Lane, LA},
title = {Ultrasound Activated Hybrid-Biomimetic Nanocarriers That Combine Tumor-Confined CRISPR/Cas9 Metabolic Reprogramming and Cuproptosis With Anticancer Macrophage Polarization.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {10},
pages = {e10436},
doi = {10.1002/smll.202510436},
pmid = {41474015},
issn = {1613-6829},
support = {113-2221-E-038-004//National Science and Technology Council/ ; 114-2221-E-038-019//National Science and Technology Council/ ; TMU111-AE1-B12//Taipei Medical University/ ; 113FRP-28//Taipei Medical University Shuang Ho Hospital Special Research Plan/ ; 114FRP-26//Taipei Medical University Shuang Ho Hospital Special Research Plan/ ; 82127806//National Natural Science Foundation of China/ ; 2022300326//Fundamental Research Funds for the Central Universities/ ; 0213-14380238//Fundamental Research Funds for the Central Universities/ ; 202205033//Nanjing Life and Health Science and Technology Special Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Macrophages/metabolism/drug effects ; Humans ; Animals ; *Nanoparticles/chemistry ; Metal-Organic Frameworks/chemistry ; Cell Line, Tumor ; *Copper/chemistry ; Mice ; *Biomimetic Materials/chemistry ; *Ultrasonic Waves ; *Neoplasms/pathology ; *Drug Carriers/chemistry ; *Biomimetics/methods ; Metabolic Reprogramming ; },
abstract = {Nanomedicine aims to develop nanocarriers that provide strong cell selectivity and efficient intracellular delivery. Additionally, therapeutic strategies are expanding to include metabolic pathways to trigger apoptosis and reduce tumor growth, especially in cases resistant to conventional chemotherapy. Here, we have created nanocarriers with hybrid-biomimetic coatings that, upon ultrasound activation, release encapsulated copper-based metal-organic frameworks (MOFs) and COP1 gene knockout Cas9 ribonucleoproteins (RNPs). This hybrid-membrane coating, which combines tumor and immune cell membranes with perfluorocarbons, enhances tumor-to-normal cell uptake and allows for controlled release and cytolytic entry of the nanocarrier contents. We observe that the RNPs efficiently knockout the COP1 gene, thereby arresting the cancer cell cycle in the G0/G1 phase and promoting mitochondrial respiration over anaerobic glycolysis. This increased respiration makes cancer cells more susceptible to cuproptosis triggered by the MOFs and decreases tumor lactate levels, preventing lactate-driven M2 polarization of tumor-infiltrating macrophages. Furthermore, the nanocarriers' cellular selectivity leaves macrophages unharmed. These effects enable infiltrating macrophages to retain an anticancer M1 polarization and continue to foster a more active immune response. The combination of tumor-specific genetic metabolic reprogramming and enhanced cuproptosis activity, along with increased immune activity, results in significant tumor growth suppression and improved survival rates.},
}
@article {pmid41457810,
year = {2026},
author = {Zhao, J and Long, Y and Zhang, Y and Hou, C and Huo, D},
title = {Nano-Mechanical DNA Devices Coupled With CRISPR-Cas12a for CA15-3 Detection.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {10},
pages = {e11023},
doi = {10.1002/smll.202511023},
pmid = {41457810},
issn = {1613-6829},
support = {81772290//National Natural Science Foundation of China/ ; CYB240064//Graduate Scientific Research and Innovation Foundation of Chongqing, China/ ; 2023-K08//Open Research Fund of State Key Laboratory of Digital Medical Engineering/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/chemistry ; Humans ; Biosensing Techniques/methods ; *Nanotechnology/methods ; Electrochemical Techniques ; },
abstract = {Accurate monitoring of cancer markers is crucial for clinical treatment and prognosis. CA15-3 activity levels are strongly associated with clinical progression of breast cancer, but their monitoring often relies on large instruments and professionals, and the process is time-consuming and costly. To address these concerns, we proposed an electrochemical biosensing strategy that integrated nano-mechanical DNA devices coupled with the CRISPR-Cas12a to drive molecularly gated functionalized substrates for the ultrasensitive detection of CA15-3. Specifically, Triple helical molecular switch (THMS) as a signal input switch to ensure target recognition specificity and the diffusion-limited 3D DNA walking machine coupled with CRISPR-Cas12a technology as signal amplification means. Based on the bimolecular dynamics model, the rate constants k1 (1.40 × 10[5] M[-1]sec[-1]) and k2 (2.5 × 10[4] M[-1]sec[-1]) of the GNP-PEG(+)/T 3D orbitals modified with positively charged SH-PEG-NH2 are larger than those of unmodified orbitals, proving that nanointerface diffusion restriction effect can accelerate the toehold-mediated chain displacement reaction (TMDR). With the assistance of Co-N/C modified screen-printed electrode (SPE-Co-N/C) sensing interface, the calculated detection limit of CA15-3 is as low as 7.14 × 10[-6] U mL[-1]. The proposed assay, which demonstrated satisfactory selectivity and reproducibility, and correlated highly with ELISA kit results, offered a promising tool for breast cancer early detection and therapeutic monitoring.},
}
@article {pmid41289157,
year = {2026},
author = {van der Meulen, SA and Roemhild, K and Driessen, M and van den Akker, E and Nethe, M},
title = {In vivo modeling of stress erythropoiesis through targeted gene editing of rat hematopoietic stem cells.},
journal = {Blood advances},
volume = {10},
number = {4},
pages = {1281-1292},
doi = {10.1182/bloodadvances.2025017433},
pmid = {41289157},
issn = {2473-9537},
mesh = {Animals ; *Erythropoiesis/genetics ; Rats ; *Gene Editing/methods ; *Hematopoietic Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Anemia/etiology/genetics/metabolism ; Cadherins/genetics/metabolism ; Disease Models, Animal ; *Stress, Physiological ; },
abstract = {In response to anemia, the erythroid lineage significantly expands. This growth is driven by extramedullary erythropoiesis in mice, but is additionally regulated within the bone marrow (BM) of rats, a process likely conserved in humans due to similar BM architecture. This process is, however, mostly elusive. We identified E-cadherin to mark the expansion of the erythroid lineage in BM from anemic rats. To explore the regulation of erythropoiesis in the BM in response to anemia, we studied the role of E-cadherin in the erythroid lineage of rats. As genetic methods to model erythropoiesis in rats are limited, we established a rat BM transplant model that, combined with CRISPR/Cas9 genome editing, enabled us to examine the control of E-cadherin in BM in response to anemia. We identified CD90+CD44+CD45R- cells to contain hematopoietic stem and progenitor cells (HSPCs) in rats. CD90+CD44+CD45R--enriched HSPCs can be efficiently edited using CRISPR/Cas9, which, upon transplant, induce high BM chimerism. Importantly, we identified that recovery from irradiation-induced anemia involves 2 phases. Phase 1 is marked by expansion of erythroid precursors in the BM, supported by extramedullary erythropoiesis in the spleen. This phase is followed by a second phase, characterized by accelerated terminal differentiation, which is selectively controlled in the BM. Finally, we discovered that genetic inactivation of hematopoietic-expressed E-cadherin delays recovery from radiation-induced anemia. Our work provides novel means to expand our knowledge on hematology, and the opportunity to dissect the molecular regulation underlying the erythroid response(s) to anemia in BM, using rat models.},
}
@article {pmid41683796,
year = {2026},
author = {Jiang, Y and Chen, Y and Huang, Z and Chen, L and Huang, X},
title = {Tyrosinase-Deficient Skin Melanophore Lineage in Xenopus tropicalis Tadpoles Shows Strong Autofluorescence.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683796},
issn = {1422-0067},
support = {LY20C120003//Zhejiang Provincial Natural Science Foundation/ ; },
mesh = {Animals ; *Monophenol Monooxygenase/genetics/deficiency/metabolism ; *Xenopus/metabolism/genetics ; Larva/metabolism/genetics ; *Melanophores/metabolism ; *Skin/metabolism ; CRISPR-Cas Systems ; Melanins ; Optical Imaging ; Xenopus Proteins/genetics/metabolism ; Skin Pigmentation ; },
abstract = {Tyrosinase, encoded by Tyr, is a key rate-limiting enzyme in melanin biosynthesis. Knockout of Tyr results in a distinct albino phenotype, making it a widely used target for evaluating gene-editing efficiency. Here, we found that the tyrosinase-deficient skin melanophore lineage of Xenopus tropicalis (X. tropicalis) tadpoles shows strong autofluorescence under the GFP filter, which may interfere with in vivo fluorescence imaging. Through spectral scanning analysis, we characterized the emission spectrum of the autofluorescence under commonly used excitation wavelengths for fluorescent proteins. Based on this, we established a reference protocol for identifying and excluding such interference in Tyr-targeted knockin studies. Furthermore, knockout of the GTP cyclohydrolase 2 gene (Gch2) using CRISPR-Cas9 significantly reduced the fluorescence intensity induced by tyrosinase deficiency, indicating an essential role of the enzyme and its mediated pterine biosynthesis in the generation of the autofluorescence. This study systematically characterized these fluorescent mutant melanophores in X. tropicalis tadpoles, providing a practical basis for avoiding fluorescent interference in experimental science and a new perspective on pigment cell development and evolution.},
}
@article {pmid41683697,
year = {2026},
author = {Kowalik, S and Samoń, M and Przyborowski, M},
title = {Molecular Regulators of In Vitro Regeneration in Wheat: Roles of Morphogenic Factors in Transformation, Genome Editing, and Breeding.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683697},
issn = {1422-0067},
support = {Dotacja Celowa task 4.1//Ministry of Agriculture and Rural Development/ ; },
mesh = {*Triticum/genetics/physiology/growth &amp; development ; *Gene Editing/methods ; *Plant Breeding/methods ; *Regeneration/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; *Transformation, Genetic ; Transcription Factors/genetics/metabolism ; },
abstract = {Efficient in vitro regeneration remains a major constraint in the genetic transformation, genome editing, and molecular breeding of wheat (Triticum aestivum L.), largely due to strong genotype-dependent recalcitrance and limited activation of developmental programs required for somatic embryogenesis. Plant regeneration relies on extensive transcriptional reprogramming and epigenetic remodeling orchestrated by morphogenic regulators that modulate meristem identity, as well as cellular pluri- and totipotency. In this review, we synthesize current molecular knowledge on key transcription factors (BBM, WUS/WUS2, GRF-GIF, WOX, LAX1, SERK, WIND1/ERF115) and signaling peptides (CLE/CLV-WUS module, phytosulfokine/PSK) that regulate embryogenic competence in monocot cereals, with emphasis on their orthologs and functional relevance in wheat. We highlight how controlled expression of these morphogenic genes, promoter engineering, and transient or excisable induction systems can significantly enhance regeneration capacity, reduce chimerism in CRISPR-Cas-edited plants, and facilitate genotype-independent transformation. We also discuss epigenetic and metabolic constraints underlying wheat recalcitrance and their potential modulation to improve culture responsiveness. By integrating evidence from wheat, rice, maize, and barley, we outline conserved gene-regulatory networks that reinitiate totipotency and propose strategies to accelerate doubled haploid production and speed-breeding pipelines. Collectively, morphogenic factors emerge as central molecular tools for overcoming regeneration bottlenecks and enabling next-generation wheat improvement. The objective of this review is to synthesize and critically evaluate current molecular knowledge on morphogenic regulators controlling in vitro regeneration in wheat (Triticum aestivum L.), with particular emphasis on their roles in genetic transformation and genome editing.},
}
@article {pmid41683605,
year = {2026},
author = {Blazyte, A and Lee, H and Yoon, C and Jeon, S and Lee, J and Bayarsaikhan, D and Kim, J and Park, S and Cho, J and Baek, SA and Byun, G and Lee, B and Bhak, J},
title = {Neurofibromin 1 (NF1) Splicing Mutation c.61-2A&gt;G: From Aberrant mRNA Processing to Therapeutic Implications In Silico.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683605},
issn = {1422-0067},
support = {1.200047.01//Ulsan City Research Fund/ ; RS-2023-00263429//Genome Editing Research Program/ ; KEIT 20018560//Alchemist Project of the Korea Evaluation Institute of Industrial Technology/ ; RS-2024-00435468//Korea Planning &amp; Evaluation Institute of Industrial Technology with support from the Ministry of Trade, Industry and Energy/ ; },
mesh = {*Neurofibromin 1/genetics ; Humans ; *Neurofibromatosis 1/genetics/therapy ; *RNA Splicing/genetics ; *Mutation ; *RNA, Messenger/genetics ; RNA Splice Sites/genetics ; Gene Editing ; Computer Simulation ; DNA Methylation ; CRISPR-Cas Systems ; Male ; },
abstract = {The neurofibromin 1 (NF1) splice-site mutation c.61-2A>G (rs1131691100) is a rare, pathogenic, autosomal dominant variant that disrupts NF1 tumor-suppressor function, causing neurofibromatosis type 1 (NF1). Its pathogenic mechanism is poorly understood, and the potential for personalized therapeutic genome editing remains unknown due to the absence of a standard framework for investigating splicing disorders. Here, we performed a comprehensive multi-omics analysis of a de novo c.61-2A>G case from South Korea, integrating short- and long-read whole genome sequencing, whole transcriptome sequencing, and methylation profiling. We confirm that c.61-2A>G abolishes the canonical splice acceptor site, activating a cryptic splice acceptor 16 nucleotides downstream in exon 2. This splicing shift generates a 16-nucleotide deletion, causing a frameshift and premature stop codon that truncates the protein's N-terminal region. Long-read sequencing further reveals that the mutation creates a novel CpG dinucleotide, which is methylated in the majority of reads. Finally, we assessed therapeutic correction strategies, revealing that CRISPR-Cas9 prime editing is the only viable approach for in vivo correction. This study provides the first comprehensive multi-omics characterization of the NF1 c.61-2A>G mutation and establishes a minimal framework for precision therapeutic development in silico in monogenic splicing disorders.},
}
@article {pmid41683574,
year = {2026},
author = {Sopel, J and Sarad, K and Kozinska, A and Mokrzyński, K and Szczygieł, D and Murzyn, A and Drzał, A and Słomiński, A and Szczygieł, M and Elas, M},
title = {Pmel17 Deficiency Affects Melanogenesis and Promotes Tumor Vascularization.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683574},
issn = {1422-0067},
mesh = {Animals ; *Melanins/metabolism/biosynthesis ; Mice ; *Neovascularization, Pathologic/metabolism/genetics/pathology ; *Melanoma, Experimental/metabolism/pathology/genetics/blood supply ; Reactive Oxygen Species/metabolism ; Cell Line, Tumor ; *gp100 Melanoma Antigen/genetics/metabolism/deficiency ; CRISPR-Cas Systems ; Cell Movement ; Melanosomes/metabolism ; Cell Cycle ; Melanogenesis ; },
abstract = {Premelanosomal protein (Pmel, also known as Pmel17) is the major component of melanosomal fibrils and plays a key role in melanin polymerization, making it an important factor in melanogenesis. We investigated how the absence of Pmel affects the properties of B16F10 melanoma cells. Pmel-knockout B16F10 cells were generated using CRISPR/Cas9-mediated genome editing. A viability assay revealed no significant differences between wild-type (WT) and Pmel-knockout (KO) sublines; however, melanosome maturation was impaired. In Pmel KO cells, the cell cycle was disrupted, and higher levels of reactive oxygen species (ROS) were observed compared with WT cells. Moreover, the migration capacity and tube formation of melanoma cells were increased. Tumors derived from Pmel KO cells exhibited unchanged growth kinetics but reduced melanin content, along with enhanced vascularization and oxygenation. Thus, knockout of the Pmel17 gene in melanoma cells alters pigmentation, vascularization, and oxygenation of tumors. These parameters are crucial for both tumor progression and therapeutic response.},
}
@article {pmid41638522,
year = {2026},
author = {Niles, A and Kroening, K and Lauer, A and Chakravorty, A},
title = {Identity of protease-based biomarkers for viability and cytotoxicity revealed by CRISPR knockouts.},
journal = {Analytical biochemistry},
volume = {712},
number = {},
pages = {116073},
doi = {10.1016/j.ab.2026.116073},
pmid = {41638522},
issn = {1096-0309},
mesh = {Humans ; Cell Survival/drug effects ; *Gene Knockout Techniques ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Biomarkers/metabolism/analysis ; *Peptide Hydrolases/metabolism/genetics ; Gene Editing ; },
abstract = {A wide variety of assay chemistries are routinely employed to determine cell health within an in vitro test population. Each method relies on the consistent and robust measurement of specific biological surrogates for cell viability or cytotoxicity. Unfortunately, the precise cellular origin or identity of many of these biomarkers remain poorly characterized or unknown, and thus subject to a host of undetermined biological and chemical interferences. This work details efforts to pinpoint the enzymatic sources of a set of proteolytic activity profiles previously discovered in a phenotypic activity screen and measured in a multiplexed viability ("live cell") and cytotoxicity ("dead cell") assay. First, Clustered Regularly Interspaced Short Palindromic Repeats gene editing (CRISPR) was utilized to knockout (KO) genes encoding candidate enzymes in a human cell background to create clones for identity testing. Next, clones demonstrating discrete reduction of either the live or dead cell signals were further characterized by Western blot analysis for presence of immunogenic protein and by Sanger Sequencing of the targeted edit site. The KO data directed the sourcing of potent and selective inhibitors for orthogonal activity studies of the proteases in a parental population. Last, the utility of the multiplexed assay reagent was further explored in both non-human and human primary cell lines to characterize the universality of the application. Collectively, the positive identification of Cathepsin C (CatC) and Tripeptidyl peptidase II (TPP II) informs assay users about potential modulators of activity leading to possible interferences. Last, the work provides new information about assay performance in previously untested cell types.},
}
@article {pmid41581373,
year = {2026},
author = {Akla, N and Boudah, A and Bertomeu, T and Chatr-Aryamontri, A and Desjarlais, M and Annabi, B},
title = {CRISPR-based chemogenomic profiling reveals redox vulnerabilities to epigallocatechin-3-gallate and green tea polyphenol extract.},
journal = {Redox biology},
volume = {90},
number = {},
pages = {104047},
pmid = {41581373},
issn = {2213-2317},
mesh = {*Catechin/analogs &amp; derivatives/pharmacology ; Humans ; Oxidation-Reduction/drug effects ; *Tea/chemistry ; *CRISPR-Cas Systems ; *Polyphenols/pharmacology ; *Plant Extracts/pharmacology/chemistry ; Oxidative Stress/drug effects ; Antioxidants/pharmacology ; Cell Line, Tumor ; },
abstract = {Green tea polyphenols, particularly epigallocatechin-3-gallate (EGCG), are widely recognized for their beneficial preventive effects against chronic diseases including cancer and obesity. These effects are traditionally attributed to EGCG's antioxidant, anti-inflammatory, and metabolic regulatory properties. In conditions characterized by persistent oxidative stress, the disrupted redox signaling further creates a unique vulnerability that EGCG may exploit through a dual redox mechanism. Emerging evidence therefore suggests that EGCG not only mitigates oxidative damage but could also induce selective pro-oxidant stress in cancer cells, enhancing its therapeutic potential. To investigate this duality, we performed a genome-wide CRISPR/Cas9 knockout screen to identify genetic determinants of EGCG sensitivity and resistance. Our chemogenomic analysis revealed that loss of key antioxidant genes, including PRDX1, CAT, GSS, GCLM, and GCLC, significantly heightened cellular susceptibility to EGCG and green tea extract (GTE), underscoring the critical role of glutathione biosynthesis and redox homeostasis in mediating cytotoxicity. In contrast, knockouts of Kelch-like ECH-associated Protein 1 (KEAP1) and peroxisome-associated PEX genes conferred resistance, implicating in part NRF2 (also known as nuclear factor erythroid-derived 2-like 2; NFE2L2) activation and peroxisomal reactive oxygen species clearance in protective responses. Comparative profiling with gallic acid (GA), which lacks EGCG's catechin structure, further highlighted the gallate moiety's contribution to glutathione-dependent antioxidant mechanisms. Altogether, these findings illuminate the complex redox biology of EGCG and identify novel genetic vulnerabilities that may be leveraged to enhance its anticancer efficacy, particularly in obesity-associated cancers. Clinically, this work could support the development of EGCG-based interventions tailored to individual redox profiles, offering a precise chemopreventive strategy for patients at high risk of malignancies driven by metabolic and oxidative dysregulation. Furthermore, the identification of new genetic markers of EGCG sensitivity and resistance may inform future exploration of patient stratification.},
}
@article {pmid41526374,
year = {2026},
author = {Xie, N and Pan, Y and Tong, H and Lin, Y and Jiang, Y and Wang, Z and Wan, J and Zhang, W and Wang, X and Sun, X and Yan, S and Yin, P and Sun, Q and Qi, C and Tian, Y and Shen, L and Jiang, H and Liang, D and Tang, B and Li, S and Li, XJ and Liu, Q},
title = {Precise excision of expanded GGC repeats in NOTCH2NLC via CRISPR/Cas9 for treating neuronal intranuclear inclusion disease.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1683},
pmid = {41526374},
issn = {2041-1723},
support = {32071037//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Gene Editing/methods ; *Intranuclear Inclusion Bodies/genetics/pathology/metabolism ; *Neurodegenerative Diseases/genetics/therapy/pathology ; *Trinucleotide Repeat Expansion/genetics ; Disease Models, Animal ; Mice, Transgenic ; *Receptor, Notch2/genetics ; Cell Line ; Male ; Nerve Tissue Proteins ; Intercellular Signaling Peptides and Proteins ; },
abstract = {Neuronal intranuclear inclusion disease (NIID) is an adult-onset neurodegenerative disease caused by expanded GGC repeats in the 5' untranslated region of the human-specific NOTCH2NLC gene. The high sequence similarity between NOTCH2NLC and its paralogs poses a significant challenge for precise gene editing. Here, we develop a CRISPR/spCas9-based gene-editing strategy that precisely excises the expanded GGC repeats in NOTCH2NLC without detectable off-target effects on the highly homologous NOTCH2/NOTCH2NL family genes (<2% sequence divergence at this locus). The efficacy, specificity and safety of this approach are rigorously validated across multiple experimental models, including human cell lines, NIID iPSCs, and our previously established transgenic NIID mouse model. Our results demonstrate that precise excision of the expanded GGC repeats effectively alleviates NIID-related neuropathological, molecular and behavioral abnormalities. This study establishes the proof of concept for genome editing as a therapeutic strategy for NIID and other related repeat expansion disorders.},
}
@article {pmid41248420,
year = {2026},
author = {Long, SA and Todd, H and Goodhart, G and Chang, WH and Amparo, AM and Bridgens, R and Dilly, J and Park, SJ and Beal, RM and Shehadeh, SM and Satyadi, MA and Trivedi, VK and Ackermann, SE and Mukherjee, R and Goodwin, CM and Edwards, AC and Stalnecker, CA and Greis, KD and Aguirre, AJ and Hobbs, GA and Bryant, KL and Ahmad, SA and Cox, AD and Der, CJ and Waters, AM},
title = {CRISPR-Cas9 Screening Identifies Resistance Mechanisms to KRAS Inhibition in Pancreatic Cancer.},
journal = {Cancer research},
volume = {86},
number = {4},
pages = {1035-1053},
doi = {10.1158/0008-5472.CAN-25-1835},
pmid = {41248420},
issn = {1538-7445},
support = {K22CA276632//National Cancer Institute (NCI)/ ; IRG-23-1141524//American Cancer Society (ACS)/ ; PF-23-1072348-01-CDP//American Cancer Society (ACS)/ ; 22-WG_DERB//Pancreatic Cancer Action Network (PCAN)/ ; T32CA244125//National Cancer Institute (NCI)/ ; 23-MF-DILL//Pancreatic Cancer Action Network (PCAN)/ ; R25CA261610//National Cancer Institute (NCI)/ ; R01CA42978//National Cancer Institute (NCI)/ ; P50CA196510//National Cancer Institute (NCI)/ ; U01CA199235//National Cancer Institute (NCI)/ ; P01CA203657//National Cancer Institute (NCI)/ ; R35CA232113//National Cancer Institute (NCI)/ ; P50CA257911//National Cancer Institute (NCI)/ ; R37CA251877//National Cancer Institute (NCI)/ ; 15-90-25-DER//Pancreatic Cancer Action Network (PCAN)/ ; W81XWH2110692//U.S. Department of Defense (DOD)/ ; S10OD026717//National Institutes of Health (NIH)/ ; },
mesh = {Humans ; *Pancreatic Neoplasms/genetics/drug therapy/pathology ; *Proto-Oncogene Proteins p21(ras)/antagonists &amp; inhibitors/genetics ; *CRISPR-Cas Systems ; *Drug Resistance, Neoplasm/genetics ; *Carcinoma, Pancreatic Ductal/genetics/drug therapy/pathology ; Cell Line, Tumor ; Mutation ; *Protein Kinase Inhibitors/pharmacology ; Erlotinib Hydrochloride/pharmacology ; },
abstract = {UNLABELLED: KRAS inhibitors (KRASi) targeting various KRAS mutations have entered clinical trials for pancreatic cancer. Despite promising preliminary clinical responses, most patients relapse due to intrinsic or acquired resistance. Thus, combination treatments are essential to extend the efficacy of KRAS-targeted therapies. To further determine the genetic mechanisms of KRASi resistance, we performed KRASi-anchored CRISPR-Cas9 loss-of-function screens in KRASG12D-, KRASG12C-, KRASG12R-, and KRASQ61H-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines, using six KRASi, to identify genes that modulate sensitivity to KRAS inhibition. Several hits from the screens, including EGFR, CK2, p110α, p110γ, and YAP, were validated by combining targeted inhibitors with KRASi. KRASQ61H-mutant PDAC cell lines were intrinsically less dependent on KRAS for survival than other KRAS mutational subtypes. Furthermore, the EGFR inhibitor erlotinib synergized with the RAS(ON) multiselective inhibitor RMC-7977 in KRASQ61H-mutant PDAC cell lines and in cell lines with highly active EGFR by mitigating ERK rebound activity. KRASi-resistant cell lines featured sustained ERK/MAPK dependence despite decreased ERK activity. Together, these findings enhance the understanding of intrinsic and acquired resistance to KRASi and identify therapeutic vulnerabilities that can potentially be exploited for KRASi combination therapies in patients with pancreatic cancer.<br><br>SIGNIFICANCE: A comprehensive assessment of genetic modulators of KRAS inhibitor sensitivity identifies combination approaches to increase the efficacy of KRAS inhibitors and demonstrates the limited response of KRASQ61H-mutant cancer cells to KRAS inhibition.},
}
@article {pmid41035413,
year = {2026},
author = {Mazzei, A and Martewicz, S and Amiri, R and Cui, M and Elvassore, N and Luni, C},
title = {Functional CRISPR-Cas9 knockout screening of the genetic determinants of human fibroblast migration propensity.},
journal = {Biotechnology progress},
volume = {42},
number = {1},
pages = {e70076},
doi = {10.1002/btpr.70076},
pmid = {41035413},
issn = {1520-6033},
support = {CUP J45F21002000001//Universit&#xe0; di Bologna/ ; F-0301-15-009//ShanghaiTech University/ ; },
mesh = {Humans ; *Cell Movement/genetics ; *CRISPR-Cas Systems/genetics ; *Fibroblasts/cytology/metabolism ; *Gene Knockout Techniques/methods ; MicroRNAs/genetics ; Gene Editing ; },
abstract = {Directional cell migration plays a central role in a wide range of physiological and pathological conditions, such as embryonic development or tumor metastasis. Steps involved in cell migration include cell polarization, formation of membrane protrusions at the cell front side and adhesion disassembly at the rear side, and a general cytoskeletal rearrangement. Overall, it is a complex phenomenon at the interface between mechanical forces and biochemical signaling, with cell-specific and context-specific molecular events acting in the process. Here, we focus on human fibroblast migration induced by a biochemical gradient with an approach that connects the identification of molecular players with the actual mechanical function. We show how to screen for genes and miRNAs involved in migration by the direct integration of a high-throughput gene editing method, the CRISPR-Cas9 knockout pool screening, and a well-established functional assay, the transwell migration assay. Moreover, the screening has been performed after an expansion step aiming at the removal of all the essential genes and miRNAs, so as to identify targets related to the cell migratory ability without affecting other major cellular functions. The results confirm known genes involved in migration, but also highlight new candidates. This work establishes a methodological advancement in the use of CRISPR technology for functional screening and represents a resource for candidate genes and miRNAs playing a role in human fibroblast directional migration under biochemical gradient.},
}
@article {pmid41683064,
year = {2026},
author = {Chen, Z and He, D and Yu, W and Fu, X and Zhang, L and Zhang, M and Yu, X and Ye, Z},
title = {Advancing Bongkrekic Acid Detection: From Conventional Instrumental Analysis to Advanced Biosensing for Cross-Toxin Applications.},
journal = {Foods (Basel, Switzerland)},
volume = {15},
number = {3},
pages = {},
pmid = {41683064},
issn = {2304-8158},
abstract = {Bongkrekic acid (BKA), a highly lethal toxin, has been implicated in frequent poisoning incidents in recent years, posing a serious threat to global food safety and creating an urgent need for rapid and sensitive detection methods. This review provides a systematic analysis of the entire BKA detection technologies, covering sample pretreatment techniques, instrumental analysis, immunoassays, and biosensing methods. It assesses the merits of key methods and also explores the strategic cross-application of detection paradigms developed for analogous toxins. This review delivers a comprehensive and critical evaluation of BKA detection technologies. First, it discusses sample pretreatment strategies, notably solid-phase extraction (SPE) and QuEChERS. Subsequently, it analyzes the principles, performance, and applications of core detection methods, including high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), high-resolution mass spectrometry (HRMS), time-resolved fluorescence immunoassay (TRFIA), dual-mode immunosensors and nanomaterial-based sensors. Instrumental methods (e.g., HRMS) offer unmatched sensitivity [with a limit of detection (LOD) as low as 0.01 μg/kg], yet remain costly and laboratory-dependent. Immunoassay and biosensor approaches (TRFIA and dual-mode sensors) enable rapid on-site detection with high sensitivity (ng/mL to pg/mL), though challenges in stability and specificity remain. Looking forward, the development of next-generation BKA detection could be accelerated by cross-applying cutting-edge strategies proven for toxins-such as Fumonisin B1 (FB1), Ochratoxin A (OTA), and Aflatoxin B1 (AFB1)-including nanobody technology, CRISPR-Cas-mediated signal amplification, and multimodal integrated platforms. To translate this potential into practical tools, future research should prioritize the synthesis of high-specificity recognition elements, innovative signal amplification strategies, and integrated portable devices, aiming to establish end-to-end biosensing systems capable of on-site rapid detection through multitechnology integration.},
}
@article {pmid41680629,
year = {2026},
author = {Selhorst, P and Van Vyve, E and Falconi-Agapito, F and Mariën, J and Ariën, KK},
title = {Sensitive, flexible, and affordable serum RNA sequencing for pathogen detection on the Oxford Nanopore platform.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {188},
pmid = {41680629},
issn = {1471-2164},
support = {U01AI151378/NH/NIH HHS/United States ; },
abstract = {UNLABELLED: Metagenomic sequencing for pathogen detection has traditionally suffered from low sensitivity due to the overwhelming presence of host nucleic acids. Commercial host-depletion kits are often prohibitively expensive and limited to specific species, hindering adoption in resource-limited settings, where the burden of zoonotic diseases is highest. To address this, we optimized and combined Sequence-Independent Single Primer Amplification (SISPA) with Depletion of Abundant Sequences by Hybridization (DASH), establishing a low-cost metagenomic protocol on the Oxford Nanopore sequencing platform. Our approach can be adapted to any species to detect microbial RNAs in serum samples at PCR-range sensitivity, outperforming existing methods in the field.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-12268-4.},
}
@article {pmid41605411,
year = {2026},
author = {Zhang, Q and Yang, Y and Huang, X and Ma, J and Duan, Y and Ma, G and Lei, H},
title = {Prime editing for ocular gene therapy and disease modeling: a narrative review of advances, delivery, and translational readiness.},
journal = {Experimental eye research},
volume = {265},
number = {},
pages = {110891},
doi = {10.1016/j.exer.2026.110891},
pmid = {41605411},
issn = {1096-0007},
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; Animals ; CRISPR-Cas Systems ; Translational Research, Biomedical ; *Eye Diseases/therapy/genetics ; Disease Models, Animal ; Gene Transfer Techniques ; },
abstract = {Prime editing is a versatile "search-and-replace" genome-editing technology that enables precise and flexible genome correction of genetic sequences by reverse-transcribing an RNA template encoded at the 3' end of a prime editing guide RNA (pegRNA). It supports the introduction of nucleotide substitutions, and insertions or/and deletions (indels) in living cells without requiring double-stranded DNA breaks or exogenous donor templates. Since its introduction in 2019, prime editing has advanced rapidly-from the first-generation prime editor (PE1) to PE7 and other next-generation variants-with editing efficiencies increasing from 0.7 to 5.5 % to more than 50 % in vitro. Optimization strategies including engineering of the Cas9 and reverse transcriptase domains, refinement of pegRNA architecture, recruitment of auxiliary proteins, and modulation of DNA repair pathways have substantially enhanced editing efficiency, product purity, and target scope across diverse cell types and tissues. These developments are particularly relevant to ophthalmology, where many blinding disorders arise from point mutations or small indels ideally suited for prime editing-based correction. Recent work in retinal cells and animal models has demonstrated the growing feasibility of prime editing to treat inherited retinal diseases, modulate pathological angiogenesis, and achieve precise gene repair in post-mitotic photoreceptors and retinal pigment epithelial cells. As delivery vectors and newer PE variants improve, prime editing is a plausible next-generation platform for a wide range of ocular diseases.},
}
@article {pmid41485557,
year = {2026},
author = {Li, D},
title = {Rising Star Engineering the Genome for Curative Futures.},
journal = {Journal of molecular biology},
volume = {438},
number = {5},
pages = {169618},
doi = {10.1016/j.jmb.2025.169618},
pmid = {41485557},
issn = {1089-8638},
mesh = {*Gene Editing/methods ; Humans ; Animals ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; *Genetic Engineering/methods ; Mice ; Neoplasms/therapy/genetics ; Disease Models, Animal ; },
abstract = {As a professor of biomedicine in the School of Life Sciences at East China Normal University (ECNU), I am dedicated to developing advanced genome editing technologies for disease modeling and precise gene therapy. My foundational training at Hunan Normal University and Texas A&amp;M University cultivated a deep interest in using engineered cellular and animal models to understand human diseases. Since 2013, my laboratory at ECNU has pioneered the use of TALEN and CRISPR/Cas9 for the rapid generation of knockout mouse and rat models for disease studies. Once stepped in genome editing field, I shifted my focus to advancing editing tools and developing gene therapy strategies for genetic disorders and cancer. My team has developed a suite of high-performance base editors for nuclear DNA, mitochondrial DNA, and RNA, broadening editing capabilities while enhancing precision and safety. Leveraging these technologies, we have designed several therapeutic strategies that have shown efficacy in cellular and animal models of genetic diseases. Through collaborative efforts, we have successfully translated genome editing into clinical applications, contributing to the treatment of patients with β-thalassemia. Additionally, we have developed a non-viral, site-specific CAR-T platform for lymphoma therapy. Looking forward, I aim to develop the next generation of long-fragment, site-specific integration technologies and accelerate clinical translation to bring transformative cures to more patients.},
}
@article {pmid41047302,
year = {2026},
author = {Boultwood, J},
title = {Induced pluripotent stem cell-based modelling of disease evolution in myeloid leukemia: MDS to AML.},
journal = {Advances in biological regulation},
volume = {99},
number = {},
pages = {101119},
doi = {10.1016/j.jbior.2025.101119},
pmid = {41047302},
issn = {2212-4934},
mesh = {Humans ; *Myelodysplastic Syndromes/pathology/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *Leukemia, Myeloid, Acute/pathology/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Animals ; },
abstract = {The myelodysplastic syndromes (MDS) are common myeloid malignancies that develop from the successive acquisition of driver mutations in hematopoietic stem cells residing in the bone marrow. Around a third of MDS patients will develop secondary acute myeloid leukemia (sAML) and patients with high-risk MDS or sAML have a dismal prognosis. The study of disease progression in myeloid malignancy has been enhanced in recent years by the use of induced pluripotent stem cells (iPSCs) technology. iPSCs offer the advantage of indefinite expansion and the potential for genetic modification, with reprogramming enabling the capture of the full complement of genetic lesions found in primary patient bone marrow samples. The power of iPSC and CRISPR-Cas9 gene editing technologies have been harnessed to generate a range of iPSC-based cellular models of MDS, reflecting the genetic and biologic heterogeneity of the disease. Stage-specific patient iPSC lines have been produced and sequential gene editing in normal human iPSCs has been performed to map the evolution of MDS to AML. These studies have increased our understanding of the impact of driver mutations, and co-mutations, on disease phenotype and revealed mechanisms underlying disease stage transitions in myeloid malignancy. iPSC-based models of MDS have also proven important tools in high throughput drug screening and have empowered drug testing and drug discovery, offering a new platform to develop personalized therapy.},
}
@article {pmid40923291,
year = {2026},
author = {Guiltinan, MJ and Landherr, L and Maximova, SN and DelVecchio, D and Sebastian, A and Albert, I},
title = {Reduced Susceptibility to Phytophthora in Non-Transgenic Cacao Progeny Through CRISPR-Cas9 Mediated TcNPR3 Mutagenesis.},
journal = {Plant biotechnology journal},
volume = {24},
number = {2},
pages = {442-454},
doi = {10.1111/pbi.70365},
pmid = {40923291},
issn = {1467-7652},
support = {//Pennsylvania State University College of Agriculture/ ; //Huck Institutes of the Life Sciences/ ; PEN05003//Endowed Program in the Molecular Biology of Cacao and USDA Hatch/ ; PEN4879//Endowed Program in the Molecular Biology of Cacao and USDA Hatch/ ; },
mesh = {*Phytophthora/pathogenicity/physiology ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology/genetics ; *Cacao/genetics/microbiology ; Gene Editing ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Mutagenesis ; Disease Resistance/genetics ; },
abstract = {Black pod disease, caused by a complex of Phytophthora species, poses a severe threat to global cacao production. This study explores the use of CRISPR-Cas9 genome editing to reduce disease susceptibility in Theobroma cacao L. by targeting the TcNPR3 gene, a known negative regulator of plant defence. Transgenic T0 lines carrying mutations predicted to disrupt TcNPR3 function exhibited reduced susceptibility to Phytophthora infection in in vitro foliar assays. These T0 plants were advanced to maturity and outcrossed with non-transgenic cacao to eliminate T-DNA sequences associated with the CRISPR-Cas9 transgene. Whole-genome sequencing of the T0 parents and 22 progeny revealed single T-DNA insertion sites in each T0 line; seven progeny retained the edited npr3 alleles but lacked T-DNA insertions. Transcriptome analysis of the mutant lines showed upregulation of genes associated with reactive oxygen species (ROS) generation, defence-related transcription factors and pathogenesis-related proteins. Several genes were also downregulated, suggesting that TcNPR3 may function as both a repressor and an activator in regulating basal transcriptional states. Genome-edited plants were phenotypically comparable to wild-type controls and displayed a 42% reduction in lesion size upon Phytophthora challenge. These findings demonstrate the feasibility of generating non-transgenic cacao with reduced susceptibility to Phytophthora through CRISPR-Cas9-mediated genome editing, offering a promising strategy for sustainable cacao cultivation and improved farmer livelihoods. Field trials are underway to evaluate long-term agronomic performance under natural conditions.},
}
@article {pmid41680487,
year = {2026},
author = {Kroell, AS and Hoffmann, KH and Motzkus, NA and Lemmen, N and Happ, N and Wolf, B and von Bachmann, AL and Southern, N and Vogd, F and Aschenbrenner, S and Niopek, D and Mathony, J},
title = {Modular engineering of thermoresponsive allosteric proteins.},
journal = {Nature chemical biology},
volume = {},
number = {},
pages = {},
pmid = {41680487},
issn = {1552-4469},
support = {520612620//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 453202693//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
abstract = {Thermogenetics enables noninvasive spatiotemporal control over protein activity in living cells and tissues, yet its applications have largely been restricted to transcriptional regulation and membrane recruitment. Here, we present a generalizable strategy for engineering thermosensitive allosteric proteins through the insertion of optimized Avena sativa LOV2 domain variants. Applying this approach to a diverse set of structurally and functionally unrelated proteins in Escherichia coli, we generated potent, thermoswitchable chimeric variants that can be tightly controlled within narrow temperature ranges (37-41 °C). Extending this strategy to mammalian systems, we engineered CRISPR-Cas genome editors directly modulated by subtle temperature changes within the physiological range. Lastly, we showcase the incorporation of a chemoreceptor domain as an alternative thermosensing module, suggesting thermosensitivity to be a widespread feature in receptor domains. This work expands the toolkit of thermogenetics, providing a blueprint for temperature-dependent control of virtually any protein of interest.},
}
@article {pmid41680202,
year = {2026},
author = {Garmendia-Antoñana, N and Dorado-Morales, P and Gil, C and García, B and Echeverz, M and Solano, C and Penadés, JR and Lasa, I},
title = {Targeted elimination of Staphylococcus aureus mastitis infections with synthetic phage-based CRISPR-Cas delivery systems.},
journal = {NPJ biofilms and microbiomes},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41522-026-00931-x},
pmid = {41680202},
issn = {2055-5008},
support = {PRE2021-097385//Spanish Ministry of Science, Innovation and Universities/ ; PID2020-113494RB-I00/ AEI//Spanish Ministry of Science, Innovation and Universities/ ; BES-2015-07285//the Spanish Ministry of Science, Innovation and Universities/ ; },
abstract = {Treatment options for Staphylococcus aureus infections are increasingly limited, particularly in livestock, where S. aureus causes mastitis requiring prolonged antibiotic therapy. This study engineered Phage Inducible Chromosomal Islands (ePICIs) to deliver CRISPR-Cas9 modules targeting small RNA genes. ePICIs exhibit bactericidal activity without chromosomal integration, an expanded host range compared to their parental phages, and biofilm-dependent efficacy influenced by the extracellular matrix composition. Biofilms mediated by the Bap protein strongly protect bacteria from ePICIs, whereas PIA/PNAG-based biofilms do not. Despite Bap-mediated protection in vitro, ePICIs achieved bactericidal effects comparable to vancomycin in a mouse mastitis model caused by Bap-producing strains. These findings reveal key factors affecting phage-delivered CRISPR-Cas efficacy and highlight that antibiofilm therapies should not be dismissed based solely on in vitro performance. Non-replicative ePICIs thus represent a promising alternative for treating localized infections such as mastitis.},
}
@article {pmid41679307,
year = {2026},
author = {Zhao, J and Wang, Z and Lu, L and Bu, G and Miao, Z and Zhang, Y and Guo, Y and Yang, Z and Ma, J and Jiao, J and Ma, X},
title = {An orthogonal CRISPR/Cpf1 platform for precise spatiotemporal gene regulation and osteoporotic fracture repair.},
journal = {Cell reports methods},
volume = {},
number = {},
pages = {101299},
doi = {10.1016/j.crmeth.2025.101299},
pmid = {41679307},
issn = {2667-2375},
abstract = {CRISPR-Cas systems enable powerful gene editing and regulation, yet single-modality control often fails to achieve orthogonal, spatiotemporally precise regulation of multiple endogenous genes. We engineered OREC, an orthogonal platform integrating chemogenetic and optogenetic modalities for precise, reversible, multiplex gene control. OREC comprises two components: OREC[C] regulated by doxycycline (Dox) and OREC[o] controlled by light. By assembling catalytically dead Cpf1 (dCpf1), gene regulatory elements, and crRNA arrays on single transcripts, OREC enables robust simultaneous manipulation of multiple genes. We demonstrated OREC's therapeutic potential in vitro for osteoblast function modulation and in vivo for osteoporotic fracture repair. OREC effectively activated Bmp2 while inhibiting Dkk1, significantly enhancing bone formation and fracture healing in mouse models. These results establish OREC as a versatile platform for precise multiplex gene regulation, offering significant advancement for CRISPR-based gene therapy applications in complex tissues where coordinated control of multiple therapeutic targets is essential.},
}
@article {pmid41678638,
year = {2026},
author = {El-Brolosy, MA and Oak, A and Hoang, AT and Damergi, Y and Fischer, A and Saunders, RA and Luo, J and Balabaki, A and Guez, J and Whitfield, TW and Goldman, SR and Latifkar, A and Lu, YR and Stainier, DYR and Karczewski, KJ and Corradin, O and Weissman, JS},
title = {Mechanisms linking cytoplasmic decay of translation-defective mRNA to transcriptional adaptation.},
journal = {Science (New York, N.Y.)},
volume = {391},
number = {6786},
pages = {eaea1272},
doi = {10.1126/science.aea1272},
pmid = {41678638},
issn = {1095-9203},
mesh = {*RNA Stability ; *RNA, Messenger/metabolism/genetics ; *Cytoplasm/metabolism ; Humans ; *Transcription, Genetic ; *Protein Biosynthesis ; *RNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Transcriptional adaptation (TA) is a genetic robustness mechanism through which mutant messenger RNA (mRNA) decay induces sequence-dependent up-regulation of so-called adapting genes. How cytoplasmically generated mRNA fragments affect nuclear transcription remains poorly understood. Using genome-wide CRISPR screens, we uncover ILF3 as an RNA binding protein connecting cytoplasmic mRNA decay and transcription during TA and show that it is required for a range of TA substrates. ILF3 is enriched at adapting genes' RNAs, and its artificial recruitment through dCas13 promotes gene expression. Using tiling oligonucleotide screens, we identify trigger RNA fragments that activate adapting genes when introduced into cells. Further functional dissection reveals a critical role for homology between trigger and target sequences. These findings enhance our molecular understanding of TA and inform the design of programmable oligonucleotides for gene expression augmentation.},
}
@article {pmid41678468,
year = {2026},
author = {Mendoza-Garcia, P and Keith, B and Nordberg, M and Quist, E and Ferrás, C and Hamza, GM and Elgendy, R and Ashenden, SK and Chi, J and van Zuydam, NR and Hattersley, N and Zhang, X},
title = {Omics-aided design genome editing strategy for challenging human immortalized cell models.},
journal = {PloS one},
volume = {21},
number = {2},
pages = {e0341124},
pmid = {41678468},
issn = {1932-6203},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; MCF-7 Cells ; Hep G2 Cells ; DNA End-Joining Repair ; Proliferating Cell Nuclear Antigen/genetics/metabolism ; Genomics/methods ; Gene Knock-In Techniques ; },
abstract = {CRISPR-Cas9 has become a popular genome editing tool for biomedical research and drug development due to its capability to enable precise correction or integration of genetic mutations in the genome. However, precise genome editing competency varies dramatically between cell types depending on their capabilities for DNA damage. In this proof-of-concept study, we took the example of HepG2 and MCF7 to show that omics profiling identifies bottlenecks that are associated with poor precise knock-in (KI) efficiency in hard-to-engineer cells. These bottlenecks include previously described factors such as the predominance of non-homologous end joining (NHEJ) repair and impaired homologous recombination (HR) capability, but also reveals apoptotic priming status of the cells as a limiting factor. Upon further comparative analysis between HepG2 and MCF7 cells, we pinpointed and validated the proliferating cell nuclear antigen (PCNA) as a target to overexpress to enhance precise KI efficiency in MCF7. Overall, we describe how employing a multi-omics approach to characterize cell models of interest can facilitate an in-depth understanding of their editability molecular signature, empowering us to manipulate the activity of key pathways for precise editing, and therefore increase efficiency of desired editing outcomes.},
}
@article {pmid41678341,
year = {2026},
author = {Wang, Z and Wang, Y and Ji, Q},
title = {Bacterial Cofactors for CRISPR Activation.},
journal = {Biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.biochem.5c00738},
pmid = {41678341},
issn = {1520-4995},
abstract = {Anti-CRISPR (Acr) proteins have long exemplified the viral counterattack against CRISPR-Cas immunity. By contrast, comparatively little is known about host proteins that may increase Cas effector activity. Recent work on a compact type V nuclease, Cas12p, demonstrates that this phage-associated effector depends on the bacterial thioredoxin TrxA for efficient DNA cleavage. TrxA binds a dedicated thioredoxin-binding (TB) domain on Cas12p through a redox-sensitive interaction, promoting an active conformation competent for DNA cleavage. This finding adds to a small but growing set of CRISPR activators and highlights that CRISPR-Cas systems are not static defense modules but dynamic networks shaped by auxiliary factors that can fine-tune their activity.},
}
@article {pmid41678334,
year = {2026},
author = {Wang, Y and Su, X and Chen, Y and Chen, Y and Shi, C and Liu, F and Ye, Y and Sun, P and Tan, M and Yu, M and Wang, Y and Xie, S and Liu, J and Yan, Q and Sun, Q and Neculai, D and Liu, W and Shao, J and Liu, Y and Lin, W and Lin, A},
title = {A CRISPR-based mitochondrial gene therapy tool derived by engineering guide RNAs.},
journal = {Cell reports},
volume = {45},
number = {2},
pages = {116958},
doi = {10.1016/j.celrep.2026.116958},
pmid = {41678334},
issn = {2211-1247},
abstract = {Mitochondrial genetic diseases arise from mitochondrial DNA (mtDNA) defects, which gene therapy tools may rectify. However, delivering single-guide RNAs (sgRNAs) into mitochondria remains a challenge limiting CRISPR-mediated mtDNA therapy. Here, through network analysis of mitochondrion-localized long noncoding RNAs (lncRNAs) and RNA-binding proteins (RBPs), we found that lncRNA RP11-46H11.3 translocates into mitochondria via binding mitochondria-associated RBPs using its key RNA recognition motifs (RRMs); its derived 30 nt ST2-RNA mitochondrial targeting sequence (RMTS) showed the highest mitochondrial localization efficiency. We engineered the RMTS-CRISPR tool by fusing ST2-RMTS to sgRNA, verifying its ability to target and cleave mtDNA. Strikingly, our results demonstrated that RMTS-CRISPR could achieve heteroplasmic mtDNA shifting efficiencies of up to 26.37% in m.3243A>G mutant cell models and 26.79% in vivo, offering a technological approach for the correction of heterogeneous mtDNA mutations. Taken together, our findings reveal a CRISPR-based mitochondrial gene intervention strategy that may have applications in mitochondrial disorders.},
}
@article {pmid41530154,
year = {2026},
author = {Li, H and Liu, F and Li, J and Shi, C and Lin, Z and Qin, Y and Pan, R and Wu, X and Peng, Y and Xing, C and Wang, Y and Qu, Q and Li, G},
title = {Evolutionary dynamics of sex determination in Branchiostoma belcheri driven by repeated transposition of a single novel gene.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1616},
pmid = {41530154},
issn = {2041-1723},
support = {Start-up//Xiamen University (XMU)/ ; 32300346//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32200411//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32270439//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32061160471//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2022J06004//Natural Science Foundation of Fujian Province (Fujian Provincial Natural Science Foundation)/ ; },
mesh = {Animals ; *Sex Determination Processes/genetics ; Female ; Male ; *Lancelets/genetics ; *Evolution, Molecular ; CRISPR-Cas Systems ; *DNA Transposable Elements/genetics ; Sex Chromosomes/genetics ; RNA, Long Noncoding/genetics ; },
abstract = {Sex determination systems display striking evolutionary flexibility, yet the mechanisms underlying their transitions remain poorly understood. Using newly generated genome assemblies, we investigated the evolving sex-determining system in the amphioxus Branchiostoma belcheri. We identified two female-specific sex-determining regions (SDRs) on chromosome 13, both derived from independent transpositions of the autosomal gene tesD, which shows testis-specific expression in amphioxus species. CRISPR/Cas9 knockout experiments in Branchiostoma floridae confirmed that tesD functions as a male-determination gene, with loss of function producing an all-female phenotype. In B. belcheri, the older SDR (tesDwa) inserted into the coding region of twai, while the younger SDR (tesDwb), flanked by active Zator-1 transposons, inserted into the 3' UTR of vps9c and later translocated to autosomes in ~10% of individuals. Transcriptomic analyses revealed that W-linked tesDwa and tesDwb produce antisense long non-coding RNAs that likely suppress tesD transcription in females, whereas autosomal tesDwb is not expressed and appears non-functional. The insertion sites and co-transcription with host genes suggest promoter hijacking. Together, these findings demonstrate that recurrent transpositions can generate new functional SDRs that coexist with older ones, driving dynamic turnover of sex determination in B. belcheri.},
}
@article {pmid41500180,
year = {2026},
author = {Sheng, C and Wang, J and Tan, M and Zhang, J and Sun, M and Sun, J and Shao, Y and Tu, J and Zhu, L and Song, X},
title = {Establishment of detection method of chicken infectious anemia virus based on CRISPR/Cas12a system.},
journal = {Research in veterinary science},
volume = {201},
number = {},
pages = {106046},
doi = {10.1016/j.rvsc.2025.106046},
pmid = {41500180},
issn = {1532-2661},
mesh = {*Chicken anemia virus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; Animals ; Chickens ; *Poultry Diseases/diagnosis/virology ; *Circoviridae Infections/veterinary/diagnosis/virology ; Sensitivity and Specificity ; },
abstract = {Chicken Infectious Anemia Virus (CIAV) causes chicken infectious anemia, characterized by anemia and immune dysfunction. The rapid dissemination of this virus is generating substantial economic consequences for poultry producers. The CRISPR/Cas12a system is widely used for virus detection through crRNA-guided target recognition and the paracrine activity of Cas12a. To enable rapid and highly sensitive detection of Chicken Infectious Anemia Virus (CIAV), a CRISPR-Cas12a-based fluorescence assay was refined. Through optimization of the CRISPR/Cas12a system and integration of enzymatic recombinase amplification (ERA), the assay achieved a detection limit of 1 copy/μL, demonstrating its significant utility for CIAV diagnostics. In addition, a CRISPR/Cas12a lateral flow assay was developed and optimized, achieving a sensitivity of 10^3 copies/μL for the rapid and visual detection of target analytes. This technique exhibits high specificity for CIAV, showing no cross-reactivity with other chicken viruses. Overall, the system enables rapid CIAV detection with cost-effective equipment, making it suitable for virus monitoring.},
}
@article {pmid41475537,
year = {2026},
author = {Tang, W and Ma, M and Song, W and Kou, M and Wang, X and Yan, H and Li, C and Zhang, A and Gao, T and Gao, R and Zhang, Y and Li, Q},
title = {An efficient CRISPR/Cas9-mediated editing of phytoene desaturase in hexaploid sweetpotato.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {364},
number = {},
pages = {112967},
doi = {10.1016/j.plantsci.2025.112967},
pmid = {41475537},
issn = {1873-2259},
mesh = {*Ipomoea batatas/genetics/enzymology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Oxidoreductases/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Polyploidy ; Chlorophyll/metabolism ; },
abstract = {CRISPR/Cas9-mediated gene editing has emerged as a pivotal tool for functional genomics and crop improvement. For the first time, we applied CRISPR/Cas9-mediated editing to the IbPDS gene in the purple-fleshed cultivar 'XZS-8', achieving mutation efficiencies up to 98.18 %. Loss-of-function mutations in IbPDS induced visually discernible albino phenotypes. Hi-TOM sequencing confirmed deletion mutations within the target locus, with editing efficiencies ranging from 17.77 % to 65.90 % (gRNA1) and 87.87-98.18 % (gRNA2). Knockout lines showed significant reductions in chlorophyll a and b content, confirming functional disruption of IbPDS. Collectively, our results demonstrate efficient CRISPR/Cas9-mediated genome editing for generating mutants in the hexaploid sweetpotato.},
}
@article {pmid41420106,
year = {2026},
author = {Ge, J and Hirosue, S and Castillon, L and Patel, SA and Wesolowski, L and Dyas, A and Yong, C and de Haan, S and Drost, J and Stewart, GD and Obenauf, AC and Muñoz-Espín, D and Vanharanta, S},
title = {Mechanisms of resistance to VHL loss-induced genetic and pharmacological vulnerabilities.},
journal = {EMBO molecular medicine},
volume = {18},
number = {2},
pages = {599-619},
pmid = {41420106},
issn = {1757-4684},
support = {C9685/A25177//Cancer Research UK (CRUK)/ ; C62187/A29760//Cancer Research UK (CRUK)/ ; BRC-1215-20014//NIHR | NIHR Cambridge Biomedical Research Centre (NIHR Cambridge BRC)/ ; 955951//European Commission (EC)/ ; MC_UU_12022/7//UKRI | Medical Research Council (MRC)/ ; RP_033_20170303//Kidney Research UK/ ; 338420//Research Council of Finland (AKA)/ ; },
mesh = {*Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; CRISPR-Cas Systems ; *Drug Resistance, Neoplasm ; Cell Line, Tumor ; *Antineoplastic Agents/pharmacology ; Epithelial Cells ; },
abstract = {The von Hippel-Lindau tumor suppressor (VHL) is a component of a ubiquitin ligase complex that controls cellular responses to hypoxia. Endogenous VHL is also utilized by proteolysis-targeting chimera (PROTAC) protein degraders, a promising class of anti-cancer agents. VHL is broadly essential for cell proliferation, yet it is a key tumor suppressor in renal cell carcinoma. To understand the functional consequences of VHL loss, and to identify targeted approaches for the elimination of VHL null cells, we have used genome-wide CRISPR-Cas9 screening in human renal epithelial cells. We find that, upon VHL loss, the HIF1A/ARNT complex is the central inhibitor of cellular fitness, suppressing mitochondrial respiration, and that VHL null cells show HIF1A-dependent molecular vulnerabilities that can be targeted pharmacologically. Combined VHL/HIF1A inactivation in breast and esophageal cancer cells can also provide resistance to ARV-771, a VHL-based bromodomain degrader that has anti-cancer activity. HIF1A stabilization can thus provide opportunities for early intervention in neoplastic VHL clones, and the VHL-HIF1A axis may be relevant for the development of resistance to the emerging class of PROTAC-based cancer therapies.},
}
@article {pmid41201433,
year = {2026},
author = {Jetley, U and Balwani, I and Sharma, P and Miller, IC and Luther, A and Dutta, I and Saravanan, N and Goel, S and Zhang, Q and Zhang, B and Kilic, O and Liu, B and Han, BW and Liu, D and Schultes, B and Prodeus, A and Zhang, Y},
title = {A differentiated and durable allogeneic strategy applicable to cell therapies.},
journal = {Cytotherapy},
volume = {28},
number = {3},
pages = {101991},
doi = {10.1016/j.jcyt.2025.10.001},
pmid = {41201433},
issn = {1477-2566},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Cell- and Tissue-Based Therapy/methods ; Gene Editing/methods ; Killer Cells, Natural/immunology ; *T-Lymphocytes/immunology ; Transplantation, Homologous ; Cell Differentiation ; *Immunotherapy, Adoptive/methods ; },
abstract = {BACKGROUND AIMS: Autologous T-cell therapies have shown profound clinical responses; however, their widespread use has been limited primarily as the result of their individualized manufacturing requirements.<br><br>METHODS: To develop a persistent "off-the-shelf" allogeneic (Allo) approach, a multiplex Nme2Cas9-based cytosine base editor was deployed to knockout select human leukocyte antigens (HLA) class I and II alleles (HLA-A, HLA-B and the class II transactivator [CIITA]) while retaining HLA-C to protect from natural killer (NK) cell rejection.<br><br>RESULTS AND CONCLUSION: Matching the residual HLA-C allele from homozygous donors to the host prevented rejection of the donor T cells by allogeneic host T and NK cells. Site-specific integration of a tumor-specific CAR or TCR into the TRAC locus using SpyCas9 nuclease and an adeno-associated virus template allowed for a high localized insertion rate while simultaneously removing the endogenous TCR and preventing graft-versus-host disease. Using an optimized T-cell engineering process involving orthogonal CRISPR/Cas9 cleavage and base editors coupled with lipid nanoparticle delivery, we achieved efficient production of Allo-CAR T cells with high editing rates and cell expansion in a scalable manner. These allogeneic T cells demonstrated comparable functional activity to their autologous counterparts in preclinical assays. Moreover, this gene-editing approach significantly minimized the occurrence of chromosomal aberrations. This promising allogeneic approach also has been applied to induced pluripotent stem cells (iPSCs) with triple edits targeting HLA-A, HLA-B and CIITA (TKO). Pancreatic progenitor cells or cardiomyocytes derived from TKO iPSCs were protected from host peripheral blood mononuclear cell-mediated rejection when matched for HLA-C, suggesting potential applications in regenerative medicine applications.},
}
@article {pmid39973047,
year = {2026},
author = {Ling, X and Zhou, C and Hong, JF and Jiang, YP and Sun, Q and Li, LY and Wang, SY and Xie, XY and Zou, QL and Yang, XL and Xiang, K and Ma, J and Qiao, L and Chen, B and Sun, W},
title = {Increased versatility and convenience: Advances and strategy optimization of Receptor-Mediated Ovary Transduction of Cargo-mediated genetic modification in insects.},
journal = {Insect science},
volume = {33},
number = {1},
pages = {2-12},
doi = {10.1111/1744-7917.70003},
pmid = {39973047},
issn = {1744-7917},
support = {No.KJZD-K202200507;No.KJQN202200533//Scientific and Technological Research Program of Chongqing Municipal Education Commission/ ; No. cx2022052//Venture and Innovation Support Program for Chongqing Overseas Returnees/ ; No.CYS23399;No.CYS240374//Graduate Research and Innovation Foundation of Chongqing under Grant/ ; No. CSTB2022BSXM-JCX0067//Chongqing "Express" Science and Research Program for PhD/ ; No. 202410637012//College Students' Innovation and Entrepreneurship Training Plan Program/ ; Nos.31772527;31872262//National Natural Science Foundation of China/ ; },
mesh = {Animals ; Ovary/metabolism ; *Gene Editing/methods ; Female ; *Insecta/genetics ; CRISPR-Cas Systems ; },
abstract = {Genetic modification via gene editing has become a widely adopted and demonstrably effective method in functional gene research within entomology. However, the optimal efficiency and simplicity of delivering exogenous guide RNA-clustered regularly interspaced short palindromic repeats-associated protein 9 complexes into target tissues are crucial for successful gene editing. The Receptor-Mediated Ovary Transduction of Cargo (ReMOT) strategy, which simplifies the delivery process, target-site selection, technical requirements, and delivery cost compared with embryonic microinjection, enabling efficient editing at the germline level, is gaining increasing attention. Although the feasibility and advantages of this technique have been demonstrated in various insect species, further optimization of operational details and the overcoming of further bottlenecks are still required. This review focuses on advances in developing ReMOT as a valuable technology, exploring its applicability, rationale for selecting the ovary as a delivery target site, factors influencing its efficiency, and improvement recommendations. The versatility and effectiveness of ReMOT make it a promising method for researchers looking to make precise genetic modifications with greater ease and efficiency.},
}
@article {pmid39829059,
year = {2026},
author = {Shi, W and He, L and Li, R and Cao, J},
title = {Role of mitochondrial complex I genes in host plant expansion of Bactrocera tau (Tephritidae: Diptera) by CRISPR/Cas9 system.},
journal = {Insect science},
volume = {33},
number = {1},
pages = {147-158},
doi = {10.1111/1744-7917.13495},
pmid = {39829059},
issn = {1744-7917},
support = {//National Science Foundation of China/ ; //Science and Technology Program of Yunnan Province/ ; },
mesh = {Animals ; *Tephritidae/genetics/growth &amp; development/physiology ; CRISPR-Cas Systems ; *Electron Transport Complex I/genetics/metabolism ; Larva/growth &amp; development/genetics/physiology ; Pupa/growth &amp; development/genetics/physiology ; *Insect Proteins/genetics/metabolism ; Gene Knockout Techniques ; },
abstract = {Host expansion facilitates tephritid flies to expand their ranges. Unraveling the mechanisms of host expansion will help to efficiently control these pests. Our previous works showed mitochondrial complex I genes Ndufs1, Ndufs3, and Ndufa7 being upregulated during host expansion of Bactrocera tau (Walker), one of the highly hazardous species of tephritids. However, their roles in the host expansion of B. tau remain unknown. Here, using clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nuclease 9 (Cas9) editing system for the first time, a stable homozygous Ndufa7 strain (Btndufa7[-/-]), heterozygous Ndufs1 (Btndufs1[+/-]), and Ndufs3 strains (Btndufs3[+/-]) were obtained from F3 generation of B. tau, after gene knockout. Reduced sizes of larvae and pupae of the Ndufa7 knockout strain were first observed. Notably, the mean values of fitness estimation (pupal numbers, single-pupal weight and emergence rate) and Ndufa7 gene expression in the Ndufa7 knockout strain were slightly reduced on 2 native hosts (summer squash and cucumber), while it sharply decreased on the novel host banana and the potential host pitaya, compared with those of the wild-type strain. Furthermore, the Ndufa7 knockout strain did not survive on the novel host guava. These results suggested that Ndufa7 disturbs the survival on native hosts, expansion to novel hosts, and further expansion to potential hosts of B. tau. Homozygous lethality occurred after the knockout of Ndufs1 or Ndufs3, suggesting that these 2 genes play a role in the early development of B. tau. This study revealed that Ndufa7 is a target gene for the management of tephritids and opens a new avenue for pest control research.},
}
@article {pmid41676858,
year = {2026},
author = {Niu, Y and Wu, S and Su, J},
title = {Harnessing CRISPR/Cas systems for food safety detection: biosensor design and emerging applications for food safety detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay01791b},
pmid = {41676858},
issn = {1759-9679},
abstract = {Food safety has become a critical global concern, with foodborne diseases affecting approximately 600 million people annually and causing 420 000 deaths each year, posing significant risks to human health and well-being. Rapid, efficient, and reliable detection methods are essential to mitigate these risks. Traditional detection methods, such as PCR and culture-based assays, while widely used, often face challenges related to speed, accuracy, and portability. Over the past 5 years (2020-2025), the (CRISPR)/Cas system has emerged as a powerful tool for food safety detection due to its high sensitivity, specificity, and versatility. This review highlights recent advances in CRISPR/Cas-based biosensors and their applications in food safety. First, we discuss the key challenges in food safety detection and the design principles of CRISPR/Cas biosensors. Next, we comprehensively summarize their applications in detecting foodborne pathogens (viruses and bacteria), food fraud, genetically modified organisms (GMOs), toxins, heavy metals, antibiotic residues, and pesticides. Finally, we address the current limitations and future prospects of CRISPR/Cas biosensors, providing insights into their potential for next-generation food safety solutions.},
}
@article {pmid41674784,
year = {2025},
author = {Yashooa, RK and Nabi, AQ and Smail, SW and Azeez, SS and Nooh, WA and Mustafa, SA and Al-Farha, AA and Capitanio, N and Shekha, MS},
title = {CRISPR-Cas technologies in neurodegenerative disorders: mechanistic insights, therapeutic potential, and translational challenges.},
journal = {Frontiers in neurology},
volume = {16},
number = {},
pages = {1737468},
pmid = {41674784},
issn = {1664-2295},
abstract = {CRISPR-Cas genome-editing technologies have emerged as powerful tools for precise DNA and RNA modulation, offering promising therapeutic strategies for neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). This review critically evaluates current CRISPR/Cas applications in neurodegeneration, with emphasis on mechanistic insights, therapeutic outcomes, and translational feasibility. Preclinical and early translational studies demonstrate that CRISPR-Cas platforms can correct pathogenic mutations, suppress toxic gene expression, and restore neuronal function. Advanced modalities, including base and prime editing, CRISPRi/a, and RNA-targeting Cas systems, improve precision and reduce genomic damage, which is particularly advantageous in post-mitotic neurons. Emerging CRISPR-based diagnostics (e.g., SHERLOCK and DETECTR), AI-assisted sgRNA design, and machine-learning approaches for predicting off-target effects further enhance the safety, stratification, and monitoring of CRISPR therapeutics. In parallel, patient-derived brain organoids and assembloids provide scalable human-relevant platforms for mechanistic studies and preclinical validation. Despite this progress, major challenges remain, including efficient delivery across the blood-brain barrier, immune responses, long-term safety, and ethical and regulatory considerations. Overall, CRISPR-Cas technologies hold strong potential as disease-modifying interventions for neurodegenerative disorders, provided that advances in delivery systems, artificial intelligence integration, and regulatory oversight continue to evolve toward clinical translation.},
}
@article {pmid41674724,
year = {2026},
author = {Baum, R and Telugu, N and Bruyneel, AAN and Kay, M and Nair, P and Perea-Gil, I and Termglinchan, V and Bharucha, N and Lee, E and Mercola, M and Diecke, S and Karakikes, I},
title = {CRISPR-MiX: A pooled single-stranded donor strategy to enhance HDR efficiency in human iPSCs.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {1},
pages = {102820},
pmid = {41674724},
issn = {2162-2531},
abstract = {CRISPR-Cas9 is widely used to model genetic disorders by introducing or correcting disease-associated mutations in induced pluripotent stem cells (iPSCs) through homology-directed repair (HDR). However, HDR efficiency in iPSCs remains low and is highly dependent on the target locus. Here, we developed CRISPR-MiX, an improved protocol to enhance HDR efficiency in human iPSCs. Using a GFP-to-BFP reporter system, we identified key single-stranded oligodeoxynucleotide (ssODN) donor design parameters, including homology arm symmetry, CRISPR/Cas-blocking mutations, and strand complementarity, which significantly influence HDR outcomes. We applied this approach to introduce pathogenic variants into five genes related to genetic cardiomyopathies. Quantitative analysis of HDR events showed that both the target locus and ssODN design strongly affect HDR efficiency. To address the locus- and design-specific limitations, we established CRISPR-MiX, a pooled ssODN-based method for scarless genome editing using ribonucleoproteins (RNPs) that does not require selection. CRISPR-MiX consistently improved HDR efficiency across multiple loci. This strategy offers a simple, robust, and versatile approach for precise genome engineering in iPSCs, supporting broad applications in disease modeling and functional genomics.},
}
@article {pmid41674468,
year = {2026},
author = {Takeda, T and Aso, M and Ueda, H and Yuzawa, S},
title = {Direct Pathway Synthesis and Editing (DiPaSE): A One-Pot DNA Assembly Method for Accurate and Efficient Refactoring of High-GC Biosynthetic Gene Clusters.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00934},
pmid = {41674468},
issn = {2161-5063},
abstract = {Natural products (NPs) produced by actinobacteria, particularly Streptomyces species, represent a rich source of bioactive compounds and have yielded many clinically important compounds. Actinobacterial genomes are characterized by high GC content and typically harbor 20-40 biosynthetic gene clusters (BGCs) per genome, which encode diverse NPs such as polyketides, peptides, and glycosides. CRISPR/Cas-based genome editing has emerged as a promising tool to activate silent BGCs and engineer NP biosynthesis. However, the efficiency of multiplex editing drastically decreases as the number of targeted sites increases. Here, we report a novel one-pot DNA assembly method, termed direct pathway synthesis and editing (DiPaSE), for the efficient synthesis and multiplex editing of long, high-GC BGCs. DiPaSE accurately assembles multiple high-GC DNA fragments up to 60 kb and enables simultaneous deletions and insertions within a target BGC without compromising the assembly efficiency. Using this approach, we identified functions of previously uncharacterized genes in the aureothin BGC and significantly enhanced the titer of the corresponding NP. The workflow employs conventional polymerase chain reaction, type IIP restriction enzymes, commercially available DNA assembly reagents, and Escherichia coli, providing a simple, cost-effective, and broadly applicable platform for genome mining, BGC refactoring, and rational design of artificial biosynthetic pathways.},
}
@article {pmid41674382,
year = {2026},
author = {Zhang, Y and Xu, Y and Ding, Z and Cheng, Y and Gao, Y and Ye, J and Zhang, H and Li, P and Wu, H},
title = {Machine learning-optimized long single-stranded DNA synthesis technology empowers high-precision diagnostic-therapeutic integration in living cells.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41674382},
issn = {1362-4962},
support = {2021YFC2100600//National Key Research and Development Program of China/ ; 31872608//National Natural Science Foundation of China/ ; 31872608//National Natural Science Foundation of China/ ; },
mesh = {*DNA, Single-Stranded/chemistry/genetics/chemical synthesis/biosynthesis ; Humans ; *Machine Learning ; Gene Editing/methods ; Nanotechnology/methods ; DNA-Directed DNA Polymerase/metabolism ; CRISPR-Cas Systems ; },
abstract = {While DNA nanotechnology holds transformative potential across biomedical and information storage applications, current technologies face critical limitations in synthesizing long single-stranded DNA (ssDNA) with high purity and homogeneity. To address these challenges, we developed Ouroborosyn-ssDNA, a nicking enzymatic assisted replication (NEAR) platform that synergizes enzymatic engineering with computational optimization. By integrating phi29 DNA polymerase and Nb.BbvCI nickase in formate-based buffers, we achieved extended ssDNA synthesis up to 15 000 nt while preserving sequence fidelity, resulting in a 4.73-fold yield enhancement compared to commercial buffers. Notably, machine learning-guided parameter optimization identified magnesium ion dynamics and thermal modulation as pivotal determinants of enzymatic efficiency. Furthermore, solid-phase synthesis using thiol-gold immobilized templates demonstrated 86.38% purification recovery via automated magnetic bead systems, enabling scalable production. To validate functional utility, we engineered six-helix bundle DNA origami-CRISPR complexes that achieved nucleolin-targeted genome editing in cervical cancer cells, coupling GFP-based diagnostics with therapeutic E7 oncogene disruption. These advancements directly overcome key limitations in enzymatic stochasticity and product heterogeneity through buffer engineering and computational optimization, establishing a scalable pathway for applications in precision nanomedicine, synthetic biology, and molecular data storage. This integrated strategy advances DNA nanotechnology from proof-of-concept studies toward standardized biomanufacturing of sequence-defined macromolecular architectures.},
}
@article {pmid41674223,
year = {2026},
author = {Goudar, P and Hall, A},
title = {The influence of GMO media strategies on public perceptions of CRISPR crop technologies in Southern Ontario.},
journal = {GM crops &amp; food},
volume = {17},
number = {1},
pages = {2620131},
doi = {10.1080/21645698.2026.2620131},
pmid = {41674223},
issn = {2164-5701},
mesh = {Ontario ; *Public Opinion ; *Plants, Genetically Modified/genetics ; Humans ; Gene Editing ; *Crops, Agricultural/genetics ; *CRISPR-Cas Systems ; Adult ; Surveys and Questionnaires ; Male ; Female ; Middle Aged ; },
abstract = {Genetically modified organisms (GMOs) have often divided public opinion, one factor influencing perceptions of GMO technologies has been misunderstood or poorly communicated scientific messaging. However, advancements in gene editing tools like CRISPR/Cas9 offer new crop modification possibilities, prompting different regulatory frameworks than traditional GMO technologies. This research examines public understanding of GMOs, awareness of CRISPR crops, and how prior experiences with GMOs shape perceptions of new genetic technologies. A mixed-methods approach was employed, combining a public survey of adults in the Greater Toronto-Hamilton area and interviews with science journalists. Results show hesitance toward GMOs and CRISPR crops, with acceptance most impacted by consumer behavior and cost. Key interview themes include journalist's concerns about levels of public education, the role of social media, and the cost of goods. Our findings suggest increased transparency and effective communication could improve public acceptance of GMOs and CRISPR crops. While CRISPR crops do not come under the GMO regulatory framework in Canada, our findings show that the public does not recognize this distinction. Without increased transparency and more effective communication CRISPR crops may become widely associated with the negative media frames that have shaped perceptions of GMOs.},
}
@article {pmid41674084,
year = {2026},
author = {Ghosh, PK and Biswas, S and Malaker, R and Pham, H and Septiningsih, EM and Ravelombola, W},
title = {Optimization of CRISPR/Cas9-mediated CtPDS knockout in guar protoplasts.},
journal = {The plant genome},
volume = {19},
number = {1},
pages = {e70177},
doi = {10.1002/tpg2.70177},
pmid = {41674084},
issn = {1940-3372},
support = {//Texas A&amp;M Institute for Advancing Health through Agriculture/ ; //Texas A&amp;M Advancing Discovery to Market/ ; GSC2024075//Texas Department of Agriculture/ ; 58-6046-2-008//USDA-ARS Germplasm Evaluation/ ; 58-6046-3-005//USDA-ARS Germplasm Evaluation/ ; },
mesh = {*CRISPR-Cas Systems ; *Protoplasts/metabolism ; *Gene Editing/methods ; *Cyamopsis/genetics ; *Plant Proteins/genetics ; Gene Knockout Techniques/methods ; },
abstract = {Guar (Cyamopsis tetragonoloba L. Taub.) is a climate-resilient legume with industrial and agricultural applications. Recently, gene editing has emerged as a key genetic tool for crop improvement. Despite its recent increasing value as a commodity for various uses, there is no documented report of gene editing work in guar to date. In this study, we present the first optimized protocol for protoplast-based clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing in guar. The most intact and viable protoplasts were observed in the cotyledons of 6-day-old seedlings that were isolated using the tape-sandwich method. Enzymatic digestion with 1.5% cellulase RS and 0.3% pectinase in 600 mM mannitol, followed by 10 min of vacuum infiltration, increased protoplast release and viability. Polyethylene glycol (PEG)-mediated transformation had been tailored using 40% PEG for 5 min with green fluorescent protein plasmid, and the cestrum yellow leaf curling virus promoter at room temperature showed the highest transient expression efficiency (∼57%). A multiplexed CRISPR/Cas9 construct was designed to target the Cyamopsis tetragonoloba phytoene desaturase (CtPDS) gene in guar. Polymerase chain reaction amplification and Sanger sequencing of transfected protoplasts confirmed highly efficient editing, with fragment deletions ranging from 714 to 1061 bp in CtPDS. Overall, we achieved 100% editing efficiency, as all successfully transformed samples showed CRISPR/Cas9-induced mutations. These findings establish a reliable, transient protoplast system for functional genomics and targeted trait improvement in guar, providing a key foundation for future crop improvement.},
}
@article {pmid41673772,
year = {2026},
author = {Gao, H and Li, B and Fengler, K and Yang, M and Schroder, M and Rahe, M and Sanyour-Doyel, N and Qi, J and LIaca, V and Beatty, M and Hu, WN and Barrett, B and Norman, B and Mo, H and Leonard, A and Wilson, B and Meeley, RB and Perugini, L and Krishnamurthy, N and Habben, JE and Tabor, G},
title = {Genome-Edited Maize Expressing Two Native Genes Confers Broad-Spectrum Resistance to Northern Corn Leaf Blight.},
journal = {Molecular plant pathology},
volume = {27},
number = {2},
pages = {e70205},
pmid = {41673772},
issn = {1364-3703},
mesh = {*Zea mays/genetics/microbiology ; *Gene Editing/methods ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; *Plant Leaves/microbiology/genetics ; *Genes, Plant ; Ascomycota/pathogenicity ; },
abstract = {Northern corn leaf blight (NCLB) can result in yield losses of up to 50% in maize. The most effective strategy for managing NCLB is the deployment of resistant hybrids. Conventional breeding methods typically require 6 or 7 backcross generations to introgress a resistance locus, often bringing along undesirable traits that reduce yield. Recent advances in genome editing offer a precise alternative, enabling the targeted incorporation of resistance genes without linkage drag. In this study, we identified an NCLB resistance gene, NLB18-R, that is allelic to Htn1 and Ht2/Ht3. Using CRISPR-Cas9, we replaced the susceptible allele (NLB18-S) with NLB18-R in an elite inbred, resulting in enhanced resistance to NCLB. In a parallel experiment, we inserted both NLB18-R and the resistance gene Ht1-R into preselected, closely linked sites on chromosome 1. Through genetic crossing, we combined these edits into a stack. The resulting genome-edited plants exhibited resistance to Setosphaeria turcica races 0, 1 and 23N. Field trials under disease-free conditions showed no significant yield differences between hybrids carrying NLB18-R, Ht1-R, or the stack compared to null and wild-type controls. These findings demonstrate that CRISPR-Cas9-mediated genome editing is a powerful tool for rapidly developing commercial-grade maize hybrids with broad-spectrum resistance to NCLB, and potentially other diseases.},
}
@article {pmid41673677,
year = {2026},
author = {Baena, JC and Cabrera-Salcedo, SC and Carrera Suárez, Y and Biancha-Vasco, JM and Rios-Serna, LJ and García-Mantilla, MD and Estrada-Schweineberg, M and Victoria Hincapie, JS and Toro-Pedroza, A and Garcia-Robledo, JE and Cañas, CA and Ortiz-Guzman, J and Loukanov, A},
title = {The avatar principle: exosomal dynamics guiding tumor adaptation and next-generation therapeutic strategies.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {159},
pmid = {41673677},
issn = {1477-3155},
mesh = {*Exosomes/metabolism ; Humans ; *Neoplasms/therapy/immunology ; Tumor Microenvironment ; Immunotherapy/methods ; Animals ; Receptors, Chimeric Antigen ; Avatar ; },
abstract = {Exosomes are nanoscale extracellular vesicles that transfer proteins, nucleic acids, and lipids, reflecting the state of their parent cells. A persistent scientific challenge is that tumor-derived exosomes (TDEs) facilitate immune evasion, remodel the tumor microenvironment, and create premetastatic niches, intensifying tumor aggressiveness and undermining therapeutic efficacy, ultimately narrowing treatment options to palliative strategies in advanced settings. Yet their dual roles as suppressive agents and potential therapeutic tools remain poorly integrated within current cancer immunotherapy frameworks. This review examines the molecular mechanisms underlying TDE-mediated immune suppression and therapeutic resistance, while also highlighting engineering strategies to exploit or counteract exosome biology. Exosomes derived from chimeric antigen receptor (CAR) T cells preserve antigen specificity and cytotoxic components without the risks of uncontrolled proliferation or cytokine release, offering a safer class of cell free immunotherapies. Advances in genetic engineering, hybrid vesicle design, and nanotechnology have extended exosome applications to the delivery of CRISPR/Cas systems, chemotherapeutic agents, immunoregulatory RNAs, and vaccines, with liposome or nanoparticle integration enhancing targeting and efficacy. Remaining obstacles include the lack of standardized protocols, scalability issues in production, and unresolved regulatory frameworks. Drawing on The Art of War, exosomes can be envisioned as avatars of strategy, discreet messengers capable of undermining host defenses while simultaneously carrying the potential to redirect immunity against the tumor. By embodying both deception and counterattack, they illustrate the capacity to penetrate hidden barriers and redefine the therapeutic battlefield, opening new horizons for precision cancer immunotherapy.},
}
@article {pmid41673558,
year = {2026},
author = {Sun, X and Wu, F and Ma, Z and Liang, G and Chen, S and Hu, X and Fan, S and Zhao, Y},
title = {Systematic synthesis of CRISPR/Cas applications for enhancing salt tolerance in crops: a decade of progress and challenges.},
journal = {BMC plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12870-026-08295-2},
pmid = {41673558},
issn = {1471-2229},
support = {42320104006//National Natural Science Foundation of China for International Cooperation/ ; },
abstract = {Soil salinity is a major constraint on global crop productivity, driving the need for salt-tolerant varieties. While CRISPR-Cas genome editing offers targeted solutions for trait improvement, significant biological and technical bottlenecks limit its application in conferring salt stress resilience. This systematic summarizes findings from 83 peer-reviewed studies (2015-2024) employing CRISPR/Cas technologies to improve salt tolerance in five major crops (rice, wheat, maize, sorghum, barley). Our systematic review reveals that early single-gene edits achieved modest gains (30-50% Na[+] exclusion) but often showed limited yield gains in field settings, potentially due to compensatory regulation and environmental variation. The literature suggests that multiplex designs spanning ion homeostasis, osmoprotection, and ROS management can improve salt-tolerance outcomes and help maintain yield under severe salinity; however, the magnitude of benefit varies with crop, genotype, and transformation/regeneration context. Protein-protein interaction networks identified 12 hub genes and three functional modules, highlighting SOS3 and MPK6 as critical bottlenecks whose disruption risks pleiotropic effects. Spatial expression analysis underscored tissue-specific trade-offs, constitutive editing of root-dominant genes in shoots reduced yields by 15-28%, while tissue-optimized promoters minimized physiological conflicts. Persistent challenges include genotype-dependent transformation inefficiencies, epigenetic drift and environmental interactions under salt stress. Collectively, our synthesis consolidates and refines current best practices for salt-tolerance genome editing and highlights major bottlenecks-particularly regeneration/transformability, genotype dependence, and epigenetic constraints-that should be explicitly considered in experimental design and reporting.},
}
@article {pmid41673492,
year = {2026},
author = {Jiang, Y and Hodgson, KJ and Segos, I and Lambie, EJ and Yang, L and Pan, M and Greig, A and Conradt, B},
title = {Tagging of C. elegans apoptosis activator EGL-1 BH3-only reveals CED-9 BCL-2-dependent mitochondrial localization and dynamic control of EGL-1 synthesis and degradation in vivo.},
journal = {Cell death and differentiation},
volume = {},
number = {},
pages = {},
pmid = {41673492},
issn = {1476-5403},
support = {BB/V007572/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/V015648/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; C0204/10-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; RSWF\R1\180008//Royal Society/ ; PhD stipend//China Scholarship Council (CSC)/ ; },
abstract = {The BH3-only protein EGL-1 is the key activator of apoptosis during C. elegans development. EGL-1 protein is thought to be synthesized predominantly in cells programmed to die and to localize to mitochondria. We used CRISPR-Cas-mediated modification of the egl-1 locus to add the coding sequence for the monomeric StayGold fluorescent protein or 18 copies of the SunTag peptide to the endogenous open reading frame. We found that tagged EGL-1 protein colocalizes with mitochondria in vivo and that mitochondrial localization is dependent on the anti-apoptotic BCL-2-like protein CED-9. Consistent with the presence of egl-1 mRNA in cells programmed to die as well as their progenitor cells ('mother' cells), EGL-1 protein is detected in both types of cells in vivo. Furthermore, real time imaging reveals that EGL-1 protein rapidly disappears from the mother cell prior to its division and that EGL-1 protein rapidly reappears specifically in the daughter cell programmed to die. Our results demonstrate CED-9 BCL-2-dependent mitochondrial localization of EGL-1 BH3-only protein and dynamic control of EGL-1 protein synthesis and degradation. Furthermore, we have identified additional levels of control of egl-1 BH3-only function that expand our understanding of apoptosis activation in vivo.},
}
@article {pmid41671593,
year = {2026},
author = {Fattahi, M and Pirbadami, F and Motallebirad, T and Beheshti, Z and Azadi, D},
title = {Next-generation gene therapy for infectious disease: Advances, challenges, and future directions.},
journal = {Journal of infection and public health},
volume = {19},
number = {4},
pages = {103164},
doi = {10.1016/j.jiph.2026.103164},
pmid = {41671593},
issn = {1876-035X},
abstract = {Infectious diseases, worsened by antimicrobial resistance and limitations of conventional treatments, demand innovative solutions. This systematic review evaluates recent advances in next-generation gene therapy for HIV, HBV, HPV, and multidrug-resistant(MDR) pathogens, while addressing key challenges and future directions. Following PRISMA guidelines, major databases were searched (2015-2025), yielding 1250 records. the114 peer-reviewed studies were included, assessed using the MMAT, and thematically synthesized for therapeutic strategies, efficacy, challenges, and prospects. Findings show CRISPR-based genome editing achieved HIV remission in preclinical and early clinical trials, engineered bacteriophages demonstrated strong efficacy against MDR bacteria, RNAi effectively silenced HBV, and phage-derived depolymerases reduced MDR biofilms. Adverse events included off-target effects, immunogenicity, and scalability issues. Efficacy varied, with CRISPR offering high specificity and phage therapies showing robust lysis. In conclusion, Gene therapy shows strong potential against resistant pathogens, but faces challenges like study heterogeneity, preclinical reliance, delivery barriers, and unequal access in low-and middle-income countries.},
}
@article {pmid41671414,
year = {2026},
author = {Yu, J and Park, JC and Uhm, H and Kim, YW and Im, HW and Bae, S},
title = {Evolution of Prime Editing: Enhancing Efficiency and Expanding Capacity.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e21015},
doi = {10.1002/advs.202521015},
pmid = {41671414},
issn = {2198-3844},
support = {RS-2024-00332601//Korean Fund for Regenerative Medicine/ ; 25202MFDS003//Ministry of Food and Drug Safety/ ; 25B-001-0700//SNUH Lee Kun-hee Child Cancer and Rare Disease/ ; 2021M3A9H3015389//National Research Foundation of Korea/ ; RS-2024-00451880//National Research Foundation of Korea/ ; RS-2024-00455559//National Research Foundation of Korea/ ; SRC-NRF2022R1A5A102641311//National Research Foundation of Korea/ ; RS-2024-00404132//Ministry of Health &amp; Welfare/ ; },
abstract = {Genetic mutations cause approximately 80% of rare human diseases, highlighting the urgent need for precise genome editing. Since clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated 9 (Cas9) nucleases were first used for genome editing in 2012, genome editing technologies have rapidly advanced. Base editors, derived from the CRISPR-Cas system, were developed to introduce specific point mutations without requiring DNA double-strand breaks, and subsequently, prime editing (PE) technology was created to enable insertions, deletions, and all types of point mutations. The precision and versatility of PE make it a promising tool for clinical applications. However, PE has potential limitations, including low editing efficiency and limited capacity for large-scale manipulation. To overcome these limitations, research has been continuously conducted to improve PE efficiency and expand its capabilities. Therefore, this review aims to highlight current efforts and future directions for developing and improving PE-related tools.},
}
@article {pmid41671402,
year = {2026},
author = {Shi, M and Ge, W and Li, C and Liu, B and Deng, X and Liu, C and Zheng, M and Zhang, P and Li, L and Guo, Y and Han, Y and Yang, Y and Yu, YV and Jin, YN},
title = {Versatile CRISPR-Cas Tools for Gene Regulation in Zebrafish via an Enhanced Q Binary System.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e11485},
doi = {10.1002/advs.202511485},
pmid = {41671402},
issn = {2198-3844},
support = {32070832//National Natural Science Foundation of China/ ; 32150610476//National Natural Science Foundation of China/ ; 2042022dx0003//Fundamental Research Funds for the Central Universities/ ; },
abstract = {CRISPR-Cas systems revolutionize gene regulation across diverse organisms, including zebrafish. However, most zebrafish studies still rely on transient delivery of CRISPR components, with limited use of transgenic models, primarily restricted to Cas9-mediated knockouts. This limitation arises from challenges in achieving sustained, tissue-specific, and efficient expression of transgenic CRISPR effectors. To address these challenges, we introduce CRISPR-Q, a transgenic system that combines the QFvpr/QUAS binary expression platform with CRISPR-Cas technologies. CRISPR-Q overcomes the drawbacks of transient mRNA or protein delivery and circumvents the toxicity and transgene silencing issues associated with other binary systems, such as Gal4/UAS. The system enables robust and spatiotemporal expression of CasRx or dCas9vpr, allowing precise transcript knockdown (CRISPR-QKD) or gene activation (CRISPR-Qa). Using CRISPR-QKD, we achieve effective knockdown of smn1 and simultaneous knockdown of tardbp and tardbpl, modeling spinal muscular atrophy and amyotrophic lateral sclerosis, respectively. CRISPR-Qa activates endogenous lin28a and sox9b, demonstrating its functional versatility. We further validate CRISPR-Q's tissue-specific applicability in heart-specific transgenic zebrafish. Together, CRISPR-Q represents a robust and versatile platform for studying gene function and modeling human diseases in zebrafish, with broad potential for adaptation in other model organisms.},
}
@article {pmid41670257,
year = {2026},
author = {Vollmer, SK and Stetter, MG and Hensel, G},
title = {First Successful Targeted Mutagenesis Using CRISPR/Cas9 in Stably Transformed Grain Amaranth Tissue.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70590},
pmid = {41670257},
issn = {1467-7652},
support = {EXC-2048/1 project ID 390686111//Deutsche Forschungsgemeinschaft/ ; STE 2654/4//Deutsche Forschungsgemeinschaft/ ; },
abstract = {Grain amaranth is a nutritionally rich, stress-tolerant C4 dicot with considerable potential for climate-resilient agriculture; however, efficient and reproducible protocols for stable transformation, regeneration, and CRISPR/Cas9-mediated editing have not yet been established. CRISPR/Cas-based genome editing is a cornerstone technology for accelerating the development of climate-resilient, high-yielding crops. Its effective application depends on robust, stable transformation procedures and CRISPR/Cas systems optimised for the target species. The absence of such tools remains a critical constraint for the genetic improvement of many promising yet underexplored crops. In this study, we edited key genes of the betalain biosynthesis pathway in grain amaranth (Amaranthus hypochondriacus L.) using the CasCADE modular cloning system, thereby demonstrating the feasibility of targeted mutagenesis in an orphan crop. We observed successful edits in up to 49% of transformed calli, resulting in deletions or insertions in the target genes. Our CRISPR/Cas9-mediated editing paves the way for targeted molecular research and breeding of grain amaranth.},
}
@article {pmid41669806,
year = {2026},
author = {Wang, ZH and Yang, ZQ},
title = {Advances in site-specific knock-in techniques for gene editing.},
journal = {Yi chuan = Hereditas},
volume = {48},
number = {2},
pages = {128-141},
doi = {10.16288/j.yczz.25-076},
pmid = {41669806},
issn = {0253-9772},
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; Animals ; DNA Nucleotidyltransferases ; },
abstract = {Gene-targeted knock-in technology serves as a cornerstone tool in genetic engineering and gene therapy, designed to circumvent the unpredictability and heterogeneityassociated with conventional random integration methods. However, its practical application has long been constrained by off-target activity and low efficiency during the editing process. Recent advances in site-specific recombinase systems (e.g., Bxb1 integrase) and programmable nuclease systems (e.g., CRISPR/Cas9) have significantly enhanced the precision and efficiency of gene knock-in. Notably, the Cas9-Bxb1 integrase system enables targeted integration of large DNA fragments (5-43 kb) into genomic safe harbor (GSH) sites, offering a transformative platform for disease modeling, functional genomics, and clinical therapeutics. This review systematically summarizes the progress of site-specific recombinase and nuclease systems, discusses GSH screening strategies and the role of multi-omics data in optimizing predictive models, and compares the strengths and limitations of twinPE+Bxb1 and PASTE systems. Future research should focus on developing novel integrases with low off-target activity, refining DSB-free editing technologies, and establishing cross-species GSH databases to advance applications in precision medicine and synthetic biology.},
}
@article {pmid41649152,
year = {2026},
author = {Gallo, D and Bes, M and Mounier, T and Calatayud, C and Meunier, AC and Périn, C},
title = {A Streamlined CRISPRa Architecture with Dual Pol II/Pol III Promoter and Optimized scRNA Enables Robust and Tunable Gene Activation.},
journal = {The CRISPR journal},
volume = {9},
number = {1},
pages = {36-48},
doi = {10.1177/25731599251408333},
pmid = {41649152},
issn = {2573-1602},
mesh = {*Promoter Regions, Genetic ; Oryza/genetics ; *RNA Polymerase II/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *RNA Polymerase III/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcriptional Activation ; Gene Expression Regulation, Plant ; },
abstract = {CRISPR activation (CRISPRa) offers a powerful approach to upregulate endogenous genes; yet, existing systems in plants can be complex or difficult to integrate with CRISPR interference (CRISPRi). Here, we present a streamlined and flexible CRISPRa platform that enables robust gene activation. Using a dual-luciferase reporter, we benchmarked a range of guide RNA scaffolds, effector proteins, and promoters. We developed a novel single-guide RNA (sgRNA) architecture, harboring two MS2 aptamers inserted into the tetraloop and driven by a composite Pol II/Pol III promoter, as the most efficient configuration. This scaffold outperformed gR2.0- and SunTag-based constructs, reaching up to 100-fold activation of a minimal 35S promoter and up to 215-fold induction of three endogenous rice genes in protoplast assays. In contrast, scaffold RNAs (scRNAs) with aptamers at the 3' end or in excessive copy numbers were ineffective. Exploratory AlphaFold modeling supports a possible role for aptamer positioning and MCP-VP64 dimerization, although this remains a working hypothesis. This modular design enables tunable gene regulation in rice protoplasts and provides a practical platform for high-throughput screening and synthetic gene circuit prototyping in plants. Given that scRNA geometry and promoter architecture are universal features of CRISPR-based transcriptional modulation, the system is expected to be broadly portable across species. While the architecture is intended to be compatible with CRISPRi, future studies will be needed to establish its practical use in combined CRISPRa/i settings.},
}
@article {pmid41616513,
year = {2026},
author = {Xu, L and Jin, J and Lyu, W and Liang, X and Wang, Q and Zhang, J and Luo, Y and Chen, J and Lu, H and Li, X and Shen, F},
title = {Point-of-care profiling of H. pylori virulence and antibiotic resistance from endoscopic biopsies using an integrated restriction enzyme-CRISPR microfluidic platform.},
journal = {Biosensors &amp; bioelectronics},
volume = {299},
number = {},
pages = {118438},
doi = {10.1016/j.bios.2026.118438},
pmid = {41616513},
issn = {1873-4235},
mesh = {*Helicobacter pylori/genetics/pathogenicity/drug effects/isolation &amp; purification ; Humans ; *Helicobacter Infections/microbiology/diagnosis/drug therapy ; *Drug Resistance, Bacterial/genetics ; Point-of-Care Systems ; Anti-Bacterial Agents/pharmacology ; Virulence/genetics ; CRISPR-Cas Systems ; Lab-On-A-Chip Devices ; Biopsy ; Biosensing Techniques ; Clarithromycin/pharmacology ; Levofloxacin/pharmacology ; Polymorphism, Single Nucleotide ; Gastric Mucosa/microbiology/pathology ; },
abstract = {Endoscopically obtained gastric mucosal biopsies contain abundant molecular information that could inform Helicobacter pylori (H. pylori) eradication therapy. However, the lack of point-of-care tools for processing solid tissue limits rapid, on-site genotyping, leading to empirical therapy and higher eradication failure rates. Here, we present the SlipChip-based On-site and User-friendly Testing (SCOUT) together with the restriction enzyme-assisted CRISPR/Cas12a (RCut) method for point-of-care genotyping from raw endoscopic gastric biopsies. This fully automated platform enables analysis of key virulence genes and resistance-associated single-nucleotide variants (SNVs) at mutant allele frequencies down to 0.1 %. Results were available within 1 h, enabling actionable molecular profiling during the same endoscopic encounter. In 159 clinical validations, SCOUT showed concordant virulence profiling with quantitative PCR (κ = 0.978) and accurately detected clarithromycin and levofloxacin resistance mutations with up to 100 % sensitivity and specificity. These results demonstrate that SCOUT bridges the gap between diagnostic sampling and informed eradication strategies, providing a scalable framework for point-of-care molecular genotyping.},
}
@article {pmid41554880,
year = {2026},
author = {Bot, JF and Zhao, Z and Li, M and Kammeron, D and Shang, P and Geijsen, N},
title = {Temporal dynamics of collateral RNA cleavage by LbuCas13a in human cells.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {233},
pmid = {41554880},
issn = {2399-3642},
support = {NNF21CC0073729//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; W.OR18-11//Prinses Beatrix Spierfonds/ ; 15804//Netherlands Organisation for Scientific Research | Stichting voor de Technische Wetenschappen (Technology Foundation STW)/ ; 201706890022//China Scholarship Council (CSC)/ ; 202206300033//China Scholarship Council (CSC)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA Cleavage ; *CRISPR-Associated Proteins/metabolism/genetics ; *RNA/metabolism/genetics ; HEK293 Cells ; Ribonucleoproteins/metabolism/genetics ; Apoptosis/genetics ; },
abstract = {CRISPR-Cas13 exclusively targets RNA. In prokaryotes, Cas13 cleaves both target and non-target RNA indiscriminately upon activation by a specific target RNA, but in eukaryotic cells collateral cleavage activity has been limited. Here we report that LbuCas13a exhibits strong collateral RNA cleavage activity in human cells when delivered as ribonucleoprotein, independent of cell line and targeting both exogenous and endogenous transcripts. Collateral RNA cleavage starts within 50 minutes of ribonucleoprotein delivery resulting in major alterations to the total RNA profile. In response to the collateral RNA cleavage, cells upregulate genes associated with the stress and innate immune response, ultimately leading to apoptotic cell death. This enables us to use LbuCas13a as a flexible and repeatable target-RNA-specific cell elimination tool. Finally, using both total RNA sequencing and Nanopore sequencing, we find that LbuCas13a activation leads to rapid and near-global depletion of cytoplasmic RNAs, and that cleavage occurs at specific nucleotide positions.},
}
@article {pmid41468058,
year = {2026},
author = {Vasileva, A and Abramova, M and Selkova, P and Arseniev, A and Musharova, O and Malysheva, P and Demkina, A and Khodorkovskii, M and Severinov, K},
title = {Streptococcus uberis Cas9-A Compact Type II-A Nuclease Recognizing a Unique PAM and Functional in Human Cells.},
journal = {The CRISPR journal},
volume = {9},
number = {1},
pages = {21-35},
doi = {10.1177/25731599251404417},
pmid = {41468058},
issn = {2573-1602},
mesh = {Humans ; *Streptococcus/genetics/enzymology ; *CRISPR-Cas Systems ; Gene Editing/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endonucleases/metabolism/genetics ; Animals ; *Bacterial Proteins/genetics/metabolism ; Cattle ; },
abstract = {Several type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 RNA-guided nucleases are commonly used for genome engineering. Their relatively large size and requirements for specific protospacer adjacent motif (PAM) sequences flanking their targets prompt continuous searches for additional more compact Cas9 enzymes with new PAM specificities. Here, we present SuCas9, a compact nuclease from Streptococcus uberis, a bacterium inhabiting the mammary glands of dairy cattle. SuCas9 recognizes a novel 5'-NNAAA-3' PAM, efficiently cleaves DNA in vitro, and is active in human cells. SuCas9 thus expands the available genome editing toolset and may find biotechnological and medicinal applications in the future.},
}
@article {pmid41338874,
year = {2026},
author = {Villegas, NK and Tran, MH and Keller, A and Plesa, C},
title = {BAR-CAT: Targeted Recovery of Synthetic Genes via Barcode-Directed CRISPR-dCas9 Enrichment.},
journal = {The CRISPR journal},
volume = {9},
number = {1},
pages = {9-20},
doi = {10.1177/25731599251401526},
pmid = {41338874},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; *Genes, Synthetic/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Plasmids/genetics ; },
abstract = {Modern gene synthesis platforms enable investigations of protein function and genome biology at an unprecedented scale. Yet, the proportion of error-free constructs in diverse gene libraries decreases with length due to the propagation of oligo synthesis errors. To rescue these error-free constructs, we developed Barcode-Assisted Retrieval CRISPR-Activated Targeting (BAR-CAT), an in vitro method that uses multiplexed dCas9-single-guide RNA (sgRNA) complexes to extract barcodes corresponding to error-free constructs. After a 15-min incubation and wash regimen, three low-bundance targets in a 300,000-member test library were enriched 600-fold, greatly reducing downstream requirements. When applied to a 384-gene DropSynth gene library, BAR-CAT enriched 12 targets up to 122-fold and revealed practical limits imposed by sgRNA competition and library complexity, which now guide ongoing protocol scaling. By eliminating laborious clone-by-clone validation and working directly on plasmid libraries, BAR-CAT provides a platform for recovering perfect synthetic genes, subsetting large libraries, and ultimately lowering the cost of functional genomics at scale.},
}
@article {pmid41330380,
year = {2026},
author = {Feng, C and Peets, EM and Zhou, Y and Crepaldi, L and Usluer, S and Dunham, A and Braunger, JM and Su, J and Strauss, ME and Muraro, D and Xian Cheam, KA and Bonder, MJ and Nogales, EG and Cooper, S and Bassett, A and Leonard, S and Gu, Y and Fussing, B and Burke, D and Parts, L and Stegle, O and Velten, B},
title = {A genome-scale single-cell CRISPRi map of trans gene regulation across human pluripotent stem cell lines.},
journal = {Cell genomics},
volume = {6},
number = {2},
pages = {101076},
doi = {10.1016/j.xgen.2025.101076},
pmid = {41330380},
issn = {2666-979X},
mesh = {Humans ; *Single-Cell Analysis/methods ; *Pluripotent Stem Cells/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation/genetics ; *Genome, Human/genetics ; Quantitative Trait Loci/genetics ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Population-scale resources of genetic, molecular, and cellular information form the basis for understanding human genomes, charting the heritable basis of disease and tracing the effects of mutations. Pooled perturbation assays, probing the effect of many perturbations coupled with single-cell RNA sequencing (scRNA-seq) readout, are especially potent references for interpreting disease-linked mutations or gene-expression changes. However, the utility of existing maps has been limited by the comprehensiveness of perturbations conducted and the relevance of their cell-line context. Here, we present a genome-scale CRISPR interference perturbation map with scRNA-seq readout across many genetic backgrounds in human pluripotent cells. We map trans expression changes induced by knockdowns and characterize their variation across donors, with expression quantitative trait loci linked to higher genetic modulation of perturbation effects. This study pioneers population-scale CRISPR perturbations with high-dimensional readouts, which will fuel the future of effective modulation of cellular disease phenotypes.},
}
@article {pmid41669364,
year = {2025},
author = {Elkonin, LA and Gerashchenkov, GA and Borisenko, NV and Sarsenova, SK and Panin, VM},
title = {Study of the progeny of sorghum mutants obtained using the CRISPR/Cas9 genetic construct directed at inducing mutations in the α-kafirin k1C5 gene.},
journal = {Vavilovskii zhurnal genetiki i selektsii},
volume = {29},
number = {8},
pages = {1161-1168},
doi = {10.18699/vjgb-25-122},
pmid = {41669364},
issn = {2500-0462},
abstract = {Site-directed mutagenesis using genetic constructs carrying the CRISPR/Cas system is an effective technology that is actively used to solve a variety of problems in plant genetics and breeding. One of these problems is to improve the nutritional value of grain sorghum, a high-yielding heat- and drought-tolerant cereal crop that is becoming increasingly important in the conditions of climate aridization. The main reason for the relatively low nutritional value of sorghum grain is the resistance of its storage proteins, kafirins, to proteolytic digestion. We have previously obtained mutants with improved kafirin in vitro digestibility using the CRISPR/Cas technology in grain sorghum variety Avance. The nucleotide sequence of one of the genes (k1C5) of the gene family encoding the signal polypeptide of 22 kDa α-kafirin was used as a target. The aim of this study was to investigate the manifestation of the main agronomically-important traits in the progeny of these mutants and inheritance of high in vitro protein digestibility, and also sequencing nucleotide sequences encoding the 22 kDa α-kafirin signal polypeptide in a number of plants from the T0 generation and their T1 progeny. It was revealed that four of the six studied T0 plants, as well as their progeny, had the same mutation: a T→C substitution in the 23rd position of the nucleotide sequence of the k1C5 gene encoding the signal polypeptide, which led to a substitution of the coding triplet CTC→CCC (Leu→Pro). This mutation is located off-target, 3' from the PAM sequence. It is suggested that this mutation may have arisen as a result of Cas9 nuclease errors caused by the presence of multiple PAM sequences located close to each other. It was found that the progeny of two of the three studied mutants (T2 and T3 families), grown in the experimental field conditions, differed from the original variety by a reduced plant height (by 12.4-15.5 %). The peduncle length, 1,000-grain mass, and grain mass per panicle did not differ from the original variety, with the exception of the progeny of the 2C-1.2.5b mutant, which had a reduced grain yield per panicle. Unlike the original variety, plants from the T2 and T3 generations had kernels with a modified type of endosperm (completely floury, or floury with inclusions of vitreous endosperm, or with a thin vitreous layer). The level of grain protein digestibility in the progeny of mutants 2C-2.1.1 #13 and 2C-1.2.5a #14 varied from 77 to 84 %, significantly exceeding the original variety (63.4 ± 2.3 %, p < 0.05). The level of protein digestibility from kernels with modified endosperm was higher than that of kernels with normal vitreous endosperm (84-93 %, p <0.05). The reasons for the variation in endosperm texture in the progeny of the mutants and its relationship with the high digestibility of kafirins are discussed.},
}
@article {pmid41666229,
year = {2026},
author = {Fan, X and Lyu, S and Fan, W and Shu, J and Cheng, X},
title = {Precision targeting: The dawn of artificially customized disease resistance.},
journal = {PLoS pathogens},
volume = {22},
number = {2},
pages = {e1013942},
pmid = {41666229},
issn = {1553-7374},
mesh = {*Plant Diseases/immunology/prevention &amp; control/genetics ; *Disease Resistance/genetics/immunology ; *Plant Immunity/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics/immunology ; *Crops, Agricultural/genetics/immunology ; },
abstract = {Advanced plant disease management strategies are essential to sustainable agriculture and global food security. Advances in plant immunity have given rise to a variety of innovative disease control strategies, such as NLR gene transfer, RNA silencing technology, and CRISPR/Cas9-based gene disruption, as well as the use of immunity inducers. Recently, several novel resistance strategies, including the bioengineering of immunoreceptors, protease-triggered resistance design, and the sentinel approach, have enabled the customized development of disease resistance traits. These new approaches envisage a new paradigm of precision-targeted, artificially engineered resistance to enhance crop protection.},
}
@article {pmid41665762,
year = {2026},
author = {Farooq, M and Khan, A and Hassan, A and Shah, MM},
title = {Advances in CRISPR/Cas systems for engineering abiotic stress tolerance in plants: mechanisms and future prospects.},
journal = {Planta},
volume = {263},
number = {3},
pages = {72},
pmid = {41665762},
issn = {1432-2048},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Stress, Physiological/genetics ; *Crops, Agricultural/genetics/physiology ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; Droughts ; },
abstract = {Abiotic stress factors such as drought, salinity, extreme temperatures, and oxidative stress significantly limit crop productivity and threaten global food security. Traditional breeding and transgenic approaches have been employed to enhance stress tolerance, but they are often time-consuming and face regulatory hurdles. The advent of CRISPR/Cas genome editing technology has revolutionized plant genetic engineering by enabling precise modifications to stress-responsive genes. This review explores recent advancements in CRISPR/Cas-based genome editing for improving abiotic stress resilience in crops. We discuss the mechanisms of CRISPR/Cas systems, their applications in stress tolerance, and emerging approaches such as multiplex genome editing, base editing, and AI-assisted CRISPR. Furthermore, we highlight challenges, ethical considerations, and future directions for integrating CRISPR into agricultural biotechnology. This review underscores the potential of CRISPR-based strategies in developing climate-resilient crops to ensure sustainable food production in the face of global climate change.},
}
@article {pmid41664621,
year = {2026},
author = {Ullah, Q and Haider, W and Zeshan, M and Waqar, M and Arshad, MT and Parveen, H and Mukhtar, S and Zahir, A},
title = {Advancing climate adaptation in saffron through CRISPR-based modulation of stress tolerance and photoperiodic flowering control.},
journal = {GM crops &amp; food},
volume = {17},
number = {1},
pages = {2626180},
pmid = {41664621},
issn = {2164-5701},
mesh = {*Flowers/genetics/physiology/growth &amp; development ; *Crocus/genetics/physiology/growth &amp; development ; Gene Editing ; *CRISPR-Cas Systems ; Photoperiod ; Stress, Physiological ; Plants, Genetically Modified/genetics ; Adaptation, Physiological ; Droughts ; Climate Change ; },
abstract = {Saffron (Crocus sativus L.) is a high-value crop known for its intricate harvesting process and limited production due to factors like triploid sterility and specific climatic needs. This review discusses biotechnological methods, particularly CRISPR/Cas9 genome editing, aimed at improving heat and drought tolerance and achieving year-round flowering. Such genetic edits as evidenced with experimental CRISPR/Cas9 systems that reach up to 70% callus initiation in saffron. Cultivation efficiency and quality are increased in Hydroponic systems and synthetic bioreactors, which have been proven in trials in non-traditional areas such as North Bengal, India. Nonetheless, internationalization threatens the market value and cultural integrity of saffron, and such measures as fair-trade labels, GI laws, and cooperatives of stakeholders must be implemented fairly and equally.},
}
@article {pmid41663678,
year = {2026},
author = {Zhu, Y and Zhang, J and Ruan, Y and Lei, T and Li, S and Cao, L and Zou, X and He, Y and Li, Q and Chen, S and Peng, A},
title = {Application of compact CRISPR/Cas nucleases for citrus genome editing.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {7},
pmid = {41663678},
issn = {1573-9368},
support = {CSTB2024TIAD-CYKJCXX0021//Sichuan-Chongqing Science and Technology Innovation Cooperation Program Project/ ; CSTB2023TIAD-KPX0044//the Technology Innovation and Application Development Key Project of Chongqing/ ; CARS-26//Earmarked Fund for China Agriculture Research System/ ; CSTB2023NSCQ-MSX1085//Chongqing Natural Science Foundation Project/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Citrus/genetics ; *Genome, Plant ; Plants, Genetically Modified/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Gene editing technology continues to advance, and the range of available editing tools is steadily expanding. Recently, several compact and ultracompact systems have been developed, gaining considerable attention because their components can be efficiently packaged into viral vectors. To identify compact tools suitable for efficient genome editing in citrus, Casπ, CoCas9, along with their respective single guide RNAs, were synthesized, and CRISPR/Casπ and CRISPR/CoCas9 constructs were designed to assess their editing efficiency in 'Wanjincheng' orange (Citrus sinensis Osbeck). The Casπ was able to mediate genome editing in the citrus genome, although with low efficiency. In comparison, CoCas9 showed a transformation efficiency three times higher than that of the widely used SpCas9. Moreover, while the gene editing efficiency of CoCas9 was comparable to that of SpCas9, the significantly elevated transformation efficiency resulted in a significantly higher overall editing efficiency for CoCas9 relative to SpCas9. Mutation profiles generated by CoCas9 and SpCas9 were highly similar, and both nucleases displayed comparable target specificity at three potential off-target sites. These results indicate that Casπ is not suitable for application in citrus genome editing, whereas CoCas9 represents a promising alternative to SpCas9 for efficient and precise genome modification in citrus.},
}
@article {pmid41663312,
year = {2026},
author = {Eraña, H and Vidal, E and Fernández-Borges, N and Charco, JM and Díaz-Domínguez, CM and Sampedro-Torres-Quevedo, C and Galarza-Ahumada, J and Fernández-Muñoz, E and San-Juan-Ansoleaga, M and Pérez-Castro, M&#xc1; and Gonçalves-Anjo, N and Piñeiro, P and Giler, S and González-Martín, N and Lorenzo, NL and Manero-Azua, A and Perez de Nanclares, G and Geijo, M and Sánchez-Martín, MA and Requena, JR and Castilla, J},
title = {The L108I polymorphism in mouse prion protein drives spontaneous disease and enhances transmission of atypical and classical prion strains.},
journal = {Brain pathology (Zurich, Switzerland)},
volume = {},
number = {},
pages = {e70083},
doi = {10.1111/bpa.70083},
pmid = {41663312},
issn = {1750-3639},
support = {PID2024-160022OB-I00//Agencia Estatal de Investigaci&#xf3;n/ ; PID2021-122201OB-C21//Agencia Estatal de Investigaci&#xf3;n/ ; PID2020-117465GB-I00//Agencia Estatal de Investigaci&#xf3;n/ ; PID2021-1222010B-C22//Agencia Estatal de Investigaci&#xf3;n/ ; CEX2021-001136-S//Ministerio de Ciencia e Innovaci&#xf3;n/ ; EFA031/01 NEURO-COOP//Interreg/ ; PT23/00123//Instituto de Salud Carlos III/ ; BN661-FTPGB-2023//Fundaci&#xf3;n Tatiana P&#xe9;rez de Guzm&#xe1;n el Bueno/ ; //Creutzfeldt-Jakob Disease Foundation-2022/ ; },
abstract = {Prion diseases are fatal neurodegenerative disorders that can be idiopathic, associated with genetic mutations, or acquired by infection with misfolded prion protein. We developed two complementary transgenic mouse models to investigate how the L108I substitution in mouse prion protein (PrP) influences spontaneous prion formation and transmission characteristics. The transgenic mouse model overexpressing the variant at approximately three times wild-type (WT) PrP levels (TgMo(L108I)3x) consistently developed a spontaneous neurodegenerative disorder between 219 and 536 days of age with 100% penetrance. This spontaneous disease exhibited biochemical and neuropathological characteristics of atypical prion disorders, featuring a distinctive 7-10 kDa protease-resistant PrP fragment and pathology comparable to small ruminants' atypical scrapie and certain forms of Gerstmann-Sträussler-Scheinker syndrome (GSS). In contrast, the knock-in model expressing the same variant at physiological levels (TgMo(L108I)1x) showed no spontaneous disease beyond 600 days, demonstrating that both the specific amino acid substitution and elevated expression levels are necessary for spontaneous prion formation. The spontaneously generated prions transmitted efficiently to models expressing the I108 variant and to Tga20 mice overexpressing WT PrP but encountered a robust transmission barrier toward WT mice, indicating strain-specific replication requirements. The TgMo(L108I)3x model demonstrated exceptional versatility as a universal acceptor for heterogeneous prion isolates, demonstrating superior efficiency in propagating atypical variants like GSS A117V (57 ± 0.6 days) and rapid propagation of classical scrapie-derived mouse prion strains, including Rocky Mountains Laboratory mouse prion strain (RML) (85 ± 3.8 days) and 22L (95 ± 1 days). Comparative analysis revealed that the L108I substitution differentially impacts strain propagation, with greater acceleration of RML (~33% shorter incubation) than 22L (~0.5% shorter) compared to WT mice. These complementary systems offer powerful experimental platforms for investigating the molecular determinants of spontaneous prion formation, strain selection and transmission barriers, providing insights into idiopathic prion pathogenesis and developing therapeutic interventions.},
}
@article {pmid41663226,
year = {2026},
author = {Yin, X and Zhang, Z and Luo, H and Qin, X and Chen, Y and Chen, W and Zheng, H},
title = {Amplification-free one-pot RNA detection by pairing CRISPR-Cas13a with cascade amplification circuit-driven DNAzyme (RAPID).},
journal = {Analytica chimica acta},
volume = {1391},
number = {},
pages = {345138},
doi = {10.1016/j.aca.2026.345138},
pmid = {41663226},
issn = {1873-4324},
mesh = {*DNA, Catalytic/metabolism/chemistry/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; Neisseria gonorrhoeae/genetics/isolation &amp; purification ; *RNA, Bacterial/analysis/genetics ; Humans ; },
abstract = {RNA has become a versatile target for diagnosing a wide range of pathogens. The demand for rapid and accurate diagnostics in point-of-care (POC) or resource-limited settings is growing. However, most RNA-based assays depend on reverse transcription and complex instruments (e.g., RT-qPCR), restricting their use in these settings. Isothermal amplification methods provide a simpler alternative with reduced instrumentation requirements, but their high amplification efficiency raises concerns about nucleic acid carry-over contamination. To address these challenges, we developed RAPID (CRISPR-Cas13a with a cascade amplification circuit-driven DNAzyme), an isothermal, one-pot RNA detection biosensing platform that eliminates the need for sample pre-amplification. RAPID integrates the precise target recognition by CRISPR-Cas13a with robust signal amplification by a toehold-mediated strand-displacement DNA circuit, eliminating the need for reverse transcription and thermal cycling. This platform enables quantitative RNA detection within 30 min at 37 °C. By reprogramming RAPID crRNAs, we successfully detected both bacterial (e.g., Treponema pallidum and Neisseria gonorrhoeae) and viral (e.g., herpes simplex virus) targets. The RAPID platform is designed for versatile detection, being compatible with both fluorescence-based (RAPID-Flu) and lateral flow assay (RAPID-LFA) readouts. The RAPID-Flu and RAPID-LFA both demonstrated a sensitivity of 5 fM per reaction, exhibiting comparable detection limits. Both methods showed excellent specificity and high concordance with clinical diagnoses of Neisseria gonorrhoeae. In summary, the RAPID platform provides rapid, programmable, and visually interpretable solutions with strong potential for POC diagnostics. Its flexibility and portability make it particularly suitable for early diagnosis and on-site monitoring of diverse infectious pathogens.},
}
@article {pmid41663224,
year = {2026},
author = {Li, Y and Chen, X and Yang, Z and Wang, Z and Wang, R},
title = {CRISPR/Cas12a empowered electrochemical biosensor for ultrasensitive detection of Vibrio parahaemolyticus in seafood samples.},
journal = {Analytica chimica acta},
volume = {1391},
number = {},
pages = {345158},
doi = {10.1016/j.aca.2026.345158},
pmid = {41663224},
issn = {1873-4324},
mesh = {*Vibrio parahaemolyticus/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *Seafood/microbiology/analysis ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Food Contamination/analysis ; Animals ; DNA, Bacterial/genetics/analysis ; },
abstract = {Rapid and ultrasensitive detection of Vibrio parahaemolyticus (V. parahaemolyticus, Vp) is of great significance for the early prevention of foodborne disease. Traditional methods for detecting Vp are time-consuming, exhibiting low sensitivity and specificity. In this study, CRISPR/Cas12a system is integrated with electrochemical sensing and polymerase chain reaction (PCR) to establish a PCR-based E-CRISPR biosensor for Vp detection. The target DNA extracted from Vp is amplified by PCR, then activate CRISPR/Cas12a system to cleave methylene blue (MB)-labeled hairpin DNA probes on electrode, resulting in great changes in current. The employment of hairpin DNA probes reduces the steric hindrance for Cas12a trans-cleavage, acquiring a better cleavage efficiency and sensing performance. Under optimal conditions, the limit of detection reaches 1.17 copies/μL (genomic DNA), 1.23 CFU/mL (standard bacteria), and 12.3 CFU/g (artificially contaminated shrimp samples) respectively. Moreover, the PCR-based E-CRISPR biosensor demonstrates superior reproducibility and specificity. Most importantly, the E-CRISPR biosensor were in 100 % agreement with real time quantitative PCR for the detection of 18 seafood samples, which confirms the biosensor's broad applicability for monitoring Vp in complex food matrix. Our developed E-CRISPR biosensor demonstrates to be a simple, rapid and ultrasensitive method for Vp detection in the food supply chain, and can be extended to other foodborne pathogens.},
}
@article {pmid41662274,
year = {2026},
author = {Tek, MI and Budak Tek, K and Sarikaya, P and Ahmed, AR and Fidan, H},
title = {Choosing the best route: Comparative optimization of wheat transformation methods for improving yield by targeting TaARE1-D with CRISPR/Cas9.},
journal = {PloS one},
volume = {21},
number = {2},
pages = {e0342491},
pmid = {41662274},
issn = {1932-6203},
mesh = {*Triticum/genetics/growth &amp; development ; *CRISPR-Cas Systems ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *Plant Proteins/genetics ; Plant Breeding/methods ; },
abstract = {Wheat (Triticum aestivum L.) is one of the most important crops worldwide, supplying a major share of calories and protein for the global population. Incorporating gene editing into breeding programs is critical to improve yield and stress tolerance, yet wheat remains difficult to transform and regenerate efficiently. These bottlenecks limit the full application of CRISPR/Cas9 for improvement yield in wheat. To address this, transformation parameters were optimized for three methods: immature embryo transformation, callus transformation, and injection-based in planta transformation. Systematic optimization of Agrobacterium strain, bacterial density, acetosyringone concentration, and incubation conditions resulted in substantially improved transformation success. Efficiencies of 66.84% for immature embryos, 55.44% for callus, and 33.33% for in planta transformation were achieved, representing more than tenfold increase compared with previously reported rate of ~3%. A key innovation was the shortening of the callus induction stage for immature embryos, reducing the time required for plant regeneration by approximately one month while maintaining high transformation efficiency. The protocols were validated through CRISPR/Cas9-mediated knockout of TaARE1-D, a negative regulator of nitrogen uptake and yield. Generated mutants exhibited increased grain number, spike length, grain length, and thousand-grain weight, as well as the characteristic stay-green phenotype associated with loss of TaARE1-D function. The optimized protocols provide robust platforms to accelerate gene-editing in wheat to increase yield and stress-tolerance.},
}
@article {pmid41661420,
year = {2026},
author = {Zhang, H and Feng, G and Feng, Y},
title = {Quinoa as a naturally stress-resistant crop: current status and future promises.},
journal = {Stress biology},
volume = {6},
number = {1},
pages = {12},
pmid = {41661420},
issn = {2731-0450},
support = {2022B02010-1//Science and Technology Department of Xinjiang Uygur Autonomous Region/ ; 2022YFF1003403-4//Key Technologies Research and Development Program/ ; 2021YFA1300401//Key Technologies Research and Development Program/ ; 32441015//National Natural Science Foundation of China/ ; },
abstract = {Quinoa (Chenopodium quinoa Willd.), a semi-domesticated halophyte originating in the Andean region, has emerged as a promising crop for exploiting marginal lands, valued for its exceptional nutritional profile and remarkable resilience to high salinity and drought. This review analyzes the current status and future potential of quinoa as a model halophytic crop. We begin by examining the physiological mechanisms that enable quinoa to thrive in marginal environments, which have been the subject of extensive study. Thanks to the advancement in high-throughput sequencing technology, genomic resources - including the recent development of high-quality reference genomes and a Chenopodium pangenome - are rapidly expanding. Sequence-based genetic mapping techniques hold the promise to dissect the molecular basis of complex traits in combination with the utility of functional genomics tools such as virus-induced gene silencing (VIGS) and stable genetic transformation. Ultimately, the application of modern breeding technologies, such as phenomics, genomic selection (GS), and CRISPR/Cas, will expedite the development of locally adapted, climate-resilient quinoa cultivars worldwide.},
}
@article {pmid41661284,
year = {2026},
author = {Li, Z and Chen, L and Luo, J and Lu, Y and Zhang, H and Zhao, P},
title = {One-pot recombinase polymerase amplification and CRISPR/Cas12a assay for Cryptococcus neoformans.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {61},
pmid = {41661284},
issn = {1432-0614},
mesh = {*Cryptococcus neoformans/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Recombinases/genetics/metabolism ; *Cryptococcosis/diagnosis/microbiology ; Humans ; Real-Time Polymerase Chain Reaction/methods ; Point-of-Care Testing ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Cryptococcus neoformans, an opportunistic fungal pathogen, can induce central nervous system infections, posing a life-threatening risk and imposing substantial global health challenges and economic burdens. Given the significant reduction in mortality achieved through early monitoring of C. neoformans, there is an urgent demand for a rapid detection method for this pathogen. Herein, we developed a rapid, sensitive, and specific assay for the detection of C. neoformans based on a one-pot recombinase polymerase amplification (RPA) and CRISPR/Cas12a system, which can be read using a real-time fluorescent PCR instrument or lateral flow strips. This assay exhibits high sensitivity, with a detection limit of 1 copy/µL for C. neoformans, and no cross-reactivity was observed across different fungal strains. Notably, the assay can be performed in harsh environments without reliance on complex equipment, making it suitable for point-of-care testing (POCT). Collectively, this method not only provides a robust alternative for C. neoformans detection but also offers valuable insights for the identification of other fungal pathogens. KEY POINTS: • We developed a one-pot RPA and CRISPR/Cas12a assay to detect Cryptococcus neoformans. • This assay exhibits high sensitivity and specificity. • Detection results can be obtained by three ways which is suitable for POCT.},
}
@article {pmid41660305,
year = {2026},
author = {Zhang, W and Jiang, A and Jia, BK and Jin, Y and Chen, Y and Li, Z and Liao, Y and Zhang, H and Lin, Z and Fang, X and Wang, L},
title = {Progress in RNA-Targeted Therapeutics for Human Diseases.},
journal = {MedComm},
volume = {7},
number = {2},
pages = {e70607},
pmid = {41660305},
issn = {2688-2663},
abstract = {RNA-targeted therapy is reshaping molecular medicine by shifting the traditional "protein-centric" view toward an "RNA-regulatory network" paradigm. Beyond carrying genetic information, RNA plays essential roles in posttranscriptional regulation, signaling pathways, and epigenetic modulation. Advances in high-throughput sequencing, structural biology, and delivery technologies have accelerated the development of diverse RNA therapeutics, including antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA) modulators, messenger RNA (mRNA) therapeutics, aptamers, short hairpin RNA, and CRISPR/Cas-guided single-guide RNAs. However, a concise comparison of these major RNA modalities and the translational barriers that limit their broader clinical application is still lacking. This review outlines the mechanisms and representative applications of these RNA-based strategies in gene silencing, editing, protein replacement, immune activation, and targeted drug delivery. Special emphasis is placed on ASOs and siRNAs for neurological, metabolic, and infectious diseases, as well as mRNA therapeutics that are transforming vaccine development. Common challenges-such as in vivo stability, delivery efficiency, and immune activation-are also discussed. Finally, we highlight how chemical modification, nanotechnology, and artificial intelligence-assisted design are enhancing the specificity, stability, and safety of RNA therapeutics, providing a framework for advancing next-generation precision RNA medicine.},
}
@article {pmid41607132,
year = {2026},
author = {Yu, K and Li, H and Hu, Y and Yu, Y and Deng, S and Yang, Y and Guo, M and Li, M and Zhe, M and He, H and Fan, C},
title = {Integrating transcriptomics and high-throughput gene editing uncovers shoot apical meristem regulators in Brassica napus.},
journal = {Plant physiology},
volume = {200},
number = {2},
pages = {},
doi = {10.1093/plphys/kiag032},
pmid = {41607132},
issn = {1532-2548},
support = {2022YFD1200400//National Key Research and Development Program of China/ ; 2023YFF1000700//National Key Research and Development Program of China/ ; 31671279//National Natural Science Foundation of China/ ; 2024BBA001//Agricultural Gene Editing Platform Technology and Breeding Research &amp; Development/ ; },
mesh = {*Brassica napus/genetics/growth &amp; development ; *Meristem/genetics/growth &amp; development ; *Gene Editing/methods ; *Transcriptome ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Plant Shoots/genetics/growth &amp; development ; Plant Proteins/genetics/metabolism ; Mutation ; },
abstract = {The shoot apical meristem (SAM) determines plant architecture, but the key components of its regulatory network remain elusive in rapeseed (Brassica napus L.). Here, we integrated transcriptomic profiling of 3 multilocular silique mutants (Bnaclv1, Bnaclv2, and Bnaclv3) across key SAM development stages (IM, stage6, and stage8) with large-scale CRISPR/Cas9 functional screening to identify regulators of SAM maintenance. Differential gene expression and GO enrichment highlighted genes significantly associated with meristem development processes. Weighted gene co-expression network analysis of stage-specific transcriptomes identified 42 candidate genes potentially related to SAM development. To enable systematic functional screening, we established a high-throughput multiplex CRISPR/Cas9 pipeline, simultaneously targeting 198 sites across 42 candidate genes through optimized sgRNA design and pooled transformation. We successfully obtained mutants for 25 genes with homozygous mutants for 9 genes. Phenotypic analysis demonstrated that mutants of BnaSCPL family genes (SCPL29, SCPL44, and SCPL45) exhibited a multi-stem phenotype and disrupted SAM organization. Mechanistic studies revealed that BnaSCPL mutations disrupt the canonical CLV3/WUS feedback loop, uncovering their roles in SAM homeostasis. Additionally, knockout of BnaLFY homologs caused permanent vegetative state and sterility, demonstrating their conserved role in floral meristem identity in Brassica napus. Collectively, our study not only elucidates the critical function of BnaSCPLs in SAM maintenance but also establishes a regulatory framework for understanding meristem phase transitions in B. napus, providing potential targets for crop architecture improvement.},
}
@article {pmid41591771,
year = {2026},
author = {Chen, D and Zhu, B and Zhou, Y and Fang, Z and Zhu, Z and Chen, C and Shen, T},
title = {Dual-mode CRISPR/Cas12a-mediated alkaline phosphatase detection (CAD) biosensor.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {6},
pages = {1239-1247},
doi = {10.1039/d5ay01195g},
pmid = {41591771},
issn = {1759-9679},
mesh = {*Alkaline Phosphatase/analysis/blood ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Humans ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Immunoassay/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Alkaline phosphatase (ALP), a crucial biomarker for hepatobiliary disorders, bone diseases, and cancer progression, requires ultrasensitive detection methods to meet clinical diagnostic requirements. Current methodologies predominantly depend on single-readout mechanisms that fail to address the growing requirements of sensitivity, operational simplicity, and adaptability to resource-limited settings. Herein, we present a CRISPR/Cas12a-mediated ALP detection (CAD) isothermal amplification system that overcomes these challenges through a novel dual-signal (fluorescence and lateral flow immunoassay (LFIA)) readout mechanism. The system features a rationally engineered hairpin DNA probe (HPP) that initiates Klenow (exo-)-driven polymerase elongation upon ALP recognition, subsequently activating Cas12a's trans-cleavage activity for exponential signal amplification. With fluorescence readout, this cascade amplification strategy achieves unprecedented sensitivity with a detection limit of 0.1 U L[-1] and a wide linear range (0.1-10 U L[-1]), outperforming conventional colorimetric methods by one order of magnitude while maintaining exceptional specificity against biological interferents. Furthermore, the LFIA adaptation of the readout bridges the gap between laboratory-based detection and point-of-care applications. This user-friendly adaptation enables instrument-free visual detection with a clear cut-off value of about 7 U L[-1], offering the potential to effectively differentiate pathological samples from normal physiological levels with an appropriate dilution factor for clinical samples. Our dual-mode biosensing strategy not only enables high-precision quantitative analysis in clinical settings but also facilitates rapid qualitative detection under resource-limited conditions, thus offering significant potential for early disease diagnosis and long-term therapeutic monitoring.},
}
@article {pmid41590404,
year = {2026},
author = {Li, W and Wang, M and Wang, S},
title = {A label-free and universal CRISPR/Cas12a platform for the detection of hazardous substances in food.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {6},
pages = {1216-1223},
doi = {10.1039/d5ay01887k},
pmid = {41590404},
issn = {1759-9679},
mesh = {*CRISPR-Cas Systems/genetics ; *Food Contamination/analysis ; *Hazardous Substances/analysis ; *Food Analysis/methods ; Limit of Detection ; },
abstract = {The present study proposes a label-free and universal CRISPR/Cas12a-based platform for the detection of hazardous substances in food. A label-free reporter, termed the G4 reporter, was carefully engineered. In the absence of the target analyte, the catalytic hairpin assembly (CHA) remains inactive, thereby preventing activation of the CRISPR/Cas12a system and preserving the cleavage of the G4 reporter, which emits a label-free fluorescence signal. Conversely, in the presence of the target analyte, the CHA process is triggered, activating the CRISPR/Cas12a system, which cleaves the G4 reporter into fragments, leading to a decrease in the label-free fluorescence signal. This detection strategy follows a negative response model, wherein quantification is based on the net fluorescence difference between positive and negative controls. The developed label-free signal output modality for the CRISPR/Cas12a system offers advantages of simplicity and cost-effectiveness. Moreover, the proposed method incorporates a CHA process to facilitate signal transduction and activate the subsequent CRISPR system. The system upon integration of CHA with CRISPR functions in a dual-amplification mode, resulting in enhanced signal amplification efficiency and superior sensitivity compared to the standalone CRISPR system. Thus, the platform achieves highly sensitive detection of hazardous substances, with limits of detection (LODs) of 3.0 fg mL[-1] for aflatoxin B1 (AFB1) and 0.02 pg mL[-1] for acetamiprid (ACE). Moreover, trace amounts of AFB1 and ACE were successfully identified in real food samples. By altering the sequences of the detection components, this platform can be readily adapted for the detection of other hazardous substances in food matrices. Therefore, this work introduces a novel, label-free, universal, and sensitive CRISPR/Cas12a-based detection platform, demonstrating considerable potential for applications in food safety surveillance and public health protection.},
}
@article {pmid41459814,
year = {2026},
author = {Celle, M and Aniorte, S and Issa, AR and Falabregue, M and Jin, H and Sanchez-Mirasierra, I and Ding, S and Soukup, SF and Seugnet, L and Liao, L and Lesca, G and Walter, L and Mollereau, B},
title = {A dwdr45 knock-out drosophila model to decipher the role of autophagy in BPAN.},
journal = {Human molecular genetics},
volume = {35},
number = {3},
pages = {},
doi = {10.1093/hmg/ddaf198},
pmid = {41459814},
issn = {1460-2083},
support = {//French Ministry of Higher Education and Research/ ; 101067877//Marie Sklodowska-Curie Action fellowship/ ; //China Scholarship Council/ ; },
mesh = {Animals ; *Autophagy/genetics ; Disease Models, Animal ; *Drosophila Proteins/genetics/metabolism ; Gene Knockout Techniques ; Drosophila melanogaster/genetics ; CRISPR-Cas Systems ; *Neurodegenerative Diseases/genetics/pathology/metabolism ; *Carrier Proteins/genetics/metabolism ; Phenotype ; Drosophila/genetics ; Humans ; },
abstract = {Beta-propeller protein-associated neurodegeneration (BPAN) is a rare neurological disorder characterized by severe cognitive and motor impairments. BPAN is caused by de novo pathogenic variants in the WDR45 gene on the X chromosome. WDR45 gene encodes the protein WDR45/WIPI4, a known regulator of autophagy. A defective autophagy has been observed in cellular models of BPAN disease and is associated with neurological dysfunctions in wdr45 knockout (KO) mice. However, it remains unclear whether the autophagic defect directly contributes to all WDR45 loss-induced phenotypes or whether other WDR45-dependent cellular functions are involved. To investigate this, we generated a CRISPR/Cas9-mediated KO of CG11975 (dwdr45 KO), the Drosophila homolog of WDR45. Our analysis revealed that dwdr45 KO flies display BPAN-like phenotypes, including impaired locomotor function, seizure-like behavior, autophagy dysregulation and iron dyshomeostasis. Additionally, dwdr45 KO flies exhibit shortened lifespan compared to control flies. These findings demonstrate that dwdr45 KO fly is a relevant in-vivo model for investigating the key cellular and molecular mechanisms underlying BPAN-associated phenotypes. Here we showed that induction of autophagy in dwdr45 KO flies improved both the shortened lifespan and the seizure-like behavior, but did not restore locomotor function. This suggests that defective autophagy contributes to some, but not all, aspects of the phenotypes resulting from loss of dWdr45 function.},
}
@article {pmid41659011,
year = {2025},
author = {Wang, B and Lu, J and Zhang, X and Hu, R and Ma, H},
title = {Advances in nanomaterial-mediated CRISPR/Cas delivery: from lipid nanoparticles to vesicle-derived systems.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {13},
number = {},
pages = {1669104},
pmid = {41659011},
issn = {2296-4185},
abstract = {Gene and genome editing therapies are increasingly connected with nanomaterials, which protect and transport fragile nucleic acids and CRISPR/Cas systems through biological barriers safely and accurately. This review discusses how different nanocarriers, including lipid-based, polymeric, inorganic, and vesicle-derived systems, can improve delivery efficiency, cell targeting, endosomal escape, and intracellular movement for gene and genome editing. It summarizes findings from early clinical and preclinical studies, comparing several carrier types such as ionizable lipid nanoparticles, polymeric nanoparticles, micelles, gold and silica nanostructures, and engineered extracellular vesicles. The review also explains how specific design factors, such as surface ligands, charge modification, PEGylation, and stimulus-responsive behaviors, influence biodistribution, and improve on-target efficiency while lowering immune responses and off-target effects. Ethical and regulatory concerns for in vivo editing are highlighted, along with current methods used to study nano-bio interactions. Among these carriers, ionizable lipid nanoparticles show the most advanced performance for delivering nucleic acids and CRISPR systems. However, new polymer-based and exosome-inspired carriers are progressing rapidly for repeated and targeted applications. Hybrid and responsive systems may also enable better spatial and temporal control of editing. Future research should focus on stronger in vivo potency testing, improved biocompatibility evaluation, and standardized manufacturing to ensure clinical safety and reliability.},
}
@article {pmid41658853,
year = {2026},
author = {Rahimian, M and Aghazadeh-Soltan-Ahmadi, M and Panahi, B},
title = {In silico exploration of the genomic repertoire of Iranian aquatic bacteria: Prophage carriage, bioactive compound potential, CRISPR-Cas immunity, and integrated defensive-metabolic islands.},
journal = {Biochemistry and biophysics reports},
volume = {45},
number = {},
pages = {102452},
pmid = {41658853},
issn = {2405-5808},
abstract = {The unique and underexplored aquatic ecosystems of Iran represent a significant reservoir of microbial diversity. This study presents the first comprehensive genomic survey of 38 native Iranian bacterial strains from hypersaline lakes and wetlands, integrating in silico analyses of their secondary metabolome, bacteriocin potential, resident prophages, and genomic architecture. Our genome mining revealed a prolific capacity for secondary metabolite production, identifying dozens of biosynthetic gene clusters (BGCs). Ectoine biosynthesis was ubiquitous, underscoring its role as a key osmoprotectant, while diverse BGCs for terpenes, polyketides, and hybrid metabolites were also prevalent. Concurrently, we identified a wide array of ribosomally synthesized and post-translationally modified peptides (RiPPs), including known bacteriocins. Furthermore, we characterized eight high-quality prophages integrated within these genomes, encoding auxiliary genes such as carbohydrate-active enzymes (CAZymes) and putative anti-CRISPR (ACR) proteins. The bacterial hosts themselves were equipped with robust defense systems, with CRISPR-Cas loci, predominantly Type I, detected in most strains. Crucially, we identified multi-functional genomic islands that physically link BGCs with defense systems (e.g., CRISPR-Cas, restriction-modification) and prophage regions. We propose the "Fortress Hypothesis" to explain this architecture, wherein the co-localization of metabolite production and defense machinery protects metabolic investment against phage predation and genetic loss. This integrative genomic arrangement highlights a sophisticated co-evolutionary strategy for survival in extreme environments. Our findings position these indigenous bacteria as a promising genetic repository for discovering novel bioactive compounds, enzymes, and biotechnological tools, with implications for antibiotic discovery, CRISPR modulation, and understanding adaptive microbial genomics in extreme niches.},
}
@article {pmid41658434,
year = {2026},
author = {Han, Y and Chen, R and Shentu, X},
title = {Advances and challenges of CRISPR/Cas gene editing for corneal diseases.},
journal = {Advances in ophthalmology practice and research},
volume = {6},
number = {1},
pages = {68-79},
pmid = {41658434},
issn = {2667-3762},
abstract = {BACKGROUND: Corneal diseases are a major cause of global visual impairment, and current treatments remain inadequate for severe or refractory cases. The CRISPR/Cas system offers robust and precise gene-editing capabilities, yet its therapeutic potential for corneal disorders remains largely unexplored.<br><br>MAIN TEXT: This narrative review introduces the CRISPR/Cas system and summarizes its recent advances in treating various corneal diseases, including inherited corneal dystrophies, infectious keratitis, corneal injury, and pathological neovascularization. We outline emerging preclinical and clinical studies, and analyze key issues that should be addressed for translation, including administration strategies, vector platform optimization and the mitigation of off-target toxicity.<br><br>CONCLUSIONS: This review provides a comprehensive and integrated overview of the current translational directions and challenges of CRISPR/Cas technology in corneal diseases from a novel perspective. It offers valuable guidance for future research and may accelerate the development of gene-editing therapies toward clinical application.},
}
@article {pmid41656481,
year = {2026},
author = {Laxmi, V and Verma, S and Kumar, M and Venkatesh, V and Mohit, and Maury, J and Mohd, S and Tripathi, S},
title = {Lactic Acid Bacteria-derived Bacteriocins: A Promising Antimicrobial Strategy against Multidrug-resistant for Neonatal Sepsis Pathogens.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41656481},
issn = {1867-1314},
abstract = {Neonatal sepsis continues to pose rising critical challenges to global health, particularly in low and middle-income countries, where it accounts for a substantial proportion of neonatal morbidity and mortality. The increasing prevalence of multidrug-resistant (MDR) pathogens in neonatal sepsis has significantly weakened the efficacy of conventional antibiotics, necessitating urgent exploration of alternative antimicrobial therapies for better clinical outcomes. Emerging research interest is growing to develop the microbial-derived peptides as novel antimicrobial agents, particularly "bacteriocins." In comparison to traditional antibiotics, many bacteriocins exhibit narrow-spectrum action, enabling them to inhibit specific pathogens without disrupting the host microbiota. Recent studies have highlighted the remarkable potential of lactic acid bacteria (LAB) derived bacteriocins in combating MDR pathogens responsible for neonatal sepsis. In this review, we compile current literature on the in vitro antimicrobial activity of LAB-derived bacteriocins, molecular diversity, mechanisms of action and clinical potential. Special attention is given to bacteriocins produced by LAB associated with the milk-derived microbiota, whose natural protective functions could be particularly beneficial for immunocompromised newborns. Additionally, we discuss the physicochemical properties of microbial peptides, including thermal stability, enzymatic resistance, and pH tolerance, which support their suitability for pharmaceutical applications. Overall, LAB-derived bacteriocins represent a novel, biocompatible, and complementary antimicrobial strategy integrated with conventional antibiotics to fight against MDR in neonatal sepsis. Further research and multicentric clinical trials are necessary to fully explore its compliance and efficacy as a future antimicrobial agent in neonatal medicine to underscore their potential as adjunct or preventive biotherapeutics in NICU settings.},
}
@article {pmid41656299,
year = {2026},
author = {Fang, T and Bogensperger, L and Feer, L and Allam, A and Bezshapkin, V and Balázs, Z and von Mering, C and Sunagawa, S and Krauthammer, M and Schwank, G},
title = {Uncovering Cas9 PAM diversity through metagenomic mining and machine learning.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-69098-5},
pmid = {41656299},
issn = {2041-1723},
abstract = {Recognition of protospacer adjacent motifs (PAMs) is crucial for target site recognition by CRISPR-Cas systems. In genome editing applications, the requirement for specific PAM sequences at the target locus imposes substantial constraints, driving efforts to search for novel Cas9 orthologs with extended or alternative PAM compatibilities. Here, we present CRISPR-PAMdb, a comprehensive and publicly accessible database compiling Cas9 protein sequences from 3.8 million bacterial and archaeal genomes and PAM profiles from 7.4 million phage and plasmid sequences. Through spacer-protospacer alignment, we infer consensus PAM preferences for 8003 unique Cas9 clusters. To extend PAM discovery beyond traditional alignment-based approaches, we develop CICERO, a machine learning model predicting PAM preferences directly from Cas9 protein sequences. Built on the ESM2 protein language model and trained on the CRISPR-PAMdb database, CICERO achieves an average cosine similarity of 0.69 on test data and 0.75 on experimentally validated Cas9 orthologs. For Cas9 clusters where alignment-based predictions are infeasible, CICERO generates PAM profiles for an additional 50,308 Cas9 proteins, including 17,453 high-confidence predictions with CICERO confidence scores above 0.8. Together, CRISPR-PAMdb and CICERO enable large-scale exploration of PAM diversity across Cas9 proteins, accelerating design of next-generation CRISPR-Cas9 tools for precise genome engineering.},
}
@article {pmid41656257,
year = {2026},
author = {Ruis, BL and Ward, H and Myers, CL and Bielinsky, AK and Hendrickson, EA},
title = {SETDB1/ATF7IP regulate the precise genome engineering of HUSH-regulated genes.},
journal = {Epigenetics &amp; chromatin},
volume = {19},
number = {1},
pages = {10},
pmid = {41656257},
issn = {1756-8935},
support = {AG077174//National Institute of Aging/ ; AG077174//National Institute of Aging/ ; R35GM141805//National Institutes of General Medical Sciences/ ; GM088351/GM/NIGMS NIH HHS/United States ; CA154461/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Histone-Lysine N-Methyltransferase/metabolism/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Recombinational DNA Repair ; DNA Breaks, Double-Stranded ; HEK293 Cells ; },
abstract = {BACKGROUND: The use of programmable nucleases has transformed genome editing and functional genomics. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) was developed such that targeted genomic lesions [usually DNA double-stranded breaks (DSBs)] could be introduced in vivo with ease and precision. In the presence of homology donors, these lesions facilitate high-efficiency precise genome editing (PGE) via homology-directed repair (HDR) pathways. Because DSBs can lead to genomic instability, however, a large amount of effort has been invested in methodologies (e.g., base editors) that only require nicking the chromosomal DNA on one strand. Indeed, we have demonstrated in human cells that oligodeoxynucleotide (ODN)-mediated PGE using nickase variants of Cas9 can proceed by at least two HDR subpathways termed synthesis-dependent strand annealing (SDSA) and single-stranded DNA incorporation (ssDI). Which pathway is utilized is determined by which chromosomal strand (sense or anti-sense/Watson or Crick) is nicked and by the strandedness (sense or anti-sense/Watson or Crick) of the donor ODN.<br><br>RESULTS: While the mechanism of mammalian SDSA is moderately well understood, that of ssDI is not. To gain genetic insight into ssDI, we carried out a genome-wide CRISPR knockout screen to identify those genes which, when absent, enable increased ssDI. This screen identified the protein lysine methyl transferase (PKMT) Su(var)3-9, enhancer-of-zeste and trithorax (SET) domain bifurcated histone lysine methyltransferase 1 (SETDB1):activating transcription factor 7-interacting protein (ATF7IP) heterodimer and the downstream human silencing hub (HUSH) complex as strong negative regulators of ssDI. Consistent with their well-known biological effects, the negative regulation of ssDI by SETDB1/ATF7IP and HUSH was specific for transgenic reporters and for a HUSH-regulated single-copy gene, but was not observed at other (non-HUSH regulated) single-copy endogenous loci.<br><br>CONCLUSIONS: In toto, these experiments underscore the profound impact that chromatin modifiers - and by extension, chromatin structure - have on PGE outcomes. Specifically, we have identified SETDB1/ATF7IP and the HUSH complex as major negative regulators of the HDR subpathway, ssDI, when the target is a transgene. These experiments are a proof-of-principle that chromatin can act as a potent barrier to genetic recombination and they strongly support the feasibility of extending similar chromatin modulating strategies to enhance PGE efficiency at endogenous single-copy loci.},
}
@article {pmid41655694,
year = {2026},
author = {Saito, A and Tankou, S and Ishii, K and Sakao-Suzuki, M and Oh, EC and Murdoch, H and Namkung, H and Adelakun, S and Furukori, K and Fujimuro, M and Salomoni, P and Maul, GG and Hayward, GS and Tang, Q and Yolken, RH and Houslay, MD and Katsanis, N and Kosugi, I and Yang, K and Kamiya, A and Ishizuka, K and Sawa, A},
title = {Cytomegalovirus (CMV)-encoded immediate early 1 (IE1) protein perturbs neural progenitor proliferation via interfering with host PML-DISC1 interaction.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {111269},
doi = {10.1016/j.jbc.2026.111269},
pmid = {41655694},
issn = {1083-351X},
abstract = {Congenital CMV infection is the most common perinatal infection, affecting up to 0.5% of infants. This elicits long-term disabilities that include neuropsychiatric manifestations, such as intellectual disability, microcephaly. Despite its high prevalence, the underlying mechanism of how congenitally acquired CMV infection causes brain pathology remains unknown. Here we discovered the molecular interplay of key host (DISC1 and PML) and viral (IE1) proteins within the neural progenitor cells, which underlay an attenuated neural progenitor proliferation in congenital CMV infection. Abolishing the viral IE1 protein by delivering IE1-targeting CRISPR/Cas9 to fetal brain rescued this progenitor cell deficit, a key pathology in congenital CMV infection. A selective targeting to a viral-specific protein by the CRISPR/Cas9 system is minimal in off-target effects. We further observed that CMV-encoded IE1 protein interferes with host PML-DISC1 interaction, resulting in disturbance of the Notch pathway in vitro and in embryonic brains. Therefore, we believe that a pivotal role of IE1 in an attenuated neural progenitor proliferation in the developing cortex through its interfering with interaction between host DISC1 and PML proteins.},
}
@article {pmid41654278,
year = {2026},
author = {Wang, X and You, J and Li, X and Xu, Y and Li, Z and Wang, L},
title = {Limitations of traditional mycotoxin control and biotechnological advances toward sustainable solutions.},
journal = {Biotechnology advances},
volume = {88},
number = {},
pages = {108836},
doi = {10.1016/j.biotechadv.2026.108836},
pmid = {41654278},
issn = {1873-1899},
abstract = {Mycotoxins are harmful fungal metabolites that contaminate food and feed, posing serious health and economic risks worldwide. Traditional control methods often fall short due to inefficiency and safety concerns, prompting the development of innovative biotechnological approaches. This review explores recent advances in mycotoxin management, focusing on engineered microbes for targeted degradation, nanotechnology-based detection and removal systems, phage therapy targeting toxin-producing fungi, CRISPR-Cas gene editing of mycotoxin biosynthesis pathways, and plant-microbe interactions that suppress fungal growth. Additionally, enzyme immobilization strategies are highlighted for improving enzyme stability and reusability in detoxification processes. These integrated biotechnological tools offer promising, sustainable solutions to mitigate mycotoxin contamination, enhancing food safety and agricultural productivity. The review also discusses current challenges and future directions for translating these advances into practical applications. Highlighting that biotechnological tools are technically feasible and increasingly close to industrial deployment in food and feed chains.},
}
@article {pmid41621146,
year = {2026},
author = {Kundu, A and Jerala, R},
title = {Small RNAs, big potential: Engineering microRNA-based synthetic gene circuits.},
journal = {Current opinion in chemical biology},
volume = {90},
number = {},
pages = {102652},
doi = {10.1016/j.cbpa.2026.102652},
pmid = {41621146},
issn = {1879-0402},
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Gene Regulatory Networks ; *Synthetic Biology/methods ; Animals ; *Genetic Engineering/methods ; CRISPR-Cas Systems ; },
abstract = {MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression. Their dysregulation is closely associated with various diseases, positioning them as biomarkers of cellular state. Synthetic biology has leveraged these properties to engineer miR-based genetic circuits capable of sensing and interpreting endogenous miR levels. Early miR-OFF systems relied on reporter gene repression but were limited by ambiguous signal interpretation. Subsequent advances introduced miR-ON architectures, logic-based classifiers integrating multiple miRs, and layered regulatory strategies combining transcriptional, translational, and cleavage-based modules to enhance sensitivity and specificity. Recent innovations include CRISPR-associated miR-responsive systems and incoherent feed-forward loop (iFFL) architectures that stabilize gene expression amid cellular variability, shifting applications from passive sensing to therapeutic intervention. Despite challenges such as leakage, cellular resource resources, and delivery, progress in orthogonal miR toolkits, computational modeling, and RNA-based delivery platforms is rapidly driving miR-based circuits toward diagnostic and therapeutic applications.},
}
@article {pmid41577003,
year = {2026},
author = {Zade, NH and Jain, M and Garg, M and Checker, R and Ghosh, A and Khattar, E},
title = {Proteomics method for identifying POT1-associated complexes at telomeres using ChIP-Mass spectrometry.},
journal = {Methods (San Diego, Calif.)},
volume = {247},
number = {},
pages = {161-174},
doi = {10.1016/j.ymeth.2026.01.007},
pmid = {41577003},
issn = {1095-9130},
mesh = {Shelterin Complex ; *Telomere/metabolism/genetics/chemistry ; *Proteomics/methods ; Humans ; *Telomere-Binding Proteins/metabolism/genetics ; Mass Spectrometry/methods ; CRISPR-Cas Systems ; *Chromatin Immunoprecipitation/methods ; Protein Binding ; Gene Editing ; Tripeptidyl-Peptidase 1 ; },
abstract = {POT1 is the only single stranded telomere binding protein in the shelterin complex. Together with TPP1, POT1 plays a crucial role in regulating telomere length and protecting telomeres from DNA damage repair proteins. The activation of DNA damage repair proteins at telomeres can be detrimental to cells, so their activity must be suppressed. POT1 interacts with other telomeric proteins (TRF2, TRF1, TIN2 and RAP1) via its association with TPP1. These proteins function together to protect and maintain the telomeres. Despite extensive knowledge of POT1's role within the shelterin complex, the full spectrum of its interactors at the single-stranded telomeric overhang remains poorly defined. To study these interactions, we generated an endogenous Flag-tag knock-in of POT1 using the CRISPR-Cas9 gene editing system. To address the risk of unintended gene disruption associated with this technique, we conducted an in-depth characterization of the endogenously Flag-tagged POT1 clone to ensure that its telomere and TPP1 binding functions remained intact. Further, we performed proteomic profiling of the Flag-tagged POT1 within the chromatin fraction using ChIP-MS to explore its proteome. Our analysis uncovered a novel set of POT1-associated proteins at the extremes of telomeres. Given that POT1 exclusively binds to the single-stranded 3' overhang of telomeres, the proteomic data obtained indicates POT1 interactions occurring at the extreme ends of telomeres. In conclusion, our study reveals previously uncharacterized POT1 associated proteins using ChIP mass spectrometric approach, paving the way for further investigations into telomere biology and potential therapies targeting telomere regulation.},
}
@article {pmid41576935,
year = {2026},
author = {Hommersom, MP and Puvogel, S and Scheefhals, N and Carpentiero, E and Bouma, M and van Beusekom, E and Dillen, L and van de Warrenburg, BPC and Nadif Kasri, N and van Bokhoven, H},
title = {Human neuronal networks on micro-electrode arrays as a tool to assess genotype-phenotype correlation in CACNA1A-related disorders.},
journal = {Stem cell reports},
volume = {21},
number = {2},
pages = {102783},
doi = {10.1016/j.stemcr.2025.102783},
pmid = {41576935},
issn = {2213-6711},
mesh = {Humans ; *Genetic Association Studies/methods ; *Nerve Net/metabolism ; *Neurons/metabolism ; *Calcium Channels/genetics ; Phenotype ; CRISPR-Cas Systems/genetics ; Electrodes ; Genotype ; Calcium Channels, N-Type ; },
abstract = {CACNA1A-related disorders constitute a diverse group of neurological conditions, including ataxia, migraine, and epilepsy. Despite extensive genetic studies, clear genotype-phenotype correlations remain elusive. Moreover, next-generation sequencing has identified many variants of uncertain significance (VUS). Here, we leveraged patient-derived and CRISPR-Cas9-engineered human neuronal networks to explore relationships between CACNA1A variants and neurophysiological activity. CACNA1A haploinsufficiency induced subtle alterations in glutamatergic network activity, whereas missense variants had a more pronounced effect on overall network function. Network fingerprints were most affected from patients where ataxia co-occurred with migraine or epilepsy. Furthermore, we analyzed the impact of CRISPR-Cas9-induced VUS on network developmental trajectories. Although functional changes could not be directly linked to clinical phenotypes, all tested variants induced measurable alterations in neuronal network function, supporting their classification as likely pathogenic. These findings highlight the potential of human neuronal networks as a translational model for evaluating CACNA1A variant effects and improving clinical variant interpretation.},
}
@article {pmid41565761,
year = {2026},
author = {Beauchemin, KS and Supattapone, S},
title = {Genome-wide screens identify core regulators of cell surface prion protein expression.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {5895},
pmid = {41565761},
issn = {2045-2322},
support = {1R37NS125431/NS/NINDS NIH HHS/United States ; 5T32AI007519/NH/NIH HHS/United States ; 1R37NS125431/NS/NINDS NIH HHS/United States ; 1R37NS125431/NS/NINDS NIH HHS/United States ; 5T32AI007519/NH/NIH HHS/United States ; },
mesh = {*Neurons/metabolism/cytology ; Humans ; CRISPR-Cas Systems ; *Prion Proteins/genetics/metabolism ; Cell Differentiation/genetics ; *Gene Expression Regulation ; *PrPC Proteins/genetics/metabolism ; Animals ; Cell Line ; Cell Membrane/metabolism ; },
abstract = {Expression of the cellular prion protein, PrP[C], on the surface of neurons plays an important role in the pathogenesis of prion disease. We performed genome-wide CRISPR/Cas9 knockout screens in prion-infectible cells of neuronal origin (CAD5) to identify regulators of cell surface PrP[C] expression. We identified and validated 46 positive and 21 negative regulators of cell surface PrP[C] expression in undifferentiated CAD5 cells. Pathway analysis of the screening dataset showed that genes involved in the glycophosphatidylinositol (GPI) anchor and N-glycosylation biosynthetic pathways were overrepresented as positive regulators of cell surface PrP[C]. We also sought to determine whether the same or different genes regulate cell surface PrP[C] in CAD5 cells that have been differentiated to a more neuronal state and validated 41 positive and 13 negative regulators of CAD5 cell surface PrP[C] expression in the differentiated state. We identified 23 core genes as shared between the undifferentiated and differentiated cell states, including many positive regulators involved in GPI anchor biosynthesis. Intriguingly, unique regulators were also identified in the undifferentiated and differentiated cell states, suggesting that some mechanisms regulating cell surface PrP[C] expression in CAD5 cells are dependent on cell state. This list of core genes involved in regulating cell surface PrP[C] expression in a prion-susceptible, neuron-like cell type offers a valuable guide for future research and may help identify potential therapeutic targets for prion disease and other neurodegenerative diseases.},
}
@article {pmid41524400,
year = {2026},
author = {Clarke, JE and Faulkner, TR and Seipke, RF},
title = {A platform for CRISPRi-seq in Streptomyces albidoflavus.},
journal = {mBio},
volume = {17},
number = {2},
pages = {e0306525},
doi = {10.1128/mbio.03065-25},
pmid = {41524400},
issn = {2150-7511},
support = {BB/T014962/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Streptomyces/genetics ; Operon ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Bacterial ; *CRISPR-Cas Systems ; Genomics/methods ; },
abstract = {UNLABELLED: Streptomyces produce a multitude of secondary metabolites, which have been exploited in drug discovery campaigns for more than three-quarters of a century. Our understanding of microbial physiology has been revolutionized by genome sequencing and large-scale functional studies. Technology for genome-wide investigations in Streptomyces species, however, has lagged behind that for other bacterial systems, hindering exploitation of unprecedented quantities of genomic data. Here, we develop a platform for en masse clustered regularly interspaced short palindromic repeats interference sequencing (CRISPRi-seq) for Streptomyces spp. By performing CRISPRi-seq with 2,160 unique sgRNAs targeting all operons (432 operons) encoding membrane transporters (629 genes) representing 1.1Mb of the 6.8Mb genome for S. albidoflavus, combined with hit validation, we discovered that only a small proportion (13 of 432 operons, 25 kb) contribute positively to fitness. Our work provides both a first-in-class platform for high-throughput functional genomics and a generalized blueprint for en masse screens in Streptomyces species.<br><br>IMPORTANCE: Streptomyces bacteria are prolific producers of clinically essential natural products, yet high-throughput tools to systematically interrogate their genomes remain underdeveloped. By establishing a robust CRISPRi-seq platform for en masse functional screening in Streptomyces albidoflavus, our work closes a critical technological gap in Streptomyces functional genomics. Our study not only identifies a small subset of transporter operons essential for fitness but also introduces a scalable, generalizable approach for dissecting gene function. This platform will accelerate systems-level understanding of an industrially and medically important genus.},
}
@article {pmid41467797,
year = {2026},
author = {Ji, HJ and Jang, A-Y and Han, SH and Kim, M-K and Lamien, CE and Wijewardana, V and Ahn, KB and Kim, K-H and Song, JY and Seo, HS},
title = {Accurate serotype identification of Streptococcus pneumoniae using nanopore Cas9-targeted serotype identification (nCATSerotyping).},
journal = {Journal of clinical microbiology},
volume = {64},
number = {2},
pages = {e0098425},
doi = {10.1128/jcm.00984-25},
pmid = {41467797},
issn = {1098-660X},
support = {22202MFDS171//Ministry of Food and Drug Safety/ ; 2018E240600//Korea Disease Control and Prevention Agency/ ; 523140-26//Korea Atomic Energy Research Institute/ ; RS-2022-00164721//National Research Foundation of Korea/ ; CRP D32039//International Atomic Energy Agency/ ; },
mesh = {*Streptococcus pneumoniae/classification/genetics/isolation &amp; purification ; Humans ; *Serotyping/methods ; *Pneumococcal Infections/microbiology ; Serogroup ; *Nanopore Sequencing/methods ; Republic of Korea ; CRISPR-Cas Systems ; Pneumococcal Vaccines ; },
abstract = {Streptococcus pneumoniae (pneumococcus) is a leading cause of community-acquired pneumonia and invasive diseases, particularly among children and the elderly. The introduction of pneumococcal conjugate vaccines has significantly reduced invasive pneumococcal disease, but the prevalence of non-vaccine serotypes and newly emerging serotypes is increasing globally. Thus, accurate serotyping is essential for epidemiological surveillance and the development of next-generation multivalent pneumococcal vaccines. Conventional serotyping methods, including multiplex polymerase chain reaction (mPCR), monoclonal antibody (mAb) assays, and Quellung reaction using rabbit antisera, are limited by serotype coverage and cross-reactivity, making the detection of new or emerging serotypes challenging. In this study, we developed a nanopore Cas9-targeted serotyping (nCATSerotyping) platform, which employs Cas9-mediated enrichment of the capsular polysaccharide synthesis locus followed by Oxford Nanopore sequencing. Applying this method to 276 clinical pneumococcal isolates collected in South Korea (2018-2020), we achieved a serotyping success rate of 97.10% (268/276), significantly outperforming conventional methods such as mAb and mPCR, which identified only 76.45% (211/276) of isolates. Whole-genome sequencing of the remaining eight non-typeable isolates revealed them to be non-pneumococcal (oral streptococci), confirming 100% accuracy for S. pneumoniae serotyping. Importantly, our method identified emerging and underrepresented serotypes, including serotype 13 and null capsule clade strains. nCATSerotyping offers a rapid, accurate, and comprehensive solution for pneumococcal serotyping, with significant advantages in identifying novel and non-typeable strains. This scalable platform will be a valuable tool for global serotype surveillance and next-generation multivalent pneumococcal vaccine development.IMPORTANCEAccurate pneumococcal serotyping is critical for vaccine development and epidemiological surveillance, particularly as non-vaccine serotypes emerge following widespread pneumococcal conjugate vaccine implementation. Current serotyping methods face significant limitations in coverage and accuracy, identifying around 76% of pneumococcal isolates and failing to detect emerging serotypes like serotype 13 and null capsule clades. The nanopore Cas9-targeted serotyping platform addresses these critical gaps by achieving 100% serotyping accuracy for confirmed Streptococcus pneumoniae isolates while identifying previously undetectable strains that conventional methods missed. This comprehensive approach is essential for monitoring vaccine effectiveness, understanding serotype replacement patterns, and informing next-generation vaccine development strategies. Furthermore, the identification of misclassified oral streptococci highlights the diagnostic precision needed for accurate pneumococcal surveillance, ensuring that epidemiological data accurately reflect true pneumococcal disease burden and serotype distribution patterns.},
}
@article {pmid41654255,
year = {2026},
author = {Saini, H and Yadav, J and Pandey, S and Kumar, A and Nanda, D and Sachan, S and Kaushik, JJ},
title = {Field-deployable CRISPR-cas variants for rapid on-site detection of plant pathogens.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {365},
number = {},
pages = {113028},
doi = {10.1016/j.plantsci.2026.113028},
pmid = {41654255},
issn = {1873-2259},
abstract = {Rapid, field-deployable diagnostics are essential for effective plant disease management. Although CRISPR-Cas systems offer high sensitivity and programmability, their use in on-site plant pathogen detection has been hindered by the lack of standardized, practical workflows. Here we present implementable CRISPR-Cas diagnostic protocols using Cas12a, Cas13a, and miniature Cas variants for rapid detection of major plant pathogens. Three field-ready assays are described: (i) an RPA-Cas12a lateral-flow test for DNA pathogens, (ii) a Cas13a RT-RPA assay for RNA viruses, and (iii) an amplification-free Cas12a electrochemical biosensor suited for portable laboratories. Each protocol includes sample preparation steps, reagent formulations, incubation conditions, and troubleshooting guidance. Across platforms, detection limits of 1-100 copies µL[-1] were achieved within 20-45 min, demonstrating analytical sensitivity comparable to conventional PCR-based diagnostics while offering substantially reduced assay time and improved field deployability. We also address practical constraints including sample inhibitors, reagent stability, and biosafety and propose solutions for field implementation. These standardized workflows translate recent advances in CRISPR diagnostics into reproducible, field-deployable tools for plant health surveillance and rapid disease detection.},
}
@article {pmid41653672,
year = {2026},
author = {Bai, T and Qu, X and Pan, J and Tang, Y and Wang, L and Zhou, P and Hu, Z and Guo, Z and Zhu, Y and Zhang, Y},
title = {CRISPR-initiated exponential amplification on fluorescently-barcoded microspheres for deep learning-assisted multiplexed HPV detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {300},
number = {},
pages = {118488},
doi = {10.1016/j.bios.2026.118488},
pmid = {41653672},
issn = {1873-4235},
abstract = {Rapid, low-cost, and multiplexed nucleic acid testing is essential for human health but remains challenging. Although CRISPR-Cas systems offer high specificity, their integration into Multiplexed platforms suitable for near-patient testing has been limited. Here, we introduce a biosensing platform that combines CRISPR-initiated enzymatic amplification with quantum-dot encoded microbeads and deep-learning based image analysis for Multiplexed detection of human papillomavirus (HPV) DNA. The high specificity of CRISPR/Cas9 first triggers an exponential amplification. The products are then specifically captured on the microbead surface for localized fluorescent readout. A custom deep-learning algorithm automatically quantifies the bead fluorescence, enabling robust and automated analysis. The integrated approach achieves simultaneous detection of HPV16, HPV18, and HPV33 with detection limits as low as 0.2 pM. By using recognition-triggered amplification and a simple deep-learning assisted fluorescence readout, the workflow is significantly simplified. The platform establishes a universal and practical strategy for molecular diagnostics, demonstrating strong potential for near-patient testing.},
}
@article {pmid41617656,
year = {2026},
author = {He, M and Chen, J and Chao, M and Yin, F and Pan, H and Wang, Q and Cheng, W and Wang, Z and Xiang, Y},
title = {Novel One-Pot Detection of Tumor mRNA Vaccine by the Target Serving as crRNA of the Split Cas12a System and Synergistically Triggering an Entropy-Driven Reaction.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {4134-4142},
doi = {10.1021/acs.analchem.5c06932},
pmid = {41617656},
issn = {1520-6882},
mesh = {*Entropy ; *Cancer Vaccines/analysis/genetics ; *RNA, Messenger/genetics/analysis ; Humans ; *mRNA Vaccines/analysis ; *Endodeoxyribonucleases/metabolism/genetics ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {mRNA vaccines, as a new form of vaccine, are gradually revealing their potential in the field of tumor treatment. With the development of mRNA vaccines, monitoring their levels has become particularly important. In this work, a novel and innovative strategy was proposed for the first time, which directly used the target as crRNA and simultaneously triggered an entropy-driven split Cas12a system for triple signal amplification for the high-sensitivity detection of mRNA vaccines. This study broke the traditional full-length crRNA and innovatively designed a split crRNA structure, directly using the target mRNA as the variable sequence of the crRNA. This design avoided the need for additional introduction of variable sequences, thereby eliminating nonspecific signal leakage and multicomponent interference from the source. At the same time, the target molecule served as the initiating chain for the EDC reaction, allowing the split Cas12a system to be coupled with the entire entropy-driven reaction process, achieving efficient collaboration and triple signal amplification and significantly improving the detection sensitivity and specificity. Moreover, the entire detection process required only one reaction step, which was simple, fast, and efficient. The aim was to provide strategies for screening and pharmacokinetic studies of mRNA vaccines.},
}
@article {pmid41587498,
year = {2026},
author = {Marpaung, DSS and Yap Sinaga, AO},
title = {Toehold-mediated strand displacement in CRISPR/Cas12a reactions: Advances in programmable and universal biosensing strategies.},
journal = {Talanta},
volume = {302},
number = {},
pages = {129442},
doi = {10.1016/j.talanta.2026.129442},
pmid = {41587498},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; DNA/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a-based biosensors have emerged as powerful tools for nucleic acid detection due to their programmability, high sensitivity, and versatility. However, challenges such as PAM dependence, limited mismatch discrimination, and difficulty in detecting non-nucleic acid analytes constrain their universality. Toehold-mediated strand displacement (TMSD) offers a programmable mechanism to overcome these limitations by dynamically regulating hybridization kinetics and molecular interactions. This review systematically summarizes recent advances integrating TMSD into CRISPR/Cas12a systems, including crRNA release, crRNA-DNA activation, activator generation, and reporter signal modulation. By coupling TMSD's precise strand exchange capabilities with Cas12a's trans-cleavage activity, these hybrid biosensors achieve improved specificity, tunable kinetics, and multi-analyte adaptability. The review further discusses design principles, thermodynamic foundations, and application examples across biomedical, environmental, and food diagnostics. Collectively, TMSD-assisted CRISPR/Cas12a biosensing provides a universal, programmable framework for next-generation molecular diagnostics with enhanced control, sensitivity, and functional diversity.},
}
@article {pmid41586479,
year = {2026},
author = {Huang, C and Yang, Y and Yin, H and Chang, H and Gong, R and Jiang, Z and Zhang, XE and Chen, Y},
title = {Ultrasensitive CRISPR Platform Enables Rapid and Extraction-Free Detection of Viral Nucleic Acid.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {4028-4035},
doi = {10.1021/acs.analchem.5c06518},
pmid = {41586479},
issn = {1520-6882},
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *RNA, Viral/analysis/genetics ; Nucleic Acid Amplification Techniques/methods ; *COVID-19/diagnosis/virology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *COVID-19 Nucleic Acid Testing/methods ; Nasopharynx/virology ; },
abstract = {CRISPR-based molecular diagnostics offers great potential for the rapid identification of pathogens. However, existing one-pot detection systems remain constrained by their restricted versatility and operational complexity. Herein, we report a novel strategy termed interspaced phosphorothioate primer-mediated one-pot detection (iPSOT), which substitutes conventional phosphodiester primers with interspaced phosphorothioate (iPS) primers. The iPS primers demonstrate strong compatibility with AapCas12b during loop-mediated isothermal amplification, enabling robust fluorescence signal generation within 15 min. The iPSOT system achieves highly specific nucleic acid detection and reliably identifies low-copy ribonucleic acid targets at concentrations of as low as 0.5 aM (0.3 copies/μL). Clinical validation further demonstrated that iPSOT enables the direct detection of SARS-CoV-2 from nasopharyngeal swabs without RNA extraction. This method reduces reagent cost and shortens assay time, achieving sample-to-result under 20 min. Overall, iPSOT enhances both sensitivity and specificity in one-pot detection and offers a promising platform for rapid, reliable point-of-care testing and large-scale pathogen surveillance.},
}
@article {pmid41582525,
year = {2026},
author = {Ding, S and Li, Y and Wang, F and Liu, Q and Liu, L and Li, J and Wu, Y and Liu, X and Jiang, D and Xu, X and Gao, X and Huang, C},
title = {DNA Flap-Controlled CRISPR/Cas12a Trans-Cleavage Enables Mix-and-Read FEN1 Activity Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {3998-4007},
doi = {10.1021/acs.analchem.5c06447},
pmid = {41582525},
issn = {1520-6882},
mesh = {*Flap Endonucleases/metabolism/analysis ; Humans ; *CRISPR-Cas Systems ; *DNA/chemistry/metabolism/genetics ; *Biosensing Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a has emerged as a powerful biosensing tool, owing to its exceptional specificity and trans-cleavage-mediated signal amplification capability, for detecting flap endonuclease 1 (FEN1) activity. However, its reliance on additional preligation or replication steps increases operational complexity and raises the risk of false signals. Herein, we report a DNA flap-controlled CRISPR/Cas12a trans-cleavage (FCT-CRISPR) strategy that enables sensitive, accurate, and mix-and-read detection for FEN1 activity. In FCT-CRISPR, a flap-structured dumbbell DNA probe was ingeniously designed, in which the flap domain serves as a split activator, and the dumbbell scaffold acts as a steric hindrance unit. Upon FEN1 recognition and cleavage, the split activator is liberated from its constrained configuration in the dumbbell scaffold, thereby activating CRISPR/Cas12a trans-cleavage activity. FCT-CRISPR strategy avoids the reliance on exogenous DNA ligation or replication processes, allowing mix-and-read detection of FEN1 activity with a detection limit as low as 0.2 mU and excellent specificity against nontarget enzymes. In addition, the successful detection application in lysates of cancer cells demonstrates the potential of FCT-CRISPR for clinical use. This work establishes a sensitive, accurate, and mix-and-read platform for monitoring FEN1 activity and offers a promising tool for the early diagnosis of FEN1-related diseases.},
}
@article {pmid41496452,
year = {2026},
author = {Liu, J and Liu, Z and Qin, D and Qi, X and Chen, M and Cheng, D and Guo, S and Zhang, X and Zhang, L and Yan, T and Li, F and He, W and Lu, Z and Wang, H and Li, J and Yang, X and Shi, Y and Zhang, S and Zhao, H and Liu, C and Chen, S and Zhong, Y},
title = {An all-in-one visual selection system for male-sterile line production in maize and rice.},
journal = {Plant communications},
volume = {7},
number = {2},
pages = {101693},
doi = {10.1016/j.xplc.2026.101693},
pmid = {41496452},
issn = {2590-3462},
mesh = {*Zea mays/genetics/physiology ; *Oryza/genetics/physiology ; *Plant Infertility/genetics ; Seeds/genetics/growth &amp; development ; *Plant Breeding/methods ; CRISPR-Cas Systems ; Hybrid Vigor/genetics ; Plants, Genetically Modified ; },
abstract = {Heterosis has significantly improved crop yields, yet hybrid seed production remains hindered by labor-intensive manual emasculation. Although current male-sterility systems, such as cytoplasmic male sterility and environment-sensitive genic male sterility, have improved the efficiency of hybrid seed production, their limited genetic adaptability and high environmental dependence remain major challenges. Here, we report an all-in-one seed production technology (ASPT) that integrates CRISPR-Cas9, RUBY, and key seed production technology (SPT) components into a single vector, enabling efficient generation and propagation of male-sterile lines in both maize and rice. The engineered RUBY marker enables visual identification of male-sterile and maintainer lines, with an accuracy of 99.81% in automated seed sorting and 100% in secondary field screening. Notably, ASPT was successfully introduced into 21 genetically diverse elite maize inbred lines, demonstrating broad compatibility. ASPT enables scalable and precise propagation of male-sterile lines in both maize and rice, providing a broadly applicable strategy to advance hybrid seed production in crops.},
}
@article {pmid40579542,
year = {2026},
author = {Ullah, HMA and Huang, Q and Chiola, S and Wang, Y and Shcheglovitov, A},
title = {Generating and characterizing human telencephalic brain organoids from stem cell-derived single neural rosettes.},
journal = {Nature protocols},
volume = {21},
number = {2},
pages = {718-748},
pmid = {40579542},
issn = {1750-2799},
support = {R01 MH113670/MH/NIMH NIH HHS/United States ; T32HG008962//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01NS123849//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R01MH113670//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R01 NS123849/NS/NINDS NIH HHS/United States ; R21NS104963//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R21 CA279773/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Organoids/cytology ; *Telencephalon/cytology ; *Neural Stem Cells/cytology ; Nerve Tissue Proteins/genetics ; CRISPR-Cas Systems ; Neurons/cytology ; *Cell Culture Techniques/methods ; },
abstract = {We have developed a method for generating human telencephalic organoids from stem cell-derived isolated single neural rosettes. The use of single neural rosettes for generating organoids offers several important advantages. First, it mimics the development of neural tissue from a singular neural tube in vivo. Second, single neural rosette-derived organoids exhibit a relatively consistent and reproducible composition of telencephalic neural cells. Finally, single neural rosette-derived organoids demonstrate predictable organization of the identified neural cells around a single neural rosette-derived lumen and contain a large proportion of functionally mature neurons that generate action potentials and receive both excitatory and inhibitory synaptic inputs. These unique features of our protocol enable the study of the specification and organization of different neural cells in the developing human telencephalon, as well as modeling of neurodevelopmental disorders associated with disrupted neural networks. Here, we describe our protocols for generating CRISPR-Cas9-engineered human stem cells with a hemizygous SHANK3 deletion, stem cell-derived single neural rosettes and telencephalic brain organoids. We also offer insights on how to conduct single-cell RNA sequencing, immunohistochemistry and slice patch-clamp electrophysiology on these organoids. Completion of the protocols takes 5-6 months and requires experience working with cultured cells. We expect this protocol will prove useful for studies of human brain development and disease, as well as for advancing the development of new organoid-based biocomputers.},
}
@article {pmid41652748,
year = {2026},
author = {Lei, Y and Ma, W and Wang, L and Chen, Y and Chen, X},
title = {Genomic insights into prophage and CRISPR-Cas system present in Lactobacillus delbrueckii subsp. bulgaricus strains.},
journal = {Food research international (Ottawa, Ont.)},
volume = {227},
number = {},
pages = {118213},
doi = {10.1016/j.foodres.2025.118213},
pmid = {41652748},
issn = {1873-7145},
mesh = {*Prophages/genetics ; *Lactobacillus delbrueckii/virology/genetics ; *CRISPR-Cas Systems/genetics ; Genomics ; Yogurt/microbiology ; Base Composition ; Genome, Bacterial ; Genome, Viral ; },
abstract = {As a key starter culture for yogurt fermentation, Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) was subjected to bioinformatics analysis to investigate the distribution of prophages and the structure of the CRISPR-Cas system in 119 L. bulgaricus strains, and explored the targeting relationship between them and annotated the functional genes of targeted prophages. A total of 1704 prophage fragments were identified, of which 8.74 % (149/1704) were classified as complete prophages, none of them carried virulence factor genes and antibiotic resistance genes. Among all complete prophage genomes, the GC content was significantly affected by geographical sources (Asia, Europe, and North America) of host bacteria (P < 0.01). The 123 CRISPR-Cas systems detected in L. bulgaricus, type I-E (69) and type II-A (40) systems were predominant. Spacers-prophages targeting analysis revealed a negative correlation (P < 0.05), with 23.43 % (1055/4503) spacers showing homology to prophages. Functional gene annotation revealed that the diversity of prophage functional genes showed significant differences in under different grouping conditions (P < 0.05). Nevertheless, in-depth analysis of targeted prophage genomes revealed a conserved functional gene--ABC transporter. This study provides data support for exploring the genomic diversity and adaptive evolution mechanism of prophages in L. bulgaricus, and providing insights for screening phage-resistant strains and developing novel anti-phage strategies in dairy industry.},
}
@article {pmid41652223,
year = {2026},
author = {Zhang, J and Zhang, J and Tang, S and Liu, C and Cai, Y and Zeng, H and Meng, X and Liu, B and Zhang, Y and Wang, Y},
title = {Single-round evolution of RNA aptamers with GRAPE-LM.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {41652223},
issn = {1546-1696},
support = {62302311//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82273967//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023M742397//China Postdoctoral Science Foundation/ ; 2024T170585//China Postdoctoral Science Foundation/ ; },
abstract = {The directed evolution of biomolecules is an iterative process. Although advancements in language models have expedited protein evolution, effectively evolving RNA remains a challenge. RNA aptamers, selected for their binding properties, provide an ideal system to address this challenge, yet traditional aptamer discovery still relies on labor-intensive, multi-round screening. Here we introduce GRAPE-LM (generator of RNA aptamers powered by activity-guided evolution and language model), a generative artificial intelligence framework designed for the one-round evolution of RNA aptamers. GRAPE-LM integrates a transformer-based conditional autoencoder with nucleic acid language models and is guided by CRISPR-Cas-based aptamer screening data derived from intracellular environments. We validate GRAPE-LM on three disparate targets: the human T cell receptor CD3ε, the receptor-binding domain of the SARS-CoV-2 spike protein and the human oncogenic transcription factor c-Myc (an intracellular disordered protein). GRAPE-LM, informed with only a single round of CRISPR-Cas-based screening, successfully obtains RNA aptamers that outperform those driven from multiple rounds of human selection and optimization.},
}
@article {pmid41652115,
year = {2026},
author = {Omelchenko, DO and Barkovskaya, AM and Omelchenko, LV and Klepikova, AV and Penin, AA and Logacheva, MD},
title = {Optimization of functional genetics tools for a model tetraploid Capsella bursa-pastoris, with focus on homoeolog-aware gene editing.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {6},
pmid = {41652115},
issn = {1573-9368},
mesh = {*Gene Editing/methods ; *Capsella/genetics/growth &amp; development ; Tetraploidy ; *Plants, Genetically Modified/genetics/growth &amp; development ; Genome, Plant/genetics ; Agrobacterium tumefaciens/genetics ; CRISPR-Cas Systems ; },
abstract = {Capsella bursa-pastoris is a recent allotetraploid and a promising model for studying early consequences of polyploidy. One of the intriguing questions in polyploid research is how new functions arise from initially identical or nearly identical homoeologous genes. Functional genetics tools, including genetic editing, can help to understand this process, but they have not been developed for C. bursa-pastoris yet. We present here the results of our study aimed at filling this gap. In particular, we compared the efficiency of floral dip transformation in six accessions of C. bursa-pastoris representing distant populations. The Asian clade accession PGL0025 had the highest efficiency of transformation (~ 1.1%). Comparison of Agrobacterium tumefaciens strains EHA105 and GV3101 (pMP90) showed that the latter is more effective. Also, we created a genome-wide gRNA database for all pairs of homoeologs of the PGL0001 accession of C. bursa-pastoris and integrated it into publicly available genome browser: https://t2e.online/igv_capsella_bursa-pastoris/ . We assessed the possibility of differential editing for two pairs of homoeologous genes with high sequence similarity (> 90%) both in vitro and in silico. Despite the test results that indicated off-target activity, we have succeeded in obtaining lines of plants with homozygous frameshift mutations in each of the homoeologs separately in vivo. We expect that these findings and resources will promote the use of C. bursa-pastoris as a model in functional genetics experiments, in particular, the studies of the fate of duplicated gene after polyploidization event.},
}
@article {pmid41652090,
year = {2026},
author = {Pan, X and Yildiz, U and Armstrong, SK and Bissonnette, K},
title = {Status and advancement of root-knot nematode management strategies and the emerging CRISPR/Cas biotechnology application.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {38},
pmid = {41652090},
issn = {1438-7948},
mesh = {*CRISPR-Cas Systems ; Animals ; *Plant Diseases/parasitology/genetics/prevention &amp; control ; Gene Editing ; *Tylenchoidea/pathogenicity/genetics ; Crops, Agricultural/genetics/parasitology ; Disease Resistance/genetics ; Plant Roots/parasitology/genetics ; Gossypium/parasitology/genetics ; },
abstract = {Root-knot nematodes (RKNs), Meloidogyne spp., exhibit a broad host range, threatening more than 3000 species of plants, including agriculturally important crops such as cotton (Gossypium hirsutum), tomato (Lycopersicon esculentum) and rice (Oryza sativa). Among the over 90 RKN species, the four most prevalent are M. incognita, M. arenaria, M. javanica, and M. hapla, with M. incognita being the most damaging. This paper reviewed the current RKN management strategies, including chemical nematicides, biological control, crop rotation, and resistant varieties, with a focus on the application of the revolutionary CRISPR/Cas genome editing tool in developing RKN resistance in plants. CRISPR/Cas has been widely utilized for improving crop traits due to its specificity, streamline, and inheritability. Recent progress has demonstrated the simplicity and robustness of CRISPR/Cas technology in improving plant traits. Among these, the development of nematode resistance by CRISPR/Cas knocking out of plant compatibility factors in model and commercial plants, has achieved significant progress. This review summarizes the RKN parasitism mechanisms and plant compatibility factors that would be promising CRISPR/Cas targets. The fundamentals and key aspects of CRISPR/Cas genome editing technology are addressed and discussed, and an example experimental pipeline for developing nematode resistance in cotton is described.},
}
@article {pmid41651998,
year = {2026},
author = {Natsagdorj, ME and Hara, H and Uosaki, H and Nakahara, F and Inoue, M and Hanazono, Y},
title = {ATM Inhibition Enhances Knock-in Efficiency by Suppressing AAV-Induced Activation of Apoptotic Pathways.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {177},
pmid = {41651998},
issn = {2399-3642},
support = {JP23bm1123020//Japan Agency for Medical Research and Development (AMED)/ ; JP22bm0804018//Japan Agency for Medical Research and Development (AMED)/ ; JP23bm1123020//Japan Agency for Medical Research and Development (AMED)/ ; JP18am0301002//Japan Agency for Medical Research and Development (AMED)/ ; JP22ae0201007//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {*Ataxia Telangiectasia Mutated Proteins/antagonists &amp; inhibitors/genetics/metabolism ; *Dependovirus/genetics ; Animals ; Mice ; *Apoptosis/genetics ; *Gene Knock-In Techniques/methods ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; Morpholines/pharmacology ; Gene Editing/methods ; Mouse Embryonic Stem Cells/metabolism ; Thioxanthenes ; },
abstract = {CRISPR/Cas9-mediated genome editing has expanded the possibilities for precise gene modifications; however, the efficiency of targeted insertion remains suboptimal. In this study, we describe a triple-reporter system in mouse embryonic stem cells that simultaneously tracks double-strand break (DSB) induction, homology-directed repair (knock-in), and end-joining-mediated targeted insertion (EJ-TI). Using both plasmid and adeno-associated virus (AAV) donor vectors, our results demonstrate that ataxia telangiectasia and Rad3-related kinase (ATR) activity is essential for knock-in regardless of the donor type, whereas ataxia telangiectasia mutated (ATM) inhibition exhibits a donor-dependent role. In cells receiving circular plasmid donors, ATM inhibition with AZD1390 markedly reduced the knock-in and EJ-TI efficiencies, consistent with its canonical role in DSB repair. In contrast, with linear AAV donors, ATM inhibition enhanced the knock-in efficiency by suppressing the overactivation of the ATM-p53-caspase 3 apoptotic pathway and partially suppressing classical non-homologous end-joining. These findings highlight the critical influence of donor DNA configuration on DNA damage response signaling and provide a strategy for optimizing genome editing efficiency by selectively modulating the ATM pathways, an approach that may have significant implications for gene therapy, cell engineering, and other applications.},
}
@article {pmid41651087,
year = {2026},
author = {Cao, W and Lu, J and Jiang, Y and Yuan, M and Wang, M and Mu, X and Gao, Z and Yang, S and Li, T and Wei, X and Guo, L},
title = {Recent advances in metabolic engineering of purple non‑sulfur photosynthetic bacteria for enhanced biohydrogen production.},
journal = {Biotechnology advances},
volume = {88},
number = {},
pages = {108832},
doi = {10.1016/j.biotechadv.2026.108832},
pmid = {41651087},
issn = {1873-1899},
abstract = {Photo-fermentative hydrogen production from lignocellulosic biomass offers a sustainable and carbon-neutral route for bioenergy conversion, providing an effective strategy to mitigate fossil resource depletion and greenhouse gas emissions. Among diverse microbial candidates, purple non‑sulfur bacteria (PNSB) have emerged as promising photosynthetic platforms owing to their broad substrate utilization, intrinsic tolerance to lignocellulose-derived inhibitors, and high hydrogen yields under light-anaerobic conditions. Their metabolic versatility allows dynamic redistribution of carbon and electron fluxes, facilitating efficient energy conversion. Recent progress in metabolic engineering has substantially advanced the hydrogen-producing capacity of PNSB through targeted manipulation of photosynthetic metabolism, redox balance and stress response pathways. Engineering strategies have focused on optimizing pigment biosynthesis to enhance light harvesting, reinforcing redox homeostasis and adenosine triphosphate (ATP) generation, and improving tolerance to environmental stresses such as ammonia, pH, and temperature fluctuations. These efforts have led to engineered strains exhibiting extraordinary improvement in hydrogen yield, stability and robustness. This review provides an overview of the fundamental mechanisms underlying photo-fermentative hydrogen metabolism in PNSB, summarizes recent advances in the metabolic and systems-level engineering strategies, and outlines the prospects of developing strains capable of approaching the theoretical limit of hydrogen yield through integrated engineering strategies, advanced tools such as CRISPR-Cas, and adaptive laboratory evolution methods.},
}
@article {pmid41648912,
year = {2026},
author = {Wang, R and Li, J and Zheng, Y and Wei, N and Li, W},
title = {CRISPR/Cas12a platform activated by a protospacer adjacent motif-engineered DNA circuit for specific target sensing.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay02079d},
pmid = {41648912},
issn = {1759-9679},
abstract = {CRISPR/Cas systems have found extensive applications in nucleic acid diagnostics. However, the generalizability of this approach, particularly for the sensing of non-nucleic acid targets, remains a challenge. This study presents the development of a CRISPR/Cas12a platform activated by a protospacer adjacent motif (PAM)-engineered DNA circuit. Initially, the influence of the presence or absence of the PAM on the DNA circuit and the subsequent CRISPR/Cas12a system was investigated, demonstrating that a PAM-engineered DNA circuit functions as an effective activator of Cas12a, whereas a DNA circuit lacking the PAM does not induce activation. Subsequently, through the strategic design of recognition elements, sensitive and selective detection of specific targets is achieved, with limits of detection (LODs) of 0.023 fM for circulating tumor DNA (ctDNA), 0.00004 U mL[-1] for uracil-DNA glycosylase (UDG), and 0.12 fM for acetamiprid (ACE). This approach exemplifies a two-stage signal amplification mechanism, achieving improved sensitivity relative to either the CRISPR/Cas12a system or the DNA circuits alone. Moreover, quantitative assays for these targets were successfully conducted in real samples, suggesting the practical applicability of the proposed method. This research establishes a versatile sensing platform for various targets, which holds significant promise for advancements in molecular diagnostics, food safety assessment, and environmental monitoring.},
}
@article {pmid41648761,
year = {2025},
author = {Lu, Z and Bukhari, STH and Azeem, M and Tariq, N and Shabbir, MAB},
title = {A narrative review of precision and ethical considerations in cardiovascular health: CRISPR-Cas9, telemedicine, and lifestyle interventions.},
journal = {Frontiers in public health},
volume = {13},
number = {},
pages = {1737251},
pmid = {41648761},
issn = {2296-2565},
mesh = {Humans ; *Telemedicine/ethics ; *Cardiovascular Diseases/prevention &amp; control/therapy ; *CRISPR-Cas Systems ; *Life Style ; *Precision Medicine ; Gene Editing ; },
abstract = {BACKGROUND: Cardiovascular diseases (CVDs) remain the leading cause of global morbidity and mortality, influenced by lifestyle, socioeconomic status, and genetic factors. Emerging innovations, including wearable health technologies, telemedicine, and CRISPR-Cas9 gene editing, provide new possibilities for rapid prevention and personalized management.<br><br>METHODS: This narrative review collected evidence from Scopus, PubMed, and Google Scholar, using keywords such as cardiovascular (CV) prevention, lifestyle determinants, digital health, telemedicine, CRISPR-Cas9, and public health ethics. Eligible peer-reviewed studies, clinical guidelines, and policy documents were included to assess behavioral, technological, and genomic strategies for CVD care.<br><br>RESULTS: Modifications in lifestyle, such as quitting smoking, regular physical activity, following a heart-healthy diet plan, and getting adequate sleep, can significantly reduce the risk of CVD. Additionally, telemedicine and wearable devices facilitate early detection, better self-management, and treatment adherence, especially in underserved communities. CRISPR-Cas9 holds a significant potential for correcting genetic variants related to lipid disorders and inherited cardiomyopathies, but its clinical translation remains in early stages. However, existing evidence is limited by heterogeneity in study design, brief follow-up, particularly for digital health and CRISPR applications. Additional challenges, such as health inequities, digital access, data privacy, and ethical oversight, further influence their real-world implementation.<br><br>CONCLUSION: Effective integration of behavioral, digital and genomic innovations requires policy frameworks that ensure equity, ethical governance, and long-term sustainability. Combining precision medicine with efforts to address social determinants of health will be crucial in reducing the global burden of CVD and shaping the future of CV care.},
}
@article {pmid41648339,
year = {2026},
author = {Phan, PT and Ozturk, M and Dougherty, EM and Ravishankar, J and Xue, C and Sashital, DG},
title = {Mismatch type impacts interference and priming activities in the type I-E CRISPR-Cas system.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.01.24.701482},
pmid = {41648339},
issn = {2692-8205},
abstract = {Type I-E CRISPR-Cas systems direct RNA-guided interference against foreign nucleic acids using the CRISPR RNA (crRNA)-guided Cascade complex and Cas3 helicase-nuclease. DNA targeting by Cascade-Cas3 promotes priming, a mechanism that allows for rapid acquisition of new spacers within the CRISPR array. Target mutations in the PAM and PAM-proximal seed region can block interference but may still allow priming. Previous studies have suggested that target mutations to T and A are tolerated, but that C and G substitutions are deleterious to interference and priming, respectively. However, the contributions of the crRNA spacer sequence to mutational tolerance remain unclear. Here, we systematically tested the effects of crRNA seed sequences on mutational tolerance. We engineered four E. coli strains with variable spacer sequences and tested CRISPR interference and priming against a plasmid library for each strain. Consistent with prior studies, we observe that mutations to C or G in the seed can be highly deleterious, especially at positions 1, 2 and 4. However, the corresponding crRNA sequence also strongly impacts the level of defect, with rC-dC and rA/G-dG causing the largest defects in our plasmid library experiments. Using in vitro biochemistry, we observe that mismatch type at the first position of the seed affects Cas8 conformation, and results in reduction in the rates of both Cascade-target binding and Cas3 recruitment. Overall, our results reveal that although nucleotide identity of target mutations is an important determinant of type I-E CRISPR immunity, the crRNA sequence also strongly impacts immune outcomes upon target mutation.},
}
@article {pmid41637875,
year = {2026},
author = {Wu, H and Merényi, Z and Virágh, M and Liu, XB and Hegedüs, B and Hou, Z and Ábrahám, E and Fürtön, A and Kristóffy, Z and Lipinszki, Z and Nagy, LG},
title = {Functional characterization of a Con7-related transcription factor in Coprinopsis cinerea indicates evolutionary conservation of morphogenetic roles.},
journal = {Microbiological research},
volume = {306},
number = {},
pages = {128462},
doi = {10.1016/j.micres.2026.128462},
pmid = {41637875},
issn = {1618-0623},
mesh = {*Transcription Factors/genetics/metabolism ; Fruiting Bodies, Fungal/growth &amp; development/genetics ; *Fungal Proteins/genetics/metabolism ; Gene Expression Regulation, Fungal ; *Agaricales/genetics/growth &amp; development ; Morphogenesis/genetics ; CRISPR-Cas Systems ; Phylogeny ; Gene Expression Profiling ; Gene Deletion ; Evolution, Molecular ; Zinc Fingers ; },
abstract = {Fruiting bodies of mushroom-forming fungi (Agaricomycetes) exhibit the highest degree of multicellular complexity in fungi, yet the molecular underpinnings of their developmental programs remain incompletely understood. Here, we characterize gcd1, a gene encoding a transcription factor in the Con7 subfamily of C2H2-type zinc finger proteins. This subfamily has previously been implicated in pathogenic morphogenesis in Ascomycota, but its role in Agaricomycetes has not previously been addressed. In Coprinopsis cinerea, CRISPR/Cas9-mediated deletion of gcd1 resulted in strains with severely impaired fruiting body morphogenesis, with malformed cap, stipe, and gill tissues. Gcd1 deletion strains lacked universal veil, resembling species with open (gymnocarpous) development. We find that GCD1/Con7 homologs are widely distributed in most Dikarya species and are mostly encoded by a single gene in each species' genome. Transcriptome analyses identified several misregulated genes in the Δgcd1 mutant, which pinpoint potential mechanisms underlying its developmental defects as well as provided insights into the morphogenesis of mushroom fruiting bodies. These findings establish GCD1 as a key regulator of multicellular development in C. cinerea and broaden the known functions of Con7-like transcription factors to include fruiting body morphogenesis in Agaricomycetes. Overall, our results and the morphogenetic role of Con7-like transcription factors of Ascomycota suggest functional conservation over half a billion years of evolution.},
}
@article {pmid41553775,
year = {2026},
author = {Zheng, I and Learn, B and Bailey, S},
title = {Structural basis for inhibition of SpyCas9 by the anti-CRISPR protein AcrIIA26.},
journal = {The Biochemical journal},
volume = {483},
number = {3},
pages = {289-300},
doi = {10.1042/BCJ20250364},
pmid = {41553775},
issn = {1470-8728},
support = {GM097330//HHS | National Institutes of Health (NIH)/ ; },
mesh = {*Streptococcus pyogenes/genetics/enzymology/metabolism ; *CRISPR-Associated Protein 9/chemistry/metabolism/antagonists &amp; inhibitors/genetics ; *CRISPR-Cas Systems ; *Viral Proteins/chemistry/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; Cryoelectron Microscopy ; *Bacteriophages/metabolism/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Models, Molecular ; },
abstract = {CRISPR-Cas9 systems provide adaptive immunity in prokaryotes by targeting and cleaving invading phage DNA. In response, phages have evolved anti-CRISPR (Acr) proteins to inhibit Cas9 and evade this immune response. AcrIIA26 is a type II-A anti-CRISPR protein that inhibits Streptococcus pyogenes Cas9 (SpyCas9) DNA binding, but its molecular mechanism remains unclear. Here, we determined the 3.0 Å resolution cryo-EM structure of AcrIIA26 in complex with SpyCas9-single-guide RNA, revealing a dual inhibition mechanism. AcrIIA26 adopts a novel fold comprising a central β-sheet flanked by two α-helical bundles. The 5-helix bundle, which features a negatively charged surface whose shape mimics duplex DNA, occupies the same position as the protospacer adjacent motif (PAM) duplex in target-bound Cas9. This directly blocks PAM recognition by burying critical residues R1333 and R1335 in the PAM-interacting domain. Mutagenesis confirmed that residues E49 and D50 in AcrIIA26 are essential for this interaction. Simultaneously, the 4-helix bundle binds the Cas9 REC lobe and sterically prevents the conformational changes required for Cas9 activation, with mutation of AcrIIA26 F121 completely eliminating inhibitory activity. Structural comparisons reveal that despite diverse folds, multiple anti-CRISPRs convergently evolved to block PAM recognition, highlighting this as a critical vulnerability in Cas9 function. Our findings provide mechanistic insights into AcrIIA26 inhibition and offer a foundation for engineering improved Cas9 off-switches for genome editing applications.},
}
@article {pmid41507205,
year = {2026},
author = {Wan, H and Kong, D and Yan, T and Zhou, Y and Liu, M and Ma, X and Zhao, T and Zhou, W and Liu, X and Yin, J and Guan, N and Ye, H},
title = {A compact and inducible dCas12f-based CRISPRa platform for programmable in vivo gene activation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1447},
pmid = {41507205},
issn = {2041-1723},
support = {25J22800100//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; 32250010//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; Dependovirus/genetics ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; *Transcriptional Activation/genetics ; HEK293 Cells ; *Gene Editing/methods ; Genetic Therapy/methods ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Precise activation of endogenous genes is a powerful strategy for functional genomics and therapeutic development, but current CRISPR-based transcriptional activation (CRISPRa) systems are limited by the large size of Cas proteins for adeno-associated virus (AAV) delivery. Here, we present a high-efficiency dCas12f-based transcriptional activation system (HEAL), which recruits transactivators through MS2 coat protein binding to MS2 aptamers embedded within the sgRNA scaffold. Engineered to enhance DNA binding, nuclear localization, and transactivator recruitment, HEAL induces over 100,000-fold activation of endogenous genes and outperforms existing CRISPRa systems in vitro and in vivo. We further develop red-light-inducible OptoHEAL and small-molecule-inducible ChemHEAL for remote and precise transcriptional control. AAV-delivered HEAL targeting interleukin 10 alleviates acute kidney injury in mice, while ChemHEAL-mediated activation of thymic stromal lymphopoietin reduces body weight in obese mice. HEAL provides a modular, compact, and controllable platform for endogenous gene activation with strong potential for fundamental research and gene therapy.},
}
@article {pmid41370233,
year = {2026},
author = {Nomura, Y and Nomura, J and Tamada, K and Eguchi, N and Torigata, K and Tokumoto, S and Nemoto, A and Shirafuji, T and Yamamoto, K and Hishimoto, A and Nagase, H and Nishikawa, T and Takumi, T},
title = {Isogenic modeling of 1q21.1 reciprocal CNVs in human ES cells reveals divergent neurodevelopmental trajectories.},
journal = {Human molecular genetics},
volume = {35},
number = {2},
pages = {},
doi = {10.1093/hmg/ddaf184},
pmid = {41370233},
issn = {1460-2083},
support = {21&#xa0;K07820//KAKENHI/ ; 24&#xa0;K10078//KAKENHI/ ; 22&#xa0;K15750//KAKENHI/ ; 16H06316//KAKENHI/ ; 16H06463//KAKENHI/ ; 23KK0132//KAKENHI/ ; 24H00620//KAKENHI/ ; 24H01241//KAKENHI/ ; 23H04233//KAKENHI/ ; 24&#xa0;K22036//KAKENHI/ ; //Japan Society for the Promotion of Science and Ministry of Education, Culture, Sports, Science, and Technology/ ; JP21wm0425011//Japan Agency for Medical Research and Development/ ; JPMJPF2018//Japan Science and Technology Agency/ ; JPMJMS2299//Japan Science and Technology Agency/ ; JPMJMS229B//Japan Science and Technology Agency/ ; //Takeda Science Foundation/ ; //Smoking Research Foundation/ ; //Taiju Life Social Welfare Foundation/ ; },
mesh = {Humans ; *DNA Copy Number Variations/genetics ; *Chromosomes, Human, Pair 1/genetics ; Neurogenesis/genetics ; *Neurodevelopmental Disorders/genetics/pathology ; *Human Embryonic Stem Cells/metabolism ; Chromosome Deletion ; Neural Stem Cells/metabolism ; Cell Differentiation/genetics ; Neurons/metabolism ; Chromosome Duplication ; CRISPR-Cas Systems ; Intellectual Disability/genetics ; Cell Line ; Schizophrenia/genetics ; },
abstract = {Copy number variations (CNVs) in the distal 1q21.1 region, both deletion (1q del) and duplication (1q dup) are associated with various neurodevelopmental and neuropsychiatric disorders such as autism spectrum disorder, intellectual disability, epilepsy, and schizophrenia. Besides common phenotypes, 1q del and 1q dup manifest opposite clinical phenotypes, e.g. microcephaly in 1q del and macrocephaly in 1q dup. However, molecular and cellular mechanisms underlying these phenotypes are still elusive. Here, to identify molecular mechanisms associated with neurodevelopmental phenotypes from the viewpoint of neurogenesis and neurodevelopment, we generate isogenic human ES cell (hESC) lines with reciprocal 1q21.1 CNVs using CRISPR/Cas9 system and differentiate them into 2-dimensional (2-D) neurons and neural progenitor cell (NPC) spheroids. Our study recapitulates reciprocal brain size in the NPC spheroids and shows dosage-dependent differentiation changes i.e. more GABAergic components in 1q del and more proliferative state in 1q dup. These results demonstrate that 1q21.1 CNVs dramatically affect cell fate in the early neurodevelopmental periods. This is the first isogenic cell model of human 1q21.1 CNVs, and our findings provide new insights into the underlying mechanisms of neurodevelopmental disorders.},
}
@article {pmid41276946,
year = {2026},
author = {Pal, T and Liu, Z and Nair, MG and Chen, J},
title = {CRISPR in MOF Formulation with Enhanced Stability, Activity, and Altered PAM Specificity for Broad-Spectrum Diagnosis of Bacterial Sepsis.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {8},
pages = {e13439},
doi = {10.1002/advs.202513439},
pmid = {41276946},
issn = {2198-3844},
support = {R35GM147069/GM/NIGMS NIH HHS/United States ; R35GM147069/GM/NIGMS NIH HHS/United States ; },
mesh = {*Sepsis/diagnosis/microbiology/genetics ; Humans ; *Metal-Organic Frameworks/chemistry ; *CRISPR-Cas Systems/genetics ; RNA, Ribosomal, 16S/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Sepsis is a life-threatening condition caused by polymicrobial infections and remains a global health emergency that requires rapid and broad-spectrum diagnostics. Existing CRISPR-based assays face two major limitations that restrict their application for sepsis: narrow protospacer adjacent motif (PAM) site compatibility and poor enzyme stability under clinical and environmental stresses. A modular diagnostic platform is presented, CRISPR-FLEXMO (CRISPR with flexible PAM in metal-organic framework encapsulation, MOF), which integrates a PAM-relaxed Cas12a variant (K607R) with a manganese-coordinated MOF (Mn-MOF) for stable and specific detection of sepsis-causing bacteria. The system targets a conserved region upstream of the Shine-Dalgarno sequence in the 16S rRNA gene containing a universal TTCC PAM, enabling broad-spectrum detection with a single universal primer pair across Gram-negative and Gram-positive pathogens. The K607R variant shows enhanced cis- and trans-cleavage activity, while Mn-MOF encapsulation maintains enzyme functionality under ambient, thermal, and chaotropic stress. The assay detects as low as 10 CFU mL[-1] in bacterial lysates following amplification and achieves 100% sensitivity and specificity in serum samples from 15 sepsis patients and 3 healthy individuals, with no cross-reactivity to six respiratory viruses. The platform retains over 78% activity after 12 weeks of room-temperature storage, offering a field-deployable CRISPR diagnostic solution for next-generation infectious disease detection.},
}
@article {pmid41276924,
year = {2026},
author = {Yun, S and Kim, S and Kim, S and Noh, M and Kim, DK and Lee, EJ and Lee, H},
title = {Parallel Genome-Wide CRISPR Screens Reveal SORL1 and ZFYVE19 as Sequential Host Determinants of Salmonella Infection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {8},
pages = {e15042},
doi = {10.1002/advs.202515042},
pmid = {41276924},
issn = {2198-3844},
support = {NRF-2022R1A2B5B02002256//National Research Foundation of Korea/ ; NRF-2022R1A4A1025913//National Research Foundation of Korea/ ; NRF-RS-2025-00561488//National Research Foundation of Korea/ ; NRF-RS-2024-00350131//National Research Foundation of Korea/ ; NRF-RS-2025-02219421//National Research Foundation of Korea/ ; RS-2025-16652968//MSIT/ ; },
mesh = {Humans ; *Salmonella Infections/genetics/microbiology/metabolism ; *Host-Pathogen Interactions/genetics ; *Membrane Transport Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Salmonella enterica/pathogenicity/genetics ; CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {Salmonella enterica, a major cause of gastroenteritis and typhoid fever, hijacks host machinery to invade cells, and replicate within a specialized niche. While some host factors are known, a comprehensive, temporally-resolved understanding of the host-pathogen interface has been hindered by a lack of suitable genome-wide methodologies. To address this, a parallel CRISPR screening platform is developed to identify host determinants for distinct infection stages. An invasion screen captured factors for bacterial entry, while a fitness screen identified factors governing long-term survival. The screens reveal a temporal switch in host dependency, from endosomal trafficking in early infection to cell cycle and DNA damage response pathways governing host cell fitness in long-term infection. Notably, the approach uncovers two novel host factors with stage-specific roles, SORL1 as a mediator of bacterial invasion and ZFYVE19 as a factor supporting intracellular proliferation. Genetic disruption of SORL1 or ZFYVE19 validate these roles, leading to impaired invasion or replication, respectively. Importantly, antibody-mediated blockade of SORL1 effectively prevented Salmonella entry, highlighting it as a novel host-directed therapeutic target. Together, the screening strategy provides a powerful framework for the temporal dissection of host-pathogen interactions, revealing novel biology and promising therapeutic targets.},
}
@article {pmid41276909,
year = {2026},
author = {Fokin, AI and Lin, Y and Guschin, DY and Chen, HY and James, J and Yan, J and Silberzan, P and Gautreau, AM},
title = {Identification of PKN2 and MOB4 as Coordinators of Collective Cell Migration.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {8},
pages = {e02907},
doi = {10.1002/advs.202502907},
pmid = {41276909},
issn = {2198-3844},
support = {ANR-20-CE13-0016//Agence Nationale de la Recherche/ ; ANR-22-CE13-0041//Agence Nationale de la Recherche/ ; ANR-24-CE44-4957//Agence Nationale de la Recherche/ ; 2021 060003815//Fondation ARC pour la Recherche sur le Cancer/ ; INCA_16712//Institut National du Cancer/ ; ANR-11-LABX-0038//Labex Cell(n)Scales/ ; ANR-10-IDEX-0001-02//Labex Cell(n)Scales/ ; //"Institut Pierre-Gilles de Gennes" laboratoire d'excellence/ ; ANR-10-IDEX-0001-02 PSL//"Investissements d'avenir" program/ ; ANR-10-LABX-31//"Investissements d'avenir" program/ ; //French National Cancer Institute/ ; NRF-MSG-2023-0001//National Research Foundation Singapore/ ; },
mesh = {*Cell Movement/physiology/genetics ; Humans ; *Adaptor Proteins, Signal Transducing/metabolism/genetics ; Adherens Junctions/metabolism ; Wound Healing ; CRISPR-Cas Systems ; Protein Kinase C ; },
abstract = {In animals, collective cell migration is critical during development and adult life for repairing organs. It remains, however, poorly understood compared with single-cell migration. The polymerization of branched actin by the RAC1-WAVE-Arp2/3 pathway is established to power membrane protrusions at the front of migrating cells, but also to maintain cell junctions in epithelial monolayers. Here, novel regulators of collective cell migration are identified using a two-pronged approach: candidates are extracted from publicly available RAC1-WAVE-Arp2/3 dependency maps and screened in a second step using CRISPR/Cas9 genetic inactivation. In a wound healing assay, PKN2 knockout (KO) MCF10A cells display decreased collective migration due to destabilization of adherens junctions, whereas MOB4 KO cells display increased collective migration with a loss of migration orientation. Upon wound healing, PKN2 relocalizes to lateral junctions and maintains coordinated migration in the monolayer, whereas MOB4 relocalizes to the front edge of leader and follower cells collectively migrating toward the wound. The role of MOB4 in controlling collective migration requires YAP1, since MOB4 KO cells fail to activate YAP1, and their phenotype is rescued by constitutively active YAP1. Together, these findings reveal two complementary activities required for coordinating cells in collective migration.},
}
@article {pmid41230595,
year = {2026},
author = {Kim, K and Kachiprath, B and Yeo, IC and Shim, KY and Kwak, IS and Jeong, CB},
title = {Environmental DNA-based RPA-CRISPR/Cas12a assay for on-site detection of chironomid larvae in aquatic environments.},
journal = {Pest management science},
volume = {82},
number = {3},
pages = {2458-2467},
doi = {10.1002/ps.70382},
pmid = {41230595},
issn = {1526-4998},
support = {//Incheon National University Research Grant in 2021 (No. 2021-0447)/ ; },
mesh = {*Chironomidae/growth &amp; development/genetics ; Animals ; *DNA, Environmental/analysis ; Larva/growth &amp; development/genetics ; *CRISPR-Cas Systems ; *Environmental Monitoring/methods ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {BACKGROUND: Freshwater chironomid midges pose a significant threat to drinking water quality and freshwater ecosystems in South Korea by disrupting ecological balance and causing biofouling in drinking water treatment systems. To enable rapid detection and provide early warnings of chironomid presence, we developed a highly sensitive and accurate diagnostic assay based on environmental DNA (eDNA) analysis. This method integrates recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated cleavage, and visualization through either fluorescence-based detection or a lateral flow assay (LFA).<br><br>RESULTS: The RPA-CRISPR/Cas12a assays demonstrated remarkable specificity for chironomid eDNA, as confirmed through field surveys and microscopic observations. Although both fluorescence-based and LFA-based RPA-CRISPR/Cas12a assays exhibited comparable detection limits of 10 copies/&#x3bc;L, the LFA method proved more convenient for on-site applications due to its ease of deployment in the field.<br><br>CONCLUSION: Our eDNA-based RPA-CRISPR/Cas12a assay addresses the limitations of traditional biomonitoring by requiring only a small sampling volume, thereby minimizing habitat disturbance, while offering much higher sensitivity. This assay represents a valuable tool for on-site detection of invasive species and for monitoring environmental and public health threats (e.g., chironomids). &#xa9; 2025 The Author(s). Pest Management Science published by John Wiley &amp; Sons Ltd on behalf of Society of Chemical Industry.},
}
@article {pmid40675140,
year = {2026},
author = {Karimi, MA and Paryan, M and Behrouzian Fard, G and Sadeghian, H and Zarrinfar, H and Hosseini Bafghi, M},
title = {Challenges and Opportunities in the Application of CRISPR-Cas9: A Review on Genomic Editing and Therapeutic Potentials.},
journal = {Medical principles and practice : international journal of the Kuwait University, Health Science Centre},
volume = {35},
number = {1},
pages = {1-17},
pmid = {40675140},
issn = {1423-0151},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Genetic Therapy/methods ; },
abstract = {<p>Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) constitute a revolutionary gene-editing technology, allowing precise DNA modifications with vast potential for disease treatment and the creation of genetically modified organisms. This system consists of various components designed to target specific genes, requiring efficient nuclear access within target cells through diverse delivery methods, including physical techniques and carrier-based approaches. Despite its transformative promise, CRISPR faces several challenges, including efficient cellular delivery, off-target effects, immune responses, optimizing editing efficiency, and ethical concerns. Overcoming these hurdles is crucial for fully harnessing its applications. However, CRISPR-Cas9 offers remarkable opportunities for pioneering gene therapies across various disorders, including cancer, and could revolutionize agriculture by engineering disease-resistant crops. A key strength of CRIScPR lies in its adaptability to a broad spectrum of genes, significantly enhancing its versatility. The technology's potential further expands through emerging interdisciplinary integrations, such as artificial intelligence, machine learning, and biological imaging. These advancements can refine CRISPR's precision, improve efficiency, and mitigate existing limitations, positioning it as an indispensable tool in future genetic research. Overall, CRISPR-Cas9 promises transformative progress in healthcare and agriculture, solidifying its role as a cornerstone in the evolution of genetic engineering. </p>.},
}
@article {pmid41649714,
year = {2024},
author = {Dong, G and Fan, Z},
title = {CRISPR/Cas-mediated germplasm improvement and new strategies for crop protection.},
journal = {Crop health},
volume = {2},
number = {1},
pages = {2},
pmid = {41649714},
issn = {2948-1945},
support = {SYND-2021-03//Administration Bureau of Sanya Yazhou Bay Science and Technology City/ ; CARS-02//China Agricultural Research System of MOF and MARA/ ; },
abstract = {Global agriculture and food security are encountering unprecedented challenges from both the ever-growing population and rapidly changing climate conditions. CRISPR/Cas-mediated genome editing technology has revolutionized plant functional genetic research and precision crop breeding with robustness, high target specificity and programmability. Furthermore, numerous emerging biotechnologies based on the CRISPR/Cas platform provide the opportunity to create new crop germplasms with durable resistance against disease or insect pests, herbicide tolerance, and other stress-tolerant improvements, reshaping crop protection to increase agricultural resilience and sustainability. In this review, we briefly describe the CRISPR/Cas toolbox, including base editing, prime editing, compact genome manipulation, transcriptional regulation and epigenetic editing, and then overview the most important applications of CRISPR/Cas-mediated crop genetic improvement, highlighting crop protection-based stress resistance engineering. In addition, we enumerate global regulations on genome-edited crops. Finally, we discuss some bottlenecks facing this cutting-edge technology and infinite possibilities for the future.},
}
@article {pmid41642758,
year = {2026},
author = {Sun, L and Liu, Z and Dong, C and Chi, T and Chen, J and Cheng, D and Zhu, L and Hu, B},
title = {Method Development and Sensitivity Optimization Paradigm for One-Step RPA-CRISPR/Cas12a Rapid Detection of the Hard-to-Amplify Gene blaNDM.},
journal = {Environmental science &amp; technology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.est.5c10280},
pmid = {41642758},
issn = {1520-5851},
abstract = {The presence and spread of antibiotic resistance genes (ARGs) across various habitats have increased the risks of antibiotic resistance, highlighting the urgent need for effective monitoring methods. One key challenge in method development lies in balancing sensitivity, speed, and portability. To address it, a one-step assay targeting the carbapenem resistance gene blaNDM was developed based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a. A sensitivity optimization paradigm─MOSAIC (multistrategy optimized sensitive assay via integrated CRISPR/Cas12a)─was proposed, incorporating component optimization, suboptimal-PAM-mediated CRISPR inhibition, and glycerol-assisted phase separation. The glycerol-assisted strategy exhibited the largest enhancement, followed by the suboptimal-PAM strategy and component optimization. When combined, these strategies demonstrated a synergistic effect, yielding greater improvement (10 000-fold) than a single strategy alone. MOSAIC reached a limit of detection (LOD) of 260 copies/μL, comparable to that of qPCR, and enabled faster quantification of blaNDM at 37 °C within 1 h on a standard plate reader. It achieved 100% diagnostic sensitivity and 95.45% specificity in clinical isolates, and 77.41-99.73% accuracy in environmental matrix-spiked samples, comparable to that of qPCR. It provides a technological foundation for on-site detection of blaNDM and offers an optimization paradigm and new insights for the development of one-step RPA-CRISPR/Cas12a assays targeting various genes.},
}
@article {pmid41642528,
year = {2026},
author = {Shafique, MS and Liu, Y and Ji, Z},
title = {Genome Editing for Developing Disease-Resistant Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3012},
number = {},
pages = {253-264},
pmid = {41642528},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Disease Resistance/genetics ; CRISPR-Cas Systems/genetics ; *Genome, Plant ; *Plant Diseases/genetics ; Plants, Genetically Modified/genetics ; Oryza/genetics ; Plant Breeding/methods ; Crops, Agricultural/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; },
abstract = {Genome editing tools, most notably the CRISPR-Cas9 system, have transformed modern crop breeding by allowing precise and efficient modification of plant genomes, particularly for enhancing resistance to disease. In this chapter, we present two major CRISPR-based approaches that have been successfully applied to develop disease-resistant crops: (1) gene knockout of host susceptibility (S) genes to block pathogen compatibility, and (2) targeted insertion of resistance alleles or synthetic genes using the homology-directed repair (HDR) pathway. Both strategies utilize the specificity and efficiency of CRISPR-Cas9 to introduce defined edits in crops such as rice. This chapter outlines comprehensive step-by-step protocols covering key elements of the process, including target site selection, construction of sgRNA and Cas9 vectors, plant transformation methods, and mutation validation techniques. Together, these methods provide a powerful platform for engineering plant immune responses and offer practical tools to accelerate the breeding of disease-resistant cultivars.},
}
@article {pmid41641699,
year = {2026},
author = {Fu, M and Wang, J and Li, J and Zhou, Y and Huang, X and Jia, Z and Luo, Y and Tan, X and Gao, Y and Yu, B and Duan, Y and Bu, Q and Li, X and Wang, Y and Takaya, N and Zhou, S},
title = {Dual-single-guide RNA strategy improves CRISPR-mediated homology-directed repair in Aspergillus.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41641699},
issn = {1362-4962},
support = {22077032//National Natural Science Foundation of China/ ; 21672065//National Natural Science Foundation of China/ ; K202415//State Key Laboratory of Natural and Biomimetic Drugs/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *Aspergillus nidulans/genetics ; *CRISPR-Cas Systems ; *Recombinational DNA Repair ; Gene Editing/methods ; DNA Breaks, Double-Stranded ; Fungal Proteins/genetics/metabolism ; DNA, Fungal/genetics ; },
abstract = {CRISPR-Cas9 knock-in efficiency is often limited by geometric misalignment between donor DNA and the endogenous strand-invasion path. In Aspergillus nidulans, we found that integration drops sharply when the insertion site is offset from the invasion entry point, producing premature annealing or unsupported 3' ends that stall DNA synthesis. Chromatin immunoprecipitation-based profiling shows directional loading of the RAD51 homolog UvsC around Cas9-induced double-strand breaks, thereby defining the spatial origin of strand invasion. Guided by this insight, we introduce a dual-single-guide RNA design that places two cuts flanking the insertion site to create a geometry-matched strand-invasion window. This alignment consistently and markedly increases homology-directed-repair-mediated integration across insert sizes and editing tasks-including C-terminal tagging, bidirectional promoter rewiring, and long-distance dual-site mutagenesis-and generalizes across multiple fungal species. We propose a structural-docking model in which pairing fidelity between the resected chromosomal strand and donor homology arms governs knock-in outcomes, providing a practical design principle for efficient and precise genome engineering at structurally constrained loci.},
}
@article {pmid41640776,
year = {2025},
author = {Di Pasquale, G and Ottaviani, L and Camardo Leggieri, M and Giorni, P and Marocco, A and Battilani, P and Lanubile, A},
title = {Plant oxylipins: adaptation to environmental stresses and impact on mycotoxin contamination.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1739321},
pmid = {41640776},
issn = {1664-462X},
abstract = {Due to increasingly frequent changes in climatic conditions and global warming, plants consistently deal with severe weather events including extreme temperature variations, floods and drought. These abiotic stressors resulting from climate change weaken host crop resistance, making them more exposed to fungal disease insurgences and mycotoxin contamination. Oxylipins are major players in the plant-environment interaction. Their synthesis begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases (LOXs) to generate fatty acid hydroperoxides that in turn are converted into a huge assortment of bioactive compounds by specialized cytochrome P450 enzymes, known as CYP74. In the present review we focus on recent advances concerning oxylipin biosynthesis and the phylogenetic relationships among the main key enzymes of the oxylipin pathway considering five monocot and dicot plant species. Moreover, new information regarding the role of these signaling molecules on the plant physiology in response to abiotic stress and mycotoxin occurrence are provided along with the application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) (CRISPR/Cas)-based tools. Here, we report the intervention of LOX, allene oxide synthase, OPDA reductase, JASMONATE (JA) resistant and JA ZIM domain genes along with the accumulation of JA and its conjugates, 12-OPDA, ketols and green leaf volatiles in response to abiotic stress. The modulation of LOX genes and the production of several fatty acids, oxylipins and sphingolipids is also required against mycotoxin contamination.},
}
@article {pmid41575301,
year = {2026},
author = {Park, H and Bae, SM and Hong, T and Song, G and Lim, W},
title = {CRISPR/Cas9-mediated SLC2A1 gene knockout changes in energy metabolism and cellular behavior in human trophoblasts.},
journal = {Reproduction (Cambridge, England)},
volume = {171},
number = {2},
pages = {},
doi = {10.1093/reprod/xaag006},
pmid = {41575301},
issn = {1741-7899},
support = {//National Research Foundation of Korea/ ; RS-2024-00453204//Korea government/ ; RS-2025-02216962//Korea government/ ; },
mesh = {Humans ; *Trophoblasts/metabolism/cytology ; *CRISPR-Cas Systems ; *Glucose Transporter Type 1/genetics/metabolism ; *Energy Metabolism/genetics ; Endoplasmic Reticulum Chaperone BiP ; Female ; Pregnancy ; Gene Knockout Techniques ; Endoplasmic Reticulum Stress ; Mitochondria/metabolism ; Reactive Oxygen Species/metabolism ; Signal Transduction ; Autophagy ; },
abstract = {Glucose transport across the placenta is essential for fetal growth and development. Glucose transporter 1, encoded by the SLC2A1 gene, plays a central role in mediating maternal-fetal glucose exchange. Dysregulation of placental glucose transport is implicated in pregnancy-related complications, such as preeclampsia and fetal growth restriction; however, the mechanistic role of SLC2A1 in trophoblast function remains poorly defined. To functionally validate the role of SLC2A1 in human trophoblasts, we used clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9-mediated knockout of the SLC2A1 gene, enabling complete and permanent loss of SLC2A1 expression. In the resulting SLC2A1 knockout human trophoblast HTR8/SVneo cells, SLC2A1 depletion induced a metabolic shift from glycolysis to oxidative phosphorylation, leading to increased mitochondrial respiration, ATP production, mitochondrial calcium overload, and elevated mitochondrial reactive oxygen species generation. These changes were accompanied by enhanced endoplasmic reticulum stress, as shown by the upregulation of p-PERK, IRE1α, and GRP78, as well as increased autophagic activity indicated by LC3B-II and p62 accumulation. Notably, mTOR signaling was also upregulated, suggesting a feedback loop that regulates autophagy. The loss of SLC2A1 impaired the PI3K/AKT pathway, reduced trophoblast migration and 3D spheroid formation, and disrupted epithelial-mesenchymal transition-like properties. These findings demonstrate that SLC2A1 is essential for maintaining trophoblast energy homeostasis, redox balance, and invasive capacity; its deficiency triggers mitochondrial and endoplasmic reticulum stress responses that may contribute to placental dysfunction during early pregnancy.},
}
@article {pmid41640466,
year = {2026},
author = {Bharti, N and Modi, U and Bhatia, D and Solanki, R},
title = {Engineering delivery platforms for CRISPR-Cas and their applications in healthcare, agriculture and beyond.},
journal = {Nanoscale advances},
volume = {},
number = {},
pages = {},
pmid = {41640466},
issn = {2516-0230},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have transformed genome editing through unprecedented precision, and next-generation variants (base and prime editors) further enhance specificity by enabling targeted nucleotide changes without introducing double-strand DNA breaks. These technologies have unlocked broad applications in therapeutic gene correction, functional genomics, infectious disease management, diagnostics, agricultural engineering, environmental biotechnology, and synthetic biology. However, the targeted delivery of these systems remains a major challenge due to the large and chemically distinct nature of their components, including Cas protein or its base/prime editor fusions, guide RNA, and in some cases, DNA repair templates-which complicate packaging, stability, and cellular uptake. Additional hurdles arise from tissue and cell-type specificity, differential intracellular environments, variable editing efficiencies, and the persistent risk of off-target genome modifications. This review outlines the key challenges in the delivery of CRISPR technologies as well provides a comprehensive overview of both current and emerging delivery strategies, including viral vectors (adenovirus, adeno-associated virus, and lentivirus), non-viral physical approaches (microinjection, electroporation, ultrasound, and hydrodynamic tail-vein injection), and nanoparticle-based modalities (lipid and polymeric nanoparticles, gold nanoparticles, DNA nanostructures, and extracellular vesicles). We also discussed the diverse applications of CRISPR-Cas9 in gene therapy, immune cell engineering for cancer therapies, and agricultural innovation.},
}
@article {pmid41638451,
year = {2026},
author = {Asemoloye, MD},
title = {The fungal cure: Harnessing mycelial approach as sustainable green solution for industrial waste treatment.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108834},
doi = {10.1016/j.biotechadv.2026.108834},
pmid = {41638451},
issn = {1873-1899},
abstract = {Industrialization has intensified releases of complex waste streams (e.g., synthetic dyes, petroleum hydrocarbons, heavy metals, and plastics) whose treatment can be costly, energy-intensive, and often incomplete using conventional physicochemical methods. 'Mycoremediation' defined as fungi mediated remediation, or their secreted materials/enzymes offers compelling advantages. These advantages stem across the extensive mycelial networks for matrix penetration, non-specific oxidative enzyme systems that transform lignin-like xenobiotics, and cell-wall chemistries that sorb metal ions. This review synthesizes mechanistic foundations on fungal enzymes (laccases; class II peroxidases such as manganese peroxidase and lignin peroxidase; biosorption and biomineralization), bioengineering strategies (CRISPR/Cas editing, artificial consortia), process intensification (immobilized-laccase reactors; whole-cell formats), and applications across textile dye effluents, petroleum-impacted soils/sediments, heavy-metal bearing wastewaters/soils, and polymer-rich wastes. Emerging evidence shows robust lab and mesocosm performance like rapid dye decolorization in fungal cartridge systems, significant alteration of petroleum (saturate, aromatic, resin and asphaltene-SARA) fractions under estuarine salinities, and high-capacity metal biosorption, while systematic verification for plastics remains a priority. Fungi sustainability assessments identify life-cycle hot spots in enzyme production and immobilization supports; techno-economic analyses suggest feasibility pathways when biocatalyst durability and reuse are optimized. This review also delves into regulatory frameworks for contained use and deliberate environmental release of engineered fungi, shaping the near-term deployments toward contained bioreactors. It concludes by projecting the combination of bioengineering (strain/secretome control), reactorization (immobilized catalysts, modular beds), and standardized metrics (toxicity, mass balance, life-cycle assessment-LCA/techno-economic analysis-TEA) for accelerating the transition of mycoremediation from promising prototypes to field-validated, scalable technologies for industrial waste treatment.},
}
@article {pmid41634489,
year = {2026},
author = {Araújo, MRB and Dos Santos, LS and Viana, MVC and Sousa, EG and Prates, FD and Perini, HF and da Silva, MV and da Silva Sousa, J and Brenig, B and Andrade, BS and Mattos-Guaraldi, AL and de Oliveira Sant'Anna, L and Ramos, JN and de Oliveira Vinhal, AL and de Castro Soares, S and Azevedo, V},
title = {Comparative genomics and molecular characterization of a multidrug-resistant Corynebacterium glucuronolyticum isolated for the first time from the human genitourinary tract in Latin America.},
journal = {Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]},
volume = {57},
number = {1},
pages = {49},
pmid = {41634489},
issn = {1678-4405},
abstract = {UNLABELLED: Although Corynebacterium glucuronolyticum has been associated with human infections, its pathogenic potential remains poorly understood. Here, we describe the first case in Latin America of C. glucuronolyticum isolated from the human urogenital tract. The strain, designated IHP2022, was identified by MALDI-TOF MS (99% probability) and exhibited resistance to benzylpenicillin, clindamycin, and tetracycline, characterizing a multidrug-resistant (MDR) phenotype. Genomic analysis revealed a 2.88-Mb genome with 59.04% G + C content and no plasmids. Comparative genomic analysis, including 11 other publicly available genomes, demonstrated high genetic diversity and positioned IHP2022 close to strain p3-SID752 from the USA, suggesting a broad geographical distribution. The genome harbored multiple virulence and resistance genes, as well as a Type I-E CRISPR-Cas system. Functional annotation and pangenome analysis identified 4,027 gene families categorized into core, shell, and cloud components. By integrating phenotypic and genomic data, this study provides the first in-depth characterization of an MDR C. glucuronolyticum strain minimizing current knowledge gaps and contributing to a better understanding of its pathogenic potential.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-025-01822-7.},
}
@article {pmid41633991,
year = {2026},
author = {Kumari, B and Damodaran, AP and Guiblet, WM and Xiao, MS and Behera, AK and On, TA and McIntosh, CE and Teszler, M and Holloway, C and Le, S and Parab, N and Zhao, Y and Aregger, M and Gonatopoulos-Pournatzis, T},
title = {Single-cell exon deletion profiling reveals splicing events that shape gene expression and cell state dynamics.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1218},
pmid = {41633991},
issn = {2041-1723},
support = {ZIA BC012033/ImNIH/Intramural NIH HHS/United States ; ZIA BC012101/ImNIH/Intramural NIH HHS/United States ; HHSN261200800001C/RC/CCR NIH HHS/United States ; HHSN261200800001E/CA/NCI NIH HHS/United States ; 1ZIABC012101//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; 1ZIABC012033//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; 75N91019D00024/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Exons/genetics ; *Alternative Splicing/genetics ; *Single-Cell Analysis/methods ; CRISPR-Cas Systems/genetics ; Gene Expression Profiling/methods ; Cell Cycle/genetics ; Sequence Deletion ; Transcriptome ; Gene Expression Regulation ; },
abstract = {Alternative splicing is a pervasive gene regulatory mechanism critical for diversifying the human proteome. To systematically investigate its role in cell fate determination, we develop scCHyMErA-Seq, a scalable CRISPR-based exon deletion screening platform integrated with 10x Genomics single-cell transcriptomic readouts. This tool enables efficient exon deletion while simultaneously capturing Cas9/Cas12a guides and polyadenylated transcripts at single-cell resolution. Applying scCHyMErA-Seq to high-throughput profiling of alternative cassette exons, we identify numerous exons with pronounced regulatory effects on gene expression and cell cycle progression. Analysis of the alternative NRF1 exon-7 demonstrates that its inclusion modulates NRF1's regulatory function by influencing its recruitment to the promoters of target genes. Importantly, gene expression profiles generated using scCHyMErA-Seq accurately recapitulate findings from traditional, labor-intensive orthogonal methods, while offering enhanced scalability and efficiency. Overall, scCHyMErA-Seq represents a versatile platform for systematically unraveling the functional impact of alternative splicing by directly linking specific splicing variants to transcriptional phenotypes.},
}
@article {pmid41611149,
year = {2026},
author = {Setia, A and Patil, D and Randhave, NV and Vaishali, and Verma, N and Rani, K and Kale, A and Wagh, B and Kumar, V and Malik, AK and Sharon Mano Pappu, J and Deshmukh, K and Muthu, MS},
title = {CRISPR/Cas9 genome editing-based preclinical theranostics, biomarkers and drug delivery systems for cancer applications.},
journal = {International journal of biological macromolecules},
volume = {345},
number = {},
pages = {150582},
doi = {10.1016/j.ijbiomac.2026.150582},
pmid = {41611149},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; *Neoplasms/genetics/therapy/diagnosis/drug therapy ; *Drug Delivery Systems/methods ; *Biomarkers, Tumor/genetics ; *Theranostic Nanomedicine/methods ; Animals ; Genetic Therapy/methods ; },
abstract = {Cancer development is driven by diverse genetic aberrations, underscoring the importance of innovative approaches like gene therapy for effective treatment. The CRISPR/Cas9 gene-editing system has all the makings of a game-changing technique for future disease treatment, owing to its pinpoint accuracy and efficiency in deleting disease-causing genes or correcting damaging base mutations. A number of efficient Cas9 variants and derivatives were recently designed to tackle the intricate genomic modifications that accompany illnesses. In addition, CRISPR/Cas9 based systems are increasingly explored for biomarker sensing and cancer diagnostics. Early identification, real-time monitoring, and therapy stratification are made possible by CRISPR-driven biosensors, which can detect circulating tumor DNA, microRNAs, or exosomal RNA with high specificity and sensitivity. Furthermore, a variety of stimuli-responsive delivery strategies, including chemical and peptide-assisted systems, light-activated mechanisms, glutathione-sensitive carriers, and pH-responsive platforms, have been explored to improve intracellular release efficiency thereby enhancing the precision of CRISPR/Cas9-mediated gene editing in cancer therapy. The CRISPR/Cas9-enabled theranostic platforms employ engineered nanocarriers to simultaneously deliver gene-editing tools and imaging agents, thereby facilitating synchronized treatment monitoring and improved therapeutic precision. This review emphasizes the transformative potential of CRISPR/Cas9-integrated theranostics, which combine targeted gene editing with advanced imaging for enhanced therapeutic monitoring and efficacy in cancer treatment.},
}
@article {pmid41508115,
year = {2026},
author = {Xiao, Y and Li, X and Jiang, L and Zhao, Y and Wang, L and Feng, Y},
title = {Construction of Escherichia coli L-isoleucine cell factories based on propionate pathway.},
journal = {Journal of biological engineering},
volume = {20},
number = {1},
pages = {28},
pmid = {41508115},
issn = {1754-1611},
abstract = {UNLABELLED: Isoleucine, an essential branched-chain amino acid with broad applications in food, pharmaceuticals, and feed, is predominantly produced via the microbial threonine pathway, which suffers from catalytic complexity and regulatory inefficiency. The propionate pathway offers a streamlined alternative but remains unexplored for L-isoleucine biosynthesis. Here, we engineered Escherichia coli to establish the first propionate pathway-based L-isoleucine cell factory. Critical enzymes—propionyl-CoA synthase (PCS), propionyl-CoA transferase (PCT), and α-ketobutyrate synthase (OBS)—were identified for converting propionate to α-ketobutyrate. Key genes (prpE from Salmonella, pctcP from Gibberella, pctcN from Clostridium propionicum, and nifJ from Moorella thermoacetica) were integrated with the propionate transporter (prpP) and carbonic anhydrase (can) to enhance substrate utilization. ILE-5a, derived from E. coli BW25113 with deletions in brnQ, livJ, and livK, and containing specific insertions, was further modified to create ILE-5b with an additional deletion in yjiP. Plasmid-based expression in these strains, ILE-5a and ILE-5b, yielded top producers ILE-5a-P10 and ILE-5b-P10, which achieved L-isoleucine titers of 304 mg/L and 235 mg/L, respectively, in shake-flask fermentation using glucose and propionate as carbon sources. To stabilize production, the optimal gene set (pctN, nifJ, prpP, can) was genomically integrated via a transposon-encoded CRISPR-Cas system, generating mutants ILE-5a-P11 and ILE-5b-P11. Response surface methodology-optimized medium and 3-L fed-batch fermentation further elevated titers to 1.13 g/L (ILE-5a-P11) and 11.33 g/L (ILE-5b-P11). This study pioneers the propionate pathway for efficient L-isoleucine production, demonstrating its industrial potential through systematic metabolic engineering and process optimization.To our knowledge, this is the first study to design and demonstrate an E. coli platform for isoleucine production that simultaneously leverages the glutamate and propionate precursors.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13036-025-00609-6.},
}
@article {pmid41345278,
year = {2026},
author = {de la Rosa, C and Kendirli, A and Baygün, S and Bauernschmitt, F and Thomann, AS and Kisioglu, I and Beckmann, D and Carpentier Solorio, Y and Pfaffenstaller, V and Tai, YH and Mehraein, N and Sanchez, P and Spieth, L and Gerdes, LA and Beltran, E and Dornmair, K and Simons, M and Peters, A and Schmidt-Supprian, M and Kerschensteiner, M},
title = {In vivo CRISPR screen reveals regulation of macrophage states in neuroinflammation.},
journal = {Nature neuroscience},
volume = {29},
number = {2},
pages = {493-509},
pmid = {41345278},
issn = {1546-1726},
support = {259373024//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 408885537//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 239283807//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Animals ; *Macrophages/metabolism ; Mice ; Cytokines/metabolism/genetics ; Mice, Inbred C57BL ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Multiple Sclerosis/immunology/genetics ; Humans ; *Neuroinflammatory Diseases/genetics ; CRISPR-Cas Systems ; *Encephalomyelitis, Autoimmune, Experimental/immunology ; },
abstract = {Here we established an in vivo CRISPR screening pipeline using genetically editable progenitor cells to dissect macrophage regulation in mouse models of multiple sclerosis (MS). Screening over 100 cytokine receptors and signaling molecules identified interferon-γ, tumor necrosis factor, granulocyte-macrophage colony-stimulating factor and transforming growth factor-β as essential regulators of macrophage polarization in vivo. Single-cell transcriptomics confirmed that transferred progenitor cells generate all blood-derived CNS myeloid cell populations, enabling Perturb-seq analysis of cytokine actions in neuroinflammation. Combined with biosensor expression, our approach allows monitoring cytokine effects on myeloid cell migration, debris phagocytosis and oxidative activity in vivo. Comparative transcriptomic analyses revealed conserved neuroinflammatory cytokine signatures across myeloid populations, CNS compartments and species, elucidating cytokine cues shaping myeloid function in the cerebrospinal fluid and parenchyma of individuals with MS. This versatile pipeline thus provides a scalable framework for high-resolution analysis of macrophage states and uncovers the cytokine signals that underlie their regulation in MS and MS models.},
}
@article {pmid41633939,
year = {2026},
author = {Lin, L and Zhang, JJ and Liu, BH and Du, S and Zhang, YQ and Yang, Y and Li, C and Dong, CC and He, YB and Wang, Q and Wang, HY and Shao, CW},
title = {Epigenetic editing of marine medaka (Oryzias melastigma) fgf2 using CRISPR/dCas9-Tet1CD.},
journal = {Zoological research},
volume = {47},
number = {1},
pages = {263-278},
doi = {10.24272/j.issn.2095-8137.2025.089},
pmid = {41633939},
issn = {2095-8137},
mesh = {Animals ; *Oryzias/genetics ; *Gene Editing/veterinary ; *Epigenesis, Genetic ; *Fibroblast Growth Factor 2/genetics/metabolism ; *CRISPR-Cas Systems ; Epigenome Editing ; },
abstract = {CRISPR/dCas9-mediated epigenetic editing offers a versatile approach for transcriptional regulation without introducing DNA strand breaks. Although this strategy has been explored in a limited number of species, its application in aquatic vertebrates remains largely uncharacterized. In this study, ten-eleven translocation methylcytosine dioxygenase 1 (tet1) was cloned and molecularly characterized in marine medaka (Oryzias melastigma). Decitabine treatment identified fibroblast growth factor 2 (fgf2) as a methylation-sensitive gene, with a regulatory CpG island located within its promoter region. Subsequently, a CRISPR/dCas9-Tet1CD activation system was constructed by fusing the catalytic domain of Tet1 (Tet1CD, Ala1352-Thr2034) to dCas9, enabling locus-specific DNA demethylation. Targeting fgf2, this CRISPR/dCas9-Tet1CD system induced efficient and selective demethylation of the CpG island, resulting in a maximal 2.41-fold increase in fgf2 transcript levels. Whole-genome bisulfite sequencing and transcriptomic analysis confirmed high on-target precision with minimal off-target effects. Epigenetic activation of fgf2 further modulated downstream gene networks associated with growth, promoting durable transcriptional enhancement and increased cellular proliferation. Collectively, these results establish a robust and highly specific epigenetic editing platform in marine medaka, providing a powerful tool for functional genomics studies and regulatory element analysis in aquatic models.},
}
@article {pmid41632443,
year = {2026},
author = {Mašlaňová, I and Nováková, D and Švec, P and Kovařovic, V and Sedláček, I and Botka, T and Šedo, O and Neumann-Schaal, M and Vives, J and Doškař, J and Pantůček, R},
title = {Genomic and taxonomic characterization of strain CCM 2573: Uncovering unique genetic features and description of Macrococcus caseolyticus subsp. lactis subsp. nov.},
journal = {Folia microbiologica},
volume = {},
number = {},
pages = {},
pmid = {41632443},
issn = {1874-9356},
abstract = {Strain CCM 2573 is a Gram-positive bacterium that has been intensively studied in the past due to its distinct chemotaxonomic properties, but its reliable taxonomic classification has not been satisfactorily clarified. Whole-genome sequencing and comparative genomic analyses performed in this study revealed that the strain belongs to the Macrococcus caseolyticus phylogenetic clade. Genome-to-genome comparisons confirmed the closest relationship to the type strains of M. caseolyticus subsp. hominis CCM 7927[T] and M. caseolyticus subsp. caseolyticus DSM 20597[T]. However, the strain harbored unique genomic elements distinguishing it from its nearest phylogenetic neighbors. Its accessory genome contains dozens of insertion sequences, a 92-kbp composite transposon with unique palindromic repeat loci associated with a CRISPR-Cas adaptive immune system, a pseudo-staphylococcal chromosome cassette, and several additional genomic islets. Unlike other macrococci, strain CCM 2573 exhibits a specific peptidoglycan composition (L-Lys-Gly2-Ser2-Gly) and shows a higher phylogenetic divergence of aminoacyltransferases (FemABX) involved in interpeptide bridge synthesis. In addition, it reveals distinct biochemical characteristics from both subspecies of M. caseolyticus, particularly in its ability to produce acid from galactose, cellobiose, melezitose, and turanose, as well as in its susceptibility to novobiocin. The MALDI-TOF mass spectra enable differentiation of the strain from other type strains of the genus Macrococcus. The results of polyphasic taxonomy obtained in this study showed that strain CCM 2573 belongs to the species M. caseolyticus, but it is distinct from both validly named M. caseolyticus subspecies. We propose to assign the analyzed strain as a new subspecies, Macrococcus caseolyticus subsp. lactis subsp. nov. The type strain is CCM 2573[T] (= DSM 20227[T]).},
}
@article {pmid41632281,
year = {2026},
author = {Li, J and Zhu, M and Hu, G and Chen, X and Xue, H and Zhang, Y and Wang, Y and Li, Z and Xu, D and Zhai, M and Zhou, G and Cui, C and Zhao, C and Qin, R and Wu, Y and Cui, F and Sun, H},
title = {Gene editing and association analysis of circadian clock gene TaPRR59 highlights its importance in yield-related traits in wheat.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {2},
pages = {58},
pmid = {41632281},
issn = {1432-2242},
support = {32272119//National Natural Science Foundation of China/ ; 32072051//National Natural Science Foundation of China/ ; ZR2022YQ19//Shandong Provincial Fund for Excellent Young Scholars/ ; 32101726//National Natural Science Foundation for Young Scholars of China/ ; ZR2022MC119//Natural Science Foundation of Shandong Province, China/ ; 20230119//Taishan scholar young expert/ ; ZR2019ZD16//Major Basic Research Project of Natural Science Foundation of Shandong Province, China/ ; 2022LZG002-2//Key R &amp; D Program of Shandong Province, China (Major Innovation Project)/ ; 2023ZDCX023//Yantai science and technology plan project/ ; IPGS2025-083//The Innovation Project for graduate students of Ludong University/ ; },
mesh = {*Triticum/genetics/growth &amp; development ; *Gene Editing ; *Plant Proteins/genetics/metabolism ; *Circadian Clocks/genetics ; Gene Expression Regulation, Plant ; Haplotypes ; Phenotype ; Plant Breeding ; *Genes, Plant ; CRISPR-Cas Systems ; },
abstract = {Mutations in TaPRR59 impact transcript levels of some key flowering genes and show earlier heading time and reduced plant height. Favorable haplotype TaPRR59-A1-Hapla was positively selected in wheat breeding programs. The circadian clock system is a crucial endogenous rhythmic regulatory mechanism with a significant role in plant growth and development. The pseudo-response regulator (PRR) family is a pivotal component of circadian networks. In the present study, we cloned the wheat PRR family member TaPRR59 and investigated its function using gene editing, transcriptome sequencing, haplotype analysis, and association analysis. The expression profile of TaPRR59 over a 24-h period exhibited a diurnal rhythmic expression pattern. Luciferase transient transcriptional assay demonstrated that TaPRR59 acts as a transcriptional repressor in the nucleus. The taprr59-ABD-KO gene-edited lines produced using the CRISPR/Cas9 genome-editing system had earlier heading time and reduced plant height. Overexpression of TaPRR59-D1 in rice significantly delayed the heading date, reduced plant height and thousand-grain weight, and increased the number of grains per panicle. Transcriptome analysis revealed the transcript levels of several key flowering genes and chlorophyll a-b binding protein-related genes were up- or down-regulated in the taprr59 mutant plants. Association analysis showed that natural variations at TaPRR59-A1, TaPRR59-B1, and TaPRR59-D1 were significantly associated with yield traits such as plant height, thousand-grain weight, and heading date. Geographical analysis showed distinctive distribution characteristics of TaPRR59 haplotypes in different agroecological production zones. Additionally, the significant difference in frequency of the favorable haplotype TaPRR59-A1-Hapla between landraces and modern cultivars indicates that it has been subject to directional selection during wheat breeding. This research provided novel insights into the influence of the circadian clock system on agronomic traits and provided useful molecular markers and genetic resources for wheat breeding.},
}
@article {pmid41630767,
year = {2025},
author = {Molaghi, MAA and Atiyah, WR and Saeed, AAR},
title = {Novel CRISPR/Cas12-based assay for the rapid and accurate detection of donkey meat.},
journal = {Open veterinary journal},
volume = {15},
number = {11},
pages = {5682-5688},
pmid = {41630767},
issn = {2218-6050},
mesh = {Animals ; *Equidae/genetics ; *Meat/analysis ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Polymerase Chain Reaction/veterinary/methods ; },
abstract = {BACKGROUND: Precise identification of items originating from animals is required for monitoring the authenticity of livestock commodities. Nucleic acid-based detection methods, including polymerase chain reaction, are highly accurate tests for detecting meat fraud. However, these tests require costly devices and highly skilled personnel.<br><br>AIM: This study aims to develop a precise and rapid test based on deoxyribonucleic acid (DNA) for detecting meat from donkeys.<br><br>METHODS: The assay was developed by combining Clustered regularly interspaced short palindromic repeat /Cas12 with RPA and a lateral flow device. A conserved sequence of the mitochondrial D-loop gene was used as the target gene. The assessment of the assay focused on its sensitivity, specificity, and completion time.<br><br>RESULTS: The results show that the assay can detect donkey meat in the tested samples with 100% specificity with no cross-reactivity with other species, and no false-positive results were recorded. Furthermore, the assay has shown high sensitivity and to detect as little as 5 ng of input DNA. Moreover, the results, including DNA extraction, were obtained in less than 40 minutes and did not require any specialized equipment.<br><br>CONCLUSION: The assay is a good option for the rapid and precise detection of donkey meat. Further optimization and studies are needed to enable the direct detection of donkey meat without DNA extraction.},
}
@article {pmid41630633,
year = {2026},
author = {Lee, SY and Park, HH},
title = {Structural Basis of Recognition of Anti-CRISPR Operon by Aca3.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {3},
pages = {e71512},
doi = {10.1096/fj.202502389RR},
pmid = {41630633},
issn = {1530-6860},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; RS-2025-16065724//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Operon ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Viral Proteins/genetics/metabolism/chemistry ; Bacteriophages/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Promoter Regions, Genetic ; },
abstract = {The CRISPR-Cas system equips bacteria with adaptive immunity by storing fragments of invading nucleic acids in CRISPR loci and deploying Cas proteins to recognize and degrade matching sequences. In turn, bacteriophages have evolved small anti-CRISPR (Acr) that neutralize diverse CRISPR-Cas types. Acr genes are often co-encoded with transcriptional regulators called anti-CRISPR-associated (Aca) proteins, which suppress acr expression. Although 13 Aca families have been identified through bioinformatic analysis, detailed information on their target DNA-binding mechanisms and the inhibition of acr expression remains limited. Here, we report the high-resolution structure of Aca3 and delineate its DNA-binding interface. We demonstrate that Aca3 selectively recognizes inverted repeats upstream of its cognate acr gene, AcrIIC1. Mutational analyses of key helix-turn-helix residues confirm their essential roles in promoter engagement. Together, these results reveal the molecular basis for Aca3-mediated control of anti-CRISPR expression and expand our understanding of regulatory strategies that phages employ to modulate host CRISPR-Cas immunity.},
}
@article {pmid41629994,
year = {2026},
author = {Papaioannou, NY and Patsali, P and Klermund, J and Papasavva, PL and Andrieux, G and Koniali, L and Naiisseh, B and Christou, S and Sitarou, M and Kleanthous, M and Cathomen, T and Lederer, CW},
title = {Functional correction and genome integrity with duplex base editing of β-thalassemic hematopoietic stem cells.},
journal = {Genome biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s13059-026-03974-7},
pmid = {41629994},
issn = {1474-760X},
support = {EXCELLENCE/1216/0092//Research and Innovation Foundation/ ; CA21113 GenE-HumDi//COST Actions/ ; New Infrastructure for the Diagnosis and Therapy of Patients//Norway Grants 2014-2021/ ; },
abstract = {BACKGROUND: Beta-thalassemia is among the most common monogenic disorders, posing a major global health challenge. Editing of genetic modifiers, such as BCL11A erythroid enhancer and HBG promoters, enhances fetal hemoglobin expression and confers major therapeutic potential. Double-strand-break (DSB)-independent genome editing tools, such as base editors (BE), are potentially safer and better suited for multiplexed application than DSB-dependent CRISPR/Cas technology. However, harmful on- and off-target events remain a concern and must be excluded before clinical application, including chromosomal rearrangements invisible to standard detection technologies.<br><br>RESULTS: Using primary patient-derived CD34[+] cells from three donors, we investigate simplex and duplex BE-based disruption of the BCL11A erythroid enhancer and the BCL11A binding site (-115 bp) on the HBG promoter for DNA-level and functional studies at the RNA, protein, and morphological level. Analyses include direct comparison to DSB-based editing, the current clinically applied standard, and CAST-seq to assess recombination events, allowing wider inferences on relative safety. RNA-seq analyses for clones of primary CD34[+] cells across all treatments confirm peak HBG induction for duplex BE and comparable effects on apoptotic and immune response signatures. Overall, duplex BE produces robust &#x3b3;-globin and fetal hemoglobin induction, improves functional correction over simplex editing and results in low incidence of genomic alterations in both target loci.<br><br>CONCLUSIONS: Duplex BE targeting both BCL11A erythroid enhancer and HBG promoter enables functional correction and genome integrity. Our study highlights the efficacy, safety, and therapeutic potential of the present duplex BE approach.},
}
@article {pmid41629464,
year = {2026},
author = {},
title = {Reprogramming CRISPR-Cas enzymes for customized genome editing.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {41629464},
issn = {1546-1696},
}
@article {pmid41629462,
year = {2026},
author = {Nayfach, S and Bhatnagar, A and Novichkov, A and Kim, N and Hoffnagle, AM and Hussain, R and Estevam, GO and Hill, E and Ruffolo, JA and Silverstein, RA and Gallagher, J and Kleinstiver, BP and Meeske, AJ and Cameron, P and Madani, A},
title = {Customizing CRISPR-Cas PAM specificity with protein language models.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {41629462},
issn = {1546-1696},
abstract = {CRISPR-Cas enzymes must recognize a protospacer-adjacent motif (PAM) to edit a genomic site, greatly limiting the range of targetable sequences in a genome. Although engineering strategies to alter PAM specificity exist, they typically require labor-intensive, iterative experimentation. We introduce an evolution-informed deep learning model, Protein2PAM, to efficiently guide the design of Cas protein variants tailored to recognize specific PAMs. Trained on a dataset of over 45,000 CRISPR-Cas PAMs, Protein2PAM rapidly and accurately predicts PAM specificity directly from Cas proteins across type I, II and V CRISPR-Cas systems. Using in silico mutagenesis, the model identifies residues critical for PAM recognition in Cas9 without using structural information. We use Protein2PAM to computationally evolve Nme1Cas9, generating variants with broadened PAM recognition and up to a 50-fold increase in PAM cleavage rates compared to the wild type in vitro. Our machine learning approach allows Cas enzymes to target sequences that were previously inaccessible because of PAM constraints, potentially increasing target flexibility in personalized genome editing.},
}
@article {pmid41629044,
year = {2026},
author = {Sharma, S and Karna, SKL and Khanal, S and Pokharel, YR},
title = {Rapid detection of measles virus RNA from clinical specimens by using RT-LAMP coupled with CRISPR/Cas12b via fluorescence and lateral flow biosensor readouts: A proof-of-concept study.},
journal = {Analytica chimica acta},
volume = {1389},
number = {},
pages = {345081},
doi = {10.1016/j.aca.2026.345081},
pmid = {41629044},
issn = {1873-4324},
mesh = {*Measles virus/genetics/isolation &amp; purification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Fluorescence ; *Measles/diagnosis/virology ; Proof of Concept Study ; Animals ; Chlorocebus aethiops ; Vero Cells ; },
abstract = {BACKGROUND: Rapid laboratory confirmation of suspected cases is essential for measles control, but current methods require a complex laboratory infrastructure. We developed loop-mediated isothermal amplification coupled with CRISPR-Cas-mediated diagnostic (LAmCaD), a novel two-pot diagnostic platform that integrates rapid nucleic acid extraction, RT-LAMP amplification, and in-house purified AapCas12b as a rapid test to detect measles RNA that could be used in settings lacking laboratory infrastructure.<br><br>METHODS: LAmCaD is based on dual detection modalities, fluorescence and lateral flow biosensor readouts. The LAmCaD assay was evaluated for analytical sensitivity and specificity using RNA from Vero/hSLAM-grown measles virus, and diagnostic evaluation was performed using patient samples, compared with standard RT-PCR. The cross-genotype detection capability was assessed across epidemiologically relevant measles genotypes D8, D4, and B3.<br><br>RESULTS: The in-house purified protein AapCas12b from Alicyclobacillus acidiphilus exhibited strong cis and trans cleavage activities, eliminating dependence on commercial enzyme preparations. The platform enables the use of clinical samples from patients through rapid nucleic acid extraction that eliminates the need for RNA purification steps, allowing direct use of extracted material in RT-LAMP reactions. RT-LAMP alone achieved analytical sensitivity of &#x223c;10[3] copies, while the complete protocol detected measles virus at &#x223c;10[5] copies by fluorescence and &#x223c;10[4] copies by lateral flow detection. Diagnostic evaluation demonstrated sensitivity of 64.00&#xa0;%, specificity of 92.59&#xa0;%, and negative predictive value of 99.95&#xa0;% with an overall accuracy of 92.56&#xa0;%. ROC curve analysis revealed an AUC of 0.717, indicating fair discriminatory performance. The assay demonstrated moderate agreement with RT-PCR (&#x3ba;&#xa0;=&#xa0;0.6) and successfully identified genotypes D8, D4, and B3. The entire testing process took 90&#xa0;min to complete.<br><br>CONCLUSIONS: This proof-of-concept LAmCaD platform establishes the foundation for a cost-effective, field-deployable diagnostic test without relying on commercial enzymes or complex sample processing. The platform could facilitate the rapid confirmation of measles cases in resource-limited and/or remote settings, thereby contributing to global measles elimination goals. Conducted within the WHO South-East Asia Region, this study is particularly relevant to the region's 2023 elimination target, addressing current surveillance gaps and specimen transport challenges that hinder efforts to eliminate the disease.},
}
@article {pmid41628256,
year = {2026},
author = {Luo, Y and Dong, Q and Yi, S and Zhang, W and Du, Y and Fang, Q and Zhang, W and Ouyang, K and Chen, Y and Yin, Y and Wei, Z and Qin, Y and Huang, W},
title = {Genome-wide CRISPR screening identifies Annexin A1 as a facilitator of porcine astrovirus entry.},
journal = {PLoS pathogens},
volume = {22},
number = {2},
pages = {e1013943},
doi = {10.1371/journal.ppat.1013943},
pmid = {41628256},
issn = {1553-7374},
mesh = {Animals ; Swine ; *Virus Internalization ; CRISPR-Cas Systems ; *Annexin A1/metabolism/genetics ; *Swine Diseases/virology/genetics/metabolism ; *Astroviridae Infections/virology/metabolism/genetics ; *Mamastrovirus/physiology/genetics ; },
abstract = {Porcine astrovirus (PAstV) is an important and widespread pathogen in swine, linked to diarrheal outbreaks and extraintestinal disease. How PAstV enters host cells has remained unclear, and no cellular factor has been defined for PAstV entry. Here, a genome-wide CRISPR-Cas9 loss-of-function screen in porcine epithelial cells identifies Annexin A1 (ANXA1) as a host factor that facilitates PAstV entry. Genetic ablation or pharmacological/antibody blockade of ANXA1 reduces binding, lowers early viral RNA and capsid signals, and delays the rise of progeny, whereas re-expression restores susceptibility. Biochemical assays and surface plasmon resonance indicate a direct interaction between ANXA1 and the acidic C-terminal domain of the PAstV ORF2 capsid protein, and imaging shows ANXA1 co-localizes with incoming particles at the cell surface and supports attachment and uptake. Loss of ANXA1 does not alter infection by the non-astrovirus panel tested, indicating selectivity for PAstV under our conditions. Notably, infection is reduced but not abolished in ANXA1-deficient cells, consistent with additional entry factors acting alongside ANXA1. These findings position ANXA1 as an entry cofactor for PAstV and provide a mechanistic basis to refine models of astrovirus host-cell recognition.},
}
@article {pmid41605211,
year = {2026},
author = {Lin, J and Lin, Y and Liu, N and Cao, W and Zhang, J and Wen, S and Zhang, Y and Liao, W and Hong, Z and Lin, Y and Liu, Q and Liu, H and Li, Q and Chen, B and Li, M and Luo, Z and Yang, L and Yang, Y and Zheng, SH and Wang, Y and Chu, H and Hu, Y and Qin, Y and Luo, BX and Tian, S and Chen, Y and Yan, T and Yang, L and Wang, H and Liu, T and Jiang, Y and Lu, Z},
title = {AAVLINK: A potent DNA-recombination method for large cargo delivery in gene therapy.},
journal = {Cell},
volume = {189},
number = {3},
pages = {969-986.e17},
doi = {10.1016/j.cell.2025.12.039},
pmid = {41605211},
issn = {1097-4172},
mesh = {*Dependovirus/genetics ; Animals ; *Genetic Therapy/methods ; Mice ; Genetic Vectors/genetics ; Humans ; *Recombination, Genetic ; *Gene Transfer Techniques ; Nerve Tissue Proteins/genetics/metabolism ; HEK293 Cells ; CRISPR-Cas Systems ; Transduction, Genetic/methods ; },
abstract = {Delivery of therapeutic genes is essential for successful gene therapy. Adeno-associated viruses (AAVs) are a prime vector for carrying gene cargoes. However, the limited packaging capacity of AAVs poses a major challenge for large gene transduction. Here, we devised a strategy termed AAV with translocation linkage (AAVLINK), leveraging Cre/lox-mediated intermolecular DNA recombination to overcome cargo size constraints. This AAVLINK strategy enabled superior gene segmentation flexibility, robust gene reconstitution efficiency, and a marked reduction in truncated protein products. AAVLINK drove expression of intact Shank3 or SCN1A and rescued behavior and seizure phenotypes of mutant mice, respectively. Moreover, we generated AAVLINK2.0 with destabilized Cre to address biosafety concerns. Importantly, we used AAVLINK to build a vector bank for 193 large genetic-disorder-associated genes and 5 CRISPR-based tools with verified gene reconstitution. Altogether, our study establishes a robust method to facilitate delivery of large gene cargoes using AAVs.},
}
@article {pmid41519897,
year = {2026},
author = {Boutin, J and Fayet, S and Marin, V and Bergès, C and Riandière, M and Toutain, J and Lamrissi-Garcia, I and Thibault, C and Cappellen, D and Dabernat, S and Poulet, A and Francillette, M and Droin, N and Debeissat, C and Brunet de la Grange, P and Moreau-Gaudry, F and Bedel, A},
title = {Single-cell multiplex approaches deeply map ON-target CRISPR-genotoxicity and reveal its mitigation by palbociclib and long-term engraftment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1429},
pmid = {41519897},
issn = {2041-1723},
mesh = {*Piperazines/pharmacology ; Animals ; *Pyridines/pharmacology ; Humans ; *Single-Cell Analysis/methods ; Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mice ; Polymorphism, Single Nucleotide ; Hematopoietic Stem Cells/drug effects/metabolism ; Loss of Heterozygosity ; Female ; },
abstract = {Genome editing by CRISPR-Cas9-nuclease is promising for gene therapy. However, safety concerns remain. Monitoring ON-target genotoxicity is essential, especially to assay megabasic rearrangements at the targeted locus. Here, we developed a combined single-cell resolution approach with DNA sequencing focused on single nucleotide polymorphism (scSNP-DNAseq), micronuclei and LOH cytometry-reporter assays. This sensitive multiplexed strategy enables the sensitive monitoring of CRISPR-mediated genotoxicity in primary cells. Using this approach, we detect, map and characterize various types of induced-losses of heterozygosity and assess editing-associated chromosomal instability. Importantly, palbociclib prevents the appearance of such genomic rearrangements in hematopoietic stem cells without impairing cell fate or graft capability. Conversely, short-term risk is significantly increased with DNA-PKcs inhibitor AZD7648. Fortunately, targeting HBG1/2p, scSNP-DNA-seq reveals that ON-target genotoxic events are no longer detectable after long-term xenografts. This work demonstrates that scSNP-DNA-seq should be routinely implemented to monitor chromosomal rearrangements before and after CRISPR-edited cell infusions.},
}
@article {pmid41519779,
year = {2026},
author = {Lim, MYT and Tan, C and Subhramanyam, CS and Teo, SJ and DeFalco, L and Pasaribu, SK and Koh, CH and Rayamajhi, D and Chi, J and Li, S and Wee, KB and Roy, S and Huber, RG and Aw, SS},
title = {A programmable ribozyme for RNA signal transduction.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1428},
pmid = {41519779},
issn = {2041-1723},
support = {H20C6a0034//Agency for Science, Technology and Research (A*STAR)/ ; UIBR//Agency for Science, Technology and Research (A*STAR)/ ; Cell and Gene Therapy Flagship Grant C253623010//Agency for Science, Technology and Research (A*STAR)/ ; IAF-PP H20C6a0034//Agency for Science, Technology and Research (A*STAR)/ ; OFYIRG16may045//MOH | National Medical Research Council (NMRC)/ ; Core funding//A*STAR | Institute of Molecular and Cell Biology (Institute of Molecular and Cell Biology - A STAR)/ ; },
mesh = {*RNA, Catalytic/metabolism/genetics/chemistry ; Humans ; Animals ; Zebrafish/genetics/embryology ; Aptamers, Nucleotide/metabolism/genetics ; CRISPR-Cas Systems/genetics ; *Signal Transduction/genetics ; Gene Editing/methods ; HEK293 Cells ; MicroRNAs/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA/metabolism/genetics ; },
abstract = {RNA detection applications can be augmented if a sensed RNA can be directly functionally transduced. However, there is no generalisable approach that allows an RNA trigger itself to directly activate diverse non-coding RNA effectors. Here, we report engineering of a programmable, RNA trigger-activated, dual-site self-cleaving ribozyme with modular sensing domain and cleavage product. This platform, UNlocked by Activating RNA (UNBAR), is entirely encoded within one RNA strand. The ribozyme can be designed to be almost completely inactive in absence of trigger, and to exhibit single-nucleotide trigger specificity. UNBAR ribozymes carry out cell-free sensing and protein-free amplification of microRNA and viral RNA sequences, and trigger-dependent release of ncRNA effectors sgRNA, shRNA and aptamer. We demonstrate RNA detection and functional transduction by a cleaved aptamer, whose fluorescence can be directly read out as a function of trigger RNA. We further engineer the ribozyme for function in cells, and demonstrate trigger-dependent regulation of CRISPR-Cas9 editing by sgRNA-embedded ribozymes in zebrafish embryos and human cells. UNBAR is a first-in-class modality with potential to be developed into a versatile platform for synthetic biology, diagnostics and gene regulation.},
}
@article {pmid41478283,
year = {2026},
author = {Zhang, X and Zhang, Y and Liu, X and Liu, C and Liu, Y and He, Y and Qiu, Y and Sun, L and Hu, J and Gao, Y and Wei, W and Liu, J},
title = {FOCAS: Transcriptome-wide screening of individual m[6]A sites functionally dissects epitranscriptomic control of gene expression in cancer.},
journal = {Cell},
volume = {189},
number = {3},
pages = {922-938.e23},
doi = {10.1016/j.cell.2025.11.037},
pmid = {41478283},
issn = {1097-4172},
mesh = {Humans ; *Adenosine/analogs &amp; derivatives/metabolism/genetics ; *Neoplasms/genetics/metabolism ; *Transcriptome/genetics ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems/genetics ; RNA, Messenger/metabolism/genetics ; RNA, Untranslated/metabolism/genetics ; Epigenesis, Genetic ; },
abstract = {Although N[6]-methyladenosine (m[6]A) is a pervasive RNA modification essential for gene regulation, dissecting the functions of individual m[6]A sites remains technically challenging. To overcome this, we developed functional m[6]A sites detection by CRISPR-dCas13b-FTO screening (FOCAS), a CRISPR-dCas13b-based platform enabling high-throughput, site-specific functional screening of m[6]A. Applying FOCAS to four human cancer cell lines identified 4,475 m[6]A-regulated genes influencing cell fitness via both mRNAs and non-coding RNAs (ncRNAs), many of which are newly linked to cancer and exhibit dynamic developmental expression. FOCAS uncovered context-dependent and reader-specific effects of m[6]A within the same gene, revealing its intricate regulatory logic. We further uncovered universal and cell-type-specific m[6]A patterns, with unique sites enriched in ncRNAs and universal ones in transcription-related genes. In SMMC-7721 cells, we identified m[6]A-regulated transcriptional networks that demonstrated extensive epitranscriptome-transcriptome crosstalk. Overall, this study established a powerful, unbiased approach for the functional dissection of m[6]A, advancing the understanding of its complexity and therapeutic relevance in cancers.},
}
@article {pmid41418537,
year = {2026},
author = {Tan, D and Ye, Y and Miao, D and Zhao, C and Wu, S and Shi, J and Yang, J and Fang, K and Lu, F and Lv, Q and Gong, J and Yang, H and Xiao, W and Xiong, Z and Zhang, X and Ruan, H},
title = {UBL3 governs VEGFR inhibitor resistance by activating NOTCH signaling in renal cell carcinoma.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {85},
number = {},
pages = {101332},
doi = {10.1016/j.drup.2025.101332},
pmid = {41418537},
issn = {1532-2084},
mesh = {Humans ; *Carcinoma, Renal Cell/drug therapy/pathology/genetics ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Kidney Neoplasms/drug therapy/pathology/genetics ; Signal Transduction/drug effects ; Animals ; Cell Line, Tumor ; Mice ; Xenograft Model Antitumor Assays ; *Receptors, Vascular Endothelial Growth Factor/antagonists &amp; inhibitors ; *Protein Kinase Inhibitors/pharmacology ; Sunitinib/pharmacology ; Pyridines/pharmacology ; Receptors, Notch/metabolism ; Apoptosis/drug effects ; Mice, Nude ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Anilides ; },
abstract = {BACKGROUND: Targeted therapy is the first-line treatment for patients with metastatic renal cell carcinoma (RCC), with vascular endothelial growth factor receptor inhibitors (VEGFRis) constituting the bulk of regimens used. Although the repertoire of VEGFRis for RCC now spans from sunitinib to cabozantinib, resistance to treatments has emerged as a common and prominent challenge. Thus, identifying novel therapeutic targets has become essential for enhancing the antitumor efficacy of current treatments and inhibiting RCC progression.<br><br>METHOD: To investigate the potential mechanisms underlying VEGFRi resistance in RCC, we performed a genome-wide CRISPR/Cas9 library screen under sunitinib and cabozantinib treatment and identified UBL3 as a key driver of VEGFRi resistance in RCC cells. The critical role of UBL3 in promoting VEGFRi resistance was validated using CCK8 assays, flow cytometry, TUNEL assays, and bioinformatics analyses. To elucidate the molecular mechanisms underlying UBL3, we utilized western blotting, RNA sequencing, chromatin immunoprecipitation, small extracellular vesicles (sEVs) isolation, and Astral-DIA proteomics. The contribution of UBL3 to VEGFRi resistance was further confirmed through comprehensive in vitro and in vivo experiments.<br><br>RESULTS: UBL3 was confirmed to suppress apoptosis and promote VEGFRi resistance through NOTCH signaling activation. Further investigations highlighted the importance of NOTCH signaling in VEGFRi resistance in RCC via the NOTCH-PTEN-AKT and NOTCH-FOS pathways and revealed the mechanisms by which UBL3 activated NOTCH signaling. On the one hand, UBL3 formed complex with NOTCH2 and ADAM17 simultaneously, accelerating ADAM17-mediated cleavage of NOTCH2. On the other hand, UBL3-modified NOTCH2 was sorted into sEVs, which were taken up by recipient cells, activating NOTCH signaling and thereby transmitting VEGFRi resistance. Finally, lipid nanoparticle-mediated delivery of the CRISPR/Cas9 knockout system targeting UBL3 effectively restored the sensitivity of RCC tumors to VEGFRis.<br><br>CONCLUSION: This study emphasized the importance of UBL3 in VEGFRi resistance in RCC and proposed that UBL3 activated NOTCH signaling through two distinct pathways, thereby suppressing cancer apoptosis and promoting resistance to VEGFRis. These findings provided a solid scientific foundation and paved the way for the development of novel therapeutic strategies for patients with advanced RCC.},
}
@article {pmid41416398,
year = {2026},
author = {Hoang, TS and Faraji, F and Mendez-Molina, AN and Adame-Garcia, SR and Sato, K and Ishikawa, T and Vo, PTT and Ramirez, SI and Anguiano Quiroz, PY and Guo, T and Fan, K and Wu, X and Molinolo, AA and Cohen, EEW and Mali, P and Lippman, SM and Gutkind, JS},
title = {Genome-wide CRISPR Screening Reveals a PKA-Driven Resistance Mechanism to Metformin for Oral Cancer Prevention That Can Be Exploited by Combination with NSAIDs.},
journal = {Cancer prevention research (Philadelphia, Pa.)},
volume = {19},
number = {2},
pages = {79-92},
pmid = {41416398},
issn = {1940-6215},
support = {T32 CA121938/CA/NCI NIH HHS/United States ; R01 DE035393/DE/NIDCR NIH HHS/United States ; R25 CA221779/CA/NCI NIH HHS/United States ; U54 CA274502/CA/NCI NIH HHS/United States ; U01 CA290479/CA/NCI NIH HHS/United States ; R01 DE026644/DE/NIDCR NIH HHS/United States ; T32 DC000028/DC/NIDCD NIH HHS/United States ; R01DE026644//National Institute of Dental and Craniofacial Research (NIDR)/ ; U01CA290479//National Cancer Institute (NCI)/ ; SU2C-FARF-FFF//Stand Up To Cancer (SU2C)/ ; 308268//Stand Up To Cancer (SU2C)/ ; T32DT4965//Tobacco-Related Disease Research Program (TRDRP)/ ; T32CA121938//National Cancer Institute (NCI)/ ; T32DC000028//National Institute on Deafness and Other Communication Disorders (NIDCD)/ ; 1061310//American Head and Neck Society (AHNS)/ ; //Takeda Science Foundation (TSF)/ ; //Japan Society for the Promotion of Science (JSPS)/ ; //Rotary Foundation (Rotary)/ ; R25CA221779//National Cancer Institute (NCI)/ ; T34DT8340//Tobacco-Related Disease Research Program (TRDRP)/ ; },
mesh = {*Metformin/pharmacology/therapeutic use ; Humans ; *Anti-Inflammatory Agents, Non-Steroidal/pharmacology/therapeutic use ; *Cyclic AMP-Dependent Protein Kinases/metabolism ; *Mouth Neoplasms/prevention &amp; control/genetics/pathology ; *Drug Resistance, Neoplasm/genetics/drug effects ; CRISPR-Cas Systems ; *Squamous Cell Carcinoma of Head and Neck/genetics/prevention &amp; control/pathology/drug therapy ; Cell Line, Tumor ; Mice ; Drug Synergism ; Animals ; Cell Proliferation/drug effects ; *Antineoplastic Combined Chemotherapy Protocols/pharmacology/therapeutic use ; Signal Transduction/drug effects ; Cyclooxygenase 2 Inhibitors/pharmacology ; },
abstract = {UNLABELLED: Head and neck squamous cell carcinoma (HNSCC) is among the 10 most common cancers worldwide and is associated with high morbidity and poor survival. Diminished HNSCC outcomes are often related to delayed diagnosis and treatment of occult progression of premalignant lesions, underscoring the need for effective and low-risk chemoprevention strategies. In this regard, metformin has shown promising clinical activity for HNSCC prevention. In this study, we performed a genome-wide CRISPR/Cas9 screen of metformin-treated HNSCC cells and identified the activation of PKA signaling as the top resistance pathway. We show that metformin mediates PKA activation in HNSCC cells and that PKA inhibition, when combined with metformin treatment, synergistically inhibits HNSCC growth. We found that metformin-induced PKA activation is mediated by a prostaglandin E2 autocrine loop, which can be blocked using cyclooxygenase-2 (COX2) inhibitors. Importantly, COX2 inhibition using nonsteroidal anti-inflammatory drugs (NSAID) combined with metformin treatment synergistically inhibits HNSCC cell growth and prevents the progression of oral premalignant lesions into invasive HNSCC in a model of tobacco-driven oral carcinogenesis. Together, these findings demonstrate that metformin and NSAID combination therapy may represent a promising therapeutic strategy for HNSCC chemoprevention.<br><br>PREVENTION RELEVANCE: Our findings reveal that using metformin for head and neck cancer chemoprevention leads to compensatory activation of a PKA-driven resistance mechanism that can be blocked by cotreatment with NSAIDs. These findings provide a rationale for combining metformin with NSAIDs as a precision head and neck cancer chemoprevention strategy.},
}
@article {pmid41317988,
year = {2026},
author = {Arya, SK and Goodman, CL and Palli, SR},
title = {The expanding toolkit of insect cell culture: a new era in biotechnology.},
journal = {Current opinion in insect science},
volume = {74},
number = {},
pages = {101465},
doi = {10.1016/j.cois.2025.101465},
pmid = {41317988},
issn = {2214-5753},
mesh = {Animals ; *Insecta/genetics/cytology ; *Biotechnology/methods ; *Cell Culture Techniques/methods ; Cell Line ; Baculoviridae/genetics ; CRISPR-Cas Systems ; },
abstract = {Insect cell culture has become an essential platform in modern biotechnology, valued for its safety, scalability, and ability to perform complex post-translational modifications. This review highlights the latest and most important advances in the field. We focus on efforts at developing and engineering new insect cell lines, innovations in expression systems, especially the baculovirus expression vector system and the transformative impact of CRISPR/Cas9-based genome editing. Additionally, we explore breakthroughs that improve the efficiency of recombinant protein production and discuss key challenges such as viral contamination and expression instability. Collectively, these developments mark an important step forward in insect cell biotechnology and are expected to enhance the efficiency and scalability of producing vaccines and biopharmaceuticals. Together, these innovations illustrate a transition from cataloging cell line development to understanding the mechanisms and engineering principles driving these advances. This review not only summarizes recent progress but also provides perspective on how foundational lepidopteran models have guided innovations now extending into dipteran, hemipteran, and hymenopteran systems, shaping the future of insect biotechnology.},
}
@article {pmid41629010,
year = {2026},
author = {Saini, A and Sharma, N and Sharma, N and Kumari, N and Sharma, M and Singh, B and Thakur, AK},
title = {Precision pest management: Genome editing tools, specifically CRISPR/Cas9 and future prospects.},
journal = {Pesticide biochemistry and physiology},
volume = {218},
number = {},
pages = {106941},
doi = {10.1016/j.pestbp.2026.106941},
pmid = {41629010},
issn = {1095-9939},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Insecta/genetics ; *Pest Control, Biological/methods ; *Pest Control/methods ; Insect Control/methods ; },
abstract = {The growing resistance to synthetic insecticides and Bt toxins, alongside persistent crop losses despite heavy pesticide application, highlights the urgent need for safer, sustainable and efficient pest management strategies. This review presents genome editing as a precise and versatile approach to reduce pest impact by altering fertility, feeding patterns or vulnerability, while protecting beneficial organisms. Among the genome editing tools, CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) is one of the most promising genome editing techniques in insects. It facilitates targeted functional studies, integration with RNAi and dual-expression systems and gene drive applications. Deployment is envisioned in two phases, initial laboratory modification followed by regulated field release, with a strong emphasis on biosafety through terminator genes, marked individuals for gene flow monitoring, optimized dosages, stringent screening and long-term ecological surveillance, along with transparency and adherence to international safety protocols. Significant challenges encompass delivery efficiency, identification of edits, off-target mutations, dose-related efficacy and sterility, unstable transmission and resistance development. Innovations such as base and prime editing minimize unintended mutations by circumventing double-stranded breaks (DSBs), while paratransgenic strategies targeting gut symbionts offer supplementary avenues; plant-mediated insect gene editing emerges as a promising frontier. Overall, carefully regulated trials aligned with policy frameworks and stakeholder involvement are vital to assess effectiveness in natural environments and achieve targeted, dependable and ecologically responsible pest control.},
}
@article {pmid41628563,
year = {2026},
author = {Liang, L and Xu, B and Xiao, S and Mu, X and Zhao, S and Tian, J},
title = {A new split DNA-based activation of CRISPR/Cas12a for amplification-free and dual-stimulus responsive detection and precise imaging of miRNA-221.},
journal = {Talanta},
volume = {303},
number = {},
pages = {129443},
doi = {10.1016/j.talanta.2026.129443},
pmid = {41628563},
issn = {1873-3573},
abstract = {The CRISPR/Cas12a system is a genome editing technology that has been widely applied in biosensing and molecular diagnostics. However, the detection and regulation of its core components remain challenging. Therefore, we constructed a new split DNA-based activation method for the regulation of CRISPR/Cas12a, and based on that, an APE1-assisted activation CRISPR/Cas12a system for miRNA detection and a precise imaging method was also developed without amplification and complex design. Two split DNA were used as activators and embedded in two hairpins. When APE1 and miRNA-221 were simultaneously input, the DNA logic gate was started, thus releasing the determinant activation chain to activate the trans-shearing activity of the CRISPR/Cas12a system, so that the fluorescent probe signal can be significantly recovered. Different cleavage-activated chain hairpins were designed, and the influence on the trans-shear activity of CRISPR/Cas12a and the activation effect were discussed. And the method was successfully applied to detect the expression levels of miRNA-221 in cell lysates. The detection limit for miRNA-221 is 9.71 pmol/L (S/N = 3). At the same time, the method was applied for precise imaging of miRNA-221 within different cells and can effectively distinguish tumor cells. This study combines the regulation of the CRISPR/Cas system by split activators with the advantages of dual-responsive DNA logic circuits. The dual-response activation design effectively reduces false positive signals, thereby enhancing the detection and imaging accuracy. This method provides a novel design concept for utilizing split-DNA activation of the CRISPR/Cas system for nucleic acid detection and cell imaging.},
}
@article {pmid41627753,
year = {2026},
author = {Arnould, K and El Kadri, M and Hervé, P and Asencio, C and Plazolles, N and Monic, S and Morel, CA and Rivière, L and Bringaud, F and Tetaud, E},
title = {Precision Without Selection: A Marker-Free CRISPR/Cas9-Based Protocol for Multiplexed Genome Editing in Trypanosomatids.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3014},
number = {},
pages = {65-78},
pmid = {41627753},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Protozoan ; Electroporation ; *Trypanosomatina/genetics ; Ribonucleoproteins/genetics ; },
abstract = {The CRISPR-Cas9 system has become a valuable tool for genome editing in trypanosomatid parasites such as Trypanosoma and Leishmania species. Although these organisms have been genetically engineered for a long time using homologous recombination, CRISPR/Cas9 offers improved efficiency for genome editing. However, conventional strategies employing stable Cas9 expression require the persistent use of a specific genetic background (i.e., strains expressing Cas9), depend on selectable resistance markers, compromise genomic stability, and are not readily applicable to diverse strain backgrounds. Herein, we report an optimized marker-free CRISPR/Cas9 method based on transient ribonucleoprotein (RNP) delivery that overcomes these drawbacks. Our method eliminates the need for plasmid integration or antibiotic selection while maintaining high editing efficiency. The protocol comprises the following steps: (1) design of the guide RNA (gRNA), (2) design of the repair template (cassette), (3) assembly of the ribonucleoprotein (RNP) complex, (4) delivery by electroporation, and (5) clonal screening through PCR and sequencing. The procedure permits rapid (≤3 weeks) production of homozygous mutant lines in wild-type strains, including low-density culture strains. The reproducibility and ease of the technique render it particularly suited for multiplexed editing of polyploid genomes, multi-gene families, and several different genes at once, as well as validation of the essential nature of genes. Although designed for trypanosomatids, the workflow can be adapted to other kinetoplastids, offering a flexible platform for functional genomics.},
}
@article {pmid41627331,
year = {2026},
author = {Roy, N and Debnath, P and Srivastava, S and Gaur, HS},
title = {Recent developments in CRISPR/Cas9 genome editing research for edible fungiculture.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {36},
pmid = {41627331},
issn = {1438-7948},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Genome, Fungal ; *Agaricales/genetics/growth &amp; development ; },
abstract = {Fungiculture refers to the deliberate cultivation or agricultural practice involving the growth and management of fungi. The practice encompasses the intentional culture of diverse species of macrofungi, including mushrooms and truffles, within controlled habitats or under specified conditions, in order to fulfill human requirements especially for food purpose. As the global market for edible mushrooms grows quickly, it is becoming increasingly necessary to grow novel and improved strains of edible fungi. Growing and breeding edible fungi using traditional methods is both time-consuming and difficult. So, there is a need for evolving advanced techniques at a molecular level which can help breeding of edible fungi with much better efficiency. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system is one of the most effective techniques for accurately cutting and modifying the genomes of edible fungi. In this review, we discuss how genome editing using CRISPR/Cas9 has been utilized in many edible fungal species such as Pleurotus ostreatus, Agaricus bisporus, Cordyceps militaris, Ganoderma lucidum, Flammulina filiformis, Lentinula edodes, and others for their target specific breeding. We also discuss the working mechanism of the above-mentioned system in these mushroom species, and also the advantages and limitations of using this system in mushrooms.},
}
@article {pmid41627751,
year = {2026},
author = {Nagamine, S and Oishi, R and Nakazawa, M},
title = {Engineering Wax Ester Composition in Euglena gracilis Using Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3014},
number = {},
pages = {37-49},
pmid = {41627751},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Euglena gracilis/genetics/metabolism ; CRISPR-Cas Systems ; *Metabolic Engineering/methods ; *Esters/metabolism/chemistry ; *Waxes/metabolism/chemistry ; },
abstract = {Genome editing technologies have significantly expanded the potential for metabolic engineering in non-model organisms. In Euglena gracilis, genome editing methods using Cas9 and Cas12a were reported in 2019 and 2024, respectively, and are increasingly being applied to modify metabolic functions. This chapter provides a detailed protocol for CRISPR/Cas9-based genome editing that enables stable modification of wax ester composition under anaerobic conditions. By targeting key enzymes in the reversed β-oxidation pathway, the method allows the generation of knockout mutants with altered wax ester chain lengths. Beyond this application, the protocol supports reproducible and stable genetic modification of E. gracilis metabolism. It can be extended to the engineering of other biosynthetic pathways and is compatible with future integration of knock-in strategies. The approach offers a practical basis for the broader use of E. gracilis as a green chassis organism in synthetic biology and biomanufacturing.},
}
@article {pmid41581816,
year = {2026},
author = {Xiao, Y and Yang, J and Yang, W and Yuan, M and Zhang, Y and Liu, J and Zhang, Y and Zhu, H and Luo, G},
title = {A rapid on-site diagnostic method for goose parvovirus disease based on recombinase polymerase amplification and CRISPR/AsCas12a.},
journal = {International journal of biological macromolecules},
volume = {343},
number = {Pt 2},
pages = {150496},
doi = {10.1016/j.ijbiomac.2026.150496},
pmid = {41581816},
issn = {1879-0003},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Geese/virology ; *Parvoviridae Infections/diagnosis/veterinary/virology ; *Parvovirus/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; *Poultry Diseases/diagnosis/virology ; Ducks/virology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Goose parvovirus (GPV) is a highly pathogenic and lethal virus responsible for Derzsy's disease in goslings and ducklings, significantly influencing the economic viability of waterfowl farming. This necessitates the development of rapid diagnostic techniques for effective disease management. In this study, an optimized clustered regularly interspaced short palindromic repeats (CRISPR)/Acidaminococcus sp. CRISPR associated nuclease 12a (AsCas12a) system was developed for the diagnosis of GPV. The study determined that the optimal conditions for the CRISPR/Cas12a-based fluorescence assay were 20 nM AsCas12a, 5 nM crRNA, and 5 nM single-stranded DNA (ssDNA), whereas the lateral flow assay (LFA) required 20 nM AsCas12a and 4 nM crRNA. Moreover, the fluorescence-based assay and LFA achieved minimum detection limits of 7.8 copies/μL and 78 copies/μL, respectively, representing 1000-fold and 100-fold improvements over conventional PCR methods. Both detection methods exhibited high specificity and demonstrated no cross-reactivity with other prevalent waterfowl pathogens, such as duck plague virus, duck hepatitis viruses, H5 avian influenza virus, waterfowl astrovirus, reovirus, Muscovy duck parvovirus, and novel GPV. The results of the LFA were in complete concordance with laboratory qPCR analyses, thereby affirming their reliability for clinical diagnostics. In conclusion, we have successfully developed a dual-readout GPV detection system utilizing CRISPR/Cas12a technology, which holds significant promise for the early surveillance and containment of GPV outbreaks.},
}
@article {pmid41577322,
year = {2026},
author = {Wang, L and Miao, M and Bao, L and Chu, J and Zhou, J and Song, W and Cai, P and Cheng, C and Xu, H and Wang, T and Zhao, R and Wang, H and Liu, F and Xu, M and Tian, G},
title = {CRISPR/Cas9-based genome-wide screen reveals a synergistic effect of Irinotecan and USP1 inhibitor in colorectal cancer.},
journal = {European journal of pharmacology},
volume = {1015},
number = {},
pages = {178558},
doi = {10.1016/j.ejphar.2026.178558},
pmid = {41577322},
issn = {1879-0712},
mesh = {Humans ; *Irinotecan/pharmacology/therapeutic use ; *Colorectal Neoplasms/drug therapy/genetics/pathology ; Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Ubiquitin-Specific Proteases/antagonists &amp; inhibitors/metabolism ; Drug Synergism ; Cell Proliferation/drug effects ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Drug Resistance, Neoplasm/drug effects ; HCT116 Cells ; Gene Expression Regulation, Neoplastic/drug effects ; Antineoplastic Combined Chemotherapy Protocols/pharmacology ; },
abstract = {Irinotecan resistance remains a significant challenge in metastatic colorectal cancer (mCRC) therapy. To address this, we identified USP1 as a synthetic lethal partner of Irinotecan through genome-wide CRISPR/Cas9 screening in HCT-116 cells. Combining the USP1 inhibitor I-138 with Irinotecan in HCT-116, HT-29, and SW620 cell lines significantly reduced IC50, suppressed proliferation, and diminished colony formation compared to monotherapy, demonstrating a synergistic effect (combination index CI < 1). The synergistic therapeutic efficacy was further validated in the xenograft mouse model. Mechanistic studies revealed that I-138 significantly upregulated pCREB (Ser133), concurrently dynamically regulating the activity of USP1, FANCD2/FANCI, and PCNA upon DNA damage response and repair. RNA sequencing further highlighted the enrichment of cAMP, PI3K-AKT, and Wnt pathways, which are all linked to CREB activity in the combination group. These findings establish USP1 inhibition as a promising strategy to overcome Irinotecan resistance through the combination strategy, providing a novel therapeutic avenue for CRC.},
}
@article {pmid41526335,
year = {2026},
author = {Gao, J and Bader, A and Linder, MI and Cheng, J and Richter, M and da Costa, R and Zehrer, A and Mitt, K and Popper, B and Meissner, F and Wei, X and de Vega Gómez, E and Tatematsu, M and Rohlfs, M and Frenz-Wiessner, S and Kiziltug, M and Somekh, I and Yacobovich, J and Steinberg-Shemer, O and Somech, R and Soehnlein, O and Schmid, B and Klein, C and Walzog, B and Maier-Begandt, D},
title = {Mutations in VPS18 lead to a neutrophil maturation defect associated with disturbed vesicle homeostasis.},
journal = {Cell death &amp; disease},
volume = {17},
number = {1},
pages = {180},
pmid = {41526335},
issn = {2041-4889},
support = {CRC914 (project A02)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; TRR332 (#449437943; project C03)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC914 (project A13)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; TRR332 (#449437943; project A02)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; TRR332 (#449437943; project B01)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC914 (projects A02 and Z03)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Neutrophils/metabolism/pathology ; Animals ; Humans ; *Vesicular Transport Proteins/genetics/metabolism ; Zebrafish ; *Mutation/genetics ; *Homeostasis ; Cell Differentiation ; Induced Pluripotent Stem Cells/metabolism ; Autophagy ; CRISPR-Cas Systems ; Neutropenia/genetics/pathology ; Autophagosomes/metabolism ; Male ; },
abstract = {Neutrophils, the first cells to arrive at the site of inflammation, are rather short-lived cells and thus have to be constantly replenished. During neutrophil development, vesicle dynamics need to be fine-tuned and impaired vesicle trafficking has been linked to failure in neutrophil maturation. Here, we characterized the role of VPS18 as a central core component of CORVET &amp; HOPS tethering complexes for neutrophil development. Using CRISPR/Cas9-engineered Hoxb8 cells with heterozygous mutations in Vps18, we found that VPS18 deficiency interfered with neutrophil development due to tethering complex instability. As a result, vesicle dynamics were impaired with a strong increase in LC3B-II and p62 levels, indicating autophagosome accumulation and reduced autophagic flux. With transmission electron microscopy, we verified the increase in autophagosomes and also found irregularly shaped vesicular structures in Vps18 mutants. Subsequently, Vps18 mutant neutrophil progenitors underwent premature apoptosis. We described a novel patient with a heterozygous stop-gain mutation in VPS18 suffering from neutropenia and recurrent infections. To verify our findings in the human system, we used human induced pluripotent stem cells (iPSCs). Upon differentiation into neutrophils, loss of VPS18 resulted in an almost complete absence of iPSC-derived developing neutrophils. Heterozygous VPS18 mutant and patient mutation-harboring iPSCs were characterized by strongly reduced numbers of developing neutrophils. Zebrafish larvae with heterozygous mutations in vps18 were also characterized by significantly reduced neutrophil numbers. This study shows the pivotal impact of VPS18 for adequate vesicle dynamics during neutrophil development which might be relevant in the context of vesicle trafficking during granulopoiesis and congenital neutropenia.},
}
@article {pmid41520927,
year = {2026},
author = {Nishizawa, H and Daimon, T},
title = {DIL-CRISPR: a practical approach to mitigate G0 mosaic lethality in insect gene editing.},
journal = {Insect biochemistry and molecular biology},
volume = {188},
number = {},
pages = {104492},
doi = {10.1016/j.ibmb.2026.104492},
pmid = {41520927},
issn = {1879-0240},
mesh = {Animals ; *Gene Editing/methods ; *Spodoptera/genetics/growth &amp; development ; *CRISPR-Cas Systems ; *Mosaicism ; Larva/genetics/growth &amp; development ; Genes, Lethal ; },
abstract = {Genome editing in insects is typically conducted by injecting genome editing reagents into early embryos, producing generation zero (G0) individuals that develop as genetic mosaics. Targeting genes whose disruption induces mosaic lethality is therefore challenging, since most or all G0 individuals frequently fail to survive to adulthood, preventing germline transmission of edited alleles. Here, we present a straightforward and practical approach, DIL-CRISPR, to mitigate G0 mosaic lethality by systematically diluting the CRISPR/Cas9 injection mix. Using the tobacco cutworm Spodoptera litura and the juvenile hormone receptor gene Met1 as a benchmark, we demonstrate that dilution of the injection mix lessens the severity of larval-pupal mosaic phenotypes and increases G0 survival in a dose-dependent manner. Amplicon sequencing further showed that somatic mutation frequencies decline with dilution, while germline mutation rates remain sufficient to establish mutant lines. Notably, we detected a substantial discrepancy between somatic and germline editing efficiencies, likely reflecting selective loss of highly edited, lethal mosaics before they reach adulthood. We conclude that DIL-CRISPR therefore offers a reliable means to balance G0 survival with germline editing, converting an empirically used dilution practice into a generalizable strategy. Overall, this approach provides a practical solution for generating mutant lines of mosaic lethal genes and is broadly applicable across diverse insect species, facilitating functional genetic studies in non-model insects.},
}
@article {pmid41519853,
year = {2026},
author = {Michael Deans, PJ and Retallick-Townsley, KG and Li, A and Seah, C and Johnson, J and Garcia Gonzalez, J and Cao, E and Schrode, N and Yu, A and Cartwright, S and Voloudakis, G and Zhang, W and Wang, M and Fullard, JF and Girdhar, K and Stahl, E and Akbarian, S and Zhang, B and Roussos, P and O'Reilly, P and Huckins, LM and Brennand, KJ},
title = {Functional implications of polygenic risk for schizophrenia in human neurons.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1355},
pmid = {41519853},
issn = {2041-1723},
support = {R01ES033630//U.S. Department of Health &amp; Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)/ ; R56MH101454//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; },
mesh = {Humans ; *Schizophrenia/genetics ; *Multifactorial Inheritance/genetics ; *Neurons/metabolism ; Induced Pluripotent Stem Cells/metabolism ; *Genetic Predisposition to Disease ; Genome-Wide Association Study ; CRISPR-Cas Systems ; Transcriptome ; Polymorphism, Single Nucleotide ; },
abstract = {Genome wide association studies of schizophrenia reveal a complex polygenic risk architecture comprised of hundreds of risk variants; most are common in the population, non-coding, and act by genetically regulating the expression of one or more gene targets ("eGenes"). It remains unclear how the myriad genetic variants that are predicted to confer individually small effects combine to yield substantial clinical impacts in aggregate. Here, we demonstrate that convergence (i.e., the shared downstream transcriptomic changes with a common direction of effect), resulting from one-at-a-time perturbation of schizophrenia eGenes, influences the outcome when eGenes are manipulated in combination. In total, we apply pooled and arrayed CRISPR approaches to target 21 schizophrenia eGenes in human induced pluripotent stem cell-derived glutamatergic neurons, finding that functionally similar eGenes yield stronger and more specific convergent effects. Points of convergence constrain additive relationships between polygenic risk loci: consistent with a liability threshold model, combinatorial perturbations of these same schizophrenia eGenes reveal that pathway-level convergence predicts when observed effects will fail to sum to levels predicted by an additive model. Targeting points of convergence as novel therapeutic targets may prove more efficacious than individually reversing the effects of multiple risk loci.},
}
@article {pmid41507381,
year = {2026},
author = {Gonzales, F and Schneider, C and Alexe, G and Lin, S and Khalid, D and Alvarez, M and Basanthakumar, A and Ellegast, JM and Merickel, L and Salhotra, S and Taillon, A and Giaimo, M and Wunderlich, M and Ansari, M and Perry, JA and Degar, B and Pikman, Y and Stegmaier, K},
title = {Identifying targeted therapies for CBFA2T3::GLIS2 acute myeloid leukemia.},
journal = {Leukemia},
volume = {40},
number = {2},
pages = {383-396},
pmid = {41507381},
issn = {1476-5551},
mesh = {Humans ; *Leukemia, Myeloid, Acute/drug therapy/genetics/pathology/metabolism ; Animals ; Mice ; CRISPR-Cas Systems ; Janus Kinase 2/antagonists &amp; inhibitors/genetics ; Nitriles ; *Molecular Targeted Therapy ; Pyrazoles/pharmacology ; Protein Kinase Inhibitors/pharmacology ; Xenograft Model Antitumor Assays ; Cell Proliferation ; Pyrimidines ; Cell Line, Tumor ; Apoptosis ; Drug Resistance, Neoplasm ; },
abstract = {CBFA2T3::GLIS2-positive pediatric acute myeloid leukemia (AML) remains one of the worst prognostic AML subgroups. To uncover innovative targeted therapy approaches in this disease subtype we performed genome-scale CRISPR-Cas9 screening that highlighted a strong, selective dependency on JAK2 compared to other types of cancer. Using a doxycycline-inducible JAK2 knockout (KO) system, we validated JAK2 dependency in CBFA2T3::GLIS2 cell lines, observing impaired proliferation in vitro and in vivo and apoptosis induction in vitro. Both type I (ruxolitinib) and type II (CHZ868) JAK2 inhibitors showed selective in vitro activity in CBFA2T3::GLIS2-positive AML models. To identify resistance and sensitizer mechanisms to JAK2 inhibitors, we used CRISPR-Cas9 ruxolitinib anchor screening in CBFA2T3::GLIS2 AML. sgRNAs targeting negative regulators of the MAPK pathway were enriched in the ruxolitinib-treated cells. Similarly, CBFA2T3::GLIS2 AML sublines grown to resistance under chronic ruxolitinib treatment expressed pathogenic NRAS mutations. Both approaches converged on MAPK pathway activation as a resistance mechanism to ruxolitinib treatment. Combining ruxolitinib with MEK inhibitors showed a synergistic effect in cell lines and patient-derived xenograft (PDX) cells expressing the fusion and in vivo activity in a CBFA2T3::GLIS2 AML PDX, suggesting a potential approach to target this signaling circuitry in this poor outcome AML subtype.},
}
@article {pmid41501085,
year = {2026},
author = {Singh, P and D'Rozario, R and Chakraborty, B and Meher, S and Raychaudhuri, D and Tannir, AJ and Li, Y and Majumdar, A and Hawkins, J and Xiong, Y and Lorenzi, P and Sharma, P and Akdemir, K and Pilie, P and Jain, AK and Lee, BHL and Goswami, S},
title = {Loss of KDM6A-mediated genomic instability and metabolic reprogramming regulates response to therapeutic perturbations in bladder cancer.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1382},
pmid = {41501085},
issn = {2041-1723},
mesh = {*Urinary Bladder Neoplasms/genetics/drug therapy/metabolism ; Animals ; *Histone Demethylases/genetics/metabolism ; Humans ; Mice ; *Genomic Instability/genetics ; Cell Line, Tumor ; Cisplatin/therapeutic use/pharmacology ; Drug Resistance, Neoplasm/genetics ; CRISPR-Cas Systems ; DNA Repair/genetics ; Female ; Mice, Knockout ; Loss of Function Mutation ; Histones/metabolism ; Metabolic Reprogramming ; },
abstract = {Mutations in epigenetic regulators are common in bladder cancer, yet their impact on therapeutic responses remains unclear. Here, we identify that loss-of-function mutations in KDM6A, a histone demethylase altered in about 26% of advanced bladder cancers, are associated with poor survival after cisplatin chemotherapy, whereas they correlate with improved outcomes with anti-PD-1 therapy. Using CRISPR-Cas9-engineered murine and human bladder cancer models, we show that KDM6A deficiency increases formation of extrachromosomal circular DNA carrying chemoresistance loci, promoting cisplatin resistance. In parallel, KDM6A loss impairs DNA repair and rewires tumor metabolism, reducing glycolysis and lactate output. This metabolic shift diminishes histone lactylation in regulatory T cells, suppressing immunoregulatory genes and limiting expansion of PD-1[hi] regulatory T cells. Collectively, our findings establish KDM6A mutation as a key regulator of therapeutic responses, providing a foundation for its use in guiding precision therapy in advanced bladder cancer.},
}
@article {pmid41495049,
year = {2026},
author = {Li, X and Shang, X and Liu, J and Zhang, Y and Jia, X and Li, H and Wang, Y and Gao, J and Ma, X and Zhang, X and Rong, X and Gan, W and Zhang, Y and Chen, J and Wang, L and Bao, Z and He, L and Yan, X and Liu, Y and Shao, J and Xiao, Z and Wang, Z and Zhu, H and Wang, Z and Wu, Y and Huang, Y},
title = {Intrathecal CRISPR-edited allogeneic IL-13Rα2 CAR T Cells for recurrent high-grade Glioma: preclinical characterization and phase I trial.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1362},
pmid = {41495049},
issn = {2041-1723},
mesh = {Humans ; Female ; Male ; *Immunotherapy, Adoptive/methods/adverse effects ; *Interleukin-13 Receptor alpha2 Subunit/genetics/immunology ; Middle Aged ; Adult ; *Glioma/therapy/immunology/pathology ; CRISPR-Cas Systems/genetics ; *Receptors, Chimeric Antigen/genetics/immunology ; *T-Lymphocytes/immunology/transplantation/metabolism ; *Brain Neoplasms/therapy/immunology/pathology ; Gene Editing ; Neoplasm Recurrence, Local/therapy ; Animals ; Graft vs Host Disease/prevention &amp; control/immunology ; Glioblastoma/therapy/immunology ; Mice ; Killer Cells, Natural/immunology ; Aged ; Treatment Outcome ; Cell Line, Tumor ; },
abstract = {Patients with recurrent high-grade glioblastoma have a median survival of 6-8 months, with limited therapeutic options. In recent years, interest has grown in applying chimeric antigen receptor T (CAR-T) cells to solid cancers, including advanced gliomas. Here we generated off-the-shelf CRISPR-Cas9-edited IL-13Rα2-specific allogeneic universal CAR-T cells (MT026) by disrupting the endogenous TCR to prevent graft-versus-host disease and knocking out HLA class I molecules to mitigate the host-versus-graft response, and observed minimal NK-cell-mediated rejection in preclinical studies. In a first-in-human, single-center, open-label investigator-initiated trial (ChiCTR2000028801) in patients with high-grade glioma with prior therapy failure and short life expectancy, intrathecal injection of MT026 via lumbar puncture (1.0-3.0×10^7 cells per dose) demonstrated favorable tolerability and safety (primary outcome), pharmacokinetic characteristics, and preliminary clinical activity (secondary outcomes). Among the five patients enrolled, one achieved a complete response and three achieved partial responses. No grade ≥3 adverse events were observed; the predominant treatment-related toxicities were grade 1-2 pyrexia, hypoxia, and vomiting. Trial enrolment was halted after enrolment of the first five patients, however these preliminary clinical data support the potential benefit of locally administered allogeneic universal CAR-T cell therapy for recurrent glioblastoma.},
}
@article {pmid41494573,
year = {2026},
author = {Pu, Z and Wang, X and Chen, Y and Li, J and He, X and Chen, W and Zhao, C},
title = {Application of CRISPR/Cas9 gene editing system in microalgal metabolic engineering and synthetic strategies of functional food ingredients.},
journal = {Biotechnology advances},
volume = {87},
number = {},
pages = {108796},
doi = {10.1016/j.biotechadv.2026.108796},
pmid = {41494573},
issn = {1873-1899},
mesh = {*Microalgae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Metabolic Engineering/methods ; *Functional Food ; *Food Ingredients ; },
abstract = {Microalgae are natural and sustainable biological resources rich in high-value nutrients such as lipids, proteins, and functional pigments, which show great potential in the fields of functional foods, dietary supplements, and natural colorants. However, the yields of target components in natural microalgae are often insufficient to meet commercialization demands. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system, a revolutionary technology, provides a precise and effective means for targeted improvement of microalgae to enhance their nutritional value and yields. This review first outlines the basic principles of the CRISPR/Cas9 system, including its core components and gene editing mechanism. It then summarizes the application of this technology in microalgae, focusing on successful cases of modifying metabolic pathways to enrich specific nutrients, such as increasing the unsaturated fatty acid content of lipids, increasing the proportion of edible proteins, and enriching natural pigments with antioxidant properties. In addition, this review discusses the main challenges faced when applying this technology to microalgae, including delivery difficulties due to strong cell walls, low efficiency of genetic transformation, and the risk of off-target effects. Finally, the paper describes cutting-edge strategies to address these challenges, such as the development of high-fidelity Cas9 enzymes and the optimization of a single-guide RNA (sgRNA) design. Continued advances in these technologies are propelling microalgae into efficient and sustainable "cell factories", providing the food industry with more natural, healthy, and high-value functional ingredients.},
}
@article {pmid41430241,
year = {2025},
author = {Nieto-Sanchez, A and Martinez-Lage, M and Puig-Serra, P and Carpintero, S and Alonso-Yanez, A and Ojeda-Walczuk, P and Ibañez-Navarro, M and Pita, G and Moya, FJ and Moreno, C and Martin, MC and Alonso, R and Nuñez-Torres, R and Sanchez-Arevalo Lobo, VJ and Alonso-Guirado, L and Malats, N and Gonzalez-Neira, A and Fernandez, L and Roda-Navarro, P and Torres-Ruiz, R and Rodriguez-Perales, S},
title = {Selective genome editing of amplified oncogenes triggers immunogenic cell death and tumor remodeling.},
journal = {Molecular cancer},
volume = {25},
number = {1},
pages = {21},
pmid = {41430241},
issn = {1476-4598},
mesh = {Humans ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; Mice ; *Oncogenes/genetics ; Cell Line, Tumor ; *Immunogenic Cell Death/genetics ; *Gene Amplification ; *Neoplasms/genetics/pathology/immunology ; Tumor Microenvironment/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {Oncogene amplifications fuel some of the most lethal, therapy‑refractory cancers, yet remain clinically untargeted. We report a single‑guide CRISPR/Cas9 strategy that converts the sheer copy‑number excess of oncogene amplicons into an Achilles' heel. A solitary intronic double‑strand break is innocuous in diploid genomes but collapses oncogene amplification‑positive cells across neuroblastoma, small‑cell lung and colorectal carcinoma models, driving > 90% loss of viability, G2/M blockade and catastrophic DNA‑damage signalling. Amplified‑locus cleavage rewires transcription toward cell death activation, necroptosis and cGAS-STING-mediated immunogenic cell death, enabling dendritic‑cell cross‑priming and T‑cell activation and proliferation. In xenografts, delivery of the intronic sgRNA shrinks tumours by 90%, prolongs survival and remodels the innate tumour microenvironment. Deep sequencing confirms negligible off‑target editing, and combination with doxorubicin achieves supra‑additive killing. These findings establish amplification density, not sequence content, as a tractable, tumour‑exclusive target and unveil a dual‑action platform that is simultaneously cytotoxic and immunostimulatory. Editing of tumor amplifications therefore offers a blueprint for translating copy‑number aberrations into precision genome‑editing therapies for treatment‑resistant cancers.},
}
@article {pmid41254880,
year = {2026},
author = {Zhang, L and Xiang, C and Zheng, N and Kang, H and Kong, L and Huang, W and Peng, H and Lian, Y and Lu, W and Gao, M and Wang, J and Meksem, K and Peng, D and Yu, F and Liu, S},
title = {GmSHMT08-Mediated Soybean Cyst Nematode Resistance Is Negatively Modulated by Two Heat Shock Protein 70s in Soybean.},
journal = {Plant, cell &amp; environment},
volume = {49},
number = {3},
pages = {1214-1228},
doi = {10.1111/pce.70296},
pmid = {41254880},
issn = {1365-3040},
support = {//This work was supported by the Joint Fund for S&amp;T Research and Development Programme of Henan, China (grant no. 222301420028), the China Postdoctoral Science Foundation (grant no. 2023M741140), the Postdoctoral Fellowship Programme of CPSF (grant no. GZC20230772), the National Natural Science Foundation of China (grant no. 31972248), and the National Key Research and Development Programme of China (grant no. 2023YFD1400400)./ ; //This work was supported by the National Key Research and Development Programme of China(grant no. 2023YFD1400400), the China Postdoctoral Science Foundation (grant no. 2023M741140), the Postdoctoral Fellowship Programme of CPSF (grant no. GZC20230772), the Joint Fund for S&amp;T Research and Development Programme of Henan, China (grant no. 222301420028), and the National Natural Science Foundation of China (grant no. 31972248)./ ; },
mesh = {*Glycine max/genetics/parasitology/metabolism/immunology ; *Disease Resistance/genetics ; Animals ; *Plant Diseases/parasitology/immunology/genetics ; *HSP70 Heat-Shock Proteins/metabolism/genetics ; *Plant Proteins/metabolism/genetics ; *Tylenchoidea/physiology ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {The soybean GmSHMT08 (Rhg4) is a major gene conferring resistance to soybean cyst nematode (SCN), which is a devastating pathogen in soybean, yet its underlying resistance mechanism remains elusive. Heat shock protein 70 (HSP70) mainly functions in maintaining protein homoeostasis and regulating plant immunity. In this study, CRISPR/Cas9-mediated knockout of two highly similar GmHSP70s [GmHSP70-13a (Glyma.13g254900) and GmHSP70-15a (Glyma.15g059900)] enhanced, whereas overexpressing either of them suppressed, SCN resistance. Both GmHSP70-13a and GmHSP70-15a interacted with the key one-carbon metabolism enzyme GmSHMT08, and promoted GmSHMT08 degradation via 26S proteasome pathway, but neither of them altered GmSHMT08 transcript levels in soybean. Furthermore, both GmHSP70-13a and GmHSP70-15a were strongly induced in susceptible soybeans, while both of them still remained low in resistant soybeans, after SCN infection. Taken together, GmSHMT08-mediated SCN resistance is shown to be negatively modulated by both GmHSP70-13a and GmHSP70-15a. The regulation is facilitated through interacting with and influencing 26S proteasome system-involved degradation of GmSHMT08. This study elucidates a SCN-resistance mechanism of GmSHMT08, expands functions of HSP70s and provides two GmHSP70s gene-editing targets for soybean SCN resistance breeding.},
}
@article {pmid41174881,
year = {2026},
author = {Hierl, M and Bischof, J and Liemberger, B and Kocher, T and Hainzl, S and Gruner, S and Trattner, L and Reichl, V and Klausegger, A and Aussel, C and Ammann, S and Raab, R and Andrieux, G and Wolf, M and Hochmann, S and Ebner, P and Wally, V and Zauner, R and Guttmann-Gruber, C and Gratz, IK and Hofbauer, JP and Cathomen, T and Strunk, D and Bauer, JW and Koller, U},
title = {Prime editing as a promising therapeutic strategy for junctional epidermolysis bullosa.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {2},
pages = {817-831},
doi = {10.1016/j.ymthe.2025.10.054},
pmid = {41174881},
issn = {1525-0024},
mesh = {*Epidermolysis Bullosa, Junctional/therapy/genetics ; Humans ; *Gene Editing/methods ; Collagen Type XVII ; *Non-Fibrillar Collagens/genetics/metabolism ; Keratinocytes/metabolism ; Animals ; Mice ; *Genetic Therapy/methods ; *Autoantigens/genetics/metabolism ; Disease Models, Animal ; CRISPR-Cas Systems ; },
abstract = {Gene therapies offer new possibilities for the precise correction of monogenic disorders. Here, we present the first prime editing (PE)-based gene repair strategy for pathogenic COL17A1 variants that cause junctional epidermolysis bullosa (JEB). Type XVII collagen (C17), encoded by COL17A1, plays a critical role in skin aging, regeneration, and the maintenance of epidermal stem cell integrity. Treatment of primary human JEB keratinocytes with PE mRNAs resulted in COL17A1 editing efficiencies of up to 60% in bulk-treated cells, leading to the restoration of full-length, accurately shed C17. Chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing analysis of gene-edited JEB keratinocytes confirmed the absence of unintended chromosomal rearrangements at potential off-target sites and only minimal on-target aberrations. Remarkably, in a xenograft model, in which C17[+] cells represented only 55.9% of the input population, COL17A1-corrected cells populated 92.2% of the basal keratinocyte layer in the resulting skin grafts after 6 weeks. These observations highlight a potential selective advantage imparted by C17 restoration, in line with its canonical role in anchoring hemidesmosomes to the basement membrane and preserving the structural integrity of the interfollicular epidermal stem cell niche. Based on our results, we envision PE as an efficient and safe option to restore gene function in EB and other genodermatoses.},
}
@article {pmid41173724,
year = {2026},
author = {Chen, Y and Duan, Q and Wang, L and He, Q and Fang, L and Xiao, L and Song, H and Cao, Y},
title = {Genome-scale CRISPRi and base-editing libraries for genetic decoding and strain engineering in Shewanella.},
journal = {Trends in biotechnology},
volume = {44},
number = {2},
pages = {496-520},
doi = {10.1016/j.tibtech.2025.10.005},
pmid = {41173724},
issn = {1879-3096},
mesh = {*Shewanella/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Bacterial/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Library ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based libraries with diverse gene-editing functions, such as gene knockdown and mutation, can significantly accelerate our understanding of complex metabolic networks in microorganisms, particularly for species beyond classical model organisms. Here, three distinct CRISPR-based libraries were designed in the electroactive microorganism Shewanella oneidensis MR-1: a CRISPR interference (CRISPRi) library covering 99.6% of genes in the genome, a protein mutation library focused on genes involved in carbon metabolism, and an inactivation library, with sizes of 30 804, 5963, and 4072 single guide (sg)RNAs, respectively. The principles for the design and construction of libraries were validated, and a conjugation-based library transformation method with high coverage and uniformity was developed. For the first time, we explored the potential essential genes of S. oneidensis MR-1, and expanded the substrate spectrum available for electricity generation, including glucose and chitin. These efforts enable deeper genomic interrogation of Shewanella, and provide a framework for applying genome-scale CRISPR-based tools to other undercharacterized microbial species.},
}
@article {pmid41130838,
year = {2026},
author = {Crooke, H and Schwindt, S and Fletcher, SL and Isken, O and Harding, S and Berkley, N and Tait-Burkard, C and Warren, C and Whitelaw, CBA and Tautz, N and Lillico, SG},
title = {DNAJC14 gene-edited pigs are resistant to classical pestiviruses.},
journal = {Trends in biotechnology},
volume = {44},
number = {2},
pages = {570-586},
doi = {10.1016/j.tibtech.2025.09.008},
pmid = {41130838},
issn = {1879-3096},
mesh = {Animals ; Swine/genetics ; *Gene Editing/methods ; *Classical Swine Fever Virus/genetics/physiology/pathogenicity ; CRISPR-Cas Systems ; *Classical Swine Fever/genetics/virology ; Diarrhea Viruses, Bovine Viral ; *Disease Resistance/genetics ; },
abstract = {Infectious diseases remain a major impediment to livestock production, negatively impacting both productivity and welfare. Where key interactions between viruses and host proteins have been identified, it is possible to rationally devise intervention strategies. In vitro studies have identified the host protein DNAJC14 as a core component of the replicative cycle of classical pestiviruses. Outbreaks caused by this group of viruses cause enormous losses in stock farming due to culling and export restrictions. Using CRISPR/Cas9 gene editing, we produced a cohort of pigs with altered DNAJC14. Primary cells from these animals did not support replication of either classical swine fever virus (CSFV) or bovine viral diarrhoea virus (BVDV) in vitro. In vivo challenge with CSFV revealed that the edited pigs displayed complete resistance to infection. This establishes gene editing as an additional strategy that can contribute to the control of classical pestiviruses.},
}
@article {pmid40915965,
year = {2026},
author = {Leech, D and Previtera, DA and Zhang, Y and Botella, JR and Crisp, PA},
title = {Precision plant epigenome editing: what, how, and why.},
journal = {Trends in plant science},
volume = {31},
number = {2},
pages = {192-204},
doi = {10.1016/j.tplants.2025.08.009},
pmid = {40915965},
issn = {1878-4372},
mesh = {*Gene Editing/methods ; *Epigenome/genetics ; *Plants/genetics ; *Genome, Plant/genetics ; *Epigenesis, Genetic ; CRISPR-Cas Systems ; Epigenome Editing ; },
abstract = {Advances in genome engineering have paved the way for targeted epigenome engineering, providing fundamental insights into the role of epigenetic modifications in trait inheritance. Engineered epialleles have already delivered stable, heritable changes in agronomic traits. Despite this capacity, progress in the field has not yet achieved its potential, leaving many avenues of research unexplored. In this review we examine the factors influencing this progress, including the advances in current epigenome editing techniques, the key research goals and translational applications, and the challenges in the selection of ideal target loci. We propose that improved tools for the selection of target loci, particularly in large and complex genomes, are needed to propel the field forward.},
}
@article {pmid40713217,
year = {2026},
author = {Getachew, H and Metais, T and Daheron, L and Garita-Hernandez, M},
title = {Advances and optimization strategies in prime editing of human pluripotent stem cells.},
journal = {Trends in biotechnology},
volume = {44},
number = {2},
pages = {351-364},
doi = {10.1016/j.tibtech.2025.06.017},
pmid = {40713217},
issn = {1879-3096},
mesh = {Humans ; *Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; },
abstract = {Prime editing, first introduced in 2019, is a precise and efficient genome-editing technique with applications across various organisms and cell lines, including human pluripotent stem cells (hPSCs). Despite its revolutionary potential, prime editing of hPSCs often shows low efficiency, hindered by current delivery methods and DNA repair mechanisms. In this review, we explore prime editing of hPSCs, emphasizing the optimization necessary for creating ex vivo and in vitro disease models, which are critical for developing personalized therapeutics. We discuss key prime-editing methods for hPSCs, optimization strategies, tools available for prime editing, and the rigorous quality control required before and after genome engineering for downstream applications.},
}
@article {pmid41626318,
year = {2025},
author = {Sustek-Sánchez, F and Eelmets, E and Nigul, L and Kärblane, K and Laasmaa, M and Balode-Sausina, M and Berzina, SA and Ducis, D and Kaktina, E and Jaškūne, K and Rognli, OA and Rostoks, N and Sarmiento, C},
title = {Isolation and transformation of perennial ryegrass (Lolium perenne L.) protoplasts for the in vivo assessment of guide RNAs editing efficiency.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1744085},
pmid = {41626318},
issn = {1664-462X},
abstract = {Protoplasts are broadly used to perform different cellular and genetic assays. Transformation of protoplasts requires isolation methods that generate a large number of intact cells suitable for downstream applications. Lolium perenne L. is an important forage grass species for which gene editing techniques are in their early stages. Using protoplasts has previously been reported as a suitable approach to test the genome editing efficiency of guide RNAs in important grass species like wheat and rice. This approach can speed up and increase the chances of generating edited plants, especially when working with species for which stable transformation methods have not been established yet. Testing two different approaches regarding the processing of L. perenne L. tillers showed that using a blender for disintegrating the tissue was easier and faster than cutting the tillers with a razor blade. Conversely, the more classical strategy (cutting with a razor) provided a higher number of viable protoplasts. The use of an enzyme solution containing 2% cellulase during 8 h was shown to be the best experimental condition for protoplast isolation. The addition of a sucrose cushion improved the purification of alive cells, which were then positively transformed with guide RNA encoding vectors using polyethylene glycol. The presence of indels induced by these vectors was then confirmed through decomposition-based analysis of their sequenced genomic DNA. These results demonstrated the suitability of using protoplasts for the in vivo assessment of guide RNAs editing efficiency.},
}
@article {pmid41623654,
year = {2026},
author = {Beh, JQ and Muzahid, NH and Mar, JH and Goh, CBS and Huët, MAL and Lim, SY and Rahman, S},
title = {Characterization of the CRISPR1-Cas array and its subtyping potential in Enterococcus faecalis from Malaysia.},
journal = {Access microbiology},
volume = {8},
number = {1},
pages = {},
pmid = {41623654},
issn = {2516-8290},
abstract = {Enterococcus faecalis is a gram-positive bacterium and a common cause of hospital-associated infections. Three major CRISPR loci have been discovered in this species, namely, CRISPR1-cas, CRISPR2 and CRISPR3-cas. We developed novel primers which target the CRISPR1-cas loci in E. faecalis and tested these primers on 26 E. faecalis isolates isolated from diverse settings from Segamat, Malaysia. Half of the isolates were found to carry the CRISPR1-cas9 locus, and the CRISPR1 array was successfully amplified in 12 out of 13 isolates that contained the cas9 gene. Characterization of the CRISPR array shows that CRISPR1-cas shares similar array length and typical repeat sequences with CRISPR2 but differs significantly in terms of spacer identities and terminal repeat (TR) sequences. Most CRISPR spacers encode for chromosomal DNA sequences. Genotype characterization based on ancestral spacer (AS) and TR sequences indicates that E. faecalis with the same CRISPR1-AS genotype do not always harbour the same CRISPR2-AS genotypes and vice versa. A combined CRISPR1-cas and CRISPR2 typing offers comparable discriminatory power to MLST, suggesting its potential to be used in short-term strain identification and epidemiological surveillance at a lower sequencing cost. Our study provides a genetic reference for future studies in Southeast Asia.},
}
@article {pmid41623178,
year = {2026},
author = {Hill, SF and Goldberg, EM},
title = {A hit for base editing: treatment of developmental epilepsy in a mouse model.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {3},
pages = {},
pmid = {41623178},
issn = {1558-8238},
mesh = {Animals ; Mice ; Disease Models, Animal ; *Gene Editing ; *CRISPR-Cas Systems ; *Epilepsy/genetics/therapy ; *NAV1.6 Voltage-Gated Sodium Channel/genetics/metabolism ; Humans ; Mutation, Missense ; *Genetic Therapy ; },
abstract = {CRISPR/Cas9 base editing holds the potential to treat disease caused by single-nucleotide variants. In contrast with conventional CRISPR/Cas9 approaches, base editing enzymatically induces precise DNA alterations and can directly correct disease-causing variants. In this issue of JCI, Reever et al. used base editing to treat a mouse model of a severe neurodevelopmental disorder caused by a pathogenic missense variant in the voltage-gated sodium channel gene SCN8A. This work represents a starting point for the further refinement of base editing to treat genetic epilepsy.},
}
@article {pmid41621837,
year = {2026},
author = {Patraskaki, M and Seyedkatouli, N and Schlicker, L and Warmoes, MO and Cordero-Maldonado, ML and Heins-Marroquin, U and Linster, CL},
title = {Unveiling Immune System Perturbations in Early Development Through Zebrafish Models of NADHX Repair Deficiency.},
journal = {Journal of inherited metabolic disease},
volume = {49},
number = {2},
pages = {e70149},
pmid = {41621837},
issn = {1573-2665},
support = {PRIDE17/12244779/PARK-QC//Fonds National de la Recherche Luxembourg/ ; PRIDE19/14063202/ACTIVE//Fonds National de la Recherche Luxembourg/ ; C22/BM/17198760//Fonds National de la Recherche Luxembourg/ ; //Fondation Juniclair/ ; },
mesh = {Animals ; Zebrafish ; Disease Models, Animal ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; NAD/metabolism ; Phenotype ; *Immune System/metabolism ; *Racemases and Epimerases/genetics/deficiency ; },
abstract = {The vital cofactors NADH and NADPH are prone to hydration, forming hydroxylated redox-inactive derivatives (NADHX and NADPHX) in cells. These damaged metabolites are repaired by two highly conserved enzymes, an NAD(P)HX dehydratase (NAXD) and an NAD(P)HX epimerase (NAXE). Mutations in NAXE or NAXD cause early onset progressive encephalopathy (PEBEL1 or PEBEL2), typically induced by fever or other triggers, and leading to premature death. To advance our comprehension of the disease mechanism and investigate potential therapeutic strategies, we generated zebrafish lines deficient in naxe or naxd using CRISPR/Cas9 technology. While both models accumulated NADHX, only naxd[-/-] larvae developed a severe phenotype, showing reduced locomotion and early death, which was partially rescued by nicotinic acid supplementation. Both mutant lines displayed signs of dysregulated immune function based on gene expression analyses and increased neutral red staining in the head region, indicating an increased number or activation of microglial cells. Our findings suggest that immune system perturbations play a role in PEBEL disease development, aligning with its inflammatory trigger-induced nature in humans. The naxd[-/-] model's responsiveness to nicotinic acid underscores its utility for preclinical drug screening. Overall, these models will be instrumental in furthering our understanding of PEBEL disease mechanisms and enhancing translational research efforts.},
}
@article {pmid41579807,
year = {2026},
author = {Zhang, Y and Wu, M and Geng, F and Wang, Y and Lü, J},
title = {Deciphering functional redundancy of lysine decarboxylases in probiotic E. coli Nissle 1917 via an integrated CRISPR-FTIR phenomics platform.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {351},
number = {},
pages = {127508},
doi = {10.1016/j.saa.2026.127508},
pmid = {41579807},
issn = {1873-3557},
mesh = {*Escherichia coli/enzymology/genetics ; Spectroscopy, Fourier Transform Infrared/methods ; *CRISPR-Cas Systems ; *Probiotics/metabolism ; *Carboxy-Lyases/metabolism/genetics ; Mutation ; *Escherichia coli Proteins/metabolism/genetics ; Phenotype ; },
abstract = {Fourier transform infrared (FTIR) microspectroscopy is a rapid, label-free tool for microbial metabolic phenotyping. Here, we integrate synchrotron-based FTIR microspectroscopy with CRISPR-Cas9 editing to decipher the functional redundancy of lysine decarboxylases (LdcC1 and LdcC2) in probiotic Escherichia coli Nissle 1917. Under lysine stress, isogenic mutants (ΔldcC1, ΔldcC1ΔldcC2) exhibited distinct FTIR fingerprints. Spectral analysis revealed: (i) CH shifts (2950-2850 cm[-1]) indicating ΔldcC1-specific membrane remodeling; (ii) Amide I band profile alterations (∼1650 cm[-1]) suggesting protein structural perturbations; and (iii) a constitutive elevation in 1220-1260 cm[-1] band area in the double mutant, revealing a basal state of metabolic frailty. Principal component analysis of second-derivative spectra revealed clear separation trends among strain phenotypes. We establish LdcC2 as a crucial functional complement, while LdcC1 uniquely contributes to membrane homeostasis. The compensatory stress response activated in the double mutant underscores metabolic redundancy as a cornerstone of intrinsic cellular robustness. Collectively, this work validates a CRISPR-FTIR phenomics platform that bridges targeted genetics with global biochemistry, offering a rapid alternative for functional genomics and metabolic engineering in microbes.},
}
@article {pmid41557883,
year = {2026},
author = {Jiang, Y and Gao, H and Ran, Q and Xian, Y and Zhang, C},
title = {Near-Infrared Light-Inducible CRISPR-Cas12a/Tetrahedral DNA Nanosystem for Spatiotemporal Control of mtDNA Mutation Imaging and Gene Therapy.},
journal = {Nano letters},
volume = {26},
number = {4},
pages = {1438-1448},
doi = {10.1021/acs.nanolett.5c05621},
pmid = {41557883},
issn = {1530-6992},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Infrared Rays ; *DNA, Mitochondrial/genetics ; Mice ; Humans ; *Genetic Therapy/methods ; *Nanoparticles/chemistry ; Mutation ; Gene Editing/methods ; Cell Line, Tumor ; *Neoplasms/therapy/genetics ; },
abstract = {Mitochondrial DNA (mtDNA) mutations are associated with tumor progression and metabolic dysregulation, yet their spatiotemporal visualization and selective therapeutic modulation remain challenging owing to their low abundance and mitochondrial double-membrane barrier. We report a near-infrared (NIR) light-inducible CRISPR-Cas12a/tetrahedral DNA nanosystem for spatiotemporally resolved imaging and editing of mtDNA mutations. The nanosystem integrates upconversion nanoparticles as NIR to ultraviolet converters, photocleavable DNA linkers, and Cas12a/crRNA modules. NIR irradiation triggers linker cleavage, activates Cas12a trans-cleavage, and generates amplified fluorescence for mtDNA mutation detection with a detection limit of 0.83 pM. In living cells and tumor-bearing mice, the nanosystem enables high-spatiotemporal resolution imaging of mtDNA mutations while inducing DNA self-assembly and CRISPR-based mtDNA editing. These disrupt the mitochondrial membrane potential, increase the level of reactive oxygen species, and promote apoptosis, resulting in effective tumor suppression. This nanosystem enables the controllable imaging of mutant mtDNA and gene modulation, offering new opportunities for spatiotemporal cancer theranostics.},
}
@article {pmid41484984,
year = {2026},
author = {Forbes, CA and Shaw, NC and Chen, KG and Hedges, M and Er, TS and Hool, L and Ward, M and Poulton, C and Baynam, G and Lassmann, T and Fear, VS},
title = {A precision medicine approach to interpret a GATA4 genetic variant in a paediatric patient with congenital heart disease.},
journal = {Human genomics},
volume = {20},
number = {1},
pages = {29},
pmid = {41484984},
issn = {1479-7364},
support = {APP2004282//National Health and Medical Research Council/ ; UWA-VCCRI Chair in Cardiovascular Research//Wesfarmers/ ; Telethon 7 Ball, Gift of Giving, 2020.//McCusker Charitable Foundation/ ; Lassmann//Feilman Foundation/ ; Perron People//Stan Perron Charitable Foundation/ ; Perron Programs and Partnership, 2025//Stan Perron Charitable Foundation/ ; },
mesh = {Humans ; *GATA4 Transcription Factor/genetics ; Induced Pluripotent Stem Cells/metabolism/pathology ; Myocytes, Cardiac/metabolism/pathology ; *Heart Defects, Congenital/genetics/pathology ; *Precision Medicine/methods ; Gene Editing ; *Genetic Variation ; Cell Differentiation/genetics ; CRISPR-Cas Systems ; Male ; Child ; Female ; },
abstract = {BACKGROUND: Patients with congenital heart disease are identified in 1% of live births. Improved surgical intervention means many patients now survive to adulthood, the corollary of which is increased mortality in the over-65-year-old congenital heart disease (CHD) population. In the clinic, genetic sequencing increasingly identifies novel genetic variants in genes related to CHD. Traditional assays for interpreting novel genetic variants are often limited by gene-specificity, whereas animal models are cumbersome and may not accurately reflect human disease. This study investigates CRISPR gene editing in induced pluripotent stem cells and cardiomyocyte-directed differentiation as a human disease model to investigate novel genetic variants identified in association with CHD.<br><br>METHODS AND RESULTS: We identified a GATA4 p.Arg284His genetic variant in a paediatric patient. This genetic variant was introduced into induced pluripotent stem cells (iPSCs) using CRISPR gene editing with homology-directed-repair. GATA4 genetic variant and isogenic control iPSCs were selected and differentiated into cardiomyocytes. Expression of the GATA4 p.Arg284His variant resulted in altered calcium transients, indicative of CHD and consistent with the patient's clinical phenotype. Transcriptomics revealed cellular pathway changes in cardiac development, calcium handling, and energy metabolism that contribute to disease aetiology, mechanism and identification of potential treatments.<br><br>CONCLUSION: Directed differentiation of iPSCs harbouring the GATA4 p.Arg284His genetic variant recapitulated the CHD phenotype, indicated disease mechanisms, and pointed to potential sites for targeting with therapy. The study highlights the utility of transcriptomics for the functional interpretation of cardiac genetic variants and is an exemplar for precision medicine approaches for the investigation of CHD.},
}
@article {pmid41469506,
year = {2025},
author = {Wang, T and Tian, Y and Yin, R and Li, M and Luo, J and Yang, Y and Zhang, C and Chen, H and Wang, Y and Lu, D},
title = {In vivo genome editing with a novel Cj4Cas9.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {152},
pmid = {41469506},
issn = {2399-3642},
support = {82070258//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; Animals ; Mice ; *CRISPR-Cas Systems ; Proprotein Convertase 9/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Humans ; },
abstract = {Natural CRISPR-Cas9 systems provides diverse properties for genome editing, yet finding compact variants remains a priority. In this study, we screened a panel of 11 CjCas9 orthologous using a GFP activation assay and identified seven active nucleases. Among these, Cj4Cas9 stood out as particularly noteworthy due to its compact genome size (985 amino acids) and unique PAM preference (5'-NNNGRY-3'). Cj4Cas9 demonstrates efficient disruption of the Tyr gene in mouse zygotes, resulting in an albino phenotype. Furthermore, when delivered via AAV8, Cj4Cas9 achieves efficient genome editing of the Pcsk9 gene in mouse liver, leading to reduced serum cholesterol and LDL-C levels. Seeking to further expand its utility, we engineered Cj4Cas9 for higher activity by introducing L58Y/D900K mutations, resulting in a variant termed enCj4Cas9. This variant exhibits a two-fold increase in nuclease activity compared to the wild-type Cj4Cas9 and recognizes a simplified N3GG PAM, considerably expanding its targeting scope. These findings establish Cj4Cas9 and its engineered variants for fundamental research and therapeutic applications.},
}
@article {pmid41466059,
year = {2025},
author = {Chen, W and He, P and Ding, L and Lou, W and Wang, Y and Shi, W and Fan, Z and Sheng, Y and Luo, J and Tan, Z and Wang, Y and Di, W and Ke, X and Yu, B},
title = {EPI-SauriCas9-based mouse ovarian cancer models recapitulating pten deletion in patients.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {159},
pmid = {41466059},
issn = {2399-3642},
support = {82403651//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; Female ; *PTEN Phosphohydrolase/genetics ; *Ovarian Neoplasms/genetics/pathology/drug therapy ; Mice ; Disease Models, Animal ; Humans ; Gene Deletion ; Tumor Suppressor Protein p53/genetics ; CRISPR-Cas Systems ; },
abstract = {Ovarian cancer remains a deadly gynecological malignancy, with PTEN loss and TP53 mutations frequently implicated in its progression. However, suitable models for studying ovarian cancers with PTEN and TP53 deletions are rare. Here we develop and validate the mouse ovarian epithelium with Pten and Trp53 deletions (MEPP) model using the EPI-SauriCas9 system. We demonstrate the role of Pten loss in promoting tumorigenicity and metastasis. Single-cell RNA sequencing reveals distinct epithelial subpopulations with varying metastatic potential. MEPP also recapitulates key features of human ovarian cancer, including its immune landscape and therapeutic responses. High-throughput drug screening identifies FK228 and thioguanine as promising therapeutic candidates, both of which show in vivo efficacy and are validated in PTEN-deleted organoids. Together, these results establish MEPP as a platform for studying PTEN-deleted ovarian cancer and provide a strategy for generating clinically relevant tumor models through targeted gene editing.},
}
@article {pmid41451985,
year = {2026},
author = {Wang, Z and Hu, T and Liu, W and Zhou, H and Lv, X and Li, H and Li, X and Huang, X and He, L},
title = {Development and clinical validation of an ERA-CRISPR/Cas12a assay for the rapid detection of 14 high-risk HPV types.},
journal = {Microbiology spectrum},
volume = {14},
number = {2},
pages = {e0303625},
doi = {10.1128/spectrum.03036-25},
pmid = {41451985},
issn = {2165-0497},
support = {82100344//National Natural Science Foundation of China/ ; 2024A22//Tongji Hospital/ ; },
mesh = {Humans ; *Papillomavirus Infections/diagnosis/virology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Female ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *Papillomaviridae/genetics/isolation &amp; purification/classification ; Uterine Cervical Neoplasms/virology/diagnosis ; Reproducibility of Results ; DNA, Viral/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {UNLABELLED: Persistent infection with high-risk human papillomavirus (HR-HPV) is the leading cause of cervical cancer, highlighting the critical need for early detection to improve prevention. Although real-time quantitative polymerase chain reaction (RT-qPCR) remains the gold standard for HR-HPV detection, its dependence on sophisticated equipment, complex procedures, and trained personnel limits accessibility. Here, we developed a simplified assay for 14 HR-HPV types by integrating direct lysis, enzyme-mediated isothermal rapid amplification (ERA), and CRISPR-Cas12a-mediated cleavage into a streamlined workflow that requires only a basic isothermal heating device. The optimized system achieved a sensitivity of 50 copies per reaction with no cross-reactivity, while a refined lysis buffer containing 20% Chelex-100 minimized inhibition from vaginal swab samples, thereby enhancing detection performance. Validation with 152 clinical samples demonstrated 97.62% sensitivity and 100% specificity, confirming the reliability of the method. This user-friendly and cost-effective assay requires minimal equipment, enabling rapid and field-deployable HR-HPV detection, and offers a practical alternative to conventional laboratory-based approaches, particularly in resource-limited settings.<br><br>IMPORTANCE: High-risk human papillomavirus (HR-HPV) is the principal etiological agent of cervical cancer, and early detection remains central to effective disease prevention. Current PCR-based assays, however, rely on specialized laboratories and trained personnel, limiting their deployment in many settings. Here, we report a streamlined CRISPR-Cas12a assay that integrates direct sample lysis, ERA, and CRISPR-based detection into a single workflow operable with only a simple heating device to determine the presence of 14 HR-HPV types. The assay achieves high analytical sensitivity, strong specificity, and robust clinical performance while maintaining low cost and ease of use. This platform enables rapid HR-HPV detection and scalable screening, particularly in resource-constrained environments, with the potential to facilitate earlier intervention and reduce cervical cancer incidence.},
}
@article {pmid41442816,
year = {2026},
author = {Zhu, Y},
title = {The potential and innovative applications of CRISPR gene editing technology in enzyme gene development.},
journal = {Enzyme and microbial technology},
volume = {195},
number = {},
pages = {110799},
doi = {10.1016/j.enzmictec.2025.110799},
pmid = {41442816},
issn = {1879-0909},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; *Enzymes/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The CRISPR gene editing technology is simple in design and highly efficient, making it the most widely used gene editing tool today. At present, CRISPR gene editing technology has shown a certain application value in enzyme development, but its application potential has not been fully developed. CRISPR gene editing technology can not only be used to knockin enzyme genes and knockout genes that are not conducive to enzyme expression, but can also be applied to single-base editing of enzyme genes, tandem sgRNA for multi-enzyme gene editing, sgRNA library for enzyme screening, endogenous enzyme gene modification, transcriptional activation or inhibition of enzyme gene expression, and fluorescence imaging of enzyme genes. Especially, this review innovatively proposes for the first time that CRISPR gene editing technology can be used for site specific fusion of enzyme genes, cell surface display of endogenous enzymes, and knockin of super long DNA for simultaneous expression of multiple enzymes, providing new ideas for maximizing the value of CRISPR gene editing technology in enzyme development in the future.},
}
@article {pmid41416796,
year = {2026},
author = {Athipanyasilp, N and Saowpak, S and Chaimayo, C and Angkasekwinai, N and Pattama, A and Athipanyasilp, A and Patchsung, M and Aphicho, K and Uttamapinant, C and Horthongkham, N},
title = {CRISPR-Cas13a SHERLOCK assay for rapid and sensitive detection of chikungunya virus.},
journal = {Microbiology spectrum},
volume = {14},
number = {2},
pages = {e0229825},
doi = {10.1128/spectrum.02298-25},
pmid = {41416796},
issn = {2165-0497},
support = {R016034012//Faculty of Medicine Siriraj Hospital, Mahidol University/ ; },
mesh = {*Chikungunya virus/genetics/isolation &amp; purification ; *Chikungunya Fever/diagnosis/virology ; Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Real-Time Polymerase Chain Reaction/methods ; },
abstract = {Chikungunya virus (CHIKV), a major cause of acute febrile illness and joint pain, remains a significant public health threat in tropical regions. Rapid and accurate detection is essential for timely clinical management and outbreak control, particularly in resource-limited settings where real-time PCR (RT-qPCR) is often impractical. We developed and validated a SHERLOCK assay coupled with recombinase polymerase amplification for CHIKV RNA detection. Analytical performance was assessed by determining the limit of detection (LOD), cross-reactivity, clinical sensitivity and specificity, and predictive values. The assay achieved an LOD of 215 copies/reaction with no cross-reactivity against other alphaviruses or flaviviruses. Clinical testing of 146 plasma samples showed a sensitivity and specificity of 94.52% and 100% with lateral-flow readout and 97.26% and 100% with fluorescence readout, respectively. This study establishes a promising CRISPR-Cas13a-based SHERLOCK platform for CHIKV detection, demonstrating high analytical performance, rapid turnaround time, and potential for future adaptation to resource-limited settings.IMPORTANCEEarly and accurate detection of chikungunya virus (CHIKV) is critical for outbreak control, especially in resource-limited settings, where real-time PCR is not feasible. This study demonstrates that the CRISPR-Cas13a-based SHERLOCK platform, combined with RPA, achieves high diagnostic accuracy and a low detection limit, comparable to RT-qPCR. The assay's rapid turnaround time and simple lateral-flow readout make it a promising tool for point-of-care diagnostics during CHIKV outbreaks, potentially improving disease surveillance and clinical decision-making.},
}
@article {pmid41367295,
year = {2026},
author = {Wyer, CAS and Amaro, IA and Pitcher, S and Ponlawat, A and Harrington, LC and Wolfner, MF and Hollis, B and Cator, LJ},
title = {Pickpocket315 affects male mating behavior in the yellow fever mosquito Aedes aegypti.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {2},
pages = {},
doi = {10.1093/g3journal/jkaf297},
pmid = {41367295},
issn = {2160-1836},
support = {R01-AI095491/NH/NIH HHS/United States ; //L.C.H and M.F.W and International Atomic Energy Association/ ; //Reproductive Biology of Male Aedes Mosquitoes for SIT Applications/ ; NE/S007415/1//Natural Environment Research Council/ ; },
mesh = {Animals ; *Aedes/genetics/physiology ; Male ; *Sexual Behavior, Animal ; Female ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; RNA Interference ; Mosquito Vectors/genetics ; Copulation ; Yellow Fever/transmission ; },
abstract = {The molecular basis of mating behavior in the important disease vector mosquito, Aedes aegypti, remains poorly characterized. We investigated the functional role of a pickpocket gene, ppk315, in male mating behavior using both RNAi-mediated knockdown and CRISPR/Cas9 approaches. Behavioral assays revealed that RNAi-treated males (dsPPK315) made fewer mating attempts, were less responsive to female acoustic cues, and were less likely to achieve copulation, though their latency to initiate contact when attempts were made was comparable to controls. Males with a CRISPR/Cas9-induced disruption to ppk315 exhibited reduced success in inseminating multiple females, consistent with previous reports from RNAi knockdown males, ruling out off-target effects as the source of behavioral changes. In contrast to the results of behavioral assays with RNAi, ppk315 mutant males (ppk315-/-) attempted copulation as frequently as wild-type males (ppk315+/+) but were slower to contact females. Despite these impairments in one-on-one interactions, both dsPPK315 and ppk315-/- males displayed normal mating success under competitive swarm-like conditions, potentially due to the socially facilitated activation of mating behavior. Collectively, our findings support a role for ppk315 in the initiation of mating behaviors via sensory detection, with context-dependent consequences for reproductive success.},
}
@article {pmid40972958,
year = {2026},
author = {Nishino, T and Ono, K},
title = {Precision cardiology: Integrating gene therapy, genome editing, and single-cell genomics.},
journal = {Journal of cardiology},
volume = {87},
number = {2},
pages = {121-127},
doi = {10.1016/j.jjcc.2025.09.009},
pmid = {40972958},
issn = {1876-4738},
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; *Genomics/methods ; *Precision Medicine/methods ; *Cardiology/methods/trends ; *Single-Cell Analysis ; *Cardiovascular Diseases/therapy/genetics ; CRISPR-Cas Systems ; },
abstract = {Gene therapy is poised to revolutionize cardiovascular medicine by targeting the molecular roots of disease. This review examines the evolution of gene therapy, highlighting its past progress and future potential with emerging technologies. We first assess foundational gene addition and silencing strategies, noting clinical progress for monogenic cardiomyopathies alongside significant setbacks in multifactorial heart failure, driven mainly by the central challenge of vector delivery. We then discuss the evolution of delivery platforms, from engineered adeno-associated virus capsids to targeted lipid nanoparticles, which are designed to enhance cardiac specificity and safety. Concurrently, the gene editing revolution-progressing from the foundational Clustered Regularly Interspaced Short Palindromic Repeats-Cas9 system to high-fidelity base and prime editors-is enabling the direct correction of pathogenic mutations with increasing precision. Catalyzing these therapeutic platforms is the recent explosion in single-cell genomics, which provides an unprecedented resolution of cardiac pathology, revealing novel cell-specific targets previously obscured by bulk analysis. We conclude that the synergistic convergence of these pillars-genomics-driven discovery, precision genome editing, and targeted delivery-is creating a new paradigm of precision cardiology, moving the field from chronic management towards durable, curative interventions.},
}
@article {pmid40748748,
year = {2026},
author = {Teubner, JP and Tümen, D and Kandulski, A and Heumann, P and Mester, P and Aschenbrenner, E and Pollinger, K and Gunckel, M and Volz, B and Hein, T and Beltzig, PL and Tengler, L and Voll, F and Kreutz, M and Kunst, C and Nicolay, JP and Müller, M and Gülow, K},
title = {CRISPR-Cas9 screen reveals that inhibition of enhancer of zeste homolog 2 sensitizes malignant T cells to dimethyl-fumarate-induced cell death.},
journal = {The FEBS journal},
volume = {293},
number = {3},
pages = {749-765},
doi = {10.1111/febs.70208},
pmid = {40748748},
issn = {1742-4658},
support = {2022.096.1//Wilhelm Sander-Stiftung/ ; },
mesh = {*Enhancer of Zeste Homolog 2 Protein/antagonists &amp; inhibitors/genetics/metabolism ; Humans ; *CRISPR-Cas Systems ; *Dimethyl Fumarate/pharmacology ; Pyridones/pharmacology ; Cell Death/drug effects ; *Lymphoma, T-Cell, Cutaneous/drug therapy/genetics/pathology/metabolism ; Cell Line, Tumor ; *Sezary Syndrome/drug therapy/genetics/pathology ; Benzamides/pharmacology ; Morpholines/pharmacology ; T-Lymphocytes/drug effects/metabolism/pathology ; Histones/metabolism ; NF-kappa B/genetics/metabolism ; Biphenyl Compounds ; },
abstract = {Constitutive activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway is a hallmark of many lymphocyte-associated cancers, including cutaneous T-cell lymphoma (CTCL) and its leukemic variant, the Sézary syndrome. Dimethyl fumarate (DMF) has been identified as a promising NF-κB-targeted therapy and has shown positive outcomes in a phase II clinical trial involving patients with Sézary syndrome. However, limited responsiveness remains a significant challenge. Through a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screen, we identified enhancer of zeste homolog 2 (EZH2; also known as histone-lysine N-methyltransferase) as a critical target for enhancing DMF-induced cell death. EZH2, the catalytic subunit of Polycomb Repressive Complex 2 (PRC2), is responsible for the methylation of histone H3 (H3K27). Combining DMF with the US Food and Drug Administration (FDA)-approved EZH2 inhibitor tazemetostat significantly increases cell death in patient-derived CTCL cells, offering a promising strategy to improve therapeutic outcomes and overcome limited responsiveness to DMF.},
}
@article {pmid41620285,
year = {2026},
author = {Sohn, M and Pantsulaia, G and Brody, J},
title = {Mouse and human T cell Cas9-RNP/nucleofection-mediated gene-editing.},
journal = {Methods in cell biology},
volume = {201},
number = {},
pages = {91-107},
doi = {10.1016/bs.mcb.2025.03.020},
pmid = {41620285},
issn = {0091-679X},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Mice ; *CD8-Positive T-Lymphocytes/metabolism ; *Ribonucleoproteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; *Transfection/methods ; },
abstract = {CRISPR/Cas9 technology has revolutionized genome editing, providing a precise and expeditious means of genetic modification. This breakthrough has enhanced our understanding of gene function, including T cell immunology. Efficient gene editing in primary T cells not only offers a robust investigative tool for dissecting gene function but also holds promise for advancing T cell-based immunotherapies, including next-generation chimeric antigen receptor T cells. In this article, we introduce a highly efficient gene editing protocol for unstimulated human CD8 T cells and unstimulated and stimulated murine CD8 T cells, utilizing transient nucleofection of ribonucleoprotein complexes composed of synthesized modified single guide RNAs (sgRNAs) and purified Cas9 protein. This protocol, initially devised for primary CD8 T cells, can be readily adapted to other primary cell cultures through optimization of nucleofection conditions as well. In essence, our method provides a practical and powerful approach for achieving precise and swift gene knockout in primary CD8 T cells.},
}
@article {pmid41618915,
year = {2026},
author = {Liu, Q and Huang, Y and Zhou, R and Ding, H and Nie, Q and Gong, X and Zuo, T and Wang, S and Liang, Y and Li, Y},
title = {A Minimal and Portable CRISPR Platform Based on Bifidobacterial Cas9 Enables Genome Editing in E. coli Nissle 1917.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00911},
pmid = {41618915},
issn = {2161-5063},
abstract = {Genetic manipulation of core gut probiotics remains challenging due to endogenous cellular barriers and a scarcity of efficient molecular tools, limiting progress in live biotherapeutic development. Here, we characterized the native type II-C CRISPR-Cas system in Bifidobacterium longum subsp. longum GNB (B. longum GNB). Through integrated bioinformatic analysis and high-throughput protospacer adjacent motif (PAM) screening, we identified a novel 5'-NNRMAT-3' (where R = A/G, M = A/C) motif recognized by its compact Cas9 nuclease (BLCas9). The stringent PAM dependency of BLCas9 was unequivocally confirmed by in vitro cleavage assays. Leveraging this endogenous mechanism, we developed a dual-plasmid editing platform for robust and multiplex genome engineering in the probiotic strain Escherichia coli Nissle 1917 (EcN). Application of this system notably enhanced extracellular γ-aminobutyric acid (GABA) production in EcN through targeted metabolic engineering. Our work provides the first molecular dissection of a type II-C system in Bifidobacterium longum and establishes a generalizable framework for the discovery and application of compact programmable nucleases, suggesting a viable strategy for modulating host physiology via the gut-brain axis.},
}
@article {pmid41617991,
year = {2026},
author = {Nagahata, N and Kato, K and Yamada, S and Kannan, S and Okazaki, S and Isayama, Y and Hiraizumi, M and Yamashita, K and Koonin, EV and Zhang, F and Nishimasu, H},
title = {Structural visualization of the molecular evolution of CRISPR-Cas9.},
journal = {Nature structural &amp; molecular biology},
volume = {},
number = {},
pages = {},
pmid = {41617991},
issn = {1545-9985},
abstract = {RNA-guided DNA nucleases Cas9 and IscB (insertion sequences Cas9-like OrfB) are components of type II CRISPR-Cas adaptive immune systems and transposon-associated OMEGA (obligate mobile element-guided activity) systems, respectively. Sequence and structural comparisons indicate that IscB (~500 residues) evolved into Cas9 (~700-1,600 residues) through protein expansion coupled with guide RNA miniaturization. However, the specific sequence of events in this evolutionary transition remains unknown. Here, we report cryo-electron microscopy structures of four phylogenetically diverse RNA-guided nucleases-two IscBs and two Cas9s-each in complex with its cognate guide RNA and target DNA. Comparisons of these four complex structures to previously reported IscB and Cas9 structures indicate that evolution from IscB to Cas9 involved the loss of the N-terminal PLMP domain and the acquisition of the zinc-finger-containing REC3 domain, followed by bridge helix extension and REC1 domain acquisition. These structural changes led to expansion of the REC lobe, increasing the target DNA cleavage specificity. Additionally, the structural conservation of the RNA scaffolds indicates that the dual CRISPR RNA (crRNA) and trans-activating crRNA guides of CRISPR-Cas9 evolved from the single ωRNA guides of OMEGA systems. Our findings provide insights into the succession of structural changes involved in the exaptation of transposon-associated RNA-guided nucleases for the role of effector nucleases in adaptive immune systems.},
}
@article {pmid41614935,
year = {2026},
author = {El Semary, NAH and Fadiel, A and Eichenbaum, KD and Alhusayni, SA},
title = {Prokaryotic Molecular Defense Mechanisms and Their Potential Applications in Cancer Biology: A Special Consideration for Cyanobacterial Systems.},
journal = {Current issues in molecular biology},
volume = {48},
number = {1},
pages = {},
pmid = {41614935},
issn = {1467-3045},
support = {KFU253867//Vice Presidency of Higher studies and Scientific research, Deanship of Scientific research, King Faisal University, AlHufuf, Al-Ahsa, Kingdom of Saudi Arabia, postcode: 31982/ ; },
abstract = {Cyanobacteria harbor sophisticated molecular defense systems that have evolved over billions of years to protect against viral invasion and foreign genetic elements. These ancient photosynthetic organisms possess a diverse array of restriction-modification (R-M) systems and CRISPR-Cas arrays that present challenges for genetic engineering, but also offer unique opportunities for cancer-targeted biotechnological applications. These systems exist in prokaryotes mainly as defense mechanisms but they are currently used in molecular applications as gene editing tools. Moreover, latest developments in nucleases such as zinc finger nucleases (ZFNs), TALENs (transcription-activator-like effector nucleases) are discussed. A comprehensive genomic analysis of 126 cyanobacterial species found 89% encode multiple R-M systems, averaging 3.2 systems per genome, creating formidable barriers to transformation but also providing molecular machinery that could be harnessed for precise recognition and targeting of cancer cells. This review critically examines the dual nature of these defense systems, their ecological functions, and the emerging strategies to translate their molecular precision into advanced anticancer therapeutics. Hence, the review main objectives are to explore the recent understanding of these mechanisms and to exploit the knowledge gained in opening new avenues for cancer-focused targeted interventions, while acknowledging the significant challenges to translate these systems from laboratory curiosities to practical applications.},
}
@article {pmid41614578,
year = {2026},
author = {Barragán-Borrero, V and de Santana Lopes, A and Rodrigues Batista, ED and Höfer, M and Elias, R and Chakraborty, A and Ponce-Mañe, A and Descombes, C and Diezma-Navas, L and Petraki, L and Huber, M and Xu, S and Marí-Ordóñez, A},
title = {Strain, procedures, and tools for reproducible genetic transformation and genome editing of the emerging plant model Spirodela polyrhiza.},
journal = {The New phytologist},
volume = {},
number = {},
pages = {},
doi = {10.1111/nph.70919},
pmid = {41614578},
issn = {1469-8137},
support = {512079118//Emmy Noether Programme/ ; //&#xd6;sterreichischen Akademie der Wissenschaften/ ; 14002787004056//Erasmus+/ ; //Johannes Gutenberg-Universit&#xe4;t Mainz/ ; 407023052//Deutsche Forschungsgemeinschaft/ ; 427577435//Deutsche Forschungsgemeinschaft/ ; 438887884//Deutsche Forschungsgemeinschaft/ ; },
abstract = {Duckweeds (Lemnaceae) have excellent potential for fundamental and applied research due to ease of cultivation, small size, and continuous fast clonal growth. However, their usage as model organisms and platforms for biotechnological applications is often limited by the lack of universal genetic manipulation methods necessary for transgene expression, gene editing, and other methods to modify gene expression. To identify suitable strains for genetic manipulation of the giant duckweed, Spirodela polyrhiza, we screened several genotypes for callus induction and regeneration and established genetic transformation. We identified SP162 to be amenable to Agrobacterium-mediated transformation via tissue culture. The procedure is robust and reproducible across laboratories, allowing stable expression of different reporter genes and selectable markers, enabling CRISPR/Cas9-mediated genome editing. In addition, due to a weak small RNA-based silencing response, S. polyrhiza sustains prolonged periods of transgene activity in transient expression assays. To promote duckweed research and encourage the adoption of S. polyrhiza, we have made SP162 (ID#: 5676) and its genome publicly available and provide here detailed procedures for its cultivation and transformation. Furthermore, we created a web server to explore its genome, retrieve gene sequences, and implement orthologous gene search and a gRNA design function for diverse CRISPR/Cas-based applications (https://agxu.uni-mainz.de/SP162/).},
}
@article {pmid41548240,
year = {2026},
author = {Fan, Y and Shen, S and Su, M and Yan, H and Dong, J and Li, Y and Li, N and Gao, Z and Xia, F},
title = {Synergistic CRISPR/Cas12a-Nanozyme System for Iontronic Sensing of Site-Specific Septin9 Methylation.},
journal = {Analytical chemistry},
volume = {98},
number = {4},
pages = {2870-2881},
doi = {10.1021/acs.analchem.5c05637},
pmid = {41548240},
issn = {1520-6882},
mesh = {*Septins/metabolism/genetics ; Humans ; *DNA Methylation ; *CRISPR-Cas Systems ; Gold/chemistry ; *DNA, Catalytic/chemistry/metabolism ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism ; Electrochemical Techniques ; Silver/chemistry ; Metal Nanoparticles/chemistry ; Platinum/chemistry ; Colorectal Neoplasms/diagnosis ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Methylated septin9 (septin9-mC) is a well-validated biomarker for colorectal cancer screening, and accurate detection of such site-specific methylated DNA holds significant clinical value for early disease diagnosis. However, conventional methods suffer from cumbersome pretreatment, DNA degradation risks, and poor performance in low-abundance samples. Herein, we report a synergistic iontronic sensing platform integrating methylation-sensitive restriction enzyme (AciI), CRISPR/Cas12a, Ag-DNAzyme, and Au/Pt heterometallic nanozyme for highly sensitive and specific detection of septin9-mC. AciI selectively cleaves unmethylated septin9 (septin9-C) while sparing septin9-mC, and intact septin9-mC activates Cas12a trans-cleavage activity to trigger catalytic hairpin assembly (CHA), generating Ag-DNAzyme. Activated Ag-DNAzyme induces detachment of Au/Pt nanoparticles from anodic aluminum oxide membranes, reducing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to positively charged oxTMB and altering ion transport fluxes in nanochannels, which is read out via current-voltage characteristics. The linear range is 100 aM to 10 nM with a detection limit of 34.0 aM. This method effectively distinguishing colorectal cancer cells from human colonic epithelial cells and colorectal cancer patients from healthy individuals, showing excellent performance in real sample analysis. The proposed method provides a dependable tool for site-specific methylation detection with promising applications in biological research and clinical diagnosis.},
}
@article {pmid41613863,
year = {2025},
author = {Safrygina, AA and Orlov, YL},
title = {Genetically engineered approaches to the treatment of cystic fibrosis.},
journal = {Biophysical reviews},
volume = {17},
number = {5},
pages = {1333-1358},
pmid = {41613863},
issn = {1867-2450},
abstract = {Failure of functions of CFTR (cystic fibrosis transmembrane conduction regulator) gene, which encodes a protein of a selective ion channel, is causing cystic fibrosis. Cystic fibrosis is a severe systemic monogenic disease with an autosomal recessive type of inheritance, which significantly reduces the duration and quality of life of patients. It is one of the most common hereditary diseases. Studying of molecular functions of CFTR protein in different types of cells, its structural and functional network interactions are critically important for the development of a new and more effective pathogenetic therapy. We are reviewing papers on the structure of the CFTR protein and its pathogenic genetic variants, as well as methods of pathogenetic therapy of cystic fibrosis by CFTR modulators and gene engineering. Recent gene engineering approaches to keep CFTR functions are discussed, such as gene-replacement therapy and genome editing, as well as viral and non-viral delivery systems and strategies of genomic editors.},
}
@article {pmid41613398,
year = {2025},
author = {Mehmood, MA and Iqbal, MM and Ashfaq, M and Chen, S and Wang, J},
title = {Advanced molecular tools for surveillance and management of tobamoviruses.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1718133},
pmid = {41613398},
issn = {1664-462X},
abstract = {Tobamoviruses are a group of plant viruses that can cause yield losses of up to 70% and reduce fruit quality by 30-50%. Historically, tobamoviruses were dominated by tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV). However, the landscape is rapidly shifting with the emergence of economically significant viruses such as tomato mottle mosaic virus (ToMMV) and tomato brown rugose fruit virus (ToBRFV). Both can circumvent the previously durable Tm-2[2] resistance in tomato and spread across multiple continents. This shift coincides with dramatic leaps in diagnostic tools, which have enhanced surveillance capabilities. Sensitive detection of tobamoviruses in the field with minimal sample preparation can be achieved using latest technologies such as isothermal amplification, CRISPR/Cas-hybrid assays or next-generation sequencing. Virus-host interactions underscore that viral proteins, including replicase components, are potent suppressors of RNA silencing (VSRs). Small RNA profiling and network analyses of viral movement proteins reveal complex mechanisms of immune evasion and resistance breakdown. These findings are largely based on dominant NB-LRR genes such as L, Tm-1, and Tm-2[2] . However, evidence indicates that ToBRFV can bypass this resistance via mutation in the movement protein, so supplementary methods should be considered. This review covers latest approaches, such as genome editing with CRISPR, targeting susceptibility genes, RNA interference (RNAi), and multi-omics approaches (transcriptomics, proteomics, metabolomics, ionomics), that can facilitate real-time surveillance and breeding for enhanced resilience. Moreover, the use of bio-formulations and nano-formulations as eco-friendly alternatives against tobamoviruses is discussed in detail. Climate change further complicates disease dynamics by undermining temperature-sensitive resistance, altering virus prevalence, and exacerbating yield losses. The rapid emergence of new tobamoviruses, which threatens the economy, necessitates a comprehensive approach. The integration of molecular diagnostics using CRISPR, omics technologies, designed protective systems, and climate-augmented disease prediction offers a detailed blueprint for the sustainable control of tobamoviruses and crop protection.},
}
@article {pmid41612768,
year = {2026},
author = {Wang, Y and Zhang, X and Lin, Y and Zhang, X and Yang, Z and Wan, Y and Negahdary, M},
title = {Single-Tube Dual-Gene Detection of Methicillin-Resistant Staphylococcus aureus via Selective Trans-Cleavage Preferences of Cas9 and Cas12a.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c05789},
pmid = {41612768},
issn = {1520-6882},
abstract = {Rapid and accurate detection of methicillin-resistant Staphylococcus aureus (MRSA) is essential for guiding clinical treatment and preventing infections. Current dual-gene detection methods based on CRISPR-Cas systems often require additional transcription steps, which increase the complexity of the assay and extend the turnaround time. Here, we report a single-tube dual-gene detection platform that leverages the orthogonal trans-cleavage preferences of Cas9 and Cas12a. By exploiting the loss of RNA cleavage activity in Cas12a when guided by split crRNA, and the inability of Cas9 to cleave DNA hairpin probes, we established a single-tube assay capable of simultaneously detecting the MRSA resistance gene (mecA) and the S. aureus-specific nuclease gene (nuc). The platform achieved attogram-level sensitivity and single-cell detection with high specificity against non-MRSA strains. Validation in an in vivo tilapia infection model demonstrated complete concordance with qPCR, reaching 100% positive and negative percent agreements. This work presents a streamlined, accurate, and practical approach for dual-gene pathogen detection, expanding the potential of Cas protein orthogonality for multiplex diagnostics.},
}
@article {pmid41612423,
year = {2026},
author = {Nizovtseva, I and Rezaykin, A and Korenskaia, A and Zakhartsev, M and Chigireva, A and Starodumov, I and Chernushkin, D},
title = {Identification and comparative genomic analysis of prophage sequences and CRISPR‒Cas immunity in Methylococcus genomes: insights into industrial methane bioconversion.},
journal = {Biotechnology for biofuels and bioproducts},
volume = {},
number = {},
pages = {},
doi = {10.1186/s13068-026-02738-6},
pmid = {41612423},
issn = {2731-3654},
support = {24-24-00454//Russian Science Support Foundation/ ; },
abstract = {BACKGROUND: Methylococcus species utilize methane as the sole carbon and energy source, converting it into biomass and other metabolic end products. Owing to this metabolic capacity, they hold particular promise in industrial C1 biotechnology, especially for the production of protein-rich feed. However, the industrial cultivation of Methylococcus-based consortia on methane is inherently nonsterile, exposing the process to potential biological risks that may compromise the stability, duration and productivity of cultivation. One of the most critical threats is bacteriophage infection, whose triggers for rapid phage-mediated lysis and resulting economic losses remain incompletely understood. Elucidating these processes is paramount for devising strategies to mitigate or prevent detrimental outcomes.<br><br>RESULTS: In this investigation, nine publicly accessible genomes of Methylococcus species were examined, culminating in the identification of eleven prophage sequences distributed variably among the genomes. Sequence annotations revealed that nine prophages are potentially functional and intact, whereas the rest carry incomplete gene sets indicative of nonviability. Phylogenetic analyses corroborated the substantial diversity of prophages, which formed distinct clusters related to &#x3b3;-proteobacteria phages. Furthermore, comparative genomic analyses demonstrated a high degree of structural conservation despite the presence of rearrangements. The annotation of the CRISPR&#x2012;Cas systems provided insights into additional dimensions of phage&#x2012;bacteria interactions. Examination of prophage integration sites did not reveal any disruption of metabolic gene structures, thus suggesting minimal risk of deleterious phenotypic outcomes.<br><br>CONCLUSIONS: These findings considerably advance the current understanding of the genetic diversity and biological properties of prophages infecting Methylococcus species, underscoring the importance of holistic approaches for the detection and analysis of these elements. Our findings underscore the need for routine prophage monitoring in industrial methanotrophic consortia, with the pipeline established here serving as a foundational framework for future refinement and industrial adaptation.},
}
@article {pmid41611506,
year = {2026},
author = {Ji, Y and Bian, X and Feng, Z and Li, F and Hu, Z and Hu, Y and Liu, P and Ma, B and Chen, X},
title = {[Map-based cloning and functional analysis of the golden panicle and brown rice gene gpr1].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {1},
pages = {356-366},
doi = {10.13345/j.cjb.250438},
pmid = {41611506},
issn = {1872-2075},
support = {32272096//the National Natural Science Foundation of China/ ; LZ24C130003//the Natural Science Foundation of Zhejiang Province/ ; 2023R404039//the Zhejiang Provincial Undergraduate Science and Technology Innovation Activity Plan ("Xinmiao" Talents Program)/ ; 2024-2-017 and 2023-2-014//the Science and Technology Key Projects of Jinhua City/ ; },
mesh = {*Oryza/genetics/metabolism ; Cloning, Molecular ; *Plant Proteins/genetics ; Chromosome Mapping ; *Genes, Plant ; CRISPR-Cas Systems ; Pigmentation/genetics ; Flavonoids/biosynthesis ; },
abstract = {The colors of panicle hull and brown rice are crucial agronomic traits in rice, which are widely used in breeding and genetic research. Here, a rice variety named 'LAL SAR' characterized by golden panicle and brown rice was identified from Nepal. Genetic analysis revealed that these phenotypes were controlled by a single recessive nuclear gene, which was designated as gpr1 (golden panicle and brown rice 1). Using the strategy of map-based cloning, we mapped gpr1 into a 47 kb interval on the rice chromosome 3 and identified its candidate gene as LOC_Os03g60509. This gene encodes a chalcone isomerase, a key enzyme in the flavonoid biosynthetic pathway. Through PCR sequencing and RT-PCR analysis, a long-fragment insertion was found in the promoter of gpr1 in 'LAL SAR', which completely suppressed the expression of this gene. Using CRISPR/Cas9 technology, we successfully knocked out GPR1 (the dominant allele of gpr1) from a Oryza sativa subsp. japonica variety 'Zhonghua 11'. The knockout mutant plants exhibited golden panicles and brown rice, with significantly increased naringenin chalcone content in the hull. Our results indicate that GPR1 participates in the flavonoid biosynthesis, providing a critical theoretical foundation and a gene resource for rice quality improvement and genetic enhancement.},
}
@article {pmid41611487,
year = {2026},
author = {Huang, X and Zeng, G and Shen, X},
title = {[Research progress and prospects in CRISPR-Cas9 gene editing in sorghum].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {1},
pages = {53-61},
doi = {10.13345/j.cjb.250419},
pmid = {41611487},
issn = {1872-2075},
support = {ZLGC201902-15//the Innovation-driven Assistance Project of China Association for Science and Technology/ ; },
mesh = {*Sorghum/genetics/growth &amp; development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Plant Breeding/methods ; },
abstract = {Sorghum is an important grain and cash crop in China, and the promotion and application of this crop have long been constrained by a shortage of genetic resources. Issues such as narrow genetic background and outdated breeding techniques have severely hindered the development and dissemination of new sorghum varieties. Although gene editing has demonstrated significant potential in the genetic improvement of crops since its inception, the application of this technology in sorghum remains lagging. This paper provides a systematic review of the latest breakthroughs in CRISPR-Cas9 in sorghum. Focusing on abiotic stress, growth and development, and quality, we explore the innovative applications of this technology in expanding genetic diversity, improving stress tolerance, optimizing plant architecture and yield potential, and enhancing quality characteristics. Additionally, we analyze the main technical challenges including low genetic transformation efficiency and insufficient adaptability of editing tools facing the gene editing in sorghum. Finally, we make an outlook on the future prospects of next-generation gene editing technologies in the genetic improvement of sorghum. This paper can provide important theoretical references for sorghum molecular breeding, and hold significant practical significance for safeguarding China's food security and enhancing the competitiveness of the sorghum industry.},
}
@article {pmid41611486,
year = {2026},
author = {Lu, Q and Teng, W and Liang, Y and Wang, Y},
title = {[Advances in genome editing and lodging resistance in sugarcane].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {1},
pages = {33-52},
doi = {10.13345/j.cjb.250555},
pmid = {41611486},
issn = {1872-2075},
support = {XDA0450000//the Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
mesh = {*Saccharum/genetics ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Gene Editing/methods ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; Zinc Finger Nucleases/genetics ; },
abstract = {Sugarcane (Saccharum spp.) is an important cash crop that provides about 90% of sugar and 40% of bioethanol in China. Due to its large genome and complicated genetic background, conventional breeding is difficult to achieve efficient genetic improvement of sugarcane. Genome editing is a disruptive technology in life sciences, enabling precise and efficient modification of target genes. From zinc-finger nucleases (ZFNs) to transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system and the derived base editing and prime editing, these technologies have greatly advanced genetic research and upgraded biological breeding. With the decoding of the sugarcane genome, genome editing has provided a new technical means for the genetic improvement of polyploid sugarcane. This article provides a comprehensive review of the trajectory of genome editing in plants, the optimization of the CRISPR/Cas system, the genetic transformation status of sugarcane, the development of sugarcane genomics, and the application of genome editing in sugarcane. It focuses on exploring the application prospects of genome editing in breeding lodging-resistant sugarcane varieties. This review aims to provide valuable references for promoting the use of genome editing in sugarcane breeding.},
}
@article {pmid41611404,
year = {2026},
author = {Park, S and Koo, B and Kim, MG and Lee, EY and Lee, HJ and Roh, Y and Lee, M and Bae, CE and Lee, SW and Kang, YA and Shin, Y},
title = {CADEM: Species-level detection of mycobacterial cfDNA via CRISPR for pulmonary disease diagnosis.},
journal = {Analytica chimica acta},
volume = {1388},
number = {},
pages = {345085},
doi = {10.1016/j.aca.2026.345085},
pmid = {41611404},
issn = {1873-4324},
mesh = {Humans ; *CRISPR-Cas Systems ; *Cell-Free Nucleic Acids/genetics/blood/analysis ; *Lung Diseases/diagnosis/microbiology ; *DNA, Bacterial/genetics/blood ; Lab-On-A-Chip Devices ; Mycobacterium avium Complex/genetics/isolation &amp; purification ; Microfluidic Analytical Techniques ; },
abstract = {BACKGROUND: Pulmonary infections caused by Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) present significant clinical challenges due to overlapping symptoms and different treatments. In particular, accurate identification of NTM species such as Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABC) is essential, as these species show drug susceptibility profiles that differ markedly from MTB. However, conventional culture-based diagnostics are time-consuming, and current molecular assays often lack resolution and rely heavily on sputum specimens. To address these limitations, liquid biopsy using bacterial-derived circulating cell-free DNA (cfDNA) offers a minimally invasive alternative, and CRISPR/Cas12a technology provides the sensitivity required to detect its low levels.<br><br>RESULTS: We developed CADEM (CRISPR-Assisted Detection via Enrichment of Mycobacterium-derived cfDNA using Microfluidic technology), a streamlined diagnostic system that integrates microfluidic cfDNA enrichment, targeted amplification, and CRISPR/Cas12a-based detection. The microfluidic platform enables high-yield recovery of cfDNA from large-volume clinical samples without the need for cell lysis. Optimized Cas12a-crRNA complexes enable highly sensitive and specific detection of MAC-, MABC-, and MTB -specific amplicons, achieving 10- to 100-fold greater sensitivity than end-point PCR and probe-based real-time PCR. In a validation set of 20 clinical specimens (7 positives and 13 healthy controls), CADEM identified all MAC, MABC, and MTB cases with full accuracy and no false positives. The CRISPR detection step produced a clear fluorescence readout within 20&#xa0;min and, together with enrichment and amplification, delivered species-level results within a 2-h workflow.<br><br>SIGNIFICANCE: CADEM offers an accurate and streamlined molecular approach for distinguishing MAC, MABC, and MTB at the species-level to support appropriate diagnosis and treatment. By combining microfluidic cfDNA enrichment with CRISPR-based detection, CADEM enables efficient analysis from liquid biopsy samples for pulmonary disease diagnosis. The system is also compatible with isothermal amplification, supporting future adaptation for point-of-care testing in resource-limited settings.},
}
@article {pmid41611402,
year = {2026},
author = {Hou, J and Wang, Y and Yuan, W and Gong, Y and Yu, Y and Qin, X and Li, H and Zhang, Y and Shi, H and Chen, Y and Zhang, X},
title = {Thermosensitive hydrogel-enhanced RPA-CRISPR/Cas12a biosensor for ultrasensitive detection of methylated loci in breast cancer ctDNA.},
journal = {Analytica chimica acta},
volume = {1388},
number = {},
pages = {345101},
doi = {10.1016/j.aca.2026.345101},
pmid = {41611402},
issn = {1873-4324},
mesh = {Humans ; *Biosensing Techniques/methods ; *Breast Neoplasms/genetics/blood/diagnosis ; *CRISPR-Cas Systems ; *DNA Methylation ; Female ; *Circulating Tumor DNA/genetics/blood ; *Hydrogels/chemistry ; *Temperature ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Methylation differences exist between breast cancer tissues and normal tissues. The release of methylated circulating tumor DNA (ctDNA) by tumor cells provides a foundation for breast cancer liquid biopsy using methylated ctDNA. However, detection of low-abundance methylated loci in ctDNA remains a significant challenge to date. Existing one-tube detection systems cannot avoid target depletion caused by CRISPR/Cas12a cleavage, leading to reduced sensitivity.<br><br>RESULTS: This study developed a thermosensitive hydrogel-based one-tube RPA-CRISPR/Cas12a detection system, combined with methylation-sensitive restriction endonucleases (MSRE), for the detection of specific methylated loci in breast cancer ctDNA. This method achieves spatial separation while maintaining connectivity of reaction phases in a single tube for the first time, and the thermosensitive hydrogel does not exert inhibitory effects on either system. The system can specifically recognize methylated target molecules with a limit of detection (LOD) as low as 1&#xa0;&#xd7;&#xa0;10[-8]&#xa0;ng/&#x3bc;L (&#x2248;70 copies/&#x3bc;L), outperforming the current glycerol-enhanced one-tube reaction system. It is capable of distinguishing methylated fractions as low as 0.05&#xa0;%, with a sensitivity twice that of the gold standard methylation-specific quantitative PCR (Methylight). Detection of genomic DNA (gDNA) from tumor tissues and paired plasma ctDNA of 15 clinical patients using this method showed both sensitivity and specificity reaching 100&#xa0;%.<br><br>SIGNIFICANCE: This novel, highly sensitive, efficient, and portable detection method innovatively resolves the target depletion issue caused by CRISPR/Cas12a cleavage in traditional RPA-CRISPR/Cas12a systems via thermosensitive hydrogel-mediated single-tube phase separation technology. It provides a new technical pathway for the accurate detection of low-abundance methylated circulating tumor DNA (ctDNA), will significantly enhance the level of breast cancer liquid biopsy, and offer strong support for the diagnosis and differential diagnosis of breast cancer.},
}
@article {pmid41610742,
year = {2026},
author = {Jiao, R and Ni, Q and Zhao, R and Zhu, X and Wang, L and Jiao, J and Jiang, H and Wu, Q and Yao, S and Yao, L and Liu, K and Qin, P},
title = {Dual CRISPR/Cas-driven amplification-free surface-enhanced Raman scattering biosensor combined with a smartphone for simultaneous detection of total and live target bacteria.},
journal = {Biosensors &amp; bioelectronics},
volume = {299},
number = {},
pages = {118446},
doi = {10.1016/j.bios.2026.118446},
pmid = {41610742},
issn = {1873-4235},
abstract = {Simultaneous detection of total and live counts of target bacteria is significant but challenging. To address this challenge, here we proposed a dual CRISPR/Cas-driven amplification-free surface-enhanced Raman scattering (SERS) biosensor, termed cc-SERS. The biosensor was constructed based on the property that DNA remains stable while some RNA degrades rapidly after bacterial death. Briefly, the target DNA and RNA from live bacteria could activate both CRISPR/Cas12a and CRISPR/Cas13a, while dead bacteria could only activate CRISPR/Cas12a through the target DNA. In the absence of the target bacteria, neither CRISPR/Cas12a nor CRISPR/Cas13a could be activated. Therefore, the characteristic Raman signal at 1079 cm[-1] generated by the target DNA-activated CRISPR/Cas12a indicated the presence of the target bacteria (the sum of dead and live), while the characteristic Raman signal at 593 cm[-1] produced by the target RNA-activated CRISPR/Cas13a indicated the presence of the live target bacteria. With this unique signaling pattern, the biosensor is capable of detecting both total and live target bacteria in a single tube with a detection limit of ∼10 CFU/mL. The introduction of a rapid pre-processing procedure and a smartphone-assisted portable Raman spectrometer enabled the entire process to be completed in the field within 45 min. Thanks to the excellent programmability of CRISPR/Cas systems, the biosensor has been successfully applied to the detection of Staphylococcus aureus and Cronobacter sakazakii, respectively. As a proof-of-concept, this work opens a promising avenue for the simultaneous detection of total and live target bacteria.},
}
@article {pmid41610326,
year = {2026},
author = {Guo, W and Cheng, Y and Yin, H and Li, S and Wan, Y and Li, C and Jiang, C and Zhou, J and Yuan, X and Wang, J},
title = {CRISPR-AD: Combinational Detection of Blood Protein and miRNA with Digital CRISPR-Based Assay Enable to Improve the Diagnostic Performance of Alzheimer's Disease.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c06080},
pmid = {41610326},
issn = {1520-6882},
abstract = {Blood-based biomarkers present a noninvasive approach for detecting and assessing Alzheimer's disease (AD) pathophysiology, always by using sophisticated instrumentation for accurate detection. Here, we introduce CRISPR-AD, a CRISPR/Cas-based digital assay designed for the combined detection of protein and microRNA in blood. This method achieves a limit of detection (LOD) as low as 60 fg/mL for phosphorylated tau217 (p-tau217) and 0.5 fM for microRNA-34a-5p (miRNA34a), enabling successful detection in both AD patients and healthy individuals. We find that the combined use of these biomarkers improves the ability to distinguish between AD patients and healthy participants, particularly in individuals with mild cognitive impairment (MCI). Additionally, we have developed a portable device that integrates a smartphone as an imaging system for point-of-care testing (POCT), offering the potential for early stage AD screening. This study represents the first effort to evaluate the combined detection of blood protein and microRNA biomarkers for AD, underscoring the potential of multiple biomarker combinations for more accurate AD diagnosis.},
}
@article {pmid41609044,
year = {2026},
author = {Anschuetz, A and Listyono, R and Vorley, T and Platt, B and Harrington, CR and Riedel, G and Schwab, K},
title = {The Icelandic Mutation in the Murine APP Gene, mAPP[A673T], on Amyloid-β Plaque Burden in the 5×FAD Alzheimer Model.},
journal = {Journal of integrative neuroscience},
volume = {25},
number = {1},
pages = {48581},
doi = {10.31083/JIN48581},
pmid = {41609044},
issn = {0219-6352},
support = {PAR1577//TauRx Therapeutics Ltd., Singapore/ ; PAR2074//TauRx Therapeutics Ltd., Singapore/ ; },
mesh = {Animals ; *Alzheimer Disease/genetics/pathology/metabolism ; *Amyloid beta-Protein Precursor/genetics/metabolism ; Disease Models, Animal ; *Plaque, Amyloid/pathology/genetics/metabolism ; Mice, Transgenic ; Male ; *Amyloid beta-Peptides/metabolism ; Mice, Inbred C57BL ; Female ; Presenilin-1/genetics ; Mice ; Humans ; Mutation ; *Brain/pathology/metabolism ; Iceland ; },
abstract = {BACKGROUND: The protective Icelandic mutation in the amyloid precursor protein (APP) gene, APP[A673T], identified in Icelandic and other Nordic populations is associated with a significantly lower risk of developing Alzheimer's disease (AD). Conflicting results have been reported for the human APP[A673T] mutation in various knock-in models of AD, but the effect of the mouse APP[A673T] form in 5× familial AD (5×FAD) mice has never been investigated.<br><br>METHODS: We crossed C57Bl6/J mice expressing a single point mutation edited into the murine APP gene via Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated (CRISPR-Cas) gene editing, termed mAPP[A673T], with 5&#xd7;FAD mice that overexpress human APP carrying the Swedish (K670N/M671L), Florida (I716V), and London (V717I) mutations as well as human presenilin-1 (PS1) with two mutations (M146L and L286V); the resulting mice were termed 5&#xd7;FAD &#xd7; mAPP[A673T] mice. We investigated amyloid beta-protein (A&#x3b2;) pathology in 5&#xd7;FAD &#xd7; mAPP[A673T], 5&#xd7;FAD and their respective controls, mAPP[A673T], and C57Bl6/J wild type mice, at 6-months of age using immunohistochemistry, immunoblotting, and enzyme-linked immunosorbent assay (ELISA).<br><br>RESULTS: We found a moderate yet significant reduction in A&#x3b2; plaque size in male 5&#xd7;FAD &#xd7; mAPP[A673T] compared with 5&#xd7;FAD mice. No differences were observed for soluble/insoluble A&#x3b2;40 and A&#x3b2;42 levels per se, but lower plaque count/area was found in 5&#xd7;FAD &#xd7; mAPP[A673T] mice when A&#x3b2;42/A&#x3b2;40 ratios were low, suggesting a genotype-dependent sensitivity to A&#x3b2; aggregation and accumulation.<br><br>CONCLUSIONS: The mAPP[A673T] mutation has the potential to modify A&#x3b2; pathology in 5&#xd7;FAD mice at the age of 6 months.},
}
@article {pmid41592354,
year = {2026},
author = {Du, T and Ding, F and Ma, X and Luo, Y and Zhu, D and Wang, L and Su, S},
title = {Ultrasensitive detection of monkeypox virus: harnessing synergistic CRISPR-driven signal amplification on a DNA tetrahedron-mediated sensing interface.},
journal = {Biosensors &amp; bioelectronics},
volume = {298},
number = {},
pages = {118441},
doi = {10.1016/j.bios.2026.118441},
pmid = {41592354},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; Limit of Detection ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; *Monkeypox virus/isolation &amp; purification/genetics ; *Mpox, Monkeypox/diagnosis/virology ; Nanostructures/chemistry ; *DNA, Viral/genetics ; Hydrogen Peroxide/chemistry ; DNA/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and ultrasensitive detection of emerging infectious diseases is critical for public health security. Herein, an electrochemical biosensor was developed for ultrasensitive detection of monkeypox virus (MPXV) by integrating CRISPR/Cas12a-driven signal amplification strategy with tetrahedral DNA nanostructure (TDN)-based sensing interface. The added MPXV DNA can efficiently activate the cleavage activity of Cas12a protein, thereby mediating the CRISPR-driven hybridization chain reaction (HCR) on TDN sensing interface. The horseradish peroxidase (HRP)-labeled HCR product can catalyze 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) reaction to generate an amplified electrochemical signal. Based on the signal change, the CRISPR-driven electrochemical biosensor exhibited better detection performance comparable to those of pre-amplification CRISPR-based biosensors for MPXV detection, including wide linear range, an ultralow detection limit, exceptional selectivity against non-target viruses (CPXV, ETCV, VZV, HSV), high reproducibility and accepted stability. Integrated with a smartphone-based portable device, the designed point-of-care testing (POCT) electrochemical biosensor can accurately detect MPXV in 10 % human saliva. This work provides a promising sensing platform for rapid, accurate and on-site detection of infectious diseases.},
}
@article {pmid41581366,
year = {2026},
author = {Yu, Y and Zhang, J and Xie, G and Lin, Y and Huang, Y and Rao, C and Hou, Y and Chen, H},
title = {A DNAzyme-CRISPR cascade strategy for preamplification-free detection of Mycobacterium tuberculosis.},
journal = {Biosensors &amp; bioelectronics},
volume = {298},
number = {},
pages = {118428},
doi = {10.1016/j.bios.2026.118428},
pmid = {41581366},
issn = {1873-4235},
mesh = {*Mycobacterium tuberculosis/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *DNA, Catalytic/chemistry/genetics ; *Tuberculosis/diagnosis/microbiology ; Humans ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; *DNA, Bacterial/genetics/isolation &amp; purification ; Nucleic Acid Amplification Techniques ; Reproducibility of Results ; },
abstract = {Tuberculosis (TB) remains one of the most fatal infectious diseases worldwide, successful treatment is often limited by insufficient diagnostic capabilities. It creates a pressing need for diagnostic methods that combine high sensitivity, specificity, and operational robustness. In this work, we developed a DNAzyme-CRISPR cascade strategy (Dz-CRISPR) for the specific identification of the IS6110 sequence of Mycobacterium tuberculosis (MTB). This system integrated a thermodynamically stabilized hairpin probe, an Arch-shaped signal transduction switch, and an allosterically activated CRISPR-Cas12a cascade. The design enabled direct target recognition and subsequent signal amplification without a preamplification step, offering a simplified workflow with enhanced stability. The assay demonstrated a detection limit of 211.3 fM and exhibited high specificity by accurately discriminating the IS6110 from specific DNA sequence of non-tuberculous mycobacteria and other common respiratory pathogens. Validation using clinical bronchoalveolar lavage fluid samples further confirmed the method's reliable performance, reproducibility, and satisfactory recovery rates. Current Dz-CRISPR detection strategy provides a reliable and practical solution for tuberculosis diagnosis with high sensitivity, high specificity, and operational robustness, thus demonstrating potential for practical use in resource-constrained areas.},
}
@article {pmid41576437,
year = {2026},
author = {Wei, L and Cheng, Z and Xu, M and Chen, H and Lan, W and Long, W and She, Y and Fu, H},
title = {Bulge DNA-driven CRISPR/Cas12a dynamic activation circuit enables highly sensitive and versatile biosensing.},
journal = {Biosensors &amp; bioelectronics},
volume = {298},
number = {},
pages = {118412},
doi = {10.1016/j.bios.2026.118412},
pmid = {41576437},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Methicillin-Resistant Staphylococcus aureus/isolation &amp; purification/genetics ; Limit of Detection ; *DNA/chemistry/genetics ; *Mycotoxins/analysis/isolation &amp; purification ; Aptamers, Nucleotide/chemistry/genetics ; Bacterial Proteins/genetics/chemistry ; *Endodeoxyribonucleases/chemistry/genetics ; Humans ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a has emerged as an innovative biosensing tool; however, its intrinsically linear accumulation of cleavage signals limits detection sensitivity. Herein, we report a bulge DNA (BD)-driven CRISPR/Cas12a dynamic activation circuit, termed CBD, as a highly sensitive and versatile biosensing platform for both nucleic acid and non-nucleic acid targets. The BD structure was rationally engineered to undergo programmable structural and functional transformation upon bulge degradation, thereby initiating exponential, self-amplifying activation of the Cas12a circuit. For nucleic acid targets, direct Cas12a recognition triggers BD cleavage and a positive feedback loop, enabling highly sensitive detection with a limit of 14 CFU/mL for methicillin-resistant Staphylococcus aureus. For non-nucleic acid targets, a universal single-stranded DNA activator was linked to the aptamer-complementary strand, enabling target-responsive release and subsequent initiation of the CBD system without altering the crRNA or BD sequence. This strategy enabled the detection of pesticides and mycotoxins at the picogram-per-milliliter level. Furthermore, an "OR" logic gate was constructed for the simultaneous detection of dual mycotoxins, highlighting the platform's capability for multiplexed hazard monitoring. Overall, CBD demonstrates significant potential as a new paradigm for next-generation biosensing technologies.},
}
@article {pmid41554314,
year = {2026},
author = {Paulo, BS and Romanowski, SB and Kadjo, AE and Lourenzon, VB and Eustáquio, AS},
title = {Genome minimization of a Burkholderia bacterial host.},
journal = {Metabolic engineering},
volume = {94},
number = {},
pages = {305-314},
doi = {10.1016/j.ymben.2026.01.008},
pmid = {41554314},
issn = {1096-7184},
mesh = {*Burkholderia/genetics/metabolism ; *Genome, Bacterial/genetics ; Plasmids/genetics ; *Metabolic Engineering/methods ; CRISPR-Cas Systems ; },
abstract = {Genome minimization, including the deletion of endogenous gene clusters that encode natural products, is a common strategy to improve the yield of heterologous products. We have been interested in developing Burkholderia sp. FERM BP-3421 as an alternative bacterial host. Instead of indiscriminately deleting gene clusters, which may have deleterious effects, we guided our efforts using transcriptomics data from production cultures. The genome of FERM BP-3421 is subdivided into two chromosomes and two plasmids. The top transcribed gene clusters were those encoding polyketide-nonribosomal peptide spliceostatins on plasmid p1 and nonribosomal peptide selethramide on chromosome 1. Deletion of the spliceostatin cluster had been shown to improve titers of the ribosomal peptide capistruin, whereas we showed that deletion of the selethramide cluster had no effect on capistruin titers. We next targeted the two endogenous plasmids using a CRISPR-Cas12a strategy, resulting in an 11 % reduction in genome size. The plasmid cured strains showed improved growth and 20-40 % increased production of capistruin depending on whether one or both plasmids were deleted. However, deletion of p2 alone negatively affected the heterologous production of two distinct polyketide-nonribosomal peptides. The p2[-] strain produced only 5-23 % of the glidobactin A and megapolipeptin A titers compared to the wild type, respectively, whereas titers were restored to wild type levels in the p1[-] p2[-] strain. The observation that p2 appears to contain functions that support polyketide-nonribosomal peptide biosynthesis was unexpected and sets the stage for future studies aimed at identifying these functions and further enabling engineering efforts that may be widely applicable to other strains.},
}
@article {pmid41547318,
year = {2026},
author = {Beckley, J and Barrangou, R},
title = {Phage-mediated delivery of CRISPR payloads.},
journal = {Current opinion in microbiology},
volume = {89},
number = {},
pages = {102704},
doi = {10.1016/j.mib.2025.102704},
pmid = {41547318},
issn = {1879-0364},
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics/virology ; Genetic Engineering/methods ; },
abstract = {Over the past decade, improvements in sequencing technologies and computational tools have advanced our understanding of the composition and function of microbial communities in various environments. Now, in order to manipulate and engineer these communities, we need technologies that enable broadly applicable and specific alterations to establish and modulate the molecular basis for their functional roles. Recent advances in bacteriophage engineering strategies, synthetic biology techniques, and in silico approaches have greatly expanded our ability to perform in situ perturbations. Clustered regularly interspaced short palindromic repeats-Cas systems in particular can provide an efficient means of engineering phages, and can also be delivered as a recombinant payload to perform precision genome editing directly in the host environment. Modified Cas effectors have been developed that allow for increasingly diverse edits with applications in the fields of medicine, food, and agriculture. In this review, we discuss recent advances in using bacteriophages to deliver various clustered regularly interspaced short palindromic repeats-Cas effectors. While challenges remain regarding the phylogenetic breadth of deployment, recombinant phages generally present a unique and effective means to rationally engineering microbial community function and composition.},
}
@article {pmid41510755,
year = {2026},
author = {Wang, L and Su, L and Gu, W and Nie, S and Luo, J and Li, Y and Pan, A and Li, Y},
title = {A photonic crystal sensing array based on a tandem CRISPR/Cas13a system for ultra-sensitive and high-throughput detection of the CVA6 virus.},
journal = {The Analyst},
volume = {151},
number = {3},
pages = {903-912},
doi = {10.1039/d5an01178g},
pmid = {41510755},
issn = {1364-5528},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; Photons ; *Biosensing Techniques/methods ; *RNA, Viral/genetics/analysis ; Fluorescent Dyes/chemistry ; High-Throughput Screening Assays/methods ; },
abstract = {Coxsackievirus A6 (CVA6) has emerged as a major cause of hand-foot-mouth disease (HFMD), yet no standardized detection method for it is currently available. Developing a simple, sensitive, and specific CVA6 test is crucial for HFMD control and safeguarding the health of at-risk children. Herein, a photonic crystal (PC) sensing array based on a tandem CRISPR/Cas13a system has been proposed for highly specific and ultra-sensitive analysis of CVA6 RNA, without the need for reverse transcription and amplification procedures. In this strategy, two crRNAs targeting CVA 6 RNA were designed and screened, and the fluorescence signal of the tandem CRISPR/Cas13a system was found to be up to 4.2 times higher than that of the non-tandem CRISPR system. The PC array with periodic nanostructures was prepared through self-deposition and further enhanced the fluorescent signal output from the tandem CRISPR system, owing to the match of the emission wavelength of the fluorescent dyes and the photonic band gap (PBG) of the PC. Benefitting from the synergistic effect of the tandem CRISPR system and PC array, as well as the high trans-cleavage activity of Cas13a protein, this engineered sensing array enables ultra-sensitive detection with a limit of detection (LOD) as low as 24.9 fM for CVA6. Meanwhile, this sensing strategy also achieved high-throughput and rapid analysis with a detection frequency of about 96 samples every 3.4 minutes. Therefore, the proposed strategy offers a simple workflow without reverse transcription or amplification, along with high sensitivity and high throughput, demonstrating strong potential for applications in biometrics and clinical diagnostics.},
}
@article {pmid41443126,
year = {2026},
author = {Rahimi, A and Rahimmanesh, I and Abedpoor, N and Boshtam, M and Bidram, E and Javanmard, SH and Khanahmad, H and Rafiee, L and Bigham, A and Rafienia, M and Karbasi, S and Shariati, L},
title = {The MCM/Lys-Cys nanodevices for the efficient gene delivery: An approach towardsMCP1gene manipulation using CRISPR technology.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {260},
number = {},
pages = {115377},
doi = {10.1016/j.colsurfb.2025.115377},
pmid = {41443126},
issn = {1873-4367},
mesh = {Humans ; *Silicon Dioxide/chemistry ; *Gene Transfer Techniques ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; *Chemokine CCL2/genetics ; Cell Proliferation ; Cell Line, Tumor ; Female ; *Cysteine/chemistry ; Breast Neoplasms/genetics/pathology/therapy ; *Lysine/chemistry ; Cell Movement ; Particle Size ; Surface Properties ; Gene Editing ; Plasmids/genetics ; },
abstract = {Breast cancer continues to be the most common malignancy among women worldwide, requiring novel therapeutic approaches. This research investigates an innovative gene delivery strategy employing mesoporous silica nanoparticles (MCM-41) modified with lysine and cysteine (Lys-Cys) for the effective delivery of CRISPR-Cas9 plasmids aimed at the monocyte chemoattractant protein-1 (MCP-1/CCL2) gene. Bioinformatics analysis of the TCGA-BRCA dataset revealed substantial deregulation of CCL2 in breast cancer, underscoring its involvement in tumor growth and inflammation. The MCM/Lys-Cys nanocarrier demonstrated remarkable biocompatibility and effectively encapsulated a plasmid containing GFP, promoting superior cellular uptake in MDA-MB-231 breast cancer cells compared to conventional techniques. Functional experiments demonstrated that CRISPR/Cas9-mediated suppression of CCL2 markedly decreased cell proliferation, migration, and invasion, highlighting the promise of this targeted gene therapy strategy in breast cancer management. The findings indicate that the MCM/Lys-Cys nanosystem presents a viable non-viral approach for precise gene editing, potentially boosting therapeutic efforts against breast cancer by modulating inflammatory pathways.},
}
@article {pmid41431758,
year = {2026},
author = {Hwang, S and Ko, H and Lee, HY and Choi, J},
title = {Nanocarriers for the delivery of the CRISPR/Cas9 system.},
journal = {Nanomedicine (London, England)},
volume = {21},
number = {3},
pages = {429-448},
doi = {10.1080/17435889.2025.2598332},
pmid = {41431758},
issn = {1748-6963},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Animals ; *Nanoparticles/chemistry ; *Gene Transfer Techniques ; *Drug Carriers/chemistry ; },
abstract = {The clustered regularly interspaced short palindromic repeat/associated protein 9 (CRISPR/Cas9) system has been used for the precise manipulation of target DNA, making efficient genome editing in cells a reality. The CRISPR/Cas9 system has shown great potential in biomedical applications, such as disease treatment, transcription regulation, and genome-wide screening, and is opening a new era in biotechnology. However, the efficient and selective delivery of the CRISPR/Cas9 system remains a critical obstacle. Literature search conducted using Web of Science, Scopus, PubMed and Google Scholar for articles published from 2015 to 2024. In this review, we discuss several delivery methods for the CRISPR/Cas9 system, focusing on techniques using nanocarriers. Specifically, we comprehensively discussed the challenges, future directions, and potential of various delivery methods for the CRISPR/Cas9 system.},
}
@article {pmid41259745,
year = {2026},
author = {Liu, L and Glaser, A and Isiaku, AI and Fairfax, K and Casolari, DA and Ristovski, A and Murphy, V and van Twest, S and Henrikus, SS and Heraud-Farlow, J and Granger, E and Novakovic, S and Monks O'Byrne, SF and Tsui, V and Conyers, R and Gonda, TJ and Crismani, W and D'Andrea, RJ and Heierhorst, J and Deans, AJ},
title = {Fancl-mutant mice reveal central role of monoubiquitination in Fanconi anemia and a model for therapeutic gene editing.},
journal = {Blood advances},
volume = {10},
number = {3},
pages = {821-836},
doi = {10.1182/bloodadvances.2025017217},
pmid = {41259745},
issn = {2473-9537},
mesh = {Animals ; *Fanconi Anemia/genetics/therapy/metabolism/pathology ; Mice ; *Gene Editing/methods ; *Ubiquitination ; Disease Models, Animal ; *Fanconi Anemia Complementation Group L Protein/genetics/metabolism ; *Mutation ; Fanconi Anemia Complementation Group D2 Protein/metabolism ; Humans ; CRISPR-Cas Systems ; },
abstract = {Fanconi anemia (FA) is a rare genetic disorder causing the progressive loss of hematopoietic stem cells (HSCs) and bone marrow failure. Most cases result from deficient monoubiquitination of FANCD2 by the FA core complex. However, given that additional functions for the complex have been proposed, it remains unclear whether loss of FANCD2 monoubiquitination is the sole cause of all FA phenotypes. Here, we generated a murine allele (FanclTATΔ) that mimics an allele from a patient with FA. This 3-bp deletion removes a catalytic cysteine in the E3 RING ligase domain of the FANCL subunit. Biochemical assays show that the mutant FA core complex retains structural integrity but lacks FANCD2 monoubiquitination activity. Homozygous FanclTATΔ/TATΔ mice phenocopy classical human FA features, including infertility, craniofacial anomalies, DNA damage hypersensitivity, and progressive HSC loss with age. Correcting the mutation using CRISPR-Cas9 or prime editing technology restores FANCD2 monoubiquitination and normal DNA damage resistance in myeloid cells. Collectively, our mouse model demonstrates that loss of RING E3 ubiquitin ligase activity of the FA core complex explains developmental defects and hematopoietic failure in FA and provides a new animal model for testing potentially therapeutic gene editing.},
}
@article {pmid41205860,
year = {2026},
author = {Yunus, IS and Carruthers, DN and Chen, Y and Gin, JW and Baidoo, EEK and Petzold, CJ and Garcia Martin, H and Adams, PD and Mukhopadhyay, A and Lee, TS},
title = {Predictive CRISPR-mediated gene downregulation for enhanced production of sustainable aviation fuel precursor in Pseudomonas putida.},
journal = {Metabolic engineering},
volume = {94},
number = {},
pages = {67-76},
doi = {10.1016/j.ymben.2025.11.007},
pmid = {41205860},
issn = {1096-7184},
mesh = {*Pseudomonas putida/genetics/metabolism ; *CRISPR-Cas Systems ; *Metabolic Engineering/methods ; *Down-Regulation ; *Bacterial Proteins/genetics/biosynthesis/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, its application is often constrained by two challenges: (i) rationally identifying effective gene targets for downregulation and (ii) efficiently constructing multiplexed CRISPRi systems. In this study, we address both challenges by integrating a computational prioritization tool with a versatile assembly method for building multiplexed CRISPRi systems. FluxRETAP (Flux-Reaction Target Prioritization) accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers in Pseudomonas putida KT2440, outperforming a conventional non-computational, pathway-guided target selection. The highest isoprenol titer of nearly 1.5 g/L was achieved by knocking down PP_4118 (a gene encoding α-ketoglutarate dehydrogenase). The use of VAMMPIRE (Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation) enabled accurate assembly of CRISPRi constructs containing up to five sgRNA arrays, reducing context dependency and achieving uniform, position-independent gene downregulation. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.},
}
@article {pmid41201956,
year = {2026},
author = {Sinha, R and Patil, RV and Romano, R and Sharma, D and Lee, E and Perriman, R and Takeda, S and Lesch, BJ and Yao, Z and Liu, YL and Cromer, MK and Porteus, MH and Bertaina, A},
title = {Novel humanized loss-of-function NF1 mouse model of juvenile myelomonocytic leukemia.},
journal = {Blood advances},
volume = {10},
number = {3},
pages = {837-849},
doi = {10.1182/bloodadvances.2024015191},
pmid = {41201956},
issn = {2473-9537},
mesh = {Animals ; *Leukemia, Myelomonocytic, Juvenile/genetics/pathology/metabolism/therapy ; Mice ; Disease Models, Animal ; Humans ; *Neurofibromin 1/genetics/metabolism ; *Loss of Function Mutation ; Granulocyte-Macrophage Colony-Stimulating Factor ; Hematopoietic Stem Cells/metabolism ; CRISPR-Cas Systems ; },
abstract = {Juvenile myelomonocytic leukemia (JMML) is a fatal pediatric cancer characterized by classical features such as splenomegaly, monocytosis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity, with RAS pathway mutations being the major drivers. Mutations causing loss-of-function of the Neurofibromin 1 gene (NF1LOF) occur in ∼20% of patients with JMML. NF1LOF drives upregulation of RAS/MAPK/PI3K pathways that leads to aggressive proliferation/differentiation of immature myeloid cells. Hematopoietic stem cell transplantation is the only curative option, but relapse occurs in ∼50% of patients, indicating an urgent need for novel and targeted therapeutic strategies. However, low patient sample availability and a lack of reliable disease models have made it difficult to study and treat JMML. Using CRISPR/Cas9, we have generated NF1LOF in human umbilical cord blood-derived hematopoietic stem and progenitor cells (HSPCs). We achieved a high gene knockout rate of ∼89% and concomitant loss of NF1 protein in the modified HSPCs. Importantly, NF1LOF cells displayed marked GM-CSF hypersensitivity in in vitro colony-forming unit assays, mirroring JMML. When transplanted into NSG-SGM3 mice, they caused rapid lethality (median survival of 32 days), myeloid expansion, tissue infiltration (spleen, liver, and lungs), and specific upregulation of RAS/MAPK pathway and STAT5 genes, consistent with patient profiles. This first humanized NF1LOF mouse model recapitulates key JMML features, enabling investigation of disease mechanisms and targeted therapies.},
}
@article {pmid40135929,
year = {2026},
author = {Ming, Z and Liu, F and Moran, HR and Lalonde, RL and Adams, M and Restrepo, NK and Joshi, P and Ekker, SC and Clark, KJ and Friedberg, I and Sumanas, S and Yin, C and Mosimann, C and Essner, JJ and McGrail, M},
title = {Lineage labeling with zebrafish hand2 Cre and CreERT2 recombinase CRISPR knock-ins.},
journal = {Developmental dynamics : an official publication of the American Association of Anatomists},
volume = {255},
number = {1},
pages = {86-105},
pmid = {40135929},
issn = {1097-0177},
support = {R01 HL168087/HL/NHLBI NIH HHS/United States ; K99 HL168148/HL/NHLBI NIH HHS/United States ; //Iowa State University of Science and Technology/ ; R24 OD036201/RI/ORIP NIH HHS/United States ; T32 GM141742/GM/NIGMS NIH HHS/United States ; R01 HL153005/HL/NHLBI NIH HHS/United States ; R24 OD020166/OD/NIH HHS/United States ; F31 HL167580/HL/NHLBI NIH HHS/United States ; R24 OD036201/OD/NIH HHS/United States ; R01 DK117266/DK/NIDDK NIH HHS/United States ; //PFIC Research Center, Cincinnati Children's Hospital/ ; //Children's Hospital Colorado/ ; R24 OD020166/RI/ORIP NIH HHS/United States ; R01 HL168097/HL/NHLBI NIH HHS/United States ; R01 DK117266/DK/NIDDK NIH HHS/United States ; T32 GM141742/GM/NIGMS NIH HHS/United States ; R24 OD020166/RI/ORIP NIH HHS/United States ; R24 OD036201/RI/ORIP NIH HHS/United States ; F31 HL167580/HL/NHLBI NIH HHS/United States ; K99 HL168148/HL/NHLBI NIH HHS/United States ; R01 HL153005/HL/NHLBI NIH HHS/United States ; R01 HL168087/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Zebrafish/embryology/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Integrases/genetics/metabolism ; *Cell Lineage/genetics ; *Basic Helix-Loop-Helix Proteins/genetics/metabolism ; Gene Knock-In Techniques/methods ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Developmental ; },
abstract = {BACKGROUND: The ability to generate endogenous Cre recombinase drivers using CRISPR-Cas9 knock-in technology allows lineage tracing, cell type-specific gene studies, and in vivo validation of inferred developmental trajectories from phenotypic and gene expression analyses. This report describes endogenous zebrafish hand2 Cre and CreERT2 drivers generated with GeneWeld CRISPR-Cas9 precision targeted integration.<br><br>RESULTS: hand2-2A-cre and hand2-2A-creERT2 knock-ins crossed with ubiquitous loxP-based Switch reporters led to broad labeling in expected mesodermal and neural crest-derived lineages in branchial arches, cardiac, fin, liver, intestine, and mesothelial tissues, as well as enteric neurons. Novel patterns of hand2 lineage tracing appeared in venous blood vessels. CreERT2 induction at 24&#x2009;h reveals hand2-expressing cells in the 24- to 48-h embryo contribute to the venous and intestinal vasculature. Induction in 3 dpf larvae restricts hand2 lineage labeling to mesoderm-derived components of the branchial arches, heart, liver, and enteric neurons.<br><br>CONCLUSIONS: hand2 progenitors from the lateral plate mesoderm and ectoderm contribute to numerous lineages in the developing embryo. At later stages, hand2-expressing cells are restricted to a subset of lineages in the larva. The endogenous hand2 Cre and CreERT2 drivers establish critical new tools to investigate hand2 lineages in zebrafish embryogenesis and larval organogenesis.},
}
@article {pmid41608958,
year = {2026},
author = {Zhang, Y and Walker, RSK and Sunna, A and Barber, TJ and Li, M},
title = {Droplet Digital CRISPR for Nucleic Acid Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e17470},
doi = {10.1002/advs.202517470},
pmid = {41608958},
issn = {2198-3844},
support = {DP200102269//Australian Research Council/ ; DP240100795//Australian Research Council/ ; GNT2017679//National Health and Medical Research Council/ ; },
abstract = {Droplet digital (dd) clustered regularly interspaced short palindromic repeats (CRISPR) integrates the high sequence specificity of CRISPR-based nucleic acid detection with the absolute quantification capability of digital droplet microfluidics, offering high sensitivity, precision, and scalability. By partitioning samples into thousands to millions of picoliter microdroplets, ddCRISPR enables single-molecule resolution and minimizes background interference. This review summarizes the principles of droplet generation, manipulation, and detection in ddCRISPR platforms, as well as recent advances in amplification-based and amplification-free detection strategies. Representative applications are highlighted for viral, bacterial, and other DNA/RNA biomarker detection. Current challenges, including workflow automation, droplet stability, multiplexing, and assay portability, are discussed alongside future perspectives such as artificial intelligence (AI)-assisted analysis, point-of-care integration, and high-throughput multiplexed detection. These insights aim to guide the translation of ddCRISPR technologies from laboratory research to robust, scalable, and accessible diagnostic solutions.},
}
@article {pmid41608579,
year = {2026},
author = {Sanchez-Pupo, RE and Kelly, JJ and Shalaby, N and Xia, Y and Martinez-Santiesteban, FM and Lau, J and Verriet, IE and Fox, MS and Hicks, JW and Thiessen, JD and Ronald, JA},
title = {Imaging CRISPR-edited CAR-T cell therapies with optical and positron emission tomography reporters.},
journal = {Theranostics},
volume = {16},
number = {7},
pages = {3227-3245},
pmid = {41608579},
issn = {1838-7640},
mesh = {*Positron-Emission Tomography/methods ; Animals ; Humans ; *Receptors, Chimeric Antigen/genetics/metabolism ; *Immunotherapy, Adoptive/methods ; Mice ; Gene Editing/methods ; *T-Lymphocytes/immunology/metabolism/transplantation ; CRISPR-Cas Systems ; Genes, Reporter ; Cell Line, Tumor ; Symporters/genetics ; Female ; Dependovirus/genetics ; Optical Imaging/methods ; },
abstract = {Rationale: Chimeric antigen receptor (CAR) T cell therapies have shown remarkable success in treating hematological cancers and are increasingly demonstrating potential for solid tumors. CRISPR-based genome editing offers a promising approach to enhance CAR-T cell potency and safety, yet challenges such as inefficient tumor homing and toxicities in normal tissues, limit broader adoption. Advanced imaging technologies, including bioluminescence imaging (BLI) and positron emission tomography (PET), provide real-time visualization of CAR-T cell behavior in vivo. Here, we developed Trackable Reporter Adaptable CRISPR-Edited CAR (tRACE-CAR) T cells, a modular system for site-specific integration of CARs and imaging reporters. Methods: The luciferase reporter AkaLuciferase (AkaLuc) or the human sodium iodide symporter (NIS) was cloned downstream of the CAR in adeno-associated virus (AAV) donors for imaging. CAR-reporter cassettes were inserted into the T-cell receptor α constant locus of primary human T cells via CRISPR editing and AAV transduction. Editing efficiency was assessed by flow cytometry. In vitro cytotoxicity was evaluated across multiple effector-to-target ratios. In vivo, BLI and PET imaging were used for tracking CAR-T cells in tumor-bearing immunodeficient mice. Results: T cell receptor (TCR) knockout efficiency exceeded 85% and CAR expression reached 70-80%. Reporter-engineered CAR-T cells exhibited significant cytotoxicity and outperformed naïve T cells. In vivo, AkaLuc BLI and [18]F-tetrafluoroborate PET enabled non-invasive tracking of viable CAR-T cells. Administration route (intravenous, peritumoral, or intraperitoneal) significantly influenced CAR-T cell distribution and therapeutic effectiveness. Conclusion: tRACE-CAR enables precise optical and PET tracking of CRISPR-edited CAR-T cells in models of leukemia and ovarian cancer, allowing dynamic, non-invasive monitoring of cell distribution in both tumors and off-target tissues. This imaging-enabled platform could lead to more personalized, effective CRISPR-edited CAR cell therapies.},
}
@article {pmid41513195,
year = {2026},
author = {Luo, Q and Huang, Y and Zheng, H and Ke, W and Zheng, H and Sun, Q and Wang, M and Weng, Z},
title = {CRISPR-engineered zebrafish expression system for human type III collagen: Therapeutic efficacy in wound healing.},
journal = {International journal of biological macromolecules},
volume = {340},
number = {Pt 1},
pages = {150161},
doi = {10.1016/j.ijbiomac.2026.150161},
pmid = {41513195},
issn = {1879-0003},
mesh = {Animals ; *Zebrafish/genetics ; Humans ; *Wound Healing/drug effects/genetics ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; *Collagen Type III/genetics/pharmacology ; Mice ; Recombinant Proteins/pharmacology/genetics ; Cell Proliferation ; },
abstract = {Human type III collagen (Col III) is a critical component for skin tissue repair and anti-aging, yet its heterologous expression often faces challenges such as incomplete structure and poor thermostability. Here, we established a transgenic zebrafish expression system via CRISPR/Cas9 technology, integrating the human Col3a1 gene into a non-functional region of zebrafish chromosome 4. The extraction yield of total zebrafish collagen (Col III-TC), a composite material comprising both recombinant human Col III and endogenous zebrafish collagens, was 45.76%. Structural analysis revealed intact fibrous architecture and a thermal shrinkage temperature of 71.3 °C, significantly superior to conventional systems. Functionally, Col III-TC exhibited remarkable free radical-scavenging capacity and suppressed LPS-induced inflammation in 3T3-L1 cells (downregulating Tnfα, Il1b, and Il6, while upregulating Il10), alongside promoting fibroblast proliferation. In a murine acute wound model, Col III-TC-based dressings achieved outstanding healing efficacy (>95% closure within 15 days), with histological analysis showing improved neoskin thickness and collagen deposition. The Col3a1 transgenic zebrafish system developed in this study not only provides a novel strategy for heterologous expression of fully functional human proteins, but also highlights the broad application potential of its high-yield collagen in biomedical fields, particularly in wound healing and anti-aging therapies.},
}
@article {pmid41062834,
year = {2026},
author = {Zhang, A and Sun, X and Wu, Y and Gao, P and Zhang, R and Zhang, M and Xie, S and Fan, W and Zhang, Y and Yin, H},
title = {Efficient and precise inversion of genomic DNA from large to chromosomal scale.},
journal = {Nature chemical biology},
volume = {22},
number = {2},
pages = {328-339},
pmid = {41062834},
issn = {1552-4469},
mesh = {Humans ; *Chromosome Inversion/genetics ; *Gene Editing/methods ; *DNA/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Plasmids/genetics ; HEK293 Cells ; Genome, Human ; },
abstract = {Chromosomal inversion is a key structural variation impacting cellular fitness and genomic integrity. Here we developed prime-editing-based inversion with enhanced performance (PIE) to efficiently induce large-scale inversions in mammalian cells. PIEv1 uses a prime-editing guide RNA (pegRNA) pair but yields one imprecise junction. PIEv2 and PIEv3 add a second pegRNA pair for precise inversion, with PIEv3b further enhancing coupling precise inversion through improved plasmid design. PIEv3b achieves inversion efficiencies up to 61.7% for 1 Mb and 14.2% for 50 Mb segments and shows 4-20-fold higher efficiency compared to twin prime editing with integrase, across ranges of 100 kb to 30 Mb. Additionally, PIEv3b outperforms nuclease-based approaches in both inversion efficiency and precision. Using PIE, we convert human chromosomes from metacentric to telocentric configurations by inverting 30-Mb and 100-Mb chromosomal segments. Our work represents a powerful tool for engineering chromosomal structural variations, with broad implications for medicine and biotechnology.},
}
@article {pmid41608243,
year = {2026},
author = {Mitrofanov, A and Beisel, CL and Baumdicker, F and Alkhnbashi, OS and Backofen, R},
title = {Comprehensive analysis of CRISPR array repeat mutations reveals subtype-specific patterns and links to spacer dynamics.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqaf050},
pmid = {41608243},
issn = {2633-6693},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-associated protein (Cas) systems are adaptive immune mechanisms in bacteria and archaea that protect against invading genetic elements by integrating short fragments of foreign DNA into CRISPR arrays. These arrays consist of repetitive sequences interspersed with unique spacers, guiding Cas proteins to recognize and degrade matching nucleic acids. The integrity of these repeat sequences is crucial for the proper function of CRISPR-Cas systems, yet their mutational dynamics remain poorly understood. In this study, we analyzed 56 343 CRISPR arrays across 25 628 diverse prokaryotic genomes to assess the mutation patterns in CRISPR array repeat sequences within and across different CRISPR subtypes. Our findings reveal, as expected to some extent, that mutation frequency is substantially higher in terminal repeat sequences compared to internal repeats consistently across system types. However, the mutation patterns exhibit an unexpected amount of variation among different CRISPR subtypes, suggesting that selective pressures and functional constraints shape repeat sequence evolution in distinct ways. Understanding these mutation dynamics provides insights into the stability and adaptability of CRISPR arrays across diverse bacterial and archaeal lineages. Additionally, we elucidate a novel relationship between repeat mutations and spacer dynamics, demonstrating that hotspots for terminal repeat mutations coincide with regions exhibiting higher spacer conservation. This observation corroborates recent findings indicating that spacer deletions occur at a frequency 374 times greater than that of mutations and are significantly influenced by repeat misalignment. Our findings suggest that repeat mutations might play a pivotal role in spacer retention or loss, or vice versa, thereby highlighting an evolutionary trade-off between the stability and adaptability of CRISPR arrays.},
}
@article {pmid41606942,
year = {2025},
author = {Fraiture, MA and D'aes, J and Gobbo, A and Delvoye, M and Meunier, AC and Frouin, J and Guiderdoni, E and Deforce, D and De Vogelaere, C and De Keersmaecker, SCJ and Vanneste, K and Roosens, NHC},
title = {Genetic fingerprints derived from genome database mining allow accurate identification of genome-edited rice in the food chain via targeted high-throughput sequencing.},
journal = {Food research international (Ottawa, Ont.)},
volume = {221},
number = {Pt 1},
pages = {117218},
doi = {10.1016/j.foodres.2025.117218},
pmid = {41606942},
issn = {1873-7145},
mesh = {*Oryza/genetics ; *High-Throughput Nucleotide Sequencing/methods ; *Plants, Genetically Modified/genetics ; *Genome, Plant ; *Gene Editing/methods ; Polymorphism, Single Nucleotide ; *Food, Genetically Modified ; Databases, Genetic ; *DNA Fingerprinting/methods ; CRISPR-Cas Systems ; },
abstract = {Genome-edited (GE) organisms are currently classified as GMOs according to European legislation, requiring traceability and labelling in the food and feed supply chain. However, unambiguous identification of a specific GE organism with one or more induced single nucleotide variations (SNVs) dispersed across the genome remains challenging. This study explored whole-genome sequencing-based characterization, public genome databases, and machine learning tools to select key genetic elements and create a unique fingerprint for distinguishing a specific GE line. As a case study, a GE Nipponbare rice line containing a single CRISPR-Cas-induced SNV was used. To experimentally assess the detection of this fingerprint, a targeted high-throughput sequencing approach, including multiplex PCR-based enrichment of key genetic elements, was developed and successfully tested. This promising proof-of-concept demonstrates the potential of combining a unique genetic fingerprint with targeted high-throughput sequencing to facilitate the accurate detection of GE organisms, thereby supporting food traceability and regulatory compliance for the development of new GE lines, as well as protecting associated intellectual property.},
}
@article {pmid41606937,
year = {2025},
author = {Jiang, W and Zhu, T and Zhou, S and Pan, L and Qiao, Z and Wang, M and Yang, D},
title = {Recent advances in electrochemical-based CRISPR/Cas biosensing for nucleic acid and non-nucleic acid pathogenic microorganism detection.},
journal = {Food research international (Ottawa, Ont.)},
volume = {221},
number = {Pt 1},
pages = {117213},
doi = {10.1016/j.foodres.2025.117213},
pmid = {41606937},
issn = {1873-7145},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; *Nucleic Acids/analysis ; *Food Microbiology/methods ; *Bacteria/genetics/isolation &amp; purification ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The widespread presence of pathogenic microorganisms in food and environmental sources poses a persistent threat to public health. Conventional detection methods-including culture, microscopy, and biochemical assays-are limited by low sensitivity, cross-reactivity, and prolonged turnaround times, particularly when microbial loads are low or phenotypic overlap occurs. These limitations underscore the urgent need for diagnostic platforms that combine speed, specificity, and sensitivity. The advent of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) systems has revolutionized microbial diagnostics, driving the emergence of electrochemical CRISPR/Cas (EC-CRISPR/Cas) biosensors. This review surveys four principal electrochemical CRISPR/Cas (EC-CRISPR/Cas) platforms-Cas9, Cas12a, Cas13a, and Cas14a-emphasizing their structural characteristics, biosensing mechanisms, and signal amplification strategies for both nucleic acid and non-nucleic acid pathogen detection. We first outline the molecular architecture and functional mechanisms of each Cas protein in the context of biosensing. EC-CRISPR/Cas detection strategies are classified as nucleic acid-based (either amplification-free or amplification-dependent) or non-nucleic acid-based, the latter primarily relying on aptamer-mediated recognition. We also provide a comparative analysis of signal enhancement techniques and application scenarios across bacterial, viral, fungal, and parasitic pathogens. Importantly, we identify key limitations of current systems-such as poor reusability, signal drift, and challenges in point-of-care deployment-and present emerging solutions including crRNA engineering, nanomaterial integration, and artificial intelligence-guided biosensor design. These innovations hold strong potential to enhance sensitivity, specificity, and real-time performance, offering a foundation for next-generation, scalable EC-CRISPR/Cas diagnostics.},
}
@article {pmid41605509,
year = {2026},
author = {Li, J and Chen, X and Yang, Y and Xu, Y and Lai, M and Shi, L and Lin, X and Chen, W and Peng, H},
title = {Electron Transfer Mechanism-Mediated Host-Guest Nanoswitch Powered Amplification-Free CRISPR/Cas12a-Electrochemiluminescence Bioassay for Alzheimer's Disease Diagnosis.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c06614},
pmid = {41605509},
issn = {1520-6882},
abstract = {Developing a high-performance amplification-free electrochemiluminescence (ECL) assay platform that operates at a low trigger potential is a promising strategy for broadening the applications of ECL sensing. In this work, we present a host-guest interaction-mediated split-type CRISPR/Cas12a-ECL assay platform by using the highly sensitive host-guest recognition between the β-cyclodextrin-functionalized gold nanoclusters (β-CD-AuNCs) probe and methylene blue (MB) system as a proof of concept. Efficient ECL quenching of β-CD-AuNCs by MB is achieved via an electron transfer mechanism based on host-guest recognition between them. By integrating the high-specific recognition and cleavage activity of the CRISPR/Cas technology, the high quantum yield, and low trigger potential β-CD-AuNCs-based ECL probes, together with the highly sensitive and selective host-guest recognition-based split-type assay design, a novel "trinity" detection platform has been successfully constructed. Using Amyloid-β oligomers (AβOs), a key biomarker for Alzheimer's disease (AD) diagnosis and therapy, as the analyte, this amplification-free CRISPR/Cas-ECL biosensing platform enables ultrasensitive and accurate detection of AβO without requiring additional signal amplification strategies. The proposed sensing platform exhibits a linear detection range from 1.0 × 10[-8] to 1.0 × 10[-1] μg/mL for AβO detection, with a detection limit as low as 0.2 fg/mL (S/N = 3). This sensitivity approaches single-molecule levels and is 3-4 orders of magnitude lower than that of traditional ELISA. Furthermore, owing to its outstanding performance including high specificity, excellent selectivity, superior sensitivity, and strong anti-interference capability, the platform demonstrates remarkable detection performance in monitoring AβO in clinical AD blood samples, showing a good Pearson's correlation between the method and ELISA results. This work provides a powerful tool for clinical diagnosis and paves the way for therapeutic development, while also offering a rational design strategy for next-generation ECL biosensing platforms.},
}
@article {pmid41605395,
year = {2026},
author = {Zhang, J and Han, B and Zhang, X and Xie, X and Zhao, F and Zhang, W and Jiang, Y and Zhang, X},
title = {A CRISPR/Cas12a-mediated marker-free fluorescent biosensor constructed based on an automated 3D DNA walker-enabled signal amplification for sensitive detection of aflatoxin B1.},
journal = {International journal of biological macromolecules},
volume = {344},
number = {Pt 2},
pages = {150549},
doi = {10.1016/j.ijbiomac.2026.150549},
pmid = {41605395},
issn = {1879-0003},
abstract = {The efficient and sensitive detection of mycotoxins is critical to ensure food safety and maintain public health worldwide. In this study, a CRISPR/Cas12a-mediated marker-free fluorescent biosensor based on an automated 3D DNA walker-enabled 'one-to-many' signal amplification was developed for sensitive detection of aflatoxin B1. Due to the efficient amplification effect of DNA walkers and the strong fluorescence properties of silver nanoclusters, a sensitive output of the amplified signal was produced by precisely regulating the activated trans-cleavage activity of the specific target DNA of Cas12a. In the established fluorescence biosensor, a small amount of aflatoxin B1 promoted the production of a large amount of activator by the established 3D DNA walker, which stimulated the trans-cleavage activity of Cas12a to degrade the single-stranded DNAs for synthesis of silver nanoclusters, leading to a decreased fluorescent signal. The established biosensor was able to achieve the sensitive detection of aflatoxin B1 under optimal conditions, obtaining a detection limit of 45.38 pg/mL in a linear range of 0.05 to 10 ng/mL. In addition, the developed biosensor showed good recoveries in spiked food samples (peanut milk and drinking water) at different concentrations. This work provided new insights for the applications of DNA walker and the development of a marker-free fluorescent biosensing platform based on CRISPR/Cas for the detection of mycotoxins.},
}
@article {pmid41605290,
year = {2026},
author = {Shi, L and Zhang, M and Zheng, R and Kwok, LY and Zhang, W},
title = {Comparative genomics reveals two major lineages of Bifidobacterium adolescentis in the human gut, driven by divergent adaptation in China and the United States.},
journal = {Journal of advanced research},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jare.2026.01.071},
pmid = {41605290},
issn = {2090-1224},
abstract = {INTRODUCTION: Bifidobacterium adolescentis is a key beneficial member of the human gut microbiota, but its genomic diversity and evolutionary drivers across human populations remain poorly characterized.<br><br>OBJECTIVES: Understanding genomic functional heterogeneity and evolutionary patterns in human gut-derived B. adolescentis.<br><br>METHODS: We performed a comparative genomic analysis of 395B. adolescentis, mainly from China (n&#x202f;=&#x202f;169) and the U.S. (n&#x202f;=&#x202f;146), with smaller sets from Australia, Italy, and the United Kingdom, to investigate functional heterogeneity and evolutionary mechanisms. Our analysis integrated core and pan-genome architecture, phylogenomics, single nucleotide polymorphism (SNP)-based population structure, carbohydrate-active enzyme profiles, CRISPR-Cas systems, antibiotic resistance genes, and recombination dynamics.<br><br>RESULTS: The pan-genome was open and highly plastic. Phylogenetic reconstruction identified two major clades with strong geographic stratification: Chinese isolates predominantly clustered in Clade B, while U.S. isolates grouped in Clade A. Functional annotation showed regional specialization in carbohydrate-active enzymes, with Chinese isolates enriched in glycosyltransferase families and U.S. isolates in carbohydrate-binding module and carboxylesterase families, likely reflecting dietary adaptations. Genomic islands were hotspots for horizontal gene transfer, harboring region-specific carbohydrate-active enzymes and antibiotic resistance genes such as tet(W/32/O) and ermX, which were frequently located in Chinese isolates. Recombination was found to be the primary driver of genetic diversity, with recombination-to-mutation ratios approaching and exceeding 3.0 in Chinese and U.S. isolates. Linkage disequilibrium decay further supported higher recombination rates in these populations.<br><br>CONCLUSION: B. adolescentis has diverged into two major genomic lineages, primarily associated with isolates from China and the U.S. This divergence reflects adaptation to distinct host-associated ecological factors, such as diet, antibiotic exposure, and lifestyle, and is predominantly driven by extensive homologous recombination rather than point mutations. These findings highlight how regional selective pressures shape the genomic and functional landscape of this key gut symbiont.},
}
@article {pmid41604096,
year = {2026},
author = {Almufarriji, FM},
title = {Nanocarrier-mediated CRISPR-Cas delivery: a novel approach against antibiotic-resistant superbugs.},
journal = {Saudi pharmaceutical journal : SPJ : the official publication of the Saudi Pharmaceutical Society},
volume = {34},
number = {1},
pages = {5},
pmid = {41604096},
issn = {1319-0164},
abstract = {Antibiotic resistance (ABR) is a leading cause of death and a major public health threat globally. Without appropriate interventions, annual ABR-associated deaths have been projected to reach 10 million by 2050 worldwide. Hence, it is critical to develop novel therapeutic interventions that would be able to tackle ABR by targeting mainly the pathogenic microbes, while lessening harm to beneficial microbes. There is an increasing research interest in CRISPR-Cas (CC) systems owing to their potential in controlling and preventing horizontal gene transfer and spread of antibiotic resistance. In addition, CC systems offer several advantages, including high efficiency, rapid turnaround time, low cost, and easy design, which allow these systems to effectively and precisely target antibiotic-resistant bacteria. CRISPR-based gene therapy offers numerous benefits; however, the major limitation in clinical translation is the safe and effective delivery of CRISPR components to target organs or cells, thus hindering its potential in therapeutic interventions. Nanocarriers (NCs) can help the CC systems to overcome their off-target effects by precisely delivering the systems to the target cells. NCs can also be engineered for target site release, payload protection, and high specificity, which can further ensure delivery of the components of CC in the target cells or regions without harming surrounding tissues. This review summarizes the principles and mechanisms of CC systems, highlights their applications against antibiotic-resistant bacteria, and discusses emerging nanocarrier-based delivery strategies that may enhance the clinical utility of CRISPR-Cas technologies in managing ABR.},
}
@article {pmid41603863,
year = {2026},
author = {Calderón, L and Schäfer, M and Rončević, M and Rauschmeier, R and Jaritz, M and Schwickert, TA and Sun, Q and Pauli, A and Zuber, J and Busslinger, M},
title = {In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {3},
pages = {},
pmid = {41603863},
issn = {1540-9538},
support = {//Boehringer Ingelheim/ ; 740349/ERC_/European Research Council/International ; LT00427/2013//Human Frontier Science Program/ ; },
mesh = {Animals ; *Cell Differentiation/genetics/immunology ; *CRISPR-Cas Systems/genetics ; *Plasma Cells/immunology/cytology ; *B-Lymphocytes/immunology/cytology ; Mice ; *Lymphocyte Activation/genetics/immunology ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Immune responses to pathogens lead to the generation of plasma cells through a complex interplay of B cells with their microenvironment in lymphoid organs. To identify new regulators of B cell activation and plasmablast differentiation in the context of the splenic microenvironment, we established an in vivo system for pooled sgRNA CRISPR/Cas9 screens in immunized mice. To improve the infection efficiency of naïve B cells, we generated Cd23-Cre Rosa26LSL-EcoR/+ mice exhibiting increased expression of the ecotropic lentivirus receptor EcoR on naïve B cells. Upon adoptive B cell transfer and immunization of recipient mice, 379 sgRNAs, targeting genes with high expression in plasma cells, were analyzed for their effects on plasmablast generation. Gene hits, encoding 23 positive and 18 negative regulators of B cell activation, plasmablast differentiation, or homeostasis, were uniquely identified in these in vivo screens. Validated genes encoded proteins involved in cell adhesion, signal transduction, protein folding, iron transport, and enzymatic processes. Hence, our in vivo screening system identified novel regulators controlling B cell-mediated immune responses.},
}
@article {pmid41603755,
year = {2026},
author = {Yuan, X and Yang, F and Chen, X and Xiao, P and Shen, L},
title = {Single-Nucleotide Variation Analysis in Oral Squamous Cell Carcinoma-Related ctDNA by dCas9/sgRNA Recognition-Mediated Proximity Ligation-Triggered Terminal Hairpin Formation and Self-Priming Amplification.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c07087},
pmid = {41603755},
issn = {1520-6882},
abstract = {Circulating tumor DNA (ctDNA) represents a promising noninvasive biomarker for cancer diagnosis, including oral cancer. However, its clinical translation is currently limited by the lack of precise and reliable detection techniques. In this study, we developed a novel fluorescent biosensor for the detection of single-nucleotide variations in ctDNA, which integrates dual dCas9/sgRNA complexes for target recognition, proximity ligation-initiated terminal hairpin formation and self-priming amplification (PS-THSP), and Cas12a/crRNA-mediated signal output. A key innovation of this design is its multilayered specificity strategy, combining mutation-specific recognition by dual dCas9/sgRNA, proximity-dependent ligation, and Cas12a/crRNA-assisted verification of PS-THSP amplicons. This integrated approach offers a significant advance over existing CRISPR/Cas-based methods that rely primarily on signal amplification. Furthermore, the biosensor achieves high sensitivity through the synergistic coupling of PS-THSP amplification and Cas12a trans-cleavage activity, enabling a broad dynamic range spanning 6 orders of magnitude and a detection limit as low as 0.12 fM within 120 min. When applied to serum samples, the biosensor reliably detected ctDNA with high accuracy, demonstrating its strong potential for clinical cancer diagnostics.},
}
@article {pmid41603736,
year = {2026},
author = {Fuglsang, A and Rout, SS and Koutna, EB and Sofos, N and Gallego, AR and Montoya, G},
title = {Conformational dynamics of CRISPR-Cas type I-F-HNH inform nickase engineering in a cascade scaffold.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41603736},
issn = {1362-4962},
support = {NNF14CC0001//Novo Nordisk Foundation Center for Protein Research (CPR)/ ; NNF14CC0001//Novo Nordisk Foundation/ ; NNF24SA0098829//Novo Nordisk Foundation/ ; NNF0024386//Novo Nordisk Foundation/ ; NNF17SA0030214//Novo Nordisk Foundation/ ; NNF18OC0055061//Novo Nordisk Foundation/ ; NNF24SA0098829//Novo Nordisk Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA Cleavage ; *Deoxyribonuclease I/genetics/chemistry/metabolism ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; Catalytic Domain ; Protein Engineering ; Humans ; Cryoelectron Microscopy ; Gene Editing ; Models, Molecular ; DNA, Single-Stranded/metabolism/genetics/chemistry ; DNA/metabolism/chemistry ; Protein Conformation ; },
abstract = {The type I-FHNH CRISPR-Cas system is a non-canonical Class 1 effector complex distinguished by the replacement of the Cas3 recruitment domain with a catalytic HNH domain in Cas8, enabling autonomous DNA cleavage without accessory nucleases. Using cryo-EM, we determined high-resolution structures of the effector complex in three catalytic states-precatalytic, NTS-cleaved, and post-catalytic-revealing a dynamic trajectory of the HNH domain through inward, middle, and outward conformations. Biochemical assays demonstrated that the complex cleaves the nontarget strand (NTS) prior to the target strand (TS), consistent with a sequential cleavage mechanism similar to Cas12 effectors but notably lacking trans-cleavage activity on single-stranded DNA. Structural comparisons confirmed a minimal PAM requirement (5'-CN) and a constrained HNH catalytic site poised for precise strand scission. We engineered a ΔLinker variant of Cas8 that repositions the HNH domain, selectively abolishing TS cleavage and converting the system into a programmable NTS-specific nickase. Importantly, we validated the functionality of both wild-type and mutant complexes in human cells. While the wild-type system induced indels and base substitutions, the ΔLinker variant triggered targeted single-strand nicks without double-stranded breaks. Together, our work establishes type I-FHNH as a compact and precise genome editing platform with in vivo efficacy.},
}
@article {pmid41603733,
year = {2026},
author = {Kwon, H and Kim, J and Zhou, L and Dean, A},
title = {LDB1 regulates gene expression and chromatin structure in pluripotency and lineage differentiation.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41603733},
issn = {1362-4962},
support = {//Intramural Program of the National Institute of Diabetes/ ; //Digestive and Kidney Diseases/ ; 075033/GF/NIH HHS/United States ; },
mesh = {Animals ; *Cell Differentiation/genetics ; Mice ; Kruppel-Like Factor 4 ; *Chromatin/chemistry/metabolism/genetics ; Cell Lineage/genetics ; *DNA-Binding Proteins/genetics/physiology/metabolism ; SOXB1 Transcription Factors/metabolism/genetics ; Embryoid Bodies/metabolism/cytology ; Erythroblasts/metabolism/cytology ; Mouse Embryonic Stem Cells/metabolism/cytology ; Kruppel-Like Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Developmental ; Mice, Knockout ; CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism/cytology ; Enhancer Elements, Genetic ; Embryonic Stem Cells/metabolism/cytology ; *Gene Expression Regulation ; LIM Domain Proteins ; },
abstract = {Chromatin organization is a pivotal factor in stem cell pluripotency and differentiation. However, the role of enhancer looping protein LIM domain-binding 1 (LDB1) in stem cells remains to be fully explored. We generated Ldb1(-/-) embryonic stem cells (ESCs) using CRISPR/Cas9 editing and observed a reduction in key stem cell factors SOX2 and KLF4 upon LDB1 loss. Differential gene expression, including of the Lin28-mediated self-renewal pathway genes, was observed between wild-type and Ldb1(-/-) ESC. LDB1 occupied super enhancers, including those of pluripotency genes, in ESC together with pluripotency factors, and LDB1 loss resulted in loss of Sox2 interactions with the SCR enhancer. Embryoid bodies (EBs) derived from Ldb1(-/-) ESC displayed reduced expression of lineage-specific markers. Ldb1(-/-) ESC had impaired ability to undergo terminal differentiation to erythroblasts, and gene dysregulation was very pronounced in Ldb1(-/-) erythroblasts. Conditional LDB1-deficient mice displayed reduced hematopoietic stem cell markers on bone marrow cells and dysregulation of the Lin28 pathway. Thus, LDB1 function is critical for ESC and EB development and becomes progressively more important for normal gene expression during differentiation to erythroblasts.},
}
@article {pmid41603729,
year = {2026},
author = {Huang, Y and Xu, H and Zhang, T and Liao, Y and Hu, J and Huang, Z and Hu, H and Li, P and Fan, L and Xie, J},
title = {Mycobacterial non-homologous end joining is required for antiphage defense.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41603729},
issn = {1362-4962},
support = {82472325//National Natural Science Foundation/ ; 82072246//National Natural Science Foundation/ ; CSTB2024NSCQ-MSX0703//Natural Science Foundation of Chongqing/ ; //Chongqing Public Health Key Specialty (Discipline) Construction Project/ ; CSTB2024NSCQ-MSX0703//Natural Science Foundation of Chongqing/ ; },
mesh = {*DNA End-Joining Repair ; *Mycobacterium smegmatis/virology/genetics ; DNA Breaks, Double-Stranded ; Oxidative Stress ; Homologous Recombination ; },
abstract = {In the ongoing arms race with phages, bacteria have evolved diverse defense systems, such as CRISPR-Cas and restriction-modification systems. The DNA double-strand break repair system represents a core mechanism for maintaining genomic integrity and is vital for cell survival. However, it remains unknown whether and how these repair systems contribute to phage resistance. This study systematically investigates the role of the non-homologous end joining (NHEJ) during phage infection in Mycobacterium smegmatis. We found that NHEJ deficiency compromises host resistance to phage SWU1, as evidenced by increased plaque counts and reduced bacterial survival. Mechanistically, phages exploit host NHEJ for genomic repair; however, the error-prone nature of NHEJ leads to imperfect repair at phage cos sites, thereby blocking replication. The host modulates the balance between NHEJ and homologous recombination (HR) to control repair fidelity: NHEJ loss shifts the balance toward high-fidelity HR, which in turn promotes phage survival. Furthermore, NHEJ deficiency exacerbates infection-induced oxidative stress, leading to a compromise in bacterial viability. Our findings reveal the multifaceted functions of NHEJ in mycobacterium-phage interactions and provide new insights into how DNA repair systems shape antiphage defense and coevolution.},
}
@article {pmid41603018,
year = {2026},
author = {Dolder, RE and Friedman, CE and Loiben, AM and Yang, KC and Glazer, AM},
title = {High-Throughput Methods for Variant Functional Assessment in Cardiac Disease.},
journal = {Circulation. Genomic and precision medicine},
volume = {},
number = {},
pages = {e005239},
doi = {10.1161/CIRCGEN.125.005239},
pmid = {41603018},
issn = {2574-8300},
abstract = {In vitro functional modeling of genetic variants has revolutionized our understanding of which variants can cause cardiac disorders, providing insights into their molecular underpinnings. This review provides an overview of high-throughput methods used for the functional assessment of variants implicated in inherited cardiac diseases. Advances in gene-editing technology now enable the efficient generation of cells expressing individual genetic variants or libraries of variants for robust functional studies. We discuss innovative assays that can evaluate dozens or hundreds of variants sequentially. For example, the electrophysiological properties of numerous cardiac ion channel variants in genes linked to inherited arrhythmias can be characterized using automated patch clamping. The mechanical properties of cardiomyocytes expressing candidate cardiomyopathy variants can be assessed using techniques such as atomic force microscopy, traction force microscopy, and impedance-based methods. Multiplexed assays of variant effect are an emerging family of techniques that use gene-specific or general assays, combined with next-generation sequencing, to characterize hundreds or thousands of pooled genetic variants. We examine the key advantages and limitations of each method and outline future goals for the field. Innovative in vitro studies of cardiac genetic variants will enhance our understanding of variant-disease relationships and improve diagnosis, screening, and treatment options for these disorders.},
}
@article {pmid41546867,
year = {2026},
author = {Nebenfuehr, B and Sanford, L and Taylor, ER and Ball, K and Woods-Killam, CE and Ghasemi, HI and Proctor, B and Ortega, R and Sempeck, C and Dowell, RD and Arnoult, N},
title = {Uncovering genetic interactions in the DNA repair network in response to endogenous damage and ionizing radiation.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116850},
doi = {10.1016/j.celrep.2025.116850},
pmid = {41546867},
issn = {2211-1247},
mesh = {*Radiation, Ionizing ; *DNA Repair/genetics/radiation effects ; Humans ; *DNA Damage/genetics ; Ku Autoantigen/metabolism/genetics ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded/radiation effects ; MRE11 Homologue Protein/metabolism/genetics ; Ubiquitin-Protein Ligases/metabolism/genetics ; },
abstract = {Genomic integrity relies on a complex network of DNA damage response (DDR) pathways that repair endogenous and exogenous lesions, yet how individual factors operate within this broader landscape remains unclear. We performed a large-scale combinatorial CRISPR-Cas9 knockout screen targeting 461 DNA repair genes, disrupting over 100,000 gene combinations under basal conditions and after ionizing radiation (IR). This approach uncovered thousands of genetic interactions spanning pathways that respond to endogenous damage and those specific to double-strand break repair. From this dataset, we validated both positive and negative interactions under basal and irradiated conditions, including a synthetic lethal relationship between MRE11A and the E3 ligase UBR5, a role for Ku70/80 in preventing unscheduled nuclease activity at telomeres, an IR-specific vulnerability upon co-disruption of CYREN and PARG, and a link between CYREN-mediated radioresistance and innate immunity. This resource enables mechanistic insight and reveals therapeutic vulnerabilities in DNA-repair-deficient cancers.},
}
@article {pmid41534496,
year = {2026},
author = {Gogate, A and Chahrour, MH},
title = {Recent advances in the neurogenomics of autism spectrum disorder.},
journal = {Current opinion in genetics &amp; development},
volume = {96},
number = {},
pages = {102431},
doi = {10.1016/j.gde.2025.102431},
pmid = {41534496},
issn = {1879-0380},
mesh = {Humans ; *Autism Spectrum Disorder/genetics/pathology ; *Gene Regulatory Networks/genetics ; *Genomics ; Induced Pluripotent Stem Cells/metabolism ; *Genetic Predisposition to Disease ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
abstract = {Neurogenomics has provided exceptional insights into the genetic architecture underlying autism spectrum disorder (ASD), which is increasingly understood as a collection of individually rare disorders. This review synthesizes current advancements in the field, examining how both rare and common genetic variants contribute to ASD etiology. To functionally interpret the convergence on biological pathways that has emerged despite this genetic heterogeneity, multiomic approaches have been applied to identify gene regulatory networks disrupted in ASD. High-throughput technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR) editing and massively parallel reporter assays, have been employed in human induced pluripotent stem cells and organoids to bridge the gap between genetic association and biological function. Finally, machine learning methods play a pivotal role in integrating and leveraging these complex datasets to inform personalized interventions.},
}
@article {pmid41468840,
year = {2026},
author = {Shi, T and Jin, X},
title = {Probing neuropsychiatric disorders through in vivo CRISPR screening.},
journal = {Current opinion in genetics &amp; development},
volume = {96},
number = {},
pages = {102424},
pmid = {41468840},
issn = {1879-0380},
support = {R01 HG012819/HG/NHGRI NIH HHS/United States ; R01 MH137042/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; Animals ; *Mental Disorders/genetics/pathology ; Mice ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Analysis/methods ; Genetic Predisposition to Disease ; Schizophrenia/genetics ; Brain/pathology/metabolism ; },
abstract = {Although there are many known risk alleles associated with adult-onset psychiatric disorders such as schizophrenia [1-4], bipolar disorder [5-7], and major depressive disorder [8-10], the mechanistic links between these risk alleles and disease pathology, especially on a circuit-level, remain unclear. In vivo pooled CRISPR screening with single‑cell readout (in vivo Perturb‑seq) has begun to fill this gap by mapping causal genes to defined cell states directly in animal tissues [11-14]. Here, we review recent developments and applications of in vivo Perturb-seq in the mouse brain and highlight the potential of utilizing human cellular systems to extend these approaches. Additionally, we discuss how in vivo Perturb-seq can couple genetic perturbation with physiological or environmental perturbations to better model psychiatric diseases with environmental triggers.},
}
@article {pmid41456843,
year = {2026},
author = {Chang, C and Yang, J and Liu, Z and Chen, J and Wang, B and Li, J and Liu, H},
title = {Layer-by-layer coated chitosan-CRISPR/Cas9 mTOR nanoparticles: A novel approach to inhibit lens epithelial cell proliferation and migration for preventing posterior capsule opacification.},
journal = {Experimental eye research},
volume = {264},
number = {},
pages = {110828},
doi = {10.1016/j.exer.2025.110828},
pmid = {41456843},
issn = {1096-0007},
mesh = {*Capsule Opacification/prevention &amp; control/pathology/metabolism ; *Chitosan/pharmacology/chemistry ; Cell Proliferation ; *TOR Serine-Threonine Kinases/antagonists &amp; inhibitors/genetics ; *Epithelial Cells/pathology/metabolism ; *Nanoparticles/chemistry ; Cell Movement ; *CRISPR-Cas Systems ; Animals ; Humans ; Rabbits ; *Lens, Crystalline/cytology ; Posterior Capsule of the Lens/pathology ; Epithelial-Mesenchymal Transition ; Coated Materials, Biocompatible ; Lenses, Intraocular ; },
abstract = {Posterior capsular opacification (PCO) is the most common complication following cataract surgery and a significant cause of vision impairment. PCO arises from the proliferation, migration, and epithelial-mesenchymal transition (EMT) of residual lens epithelial cells (LECs), driven by an activated mTOR signalling pathway. Previous research has demonstrated that inhibiting mTOR activity effectively reduces LEC proliferation and EMT in rabbit models. However, achieving sustained mTOR inhibition remains a challenge. In this study, we encapsulated the CRISPR/Cas9 system targeting mTOR into chitosan nanoparticles (Chi-gRNA) with an average size of 135 nm. These nanoparticles exhibited resistance to DNase I digestion. To prolong release duration, we incorporated these Chi-gRNA nanoparticles onto the surface of intraocular lenses (IOLs) via layer-by-layer (LbL) assembly. The LbL coatings consisted of alternating layers of positively charged polyethyleneimine (PEI) and negatively charged heparin, interspersed with Chi-gRNA nanoparticles over five consecutive cycles. Spectral analysis confirmed the successful integration and coating of nanoparticles, with characteristic peaks validating the electrostatic assembly of the layers. In vitro assays demonstrated that Chi-gRNA-coated IOLs significantly inhibited the proliferation, migration, and adhesion of human lens epithelial cells (hLECs). These findings highlight the potential of LbL-coated IOLs to deliver CRISPR/Cas9 system-targeting mTOR nanoparticles as a novel and effective strategy to prevent PCO in patients undergoing cataract surgery. This approach offers a promising avenue for the long-term management of this prevalent postoperative complication.},
}
@article {pmid41456781,
year = {2026},
author = {Puri, B and Gaikwad, AB},
title = {Targeting LncRNAs with CRISPR/Cas9 for Kidney Therapeutics: A Review.},
journal = {International journal of biological macromolecules},
volume = {339},
number = {Pt 1},
pages = {149932},
doi = {10.1016/j.ijbiomac.2025.149932},
pmid = {41456781},
issn = {1879-0003},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Long Noncoding/genetics ; Gene Editing/methods ; Animals ; *Kidney Diseases/therapy/genetics ; Genetic Therapy/methods ; },
abstract = {Long noncoding RNAs (lncRNAs) have emerged as key players in the pathogenesis of kidney diseases, including acute kidney injury (AKI), AKI-to-chronic kidney disease (CKD) transition, CKD, diabetic kidney disease (DKD), renal cell carcinoma (RCC), polycystic kidney diseases (PKD), and lupus nephritis (LN). Although the roles of lncRNAs in disease progression have been investigated in preclinical models, their underlying mechanisms remain poorly understood. The therapeutic potential of lncRNA-based therapies remains largely unexplored in clinical settings. Recently, an advancement in clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology offers a novel strategy for treating sickle cell anemia and β-thalassemia. Additionally, CRISPR/Cas9 is currently being evaluated in clinical trials for various diseases, including kidney diseases like RCC. However, the application of CRISPR/Cas9 to target lncRNAs is still in the early stages. Preclinical experiments have revealed that CRISPR/Cas9 could effectively target lncRNAs in kidney disorders. However, its clinical translation in AKI and CKD conditions remains unclear, and various biological challenges remain to be addressed. This review aims to investigate advancements in CRISPR/Cas9 that target lncRNAs in the kidney, highlighting the limitations and future directions for advancing CRISPR/Cas9-based lncRNA therapy and translating these findings into clinical applications.},
}
@article {pmid41447528,
year = {2026},
author = {Liu, P and Yuan, Q and Yang, X and Wang, Q and Chang, T and Bi, Y and Wu, P and Zhang, T and Yang, J and Guo, S and Xue, C and Zheng, Z and Xin, B and Ma, H and Wang, Y},
title = {A synthetic biology toolkit for the plasmid-dependent and thermophilic methylotroph Bacillus methanolicus.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116788},
doi = {10.1016/j.celrep.2025.116788},
pmid = {41447528},
issn = {2211-1247},
mesh = {*Plasmids/genetics/metabolism ; *Bacillus/genetics/metabolism ; *Synthetic Biology/methods ; CRISPR-Cas Systems/genetics ; Gene Editing ; Methanol/metabolism ; Arginine/metabolism ; Metabolic Engineering ; },
abstract = {Bacillus methanolicus, a unique plasmid-dependent and thermophilic methylotroph, is an ideal chassis for one-carbon (C1) biomanufacturing. Despite its evolutionary uniqueness and industrial promise, the synthetic biology toolkit remains limited in comparison to that of conventional model microorganisms. Here, we present a comprehensive toolkit comprising a high-efficiency electroporation protocol, a CRISPR-Cas9 method enabling robust and multiplex genome editing, diverse neutral loci for gene integration, and a cloud-based genome-scale metabolic model iBM822 for user-friendly biodesign. Leveraging this toolkit, we systematically dissected plasmid-dependent methylotrophy, restriction-modification machinery, and the functional significance of chromosomal methylotrophic genes. To address plasmid loss-induced strain degeneration, we integrated the large endogenous plasmid pBM19 into the chromosome for stable and intact methylotrophic growth. Finally, by integrating metabolic modeling with CRISPR-Cas9 editing, we engineered L-arginine feedback regulation to achieve L-arginine overproduction from methanol. This study establishes a synthetic biology framework for B. methanolicus, promoting mechanistic exploration of methylotrophy and C1 biomanufacturing.},
}
@article {pmid41428486,
year = {2026},
author = {Ribeiro Gomes, AR and Hamel, N and Mastwal, S and Wright, N and Ide, DC and Richie, CT and Usdin, TB and Wang, KH and Leopold, DA},
title = {Targeted gene transfer into developmentally defined cell populations of the primate brain.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116756},
doi = {10.1016/j.celrep.2025.116756},
pmid = {41428486},
issn = {2211-1247},
mesh = {Animals ; *Gene Transfer Techniques ; *Brain/metabolism/embryology ; Callithrix ; Female ; Rats ; Dependovirus/genetics ; Transgenes/genetics ; Gene Editing/methods ; Genetic Vectors ; Male ; CRISPR-Cas Systems ; },
abstract = {The primate brain possesses unique physiological and developmental features, yet its systematic investigation has been hampered by a paucity of transgenic germline models and tools. Here, we present a minimally invasive method to introduce transgenes widely across the primate cerebral cortex using ultrasound-guided fetal intracerebroventricular viral injections (FIVIs). FIVI enables efficient and long-lasting transgene expression following intrauterine delivery of recombinant adeno-associated viruses (rAAVs). In the marmoset, we demonstrate that adjusting gestational timing, rAAV serotype, and transcriptional regulatory elements enables selective targeting of defined cell populations, including layer-restricted labeling and Cre-dependent intersectional access. Pilot experiments in rats further demonstrate the potential of FIVIs for prenatal CRISPR-based gene editing and labeling of peripheral somatosensory and retinal pathways. By mimicking key desirable features of germline transgenic models, this efficient and targeted method for gene transfer into the fetal primate brain expands the experimental opportunities for basic and translational neuroscience research across the lifespan.},
}
@article {pmid41417728,
year = {2026},
author = {Huber, A and Djajawi, TM and Rivera, IS and Vervoort, SJ and Kearney, CJ},
title = {CRISPR screens define unified hallmarks of cancer cell-autonomous immune evasion.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116738},
doi = {10.1016/j.celrep.2025.116738},
pmid = {41417728},
issn = {2211-1247},
mesh = {Humans ; *Neoplasms/immunology/genetics/pathology ; *Tumor Escape/genetics ; *CRISPR-Cas Systems/genetics ; Tumor Microenvironment/immunology ; Immunotherapy ; Animals ; *Immune Evasion ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CD8-Positive T-Lymphocytes/immunology ; },
abstract = {Cancer immunotherapy has transformed cancer treatment, yet only a minority of patients achieve durable benefit. Although early efforts to enhance immunotherapy focused on boosting immune effector function, reversing T cell exhaustion, or altering the tumor microenvironment, it is now clear that cancer cell-autonomous mechanisms play a major role in immune escape. Such programs, driven by the cancer cell genome, transcriptome, and epigenome, include desensitization to cytokine signaling, such as interferon (IFN)γ and tumor necrosis factor (TNF); impaired antigen presentation; upregulation of suppressive ligands such as programmed cell death ligand 1 (PD-L1); and epigenetic silencing of immunogenic pathways. The rise of high-throughput functional genomics, especially in vitro and in vivo CRISPR-based screening, has greatly expanded our ability to map these pathways and define how tumors evade CD8[+] T cell-mediated pressure. A deeper understanding of these cancer cell-autonomous immune-evasion mechanisms will be essential for developing new therapeutic strategies that broaden the impact of immunotherapy across diverse cancers.},
}
@article {pmid41417727,
year = {2026},
author = {Higginson, LA and Wang, X and He, K and Torstrick, M and Kim, M and Wijeratne, HRS and Runnebohm, AM and Benayoun, BA and MacLean, A and Chanfreau, GF and Morton, DJ},
title = {The RNA exosome maintains cellular RNA homeostasis by controlling transcript abundance in the brain.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116729},
doi = {10.1016/j.celrep.2025.116729},
pmid = {41417727},
issn = {2211-1247},
mesh = {Animals ; *Brain/metabolism ; *Homeostasis ; *Exosome Multienzyme Ribonuclease Complex/metabolism/genetics ; Drosophila melanogaster/metabolism/genetics ; Drosophila Proteins/metabolism/genetics ; *Exosomes/metabolism ; Humans ; CRISPR-Cas Systems/genetics ; *RNA/metabolism/genetics ; Transcriptome ; Mutation ; },
abstract = {Intracellular ribonucleases (RNases) are essential for maintaining accurate RNA levels. Inherited mutations in genes encoding ubiquitous RNases are associated with human diseases, primarily affecting the nervous system. Recessive mutations in genes encoding the evolutionarily conserved RNA exosome, an RNase complex, cause syndromic neurodevelopmental disorders, such as pontocerebellar hypoplasia type 1b (PCH1b), characterized by progressive neurodegeneration. Here, we establish a CRISPR-Cas9-engineered Drosophila model of PCH1b to investigate the cell-type-specific post-transcriptional regulatory functions of the RNA exosome complex in vivo. Pathogenic variants in Rrp40, a subunit of the complex, disrupt RNA exosome activity, leading to widespread transcriptomic dysregulation in brain-enriched cell populations, including defective rRNA processing. These molecular defects coincide with progressive neurodegeneration and behavioral impairments that track with allele severity. Our findings provide a cell-type-resolved view of RNA exosome function in a fully developed animal brain and underscore the critical role of RNA surveillance in safeguarding transcriptome homeostasis and neuronal integrity.},
}
@article {pmid41411128,
year = {2026},
author = {Mochida, T and Fujimoto, N and Asahina, M and Asano, S and Araki, S and Inukai, N and Hotta, A},
title = {Muscle satellite cell editing by LNP-CRISPR-Cas9 to resist muscle injury.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116695},
doi = {10.1016/j.celrep.2025.116695},
pmid = {41411128},
issn = {2211-1247},
mesh = {Animals ; *Satellite Cells, Skeletal Muscle/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; *Muscular Dystrophy, Duchenne/genetics/therapy/pathology ; *Muscle, Skeletal/injuries/metabolism/pathology ; *Nanoparticles/chemistry ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Mice, Inbred mdx ; Male ; Exons/genetics ; Liposomes ; },
abstract = {Muscle satellite cells are essential for skeletal muscle regeneration and represent an attractive therapeutic target for gene delivery in Duchenne muscular dystrophy (DMD). However, efficient in vivo transduction of these cells has remained challenging. Here, we demonstrate that lipid nanoparticle (LNP)-mediated delivery of Streptococcus pyogenes CRISPR-Cas9 mRNA and guide RNA (LNP-CRISPR) induces exon skipping in Pax7-positive satellite cells more efficiently than adeno-associated virus (AAV) vectors following intramuscular or intravenous administration in a DMD mouse model. Furthermore, unlike AAV-CRISPR, LNP-CRISPR-mediated genome editing showed greater resistance to repeated muscle injuries, indicating successful editing of regenerative satellite cells. These results highlight the potential of LNPs as a non-viral platform for durable genome editing in skeletal muscle and lay the foundation for developing safe and sustainable genome-editing therapies for DMD.},
}
@article {pmid41397613,
year = {2026},
author = {Yin, C and Chen, B and Zheng, X and Wang, N and Wang, J and Li, R and Li, J and Yao, S and Zhai, Y and Song, X},
title = {Portable visual platform integrates polymerase spiral amplification and CRISPR/Cas12a for foodborne bacteria point-of-care testing.},
journal = {Journal of dairy science},
volume = {109},
number = {2},
pages = {1036-1051},
doi = {10.3168/jds.2025-27493},
pmid = {41397613},
issn = {1525-3198},
mesh = {*Staphylococcus aureus/isolation &amp; purification/genetics ; *Point-of-Care Testing ; CRISPR-Cas Systems ; Animals ; Nucleic Acid Amplification Techniques ; Food Microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Staphylococcus aureus, a prominent global foodborne pathogen, frequently triggers epidemics with severe public health impacts. Timely and reliable detection of S. aureus is crucial for mitigating the disease burden in low- and middle-income countries. However, conventional laboratory-based detection methods remain impractical in resource-limited settings, highlighting the urgent need for accessible point-of-care solutions. Here, we present an inner-outer-tube (IOT) assay that synergistically integrates the polymerase spiral amplification (PSR) technology for enhanced sensitivity with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 12a (Cas12a) system for sequence-specific identification. Additionally, we have created a portable all-in-one mobile detection (PAMD) device that combines all the steps needed for testing in the field, allowing for quick visual detection of S. aureus in just 60 min. The PSR-CRISPR/Cas12a-IOT method implemented with the PAMD device achieves a detection limit of 10 cfu/mL without needing extra preparation or costly equipment. The detection platform developed in this work has advantages of ease of operation, manageable costs, and robust performance, making it highly ideal for low-resource contexts and on-site detection scenarios. Furthermore, the PSR-CRISPR/Cas12a-IOT-PAMD detection platform provides global versatility through the interchangeable use of primer sets, hence broadening its applicability to various infections.},
}
@article {pmid41173524,
year = {2026},
author = {Dai, J and Molloy, EK},
title = {StarCDP: Dynamic Programming Algorithms for Fast and Accurate Cell Lineage Tree Reconstruction from CRISPR-Based Lineage Tracing Data.},
journal = {Journal of computational biology : a journal of computational molecular cell biology},
volume = {33},
number = {1},
pages = {48-66},
doi = {10.1177/15578666251386082},
pmid = {41173524},
issn = {1557-8666},
mesh = {*Cell Lineage/genetics ; *Algorithms ; Animals ; Mice ; Humans ; Phylogeny ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Software ; *Computational Biology/methods ; Lung Neoplasms/genetics/pathology ; Single-Cell Analysis/methods ; Dynamic Programming ; },
abstract = {CRISPR-based lineage tracing, coupled with single-cell RNA sequencing, has emerged as a promising approach for studying development and disease progression at the cellular level. Thus, cell lineage tree (CLT) reconstruction has attracted significant attention in recent years, including the introduction of Star Homoplasy Parsimony (SHP) to model the unique properties of CRISPR-induced mutations, along with the Startle family of methods. However, CLT reconstruction continues to be challenged by technological limitations in producing consistent phylogenetic signals across CLTs. To address these issues, we present Star-CDP, a collection of dynamic programming algorithms that enable researchers to seek, count, sample, and build consensus trees from solutions to SHP within a constrained search space, defined by subsets of cells from which a solution must draw its clades. When using our procedure to construct clade constraints, Star-CDP runs in polynomial time, enabling scalability to larger numbers of cells than Startle-ILP (integer linear programming), the leading method for SHP. In simulations, Star-CDP's strict consensus achieved the same or higher accuracy (f1-score) compared to the leading parsimony methods, with the greatest gains in accuracy occurring when the phylogenetic signal was limited due to the high ratio of cells to mutations. On lineage tracing data from a mouse model of lung adenocarcinoma, Star-CDP's strict consensus achieved the lowest SHP score and comparable numbers of metastatic reseedings compared to PAUP*'s strict consensus and Startle-NNI (nearest neighbor interchange), all benchmarked on a standard data processing pipeline (although our study also revealed that the pipeline can impact relative performance for migrations/reseedings). Star-CDP is available on GitHub: https://github.com/molloy-lab/Star-CDP.},
}
@article {pmid40914889,
year = {2025},
author = {Beran, K and Park, SH and Van den Bergh, A and Dressman, J and Hermans, E and Holm, R and Lin, Y and Sepassi, K and Yang, B and Evers, R},
title = {Canine Mdr1 Knockout MDCK Cells Reliably Estimate Human Small Intestinal Permeability (Peff) and Fraction Absorbed (fa).},
journal = {Molecular pharmaceutics},
volume = {22},
number = {10},
pages = {6067-6082},
pmid = {40914889},
issn = {1543-8392},
mesh = {Animals ; Dogs ; ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics/metabolism ; Madin Darby Canine Kidney Cells/metabolism ; Permeability ; *Intestinal Absorption/physiology ; Intestine, Small/metabolism ; Gene Knockout Techniques ; Voriconazole/pharmacokinetics ; Acetaminophen/pharmacokinetics ; Intestinal Barrier Function ; Humans ; Purines/pharmacokinetics ; Pyridines/pharmacokinetics ; Pyrrolidines/pharmacokinetics ; Benzenesulfonamides/pharmacokinetics ; Benzaldehydes/pharmacokinetics ; CRISPR-Cas Systems ; Administration, Oral ; *Pharmaceutical Preparations/metabolism ; Pyrazines ; Pyrazoles ; Pyrimidines ; ATP Binding Cassette Transporter, Subfamily B ; },
abstract = {Human intestinal permeability is a key determinant of the oral fraction absorbed (fa) of active pharmaceutical ingredients (APIs). This study evaluated the ability of an in-house canine Mdr1 (cMdr1) knockout (KO) Madin-Darby Canine Kidney (MDCK) cell line to correlate in vitro apparent permeability (Papp) with human small intestinal permeability (Peff). In vitro Papp values of 16 reference compounds with high, medium, or low permeabilities were measured in the in-house cMdr1 KO MDCK protocol under pH gradient (6.5 ⇒ 7.4) and pH equivalent conditions (7.4 ⇒ 7.4) and correlations with human Peff were established (R[2] > 0.8). The correlations were subsequently used to estimate Peff and fa for six test APIs: acetaminophen, voriconazole, fedratinib, voxelotor, lemborexant, and istradefylline. The results for these APIs were compared against literature and permeability data from other methods routinely used in drug discovery and development. The projected Peff and fa values for the test APIs aligned well with literature permeabilities derived using other methods and clinical pharmacokinetic studies, respectively. This work highlights the usefulness of cMdr1 KO MDCK cells in permeability classification, especially for highly permeable APIs, and supports its broader use in both research and regulatory contexts.},
}
@article {pmid41601654,
year = {2025},
author = {Yin, L and He, W and Wang, Y and Zhang, H and Huang, M and Yan, Y and Li, S and Feng, X and Saenz, F and Zhang, J and Zhu, D and Yang, C and Ma, T and Fu, J and Chen, J},
title = {FACS-based genome-wide CRISPR screening platform identifies modulators of CD47.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1684539},
pmid = {41601654},
issn = {1664-3224},
mesh = {*CD47 Antigen/genetics/metabolism/immunology ; Animals ; Mice ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Humans ; Flow Cytometry/methods ; Phagocytosis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Macrophages/immunology/metabolism ; Female ; Immunity, Innate ; },
abstract = {BACKGROUND: CD47 is a key innate immune checkpoint that enables tumor cells to evade macrophage-mediated clearance.<br><br>METHODS/RESULTS: To systematically identify genetic regulators of CD47 surface expression, we performed FACS-based genome-wide CRISPR screens in three murine cancer cell lines B16 (melanoma), MC38 (colon adenocarcinoma), and EMT6 (breast carcinoma).<br><br>RESULTS: Comparative analysis of cells with high or low CD47 surface expression using DrugZ revealed CD47 itself as the top hit, validating the screens. Notably, DNAJC13 emerged as a consistent and robust regulator of CD47 expression across all three cell lines. Functional validation using DNAJC13-knockout cells confirmed a significant reduction in CD47 surface levels. Furthermore, in co-culture assays with macrophages, DNAJC13-deficient tumor cells exhibited increased susceptibility to phagocytosis, supporting a functional role for DNAJC13 in innate immune evasion. Finally, we verify that DNAJC13-knockout decrease tumor burden when treated with CD47 blockade.<br><br>CONCLUSIONS: Overall, this study highlights a previously unrecognized regulator of CD47 and demonstrates the utility of high-throughput FACS-based CRISPR screening to uncover modulators of key immune checkpoint pathways.},
}
@article {pmid41601145,
year = {2026},
author = {Jiao, Y and Liu, Y and Sun, F and Wang, W and Li, H and Gao, Q and Li, Y and Lu, N and Tian, X and Ding, X and Du, J},
title = {Genetically Modified Plant Beneficial Microorganisms: A Sustainable Solution or a New Challenge for Agriculture.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c14342},
pmid = {41601145},
issn = {1520-5118},
abstract = {Plant diseases significantly impact crop yield and quality, while conventional pesticide treatments often disrupt beneficial plant microbiota essential for pathogen prevention and immune regulation. Although plant beneficial microorganisms (PBMs) show promise as disease control agents, their effectiveness is constrained by strain-dependent variations, survival challenges, and inconsistent immune responses. Recent advances in genetic engineering, particularly CRISPR-Cas systems combined with complementary technologies like RecE/T, enable precise modifications of PBMs to enhance their protective potential. Enhanced PBMs improve functionality via multiple mechanisms: targeted gene-expression-mediated colonization, specific antimicrobial activity, and immune regulation. Studies demonstrate that genetically modified PBMs can prevent and control plant diseases through competitive exclusion, antibiotic production, barrier reinforcement, and immune modulation. We analyzed the considerations for the environmental release of engineered PBMs to reduce risks. Future research should focus on optimizing PBMs for specific applications while addressing biosafety concerns, thereby unlocking their full potential in safeguarding plant health.},
}
@article {pmid41600812,
year = {2025},
author = {Tian, C and Feng, L and Zhou, X and Huang, K and Wang, F and Luo, R and Meng, F and Yang, H and Qiao, C and Wang, X and Shi, J and Chen, Y},
title = {A Portable One-Tube Assay Integrating RT-RPA and CRISPR/Cas12a for Rapid Visual Detection of Eurasian Avian-like H1N1 Swine Influenza Virus in the Field.},
journal = {Viruses},
volume = {18},
number = {1},
pages = {},
pmid = {41600812},
issn = {1999-4915},
support = {2021YFD1800200//Nnational Key Research and Development Program of China/ ; },
mesh = {*Influenza A Virus, H1N1 Subtype/isolation &amp; purification/genetics ; Animals ; *CRISPR-Cas Systems ; Swine ; Sensitivity and Specificity ; *Orthomyxoviridae Infections/diagnosis/virology/veterinary ; *Swine Diseases/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The widespread circulation of Eurasian avian-like H1N1 (EA H1N1) swine influenza virus poses significant zoonotic and pandemic risks worldwide. However, current diagnostic methods are difficult to deploy in the field, as they generally require specialized laboratory infrastructure and trained personnel. Here, we present a novel dual-signal detection platform that combines reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR/Cas12a technology for rapid, on-site EA H1N1 detection. We established an integrated one-tube assay by designing and optimizing RT-RPA primers targeting a conserved region of the hemagglutinin (HA) gene, together with engineered CRISPR/Cas12a guide RNAs exhibiting high specificity. The platform incorporates two complementary readout modes: real-time fluorescence monitoring and visual colorimetric detection using a smartphone. The assay shows excellent analytical specificity, with no cross-reactivity observed against other swine influenza virus subtypes or common swine pathogens, (including CSFV, PRRSV, PEDV, PCV, TGEV, and RV). The detection limit is 2 copies/μL, and the entire procedure can be completed within 30 mins using simple portable equipment. When evaluated on 86 clinical samples, the assay demonstrated 94.18% concordance with RT-qPCR. Compared with conventional diagnostic methods, this RT-RPA-CRISPR/Cas12a assay offers greater convenience and cost-effectiveness. Its strong potential for field-based rapid testing underscores promising application prospects in swine influenza surveillance and control programs.},
}
@article {pmid41599334,
year = {2026},
author = {Wang, X and Chen, X and Zhou, Y and Zhao, Y and Shi, C and Li, R and Liu, L and Mu, C and Song, W and Wang, C},
title = {Establishment of CRISPR-Cas9-Mediated Gene Editing in the Swimming Crab Portunus trituberculatus.},
journal = {Molecules (Basel, Switzerland)},
volume = {31},
number = {2},
pages = {},
pmid = {41599334},
issn = {1420-3049},
support = {2022YFD2400104//the National Key R&amp;D Program of China/ ; 32341058 and 31972783//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Brachyura/genetics ; Electroporation ; Myostatin/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; INDEL Mutation ; },
abstract = {Portunus trituberculatus is an economically important marine crustacean in East Asia's aquaculture industry. Nevertheless, precise genome modification has not yet been established. In this study, we evaluated the applicability of the CRISPR-Cas9 gene editing system in P. trituberculatus using electroporation for efficient delivery of the Cas9-sgRNA complex into zygotes. We systematically investigated electroporation parameters, including buffer composition, voltage, capacitance, and pulse times. Our results showed that artificial seawater was a superior buffer to phosphate-buffered saline (PBS) and identified an effective electroporation condition of 600 V, 1 μF capacitance, and two pulses, resulting in approximately 72.7% fluorescent zygotes. Under these electroporated conditions, we detected gene indels and putative insertion events at the targeted locus of myostatin (mstn) gene. These results demonstrate the feasibility of Cas9-based genome editing in P. trituberculatus and provide a proof-of-concept for functional genomics studies and future genetic improvement of this species.},
}
@article {pmid41599202,
year = {2026},
author = {Yoon, B and Kim, JA and Kang, YK},
title = {CRISPR-Cas-Mediated Reprogramming Strategies to Overcome Antimicrobial Resistance.},
journal = {Pharmaceutics},
volume = {18},
number = {1},
pages = {},
pmid = {41599202},
issn = {1999-4923},
support = {grant number RS-2024-00417430//National Research Foundation of Korea (NRF)/ ; grant number RS-2024-00399808//Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET)/ ; 2025-RISE-16-001//Regional Innovation System &amp; Education (RISE) program/ ; A26234, CTRQQR-2021\100009//CRUK Convergence Science Centre at The Institute of Cancer Research, London, and Imperial Col-lege London/ ; },
abstract = {Antimicrobial resistance (AMR) is escalating worldwide, posing a serious threat to global public health by driving infections that are no longer treatable with conventional antibiotics. CRISPR-Cas technology offers a programmable and highly specific therapeutic alternative by directly targeting the genetic determinants responsible for resistance. Various CRISPR systems can restore antibiotic susceptibility and induce selective bactericidal effects by eliminating resistance genes, disrupting biofilm formation, and inhibiting virulence pathways. Moreover, CRISPR can suppress horizontal gene transfer (HGT) by removing mobile genetic elements such as plasmids, thereby limiting the ecological spread of AMR across humans, animals, and the environment. Advances in delivery platforms-including conjugative plasmids, phagemids, and nanoparticle-based carriers-are expanding the translational potential of CRISPR-based antimicrobial strategies. Concurrent progress in Cas protein engineering, spatiotemporal activity regulation, and AI-driven optimization is expected to overcome current technical barriers. Collectively, these developments position CRISPR-based antimicrobials as next-generation precision therapeutics capable of treating refractory bacterial infections while simultaneously suppressing the dissemination of antibiotic resistance.},
}
@article {pmid41599009,
year = {2025},
author = {Lv, YR and Liu, YY and Zhang, R and Yang, B and Xue, SY and Ding, YL and Jia, JT and Bayaer, H and Bagen, A and Chen, RB and Tunala, S and Zhao, L and Liu, YH},
title = {Rapid and Simple Detection of Mycobacterium avium subsp. paratuberculosis Using a Lateral Flow Assay Based on CRISPR-Cas12a Combined with Recombinase Polymerase Amplification or Nested PCR.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {1},
pages = {},
pmid = {41599009},
issn = {2076-0817},
support = {1.(YF20240007) 2.(BR251303) 3.(YLXKZX-NND-012)//1.Ordos Science &amp; Technology Plan 2.Special Project for the Construction of Scientific Research and Innovation Teams, Class B Team 3.Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; },
mesh = {*CRISPR-Cas Systems ; *Mycobacterium avium subsp. paratuberculosis/genetics/isolation &amp; purification ; *Paratuberculosis/diagnosis/microbiology ; *Polymerase Chain Reaction/methods ; Animals ; Sensitivity and Specificity ; Feces/microbiology ; Recombinases/genetics ; DNA, Bacterial/genetics ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic intestinal disease in ruminants. PTB is difficult to diagnose, control, and eradicate, leading to substantial economic losses. Thus, sensitive and specific detection methods are urgently required. crRNA and primers targeting the MAP ATPase FtsK gene were designed for recombinase polymerase amplification (RPA) and nested PCR. Fecal DNA was amplified using RPA or nested PCR, purified with Tris-saturated phenol-chloroform-isoamyl alcohol, and detected via CRISPR-Cas12a. Moreover, signals were read using a qPCR instrument, fluorescence reader, or lateral flow strips. RPA-CRISPR-Cas12a and nested PCR-CRISPR-Cas12a assays were optimized and validated on 50 clinical samples and 7 MAP cultures. The limits of detection were 1 × 10[-10] μg/μL for RPA-CRISPR-Cas12a and 1 × 10[-14] μg/μL for nested PCR-CRISPR-Cas12a. Efficient cleavage of the ssDNA reporter occurred at DNA concentrations of ≥1 × 10[-4] μg/μL, producing a strong fluorescent signal. All three detection methods showed perfect agreement with reference assays across both sample sets. This study presents the first integration of RPA or nested PCR with CRISPR-Cas12a for MAP detection, enabling rapid, specific, and highly sensitive diagnosis. Flexible detection options allow adaptation to available resources and bacterial loads, supporting practical use in PTB control.},
}
@article {pmid41598917,
year = {2026},
author = {Pei, X and Xie, J and Liang, C and Utkina, AO},
title = {Next-Generation Precision Breeding in Peanut (Arachis hypogaea L.) for Disease and Pest Resistance: From Multi-Omics to AI-Driven Innovations.},
journal = {Insects},
volume = {17},
number = {1},
pages = {},
pmid = {41598917},
issn = {2075-4450},
abstract = {Peanut (Arachis hypogaea L.) is a globally important oilseed and food legume, yet its productivity is persistently constrained by devastating diseases and insect pests that thrive under changing climates. This review aims to provide a comprehensive synthesis of advances in precision breeding and molecular approaches for enhancing disease and pest resistance in peanut. Traditional control measures ranging from crop rotation and cultural practices to chemical protection have delivered only partial and often unsustainable relief. The narrow genetic base of cultivated peanut and its complex allotetraploid genome further hinder the introgression of durable resistance. Recent advances in precision breeding are redefining the possibilities for resilient peanut improvement. Multi-omics platforms genomics, transcriptomics, proteomics, and metabolomics have accelerated the identification of resistance loci, effector-triggered immune components, and molecular cross-talk between pathogen, pest, and host responses. Genome editing tools such as CRISPR-Cas systems now enable the precise modification of susceptibility genes and defense regulators, overcoming barriers of conventional breeding. Integration of these molecular innovations with phenomics, machine learning, and remote sensing has transformed resistance screening from manual assessment to real-time, data-driven prediction. Such AI-assisted breeding pipelines promise enhanced selection accuracy and faster deployment of multi-stress-tolerant cultivars. This review outlines current progress, technological frontiers, and persisting gaps in leveraging precision breeding for disease and pest resistance in peanut, outlining a roadmap toward climate-resilient, sustainable production systems.},
}
@article {pmid41598889,
year = {2025},
author = {Wang, Y and Zhang, C and Li, MJ and Iqbal, A and Ahmed, KS and Idrees, A and Habiba, and Yang, BM and Jiang, L},
title = {Exploring the Role of Pheromones and CRISPR/Cas9 in the Behavioral and Olfactory Mechanisms of Spodoptera frugiperda.},
journal = {Insects},
volume = {17},
number = {1},
pages = {},
pmid = {41598889},
issn = {2075-4450},
support = {2024C014-2//Innovation Capacity Building Project of the Jilin Provincial Development and Reform Commission./ ; },
abstract = {Globally, Spodoptera frugiperda is a major threat to many important crops, including maize, rice, and cotton, causing significant economic damage. To control this invasive pest, environmentally friendly pest control techniques, including pheromone detection and identification of potential molecular targets to disrupt S. frugiperda mating communication, are needed. Female moths biosynthesize pheromones and emit them from the pheromone gland, which significantly depends on the intrinsic factors of the moth. Male S. frugiperda have a sophisticated olfactory circuit on their antennae that recognizes pheromone blends via olfactory receptor neurons (ORNs). With its potential to significantly modify the insect genome, CRISPR/Cas9 offers a revolutionary strategy to control this insect pest. The impairing physiological behaviors and disrupting the S. frugiperda volatile-sensing mechanism are the main potential applications of CRISPR/Ca9 explored in this review. Furthermore, the release of mutant S. frugiperda for their long-term persistence must be integral to the adoption of this technology. Looking forward, CRISPR/Cas9-based gene drive systems have the potential to synergistically target pheromone signaling pathways in S. frugiperda by disrupting pheromone receptors and key biosynthesis genes, thereby effectively blocking intraspecific communication and reproductive success. In conclusion, CRISPR/Cas9 provides an environmentally friendly and revolutionary platform for precise, targeted pest management in S. frugiperda.},
}
@article {pmid41598172,
year = {2025},
author = {Magyar-Tábori, K and Udupa, SM and Hanász, A and Juhász, C and Mendler-Drienyovszki, N},
title = {Rising Demand for Winter Crops Under Climate Change: Breeding for Winter Hardiness in Autumn-Sown Legumes.},
journal = {Life (Basel, Switzerland)},
volume = {16},
number = {1},
pages = {},
pmid = {41598172},
issn = {2075-1729},
abstract = {Climate change in the Pannonian region is accelerating a shift toward autumn sowing of cool-season grain legumes (pea, faba bean, lentil, chickpea, lupine) to achieve higher yields, greater biomass production, enhanced nitrogen fixation, improved soil cover, and superior resource use efficiency compared with spring sowing. However, successful overwintering depends on the availability of robust winter-hardy cultivars. This review synthesizes recent breeding advances, integrating traditional approaches-such as germplasm screening, hybridization, and field-based selection-with genomics-assisted strategies, including genome-wide association studies (GWAS), quantitative trait locus (QTL) mapping, marker-assisted selection (MAS), and CRISPR/Cas-mediated editing of CBF transcription factors. Key physiological mechanisms-LT50 determination, cold acclimation, osmoprotectant accumulation (sugars, proline), and membrane stability-are assessed using field survival rates, electrolyte leakage assays, and chlorophyll fluorescence measurements. Despite challenges posed by genotype × environment interactions, variable winter severity, and polygenic trait control, the release of cultivars worldwide (e.g., 'NS-Mraz', 'Lavinia F', 'Ghab series', 'Pinklevi', and 'Rézi') and ongoing breeding programs demonstrate substantial progress. Future breeding efforts will increasingly rely on genomic selection (GS), high-throughput phenomics, pangenomics, and G×E modeling to accelerate the development of climate-resilient legume cultivars, ensuring stable and sustainable production under increasingly unpredictable winter conditions.},
}
@article {pmid41597233,
year = {2026},
author = {Bao, C and Channell, CI and Tseng, YH and Bailey, J and Sbaiti, N and Demirkol, A and Tsang, SH},
title = {Chronic In Vivo CRISPR-Cas Genome Editing: Challenges, Long-Term Safety, and Outlook.},
journal = {Cells},
volume = {15},
number = {2},
pages = {},
pmid = {41597233},
issn = {2073-4409},
support = {the Foundation Fighting Blindness TA-GT-0321-0802-COLU-TRAP, the Lynette and Richard Jaffe Foundation, NYEE Foundation, the Rosenbaum Family Foundation, the Gebroe Family Foundation, the Research to Prevent Blindness (RPB) Physician-Scientist Award, unres//Jonas Children's Vision Care is supported by the National Institute of Health U01EY030580, U01EY034590 R24EY028758, R24EY027285, R01EY033770, R01EY018213, R01EY024698,/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; Genetic Therapy/methods ; },
abstract = {CRISPR/Cas systems have transformed molecular medicine, yet the field still lacks principled guidance on when transient editing suffices versus when sustained exposure through in vivo viral delivery is necessary and how to keep prolonged exposure safe. Notably, EDIT-101 was designed for a permanent edit in post-mitotic photoreceptors with lifelong Cas9 persistence. This review addresses this gap by defining the biological and therapeutic conditions that drive benefit from extended Cas activity while minimizing risk. We will (i) examine relationships between expression window and efficacy across Cas9/Cas12/Cas13 modalities, (ii) identify genome-wide off-target liabilities alongside orthogonal assays, and (iii) discuss controllable, self-limiting, and recallable editor platforms. By separating durable edits from persistent nuclease exposure, and by providing validated control levers, this work establishes a generalizable framework for safe, higher-efficacy CRISPR medicines. Furthermore, we highlight key studies in cell lines, murine models, non-human primates, and humans that examine the long-term effects of sustained expression of CRISPR/Cas systems and discuss the safety and efficacy of such approaches. Current evidence demonstrates promising therapeutic outcomes with manageable safety profiles, although there is a need for continued monitoring as CRISPR/Cas therapies are increasingly applied in clinical contexts and therapies are developed for broader clinical applications.},
}
@article {pmid41552883,
year = {2026},
author = {Xu, Q and Ji, M},
title = {A Bst-driven Cas12a cascade amplification strategy for microRNA detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {4},
pages = {899-906},
doi = {10.1039/d5ay01957e},
pmid = {41552883},
issn = {1759-9679},
mesh = {*MicroRNAs/analysis/genetics/blood ; Humans ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Quantification of trace microRNAs is crucial for early disease diagnosis but remains technically challenging. Herein, we developed an ultrasensitive fluorescence platform for microRNA-21 (miR-21) detection by integrating Bst DNA polymerase - assisted target recycling with CRISPR/Cas12a-mediated signal amplification. In this design, the target miRNA triggers toehold-mediated opening of a hairpin probe, followed by Bst-driven primer extension that enables efficient target recycling and the generation of abundant DNA duplex activators. Subsequently, these activators induce strong trans-cleavage activity of Cas12a, producing markedly enhanced fluorescence responses. Benefiting from the dual amplification of enzymatic recycling and Cas12a activation, the proposed assay exhibits high sensitivity toward miR-21 with a detection limit down to 9.25 × 10[-12] M. Furthermore, the platform exhibited excellent sequence selectivity and was successfully applied to monitor miR-21 in both cell lysates and clinical serum samples. Considering its convenient operation, strong analytical performance, and simple readout mode, this method holds great potential for trace biomarker analysis in clinical diagnostics.},
}
@article {pmid40522814,
year = {2026},
author = {Blumberg, LC and Bakker, GM and van der Kaaij, A and Gariépy, &#xc9; and van de Geest, H and Slootweg, EJ and Los, FCO and Westerhof, LB and Wilbers, RHP},
title = {Highly efficient transgene-free ErCas12a RNP-protoplast genome editing and single-cell regeneration in Nicotiana benthamiana for glyco-engineering.},
journal = {Plant biotechnology journal},
volume = {24},
number = {1},
pages = {239-255},
doi = {10.1111/pbi.70141},
pmid = {40522814},
issn = {1467-7652},
support = {101080784//Horizon Europe/ ; 16740//The Netherlands Organization for Scientific Research/ ; //Hudson River Biotechnology B.V./ ; },
mesh = {*Nicotiana/genetics/metabolism ; *Gene Editing/methods ; *Protoplasts/metabolism ; CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; Plants, Genetically Modified ; Polysaccharides/metabolism ; Regeneration ; Transgenes ; },
abstract = {Nicotiana benthamiana serves as a unique platform for biopharmaceutical production, offering advantages such as efficient and scalable protein synthesis. In addition, custom N-glycans can be engineered on biopharmaceutical glycoproteins. Yet, plant-native glycosyltransferases and glycoside hydrolases need to be removed to prevent undesired modifications of tailored N-glycans. CRISPR-based systems offer tremendous potential; however, the ploidy of the allotetraploid N. benthamiana can make genome editing challenging when attempting to knock out multiple undesired enzymes using transgenes. Here, we report a highly efficient CRISPR ribonucleoprotein (RNP)-protoplast genome editing strategy for rapid, single-generation platform engineering. We delineate the editing characteristics of ErCas12a RNPs and apply hydrogel protoplast immobilization to characterize true single-cell regeneration. We target three β-hexosaminidases responsible for removing terminal GlcNAc and/or GalNAc residues from N-glycans and verify their inactivity via MALDI-TOF-MS N-glycan analysis. We achieve up to 89.6%, 95.3% and 86.5% on-target editing in the absence of off-target editing. We demonstrate the feasibility of low cell density (10[4] ml[-1]) regeneration of individual CRISPR-edited protoplasts in 12-14 weeks, carrying intended tetra-allelic and/or deca-allelic mutations while maintaining monoclonality. Despite the occurrence of genome duplications during the single-cell regeneration of N. benthamiana protoplasts, high-efficiency genome editing paired with shoot induction frequencies exceeding 89% facilitated the ubiquitous identification of desired β-hexosaminidase mutants. We anticipate that this genome-editing method will rapidly advance glyco-engineering in polyploids such as N. benthamiana.},
}
@article {pmid40379239,
year = {2026},
author = {Wu, J and Wang, X and Xu, J and Li, T and Shan, G and Zhang, L and Yan, T and Song, X and Sun, Y and Guo, H and Zeng, F},
title = {Overexpression of soybean flavonoid 3'-hydroxylase enhances plant salt tolerance by promoting ascorbic acid biosynthesis.},
journal = {Journal of advanced research},
volume = {80},
number = {},
pages = {111-123},
doi = {10.1016/j.jare.2025.05.009},
pmid = {40379239},
issn = {2090-1224},
mesh = {*Glycine max/genetics/metabolism/enzymology ; *Salt Tolerance/genetics ; Plants, Genetically Modified/genetics ; *Ascorbic Acid/biosynthesis ; Gene Expression Regulation, Plant ; *Cytochrome P-450 Enzyme System/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Salt Stress ; },
abstract = {INTRODUCTION: Salt stress is a major cause of crop loss. Soybean (Glycine max), a globally vital legume crop, faces mounting yield constraints due to soil salinization. It is known that the flavonoid biosynthesis pathway involving flavonoid 3'-hydroxylase (F3'H) plays an important role in salt tolerance. However, the precise molecular basis of F3'H-mediated salt tolerance remains inadequately characterized.<br><br>OBJECTIVES: This study aimed to elucidate the function and explore the pleiotropic molecular basis of F3'H protein in soybean salt tolerance. Innovation on elite new crop varieties facilitates breeding and production applications on salt tolerance.<br><br>METHODS: We employed CRISPR/Cas9-mediated knockout and Agrobacterium-based overexpression to generate GmF3'H allelic variants and ectopic expression in soybeans. Sanger sequencing and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to confirm the specificity of gene editing and quantify expression levels in overexpression transgenic plants, respectively. As well as Subcellular localization analysis, Yeast two-hybrid (Y2H) assay, LUC activity assay and plant physiological measurements were carried out to elucidate the F3'H-mediated salt tolerance molecular basis in plants.<br><br>RESULTS: In this study, we identified the flavonoid 3' hydroxylase gene (GmF3'H) in soybeans, which as a master regulator of salt stress adaptation during seed germination and seedling stages in both soybean and Arabidopsis thaliana. Furthermore, our study revealed that the evolutionarily conserved F3'H protein competitively binds to photomorphogenic factor COP9 signalosome subunit 5B (CSN5B) and disrupts its interaction with GDP-mannose pyrophosphorylase 1 (VTC1), a key enzyme in ascorbate biosynthesis. This competitive inhibition redirects metabolic flux toward the L-galactose pathway, leading to an increase in ascorbic acid (AsA) biosynthesis. The enhanced AsA production subsequently improves seedling salt stress tolerance in plants by maintaining redox homeostasis through ROS scavenging.<br><br>CONCLUSION: The discovery and characterization of F3'H-mediated salt tolerance provide a crucial framework for the genetic improvement of crops. This work provides new insights into plant salt stress tolerance and develops innovative strategies to enhance broad-spectrum salt tolerance, a crucial aspect for ensuring food security in crops.},
}
@article {pmid40274228,
year = {2026},
author = {Song, Y and Guan, C and Zhang, Y and Xu, Y and Li, P and Luo, L and Feng, C and Chen, G},
title = {A novel CRISPR-Cas9 nickase-mediated rolling circle amplification (CRIRCA) technique for gene identification and quantitative analysis of extrachromosomal DNA.},
journal = {Journal of advanced research},
volume = {80},
number = {},
pages = {239-248},
doi = {10.1016/j.jare.2025.04.031},
pmid = {40274228},
issn = {2090-1224},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *DNA, Circular/genetics ; *Deoxyribonuclease I/metabolism/genetics ; Cell Line, Tumor ; In Situ Hybridization, Fluorescence ; Microscopy, Atomic Force ; },
abstract = {INTRODUCTION: Extrachromosomal DNA (ecDNA) plays an important role in the initiation and progression of cancerous tumors. Although Circle-seq and other genetic technologies can be utilized for ecDNA analysis, they fail to provide multi-dimensional information from ecDNA, which is time-consuming and laborious.<br><br>OBJECTIVES: Herein, by combining the netlike rolling circle amplification (NRCA) with CRISPR, we developed a novel CRISPR-Cas9 nickase-mediated RCA (CRIRCA) technology that can meet the clinical analysis needs of ecDNA.<br><br>METHODS: Atomic force microscope (AFM) was applied to confirm the circular structure of the ecDNA. Agarose gel electrophoresis was performed to analyze the CRIRCA products. Fluorescent detection was applied to characterize the fluorescence signal of amplified products. qPCR and FISH techniques were applied to verify the CRIRCA results of gene identification of ecDNA.<br><br>RESULTS: Our data revealed that CRIRCA achieved more efficient signal amplification compared to traditional RCA methods, allowing it to sensitively analyze small amounts of ecDNA in single tumor cells. Utilizing computer-aided design, we successfully constructed the primer library and sgRNA library of oncogene in ecDNA, and adopted CRIRCA technology to identify the oncogenes of ecDNA in breast cancer cells.<br><br>CONCLUSION: Therefore, CRIRCA can simultaneously obtain the information from structure, sequence and quantitation of ecDNA. This work will fill the gap in the current research on the early monitoring of cancer targeting ecDNA, and provide support for the accurate diagnosis and treatment of cancer.},
}
@article {pmid40274227,
year = {2026},
author = {Wang, X and Zhang, X and Liu, Y and Ru, L and Yan, G and Xu, Y and Yu, Y and Zhu, Z and He, Y},
title = {miR398-SlCSD1 module participates in the SA-H2O2 amplifying feedback loop in Solanum lycopersicum.},
journal = {Journal of advanced research},
volume = {80},
number = {},
pages = {19-30},
doi = {10.1016/j.jare.2025.04.035},
pmid = {40274227},
issn = {2090-1224},
mesh = {*Solanum lycopersicum/metabolism/genetics ; *MicroRNAs/genetics/metabolism ; *Hydrogen Peroxide/metabolism ; *Salicylic Acid/metabolism ; Gene Expression Regulation, Plant ; *Plant Proteins/metabolism/genetics ; Signal Transduction ; Reactive Oxygen Species/metabolism ; Feedback, Physiological ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {INTRODUCTION: Salicylic acid (SA) is essential for immune response signal transduction in higher plants, with its signaling thought to be enhanced through interactions with reactive oxygen species (ROS). However, the exact mechanisms behind this SA self-amplifying signaling are still not well understood.<br><br>OBJECTIVES: In this study, we report the involvement of the miR398b-SlCSD1 module in the SA-H2O2 amplifying feedback loop in tomato (Solanum lycopersicum).<br><br>METHODS: Experiments were conducted using various concentrations of SA to assess its impact on ROS metabolism and the expression of SlCSD1 and sly-miR398. CRISPR/Cas9 was employed to knock out sly-miR398 and SlCSD1. Bioinformatics analyses, dual-luciferase reporter assays (Dual-Luc), and electrophoretic mobility shift assays (EMSA) were used to identify SA-responsive transcription factors and validate their regulation of sly-miR398b. The role of miR398 in endogenous SA synthesis was examined using overexpression and knockout tomato lines.<br><br>RESULTS: Low SA concentrations stimulated H2O2 accumulation, increased superoxide dismutase (SOD) activity, and suppressed sly-miR398 expression, effects absent in NahG plants with reduced SA levels. Knockout of SlCSD1 via CRISPR/Cas9 partially inhibited SA-induced H2O2 accumulation, confirming SlCSD1's role in SA-dependent H2O2 signaling. Furthermore, Dual-Luc and EMSA results revealed that TGACG-sequence-specific binding protein 2 (TGA2) mediated the regulation of miR398-SlCSD1 module by SA in tomato. Additionally, overexpression and mutation of sly-miR398b promoted SA synthesis via the phenylalanine ammonia-lyase (PAL) and isochorismate synthase (ICS) pathways, highlighting its regulatory role in SA biosynthesis.<br><br>CONCLUSION: Taken together, our results shed light on the involvement of the miR398-SlCSD1 module in the SA-H2O2 amplifying feedback loop, providing new insights into SA signaling in tomato. These findings contribute to understanding SA-ROS interactions and offer a potential strategy for enhancing stress tolerance in crops by targeting microRNA-regulated pathways.},
}
@article {pmid41597209,
year = {2026},
author = {Ren, Z and Zhou, J and Yang, D and Guo, Y and Zhang, J and Xu, J and Chen, YE},
title = {Gene Editing Therapies Targeting Lipid Metabolism for Cardiovascular Disease: Tools, Delivery Strategies, and Clinical Progress.},
journal = {Cells},
volume = {15},
number = {2},
pages = {},
doi = {10.3390/cells15020134},
pmid = {41597209},
issn = {2073-4409},
mesh = {Humans ; *Gene Editing/methods ; *Cardiovascular Diseases/therapy/genetics/metabolism ; *Lipid Metabolism/genetics ; *Genetic Therapy/methods ; Animals ; *Gene Transfer Techniques ; CRISPR-Cas Systems ; },
abstract = {Gene editing technologies have revolutionized therapeutic development, offering potentially curative and preventative strategies for cardiovascular disease (CVD), which remains a leading global cause of morbidity and mortality. This review provides an introduction to the state-of-the-art gene editing tools-including ZFNs, TALENs, CRISPR/Cas9 systems, base editors, and prime editors-and evaluates their application in lipid metabolic pathways central to CVD pathogenesis. Emphasis is placed on targets such as PCSK9, ANGPTL3, CETP, APOC3, ASGR1, LPA, and IDOL, supported by findings from human genetics, preclinical models, and recent first-in-human trials. Emerging delivery vehicles (AAVs, LNPs, lentivirus, virus-like particles) and their translational implications are discussed. The review highlights ongoing clinical trials employing liver-targeted in vivo editing modalities (LivGETx-CVD) and provides insights into challenges in delivery, off-target effects, genotoxicity, and immunogenicity. Collectively, this review captures the rapid progress of LivGETx-CVD from conceptual innovation to clinical application, and positions gene editing as a transformative, single-dose strategy with the potential to redefine prevention and long-term management of dyslipidemia and atherosclerotic cardiovascular disease.},
}
@article {pmid41596857,
year = {2026},
author = {Tsouggou, N and Korozi, E and Pemaj, V and Drosinos, EH and Kapolos, J and Papadelli, M and Skandamis, PN and Papadimitriou, K},
title = {Advances in Shotgun Metagenomics for Cheese Microbiology: From Microbial Dynamics to Functional Insights.},
journal = {Foods (Basel, Switzerland)},
volume = {15},
number = {2},
pages = {},
doi = {10.3390/foods15020259},
pmid = {41596857},
issn = {2304-8158},
abstract = {The cheese microbiome is a complex ecosystem strongly influenced by both technological practices and the processing environment. Moving beyond traditional cultured-based methods, the integration of shotgun metagenomics into cheese microbiology has enabled in-depth resolution of microbial communities at the species and strain levels. The aim of the present study was to review recent applications of shotgun metagenomics in cheese research, underscoring its role in tracking microbial dynamics during production and in discovering genes of technological importance. In addition, the review highlights how shotgun metagenomics enables the identification of key metabolic pathways, including amino acid catabolism, lipid metabolism, and citrate degradation, among others, which are central to flavor formation and ripening. Results of the discussed literature demonstrate how microbial composition, functional traits, and overall quality of cheese are determined by factors such as raw materials, the cheesemaking environment, and artisanal practices. Moreover, it highlights the analytical potentials of shotgun metagenomics, including metagenome-assembled genomes (MAGs) reconstruction, characterization of various genes contributing to flavor-related biosynthetic pathways, bacteriocin production, antimicrobial resistance, and virulence, as well as the identification of phages and CRISPR-Cas systems. These insights obtained are crucial for ensuring product's authenticity, enabling traceability, and improving the assessment of safety and quality. Despite shotgun metagenomics' advantages, there are still analytical restrictions concerning data handling and interpretation, which need to be addressed by importing standardization steps and moving towards integrating multi-omics approaches. Such strategies will lead to more accurate and reproducible results across studies and improved resolution of active ecosystems. Ultimately, shotgun metagenomics has shifted the field from descriptive surveys to a more detailed understanding of the underlying mechanisms shaping the overall quality and safety of cheese, thus bringing innovation in modern dairy microbiology.},
}
@article {pmid41596715,
year = {2026},
author = {Ansari, RA and Rezaee Danesh, Y and Castello, I and Vitale, A},
title = {Molecular Identification and RNA-Based Management of Fungal Plant Pathogens: From PCR to CRISPR/Cas9.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
doi = {10.3390/ijms27021073},
pmid = {41596715},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; *Plant Diseases/microbiology/prevention &amp; control/genetics ; *Fungi/genetics/pathogenicity ; Polymerase Chain Reaction/methods ; *RNA, Fungal/genetics ; Plants/microbiology ; },
abstract = {Fungal diseases continue to limit global crop production and drive major economic losses. Conventional diagnostic and control approaches depend on time-consuming culture-based methods and broad-spectrum chemicals, which offer limited precision. Advances in molecular identification have changed this landscape. PCR, qPCR, LAMP, sequencing and portable platforms enable rapid and species-level detection directly from plant tissue. These tools feed into RNA-based control strategies, where knowledge of pathogen genomes and sRNA exchange enables targeted suppression of essential fungal genes. Host-induced and spray-induced gene silencing provide selective control without the long-term environmental costs associated with chemical use. CRISPR/Cas9 based tools now refine both diagnostics and resistance development, and bioinformatics improves target gene selection. Rising integration of artificial intelligence indicates a future in which disease detection, prediction and management connect in near real time. The major challenge lies in limited field validation and the narrow range of fungal species with complete molecular datasets, yet coordinated multi-site trials and expansion of annotated genomic resources can enable wider implementation. The combined use of molecular diagnostics and RNA-based strategies marks a shift from disease reaction to disease prevention and moves crop protection towards a precise, sustainable and responsive management system. This review synthesizes the information related to current molecular identification tools and RNA-based management strategies, and evaluates how their integration supports precise and sustainable approaches for fungal disease control under diverse environmental settings.},
}
@article {pmid41596696,
year = {2026},
author = {Boren, C and Kumar, R and Gollahon, L},
title = {In Silico Design and Characterization of a Rationally Engineered Cas12j2 Gene Editing System for the Treatment of HPV-Associated Cancers.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
doi = {10.3390/ijms27021054},
pmid = {41596696},
issn = {1422-0067},
support = {NA//Texas Tech University Association of Biologists Grants-in-Aid/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Papillomavirus Infections/therapy/virology/genetics ; *Neoplasms/therapy/virology/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Oncogene Proteins, Viral/genetics ; Computer Simulation ; },
abstract = {CRISPR-Cas9 systems have enabled unprecedented advances in genome engineering, particularly in developing treatments for human diseases, like cancer. Despite potential applications, limitations of Cas9 include its relatively large size and strict targeting requirements. Cas12j2, a variant ofCasΦ-2, shows promise for overcoming these limitations. However, its effectiveness in mammalian cells remains relatively unexplored. This study sought to develop an optimized CRISPR-Cas12j2 system for targeted knockout of the E6 oncogene in HPV-associated cancers. A combination of computational tools (ColabFold, CCTop, Cas-OFFinder, HADDOCK2.4, and Amber for Molecular Dynamics) was utilized to investigate the impact of engineered modifications on structural integrity and gRNA binding of Cas12j2 fusion constructs, in potential intracellular conditions. Cas12j2_F2, a Cas12j2 variant designed and evaluated in this study, behaves similarly to the wild-type Cas12j2 structure in terms of RMSD/RMSF profiles, compact Rg values, and minimal electrostatic perturbation. The computationally validated Cas12j2 variant was incorporated into a custom expression vector, co-expressing the engineered construct along with a dual gRNA for packaging into a viral vector for targeted knockout of HPV-associated cancers. This study provides a structural and computational foundation for the rational design of Cas12j2 fusion constructs with enhanced stability and functionality, supporting their potential application for precise genome editing in mammalian cells.},
}
@article {pmid41596664,
year = {2026},
author = {Luo, X and Ding, Y and Wang, Z and Liu, J},
title = {MED12 Dictates Epithelial Ovarian Cancer Cell Ferroptosis Sensitivity via YAP-TEAD1 Signaling.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
doi = {10.3390/ijms27021020},
pmid = {41596664},
issn = {1422-0067},
support = {82102732//National Natural Science Foundation of China/ ; 82272698//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Ferroptosis/drug effects/genetics ; Female ; *Carcinoma, Ovarian Epithelial/metabolism/genetics/pathology ; *Transcription Factors/metabolism/genetics ; *Signal Transduction ; Cell Line, Tumor ; YAP-Signaling Proteins ; TEA Domain Transcription Factors ; *Ovarian Neoplasms/metabolism/genetics/pathology ; *DNA-Binding Proteins/metabolism/genetics ; *Adaptor Proteins, Signal Transducing/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; *Nuclear Proteins/metabolism/genetics ; Drug Resistance, Neoplasm ; Cysteine-Rich Protein 61/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Epithelial ovarian cancer (EOC) represents the most lethal malignancy arising from the female reproductive tract, largely due to the clinical challenge of chemotherapy resistance. Recent studies indicate that ferroptosis-a distinct form of programmed cell death driven by iron accumulation and lipid peroxidation, could potentially exploit a vulnerability in chemoresistant cancer cells. Here, we identify MED12 as a critical regulator of ferroptosis sensitivity in EOC through modulation of the YAP-TEAD1 signaling pathway. Using CRISPR/Cas9-mediated knockout and rescue experiments in EOC cell lines, we demonstrate that MED12 deficiency significantly enhances sensitivity to ferroptosis inducers (RSL3 and Erastin), as evidenced by reduced IC50 values. Transcriptomic and chromatin accessibility analyses reveal that MED12 loss activates YAP signaling through TEAD1 upregulation, increasing chromatin accessibility at YAP-TEAD1 target loci and elevating the expression of downstream effectors CYR61 and CTGF. Pharmacological inhibition of YAP with verteporfin or siRNA-mediated TEAD1 knockdown reverses ferroptosis sensitivity in MED12-deficient cells, confirming pathway specificity. These findings establish MED12 as a modulator of the YAP-TEAD1-ferroptosis axis and suggest that targeting this pathway could overcome chemoresistance in MED12-deficient EOC. Our work provides a mechanistic foundation for exploiting ferroptosis induction as a therapeutic strategy in ovarian cancer.},
}
@article {pmid41596567,
year = {2026},
author = {Kosmas, CE and Rallidis, LS and Hoursalas, I and Papakonstantinou, EJ and Kostara, CE},
title = {Angiopoietin-like Protein 3 (ANGPTL3) Targeting in the Management of Dyslipidemias.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
doi = {10.3390/ijms27020921},
pmid = {41596567},
issn = {1422-0067},
mesh = {Humans ; *Angiopoietin-Like Protein 3/antagonists &amp; inhibitors/metabolism/genetics ; *Angiopoietin-like Proteins/antagonists &amp; inhibitors/metabolism/genetics ; *Dyslipidemias/drug therapy/metabolism/genetics ; Animals ; Antibodies, Monoclonal/therapeutic use/pharmacology ; Oligonucleotides, Antisense/therapeutic use ; RNA, Small Interfering/therapeutic use ; },
abstract = {Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality, despite advances in pharmacological prevention and treatment. The burden of CVD necessitates implementing the treatment of risk factors including dyslipidemia. Pharmaceutical advancements and in depth understanding of pathophysiology have enabled innovative therapies targeting pathways underlying lipoprotein metabolism disorders. Angiopoietin protein-like 3 (ANGPTL3) plays a crucial role in the regulation of lipoprotein metabolism, therefore being a potential therapeutic target. Inhibition of ANGPTL3 has emerged as a new therapeutic strategy to reduce LDL-cholesterol levels independent of the LDL receptor function. Therapeutic approaches for ANGPTL3 inhibition range from monoclonal antibodies to nucleic acid therapeutics including antisense oligonucleotides and small interfering RNAs. In this review, we briefly explain the structure and mechanism of action of ANGPTL3 and discuss the therapeutic approaches for targeting ANGPTL3 in the clinical setting. We also discuss Evinacumab, a monoclonal antibody, its structure, mechanism of action, safety, tolerability, pharmacokinetics, and pharmacodynamics, as well as its clinical trial-derived results. The antisense oligonucleotides modify ANGPTL3 mRNA to inhibit protein production, and small interfering RNAs induce mRNA degradation; results from clinical trials were reviewed in detail. Finally, we discuss promising gene editing approaches including clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems.},
}
@article {pmid41596458,
year = {2026},
author = {Ionas, K and Vukosavljev, M and Bulić, E and Radanović, A and Jocić, S and Kondić-Špika, A and Miladinović, D},
title = {Beyond the Bottleneck: Predicting Regeneration Potential in Sunflower Through Integrated Morphological and Statistical Profiling.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
doi = {10.3390/ijms27020809},
pmid = {41596458},
issn = {1422-0067},
support = {101081974//European Commission/ ; },
mesh = {*Helianthus/genetics/physiology/growth &amp; development ; *Regeneration/genetics ; Genotype ; Plant Shoots/growth &amp; development/genetics ; Plant Roots/growth &amp; development/genetics ; Plant Breeding ; Gene Expression Regulation, Plant ; Sucrose/pharmacology ; Cluster Analysis ; },
abstract = {This study presents the first integrated analysis of genotype-medium interactions and temporal morphogenesis profiling in sunflower regeneration. It aims to characterize genotype-specific responses, identify predictive morphological markers, and develop a scalable framework for breeding and transformation. Eighteen sunflower genotypes were evaluated to assess organogenic performance. The model genotype Ha-26-PR was used for a complementary experiment, testing varying sucrose concentrations to examine their influence on morphogenic outcomes. Hierarchical Cluster Analysis (HCA), guided by the Elbow method, identified four optimal clusters (K = 4). These aligned with three biologically meaningful categories: High Regenerators (Cluster 1), Moderate/Specific Regenerators (Clusters 2 and 3), and Non-Regenerators (Cluster 4). On S1 medium, NO-SU-12 and AS-1-PR showed superior shoot regeneration, while on R4 medium, HA-26-PR-SU and NO-SU-12 performed best. Genotypes such as NO-SU-12 and AS-1-PR consistently excelled across both media, whereas AB-OR-8 and FE-7 remained non-regenerators. Medium R4 supported superior regeneration, primarily through root formation, while S1 failed to induce roots in any genotype, highlighting the importance of hormonal composition. Although sucrose promoted callus induction, it did not trigger organogenesis. Callus was consistently present across media and time points, but its correlations with shoot and root formation were weak and temporally unstable, limiting its predictive value. Root formation at 14 days (Root 14D) emerged as a robust early predictor of organogenic success. This integration of morphological, temporal, and statistical analyses offers a genotype-tailored regeneration framework with direct applications in molecular breeding and CRISPR/Cas-based genome editing.},
}
@article {pmid41596437,
year = {2026},
author = {Luo, H and Zou, H and Lin, S and Liu, J and Zhou, G and Gao, L and Huang, J and Li, J and Gao, J and Ma, C},
title = {Multiplex Editing of OsMads26, OsBsr-d1, OsELF3-2 and OsERF922 with CRISPR/Cas9 Confers Enhanced Resistance to Pathogens and Abiotic Stresses and Boosts Grain Yield in Rice (Oryza sativa).},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
doi = {10.3390/ijms27020781},
pmid = {41596437},
issn = {1422-0067},
mesh = {*Oryza/genetics/microbiology/growth &amp; development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Disease Resistance/genetics ; *Plant Proteins/genetics ; *Stress, Physiological/genetics ; Plant Diseases/microbiology/genetics ; Plants, Genetically Modified/genetics ; *Edible Grain/genetics/growth &amp; development ; Gene Expression Regulation, Plant ; Droughts ; },
abstract = {Rice (Oryza sativa) is one of the world's major staple foods. However, stable rice production is constrained by various biotic and abiotic and stresses. Breeding and cultivation of rice varieties with resistance to multiple pathogens and environmental stresses is the most effective strategy to mitigate the adverse effect of pathogen attacks and abiotic stresses. Recently, researchers have focused on the exploitation of CRISPR/Cas9 technology to manipulate some negative defense-regulator genes to generate rice varieties with broad-spectrum resistance against rice pathogens. In this study, four negative regulator genes of rice blast, OsMads26, OsBsr-1, OsELF3-2 and OsERF922, were selected as CRISPR/Cas9 targets. By simultaneously knocking out all four genes via CRISPR/Cas9 technology, we created three mads26/bsr-1/elf3-2/erf922 quadruple knockout mutants. Our results demonstrated that all quadruple mutants exhibited much higher resistance not only to rice blast and bacterial blight but also to drought and salt stresses than the wildtype. Interestingly, grain yield of all three quadruple mutants was also drastically increased by 17.35% to 21.95%. Therefore, this study provides a novel strategy to rapidly improve rice varieties with broad-spectrum resistance to pathogens, elevated tolerance to abiotic stresses and enhanced yield potential.},
}
@article {pmid41596278,
year = {2026},
author = {Bernacka, KU and Michalski, K and Wojciechowski, M and Sowa, S},
title = {Application of SNV Detection Methods for Market Control of Food Products from New Genomic Techniques.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
doi = {10.3390/ijms27020626},
pmid = {41596278},
issn = {1422-0067},
support = {101137025//Horizon Europe- European Union/ ; DHR.bz.070.2.2024//Polish Ministry of Agriculture and Rural Development/ ; },
mesh = {*Plants, Genetically Modified/genetics ; *Genomics/methods ; *Polymorphism, Single Nucleotide ; *Food, Genetically Modified ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {The detection of single-nucleotide variants (SNVs) is an important challenge in modern genomics, with broad applications in medicine, diagnostics, and agricultural biotechnology. Current detection approaches include PCR-based techniques with high-affinity probes, ligase-based strategies, and sequencing approaches, each with varying degrees of sensitivity, specificity, and practicality. Despite advances in SNV analysis in the medical field, their implementation in the official control and monitoring of genetically modified organisms (GMOs) remains limited. This challenge has gained priority with the advent of new genomic techniques (NGTs), such as CRISPR-Cas nucleases, which allow precise genome editing, including subtle changes at the nucleotide level without introducing foreign DNA. Therefore, traditional methods of GMO detection targeting transgene sequences may not be sufficient to monitor such GMOs. In the European Union, GMO legislation requires distinguishing between conventionally bred and genetically modified plants. The planned introduction of new regulatory categories of NGT plants (NGT1 and NGT2) with different surveillance requirements emphasizes the need for robust, sensitive, and cost-effective SNV detection methods suitable for distinguishing between GMOs, particularly in the context of food and feed safety, traceability, and compliance.},
}
@article {pmid41595497,
year = {2026},
author = {Guan, S and Han, Y and Zhang, J and Du, Y and Chen, Z and Miao, C and Li, J},
title = {Multiplex Gene Editing and Effect Analysis of Yield, Fragrance, and Blast Resistance Genes in Rice.},
journal = {Genes},
volume = {17},
number = {1},
pages = {},
doi = {10.3390/genes17010077},
pmid = {41595497},
issn = {2073-4425},
mesh = {*Oryza/genetics/growth &amp; development/microbiology ; *Gene Editing/methods ; *Disease Resistance/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Plant Diseases/genetics/microbiology ; Plant Proteins/genetics ; Genes, Plant ; Plant Breeding ; },
abstract = {BACKGROUND: The coordinated improvement of yield, quality and resistance is a primary goal in rice breeding. Gene editing technology is a novel method for precise multiplex gene improvement.<br><br>METHODS: In this study, we constructed a multiplex CRISPR/Cas9 vector targeting yield-related genes (GS3, OsPIL15, Gn1a), fragrance gene (OsBADH2) and rice blast resistance gene (Pi21) to pyramid traits for enhanced yield, quality, and disease resistance in rice. A tRNA-assisted CRISPR/Cas9 multiplex gene editing vector, M601-OsPIL15/GS3/Gn1a/OsBADH2/Pi21-gRNA, was constructed. Genetic transformation was performed using the Agrobacterium-mediated method with the japonica rice variety Xin Dao 53 as the recipient. Mutation editing efficiency was detected in T0 transgenic plants. Grain length, grain number per panicle, thousand-grain weight, 2-acetyl-1-pyrroline (2-AP) content, and rice blast resistance of homozygous lines were measured in the T3 generations.<br><br>RESULTS: Effectively edited plants were obtained in the T0 generation. The simultaneous editing efficiency for all five genes reached 9.38%. The individual gene editing efficiencies for Pi21, GS3, OsBADH2, Gn1a, and OsPIL15 were 78%, 63%, 56%, 54%, and 13%, respectively. Five five-gene homozygous edited lines with two genotypes were selected in the T2 generation. In the T3 generation, compared with the wild-type (WT), the edited homozygous lines showed increased grain number per panicle (14.60-25.61%), increased grain length (7.39-11.16%), increased grain length-width ratio (8.37-13.02%), increased thousand-grain weight (3.79-9.15%), a 42-64 folds increase in the fragrant substance 2-AP content, and significantly enhanced rice blast resistance. Meanwhile, there were no significant changes in other agronomic traits.<br><br>CONCLUSIONS: CRISPR/Cas9-mediated multiplex gene editing technology enabled the simultaneous editing of genes related to rice yield, quality, and disease resistance. This provides an effective approach for obtaining new japonica rice germplasm with blast resistance, long grains, and fragrance.},
}
@article {pmid41595237,
year = {2026},
author = {Karnik, M and Tulimilli, SV and Anantharaju, PG and Bettadapura, ADS and Natraj, SM and Mohideen, HS and Dovat, S and Sharma, A and Madhunapantula, SV},
title = {An Overview of the Mechanisms of HPV-Induced Cervical Cancer: The Role of Kinase Targets in Pathogenesis and Drug Resistance.},
journal = {Cancers},
volume = {18},
number = {2},
pages = {},
doi = {10.3390/cancers18020318},
pmid = {41595237},
issn = {2072-6694},
support = {No. JSSAHER/REG/RES/URG/54/2023-24//Intramural grant sanctioned by JSS AHER/ ; No. JSSAHER/REG/RES/URG/54/2025-26//JSS AHER/ ; },
abstract = {Despite a thorough understanding of the structure of human papillomavirus (HPV) and its genotypic variations (high-risk and low-risk variants), the mechanisms underlying HPV-induced cervical cancer (CC) pathogenesis and the molecular signatures of drug resistance remain to be fully understood. Accumulating evidence has shown the involvement of kinase targets in the induction of drug resistance in high-risk (HR) HPV-CC. Molecularly, the genome of high-risk HPV is reported to control the expression of host kinases. In particular, Aurora kinases A, B, and C (ARKA, ARKB, and ARKC), phosphotidylinositol-trisphosphate kinase (PI3K)-Akt, and Glycogen synthase kinase3-α/β (GSK3 α/β) promote the transformation of infected cells, and also enhance the resistance of cells to various chemotherapeutic agents such as nelfinavir and cisplatin. However, the precise mechanisms through which HPV activates these kinases are yet to be fully elucidated. Furthermore, there is still ambiguity surrounding whether targeting HPV-induced kinases along with HPV-targeted therapies (such as phytopharmaceuticals and PROTAC/CRISPR-CAS-based systems) synergistically inhibit cervical tumor growth. Given the critical role of kinases in the pathogenesis and treatment of CC, a comprehensive review of current evidence is warranted. This review aims to provide key insights into the mechanisms of HPV-induced CC development, the involvement of kinases in drug resistance induction, and the rationale for combination therapies to improve clinical outcomes.},
}
@article {pmid41594570,
year = {2025},
author = {Anschuetz, A and Robinson, L and Mondesir, M and Melis, V and Platt, B and Harrington, CR and Riedel, G and Schwab, K},
title = {Effect of the Icelandic Mutation APP[A673T] in the Murine APP Gene on Phenotype of Line 66 Tau Mice.},
journal = {Biomolecules},
volume = {16},
number = {1},
pages = {},
doi = {10.3390/biom16010028},
pmid = {41594570},
issn = {2218-273X},
support = {PAR1577 and PAR2074//TauRx Therapeutics Ltd Singapore/ ; },
mesh = {Animals ; *tau Proteins/genetics/metabolism ; Mice ; Mice, Transgenic ; Male ; Female ; *Amyloid beta-Protein Precursor/genetics/metabolism ; *Alzheimer Disease/genetics/pathology/metabolism ; Phenotype ; Iceland ; *Mutation ; Humans ; Mice, Inbred C57BL ; Brain/metabolism/pathology ; Disease Models, Animal ; },
abstract = {The Icelandic mutation in the amyloid precursor protein (APP), APP[A673T], has been identified in Icelandic and Scandinavian populations and is associated with a significantly lower risk of developing Alzheimer's disease (AD). The introduction of the human APP[A673T] form led to a reduction in amyloid β-protein (Aβ) production and tau pathology, but the effect of mouse APP[A673T] on tau and Aβ pathology is not well studied. We have crossed line 66 (L66) tau transgenic mice that overexpress the P301S aggregation-prone form of tau with C57Bl6/J mice expressing a single-point mutation edited into the murine APP gene via CRISPR-Cas gene editing, known as mAPP[A673T]. We have performed ELISA, histopathological, and behavioural analyses of heterozygous male/female L66 and L66 xmAPP[A673T] crosses at the age of 6 months to investigate the effect of the murine A673T mutation on tau brain pathology and behavioural deficits in these mice. Using immunohistochemistry, we found only a moderate, yet significant, reduction in mAb 7/51-reactive tau for female L66 x mAPP[A673T] compared to L66 mice. Quantification of tau in soluble/insoluble brain homogenate fractions by ELISA confirmed the lack of overt differences between genotypes, as did our extensive behavioural phenotyping using six different paradigms assessing motor function, olfaction, depression/apathy-like behaviour, as well as exploration and sociability. Therefore, the mAPP[A673T] mutation has a moderate impact on tau pathology but does not appear to impact motor and neuropsychiatric behaviour in L66 tau transgenic mice.},
}
@article {pmid41594550,
year = {2025},
author = {Wang, R and Guo, C and Zhang, J and Wang, Z and Jin, W and Liu, W},
title = {Evaluation of the Unintended Effects of fad2-1-Gene-Edited Soybean Line AE15 Seeds.},
journal = {Biomolecules},
volume = {16},
number = {1},
pages = {},
doi = {10.3390/biom16010008},
pmid = {41594550},
issn = {2218-273X},
support = {2023YFF1001603//the National Key Research and Development Program/ ; ZDYF2025GXJS151//the Hainan Provincial Sanya Yazhou Bay Science and Technology Innovation Joint Project/ ; },
mesh = {*Glycine max/genetics/metabolism ; *Seeds/genetics/metabolism ; *Gene Editing ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; Proteomics ; Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; },
abstract = {A data-independent acquisition (DIA)-based proteomic analysis was performed to evaluate the unintended effects of fad2-1-gene-edited soybean line AE15 seeds. A total of 561, 269, and 227 differentially expressed proteins (DEPs) were identified in seeds from three consecutive generations of AE15 soybean, respectively, and were primarily enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to carbon metabolism, protein processing in the endoplasmic reticulum, and proteasome function. Furthermore, eight commonly differentially expressed proteins (co-DEPs) were detected across all three generations of AE15 soybean seeds, among which two-beta-amylase and endoplasmic reticulum (ER) lumen protein-retaining receptor-exhibited consistently upregulated expression. In the wild-type soybean control groups, 1063, 989, and 671 DEPs were identified across the three comparisons (ZhH302E3/ZhH10, ZhH10/ZhH42, and ZhH42/ZhH302E3), among which 71 co-DEPs were observed. These findings indicate that the protein expression profile alterations resulting from fad2-1 gene editing are considerably less pronounced compared to those caused by natural genetic variation among soybean seeds.},
}
@article {pmid41593344,
year = {2026},
author = {Munir, S and Wan, S and Gao, X and Lai, M and Mu, Z and Wang, H and Liu, Z and Li, F and Xia, L and Tan, Y},
title = {Elucidating the roles of essential genes in autotrophic metabolism and cell morphology of Clostridium ljungdahlii by CRISPRi.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {44},
pmid = {41593344},
issn = {1432-0614},
mesh = {*Clostridium/genetics/metabolism/cytology/growth &amp; development ; *Autotrophic Processes/genetics ; *Genes, Essential ; Ethanol/metabolism ; Fermentation ; Gene Expression Regulation, Bacterial ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Understanding the function of essential genes in Clostridium ljungdahlii is critical for unraveling its autotrophic metabolism and optimizing its potential as a platform for syngas fermentation. However, study on essential genes of this species remains insufficient. Here, we employed an inducible CRISPR interference (CRISPRi) system to investigate the roles of key metabolic and cell division genes in C. ljungdahlii. Targeted repression of genes encoding pyruvate:ferredoxin oxidoreductase (PFOR1, PFOR2), acetaldehyde:ferredoxin oxidoreductase (AOR1, AOR2), and glyceraldehyde phosphate hydrogenase type I (GAP-I) revealed their essential contributions to autotrophic growth, as knockdown strains exhibited impaired growth and reduced ethanol production. Furthermore, downregulation of the cell division gene ftsZ resulted in elongated cell morphology, highlighting its critical role in cell shape regulation. These findings provide new insights into the functional importance of essential genes in C. ljungdahlii and demonstrate how targeted gene repression can advance our understanding of autotrophic metabolism and cellular processes.},
}
@article {pmid41592905,
year = {2026},
author = {Shabbir, R and Javed, T and Sun, SR and Wang, ZQ and Zhang, W and Gao, SJ and Wang, QN},
title = {Functional genomics in sugarcane breeding: key challenges and strategies.},
journal = {Critical reviews in biotechnology},
volume = {},
number = {},
pages = {1-21},
doi = {10.1080/07388551.2026.2614075},
pmid = {41592905},
issn = {1549-7801},
abstract = {Sugarcane, a leading source of sugar and bio-energy around the globe stands at the cross-road of genome complexity and agricultural innovation, offering the immense potential to fuel a sustainable future. Functional genomics with its precise identification and manipulation of genes could enable researchers unlock this potential and accelerate the breeding efforts. However, the polyploid genome of sugarcane with: high heterozygosity, high-repetitive DNA content, multiple copies of homo(eo)logous gene, epistatic interaction of alleles, etc., challenges the gene annotation, genome sequencing, genome editing, and phenotypic characterization. Similarly long breeding cycle, low transformation efficiency, time-consuming, and labor-intensive transformation methods further complicates the genome editing. Recent advances of functional genomics are transforming this scenario, such as current availability of reference genome "R570," has provided a significant insight of genome architect and function. Genome wide association studies (GWAS)/genome selection (GS) are enhancing trait-mapping and prediction of breeding values to accelerate the breeding cycles. The current era of smart breeding with integrative bio-informatics, advance genome editing tools, i.e., CRISPR/Cas-systems (Cas-proteins, Cas-RNPs, d-Cas-RNPs, and CRISPRa/i), and high-throughput phenomics offers a significant approach to: overcome transformation bottlenecks, explore complex trait architect and address polyploidy challenges. Therefore, this review summarizes the key challenges and focuses on elaborating recent advances and suggests optimized strategies for future improvement in functional genomics of sugarcane breeding.},
}
@article {pmid41592109,
year = {2026},
author = {Saglam, M and Tsakirpaloglou, N and Bridgeland, A and Miller, R and Thomson, MJ and Septiningsih, EM},
title = {Carbon nanotube and carbon dot mediated plasmid DNA delivery in cowpea leaves.},
journal = {PloS one},
volume = {21},
number = {1},
pages = {e0340716},
doi = {10.1371/journal.pone.0340716},
pmid = {41592109},
issn = {1932-6203},
mesh = {*Nanotubes, Carbon/chemistry ; *Plant Leaves/genetics/metabolism ; *Vigna/genetics/metabolism ; *Plasmids/genetics ; *Gene Transfer Techniques ; CRISPR-Cas Systems ; Glucuronidase/genetics/metabolism ; Plants, Genetically Modified/genetics ; Oxidoreductases/genetics ; *Quantum Dots/chemistry ; Gene Editing ; Carbon/chemistry ; },
abstract = {CRISPR-Cas9 technology has been widely used as a key molecular biology tool for crop improvement. However, the advance of this technology has been hindered by host species- or genotype-dependent tissue culture protocols and poor transformation efficiencies. Recent research has shown that plasmid DNA delivered by single-walled carbon nanotubes (SWCNTs) and carbon dots (CDs) can diffuse through plant cell walls, enabling the transient expression of genetic material in plant tissues. However, such an experiment has not been performed in legumes, most of which are considered recalcitrant species for transformation. In this study, we aim to investigate the capability of a SWCNT or CD-based plasmid delivery system in expressing a target gene in cowpea (Vigna unguiculata) leaves via infiltration using the β-glucuronidase (GUS) reporter gene. Further, we aim to see the potential of SWCNTs and CDs for a CRISPR-Cas9 gene construct delivery system, with phytoene desaturase (PDS) as the target gene. Our results showed that SWCNTs and CDs can deliver the GUS reporter gene construct in the surrounding area near the site of the infiltration, which results in the temporary expression of GUS by observing the blue color in this area. Likewise, infiltration of the CRISPR-Cas9 vectors targeting the PDS gene for the knockout resulted in multiplex editing and large deletions within the target gene. Overall, our findings pave the way for overcoming conventional DNA delivery challenges. However, further research is needed to explore optimal germline targets for plant tissues to avoid chimerism and to allow for more efficient CRISPR-Cas9 editing resulting in heritable mutations.},
}
@article {pmid41591594,
year = {2026},
author = {Singhal, P and Saini, S and Saini, O and Bishnoi, A and E R, R and Meena, BR and Singh, J and Yogendra, K},
title = {Molecular gatekeepers: eukaryotic translation factors decoding plant-virus dynamics for resistance engineering.},
journal = {Stress biology},
volume = {6},
number = {1},
pages = {9},
pmid = {41591594},
issn = {2731-0450},
abstract = {Plant viruses are among the most significant biotic stressors, posing a severe threat to crop productivity and global food security. Their success largely depends on the exploitation of host eukaryotic translation factors (eTFs), including initiation factors (eIFs) and elongation factors (eEFs), which act as molecular gatekeepers of the viral life cycle. Key members such as eIF4E, eIF(iso)4E, eIF4G, eEF1A, and eEF1B have been identified as susceptibility factors that mediate viral translation, replication, and systemic movement. Viruses have co-evolved specialized proteins and RNA elements, including VPg and IRES structures, to hijack these host factors and circumvent plant defense barriers. This review synthesizes current understanding of the mechanistic roles of eTFs in virus-host dynamics and highlights strategies to mitigate viral stress. Approaches such as natural allele mining, induced mutagenesis, TILLING/EcoTILLING, RNA interference, and precise genome editing with CRISPR/Cas systems are explored as practical tools for reducing susceptibility. Targeted manipulation of eTFs offers a promising avenue to reprogram plants for resistance while maintaining essential cellular functions. By integrating molecular biology with applied strategies, we propose an eTF-centered framework for resistance breeding within a broader stress biology perspective. Future research combining functional genomics, synthetic biology, and breeding innovation will be pivotal in delivering broad-spectrum, durable, and environmentally sustainable resistance to plant viral stress.},
}
@article {pmid41590698,
year = {2025},
author = {Antonacci, A and Masi, A and Vedi, V and Colella, S and Musella, F and Fiorentino, G and Scognamiglio, V},
title = {CRISPR-Cas Technology Turns Chlamydomonas reinhardtii into a Flagship for Algal Biotechnology.},
journal = {Marine drugs},
volume = {24},
number = {1},
pages = {},
doi = {10.3390/md24010001},
pmid = {41590698},
issn = {1660-3397},
mesh = {*Chlamydomonas reinhardtii/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Biotechnology/methods ; Gene Editing/methods ; *Microalgae/genetics/metabolism ; Biofuels ; Metabolic Engineering/methods ; },
abstract = {Microalgae represent some of the most promising eukaryotic platforms in biotechnology due to their rapid growth, simple cultivation requirements, reliance on sunlight as a primary energy source, and ability to synthesize high-value bioactive compounds. These characteristics have made microalgae attractive candidates in various fields, including biofuel production, carbon capture, and pharmaceutical development. However, several technical limitations have limited their large-scale use as sustainable biofactories. A paradigm shift is currently occurring thanks to the genetic manipulation of microalgae, driven by CRISPR-Cas technology. Significant progress has been made in the model species Chlamydomonas reinhardtii, particularly in the targeted and efficient insertion of foreign DNA. Despite this progress, key challenges remain, and further optimization of CRISPR-Cas methodologies is needed to fully unleash the genetic potential of this organism. This review provides an overview of the convergence of CRISPR-Cas technologies in microalgae research, highlighting their impact on genetic studies, metabolic engineering, and industrial applications. It summarizes recent advances in microalgal genome editing through CRISPR systems, outlines current technical challenges, and highlights future directions for improving the implementation of this innovative technology in microalgal biotechnology.},
}
@article {pmid41590485,
year = {2026},
author = {Gupta, DR and Kasfy, SH and Ali, J and Hia, FT and Hoque, MN and Rahman, M and Islam, T},
title = {Validation and Improvement of a Rapid, CRISPR-Cas-Free RPA-PCRD Strip Assay for On-Site Genomic Surveillance and Quarantine of Wheat Blast.},
journal = {Journal of fungi (Basel, Switzerland)},
volume = {12},
number = {1},
pages = {},
doi = {10.3390/jof12010073},
pmid = {41590485},
issn = {2309-608X},
support = {Grant Code: V0156.01//Bill and Melinda Gates Foundation and the Foreign, Commonwealth &amp; Development Office (FCDO), UK/ ; },
abstract = {As an emerging threat to global food security, wheat blast necessitates the development of a rapid and field-deployable detection system to facilitate early diagnosis, enable effective management, and prevent its further spread to new regions. In this study, we aimed to validate and improve a Recombinase Polymerase Amplification coupled with PCRD lateral flow detection (RPA-PCRD strip assay) kit for the rapid and specific identification of Magnaporthe oryzae pathotype Triticum (MoT) in field samples. The assay demonstrated exceptional sensitivity, detecting as low as 10 pg/µL of target DNA, and exhibited no cross-reactivity with M. oryzae Oryzae (MoO) isolates and other major fungal phytopathogens under the genera of Fusarium, Bipolaris, Colletotrichum, and Botrydiplodia. The method successfully detected MoT in wheat leaves as early as 4 days post-infection (DPI), and in infected spikes, seeds, and alternate hosts. Furthermore, by combining a simplified polyethylene glycol-NaOH method for extracting DNA from plant samples, the entire RPA-PCRD strip assay enabled the detection of MoT within 30 min with no specialized equipment and high technical skills at ambient temperature (37-39 °C). When applied to field samples, it successfully detected MoT in naturally infected diseased wheat plants from seven different fields in a wheat blast hotspot district, Meherpur, Bangladesh. Training 52 diverse stakeholders validated the kit's field readiness, with 88% of trainees endorsing its user-friendly design. This method offers a practical, low-cost, and portable point-of-care diagnostic tool suitable for on-site genomic surveillance, integrated management, seed health testing, and quarantine screening of wheat blast in resource-limited settings. Furthermore, the RPA-PCRD platform serves as an early warning modular diagnostic template that can be readily adapted to detect a wide array of phytopathogens by integrating target-specific genomic primers.},
}
@article {pmid41590284,
year = {2026},
author = {Zhang, Z and Fu, Q and Wen, T and Zheng, Y and Ma, Y and Liu, S and Liu, G},
title = {Integrated Colorimetric CRISPR/Cas12a Detection of Double-Stranded DNA on Microfluidic Paper-Based Analytical Devices.},
journal = {Biosensors},
volume = {16},
number = {1},
pages = {},
doi = {10.3390/bios16010032},
pmid = {41590284},
issn = {2079-6374},
support = {22174121, 22211530067, T2250710180//National Natural Science Foundation of China/ ; },
mesh = {*Colorimetry ; Paper ; CRISPR-Cas Systems ; *Biosensing Techniques ; Humans ; Human papillomavirus 16 ; Lab-On-A-Chip Devices ; Gold/chemistry ; *DNA/analysis ; Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques ; },
abstract = {Early detection of high-risk human papillomavirus (HPV), particularly HPV16 E7, is critical for cervical cancer prevention. Here, we report a novel, portable, and instrument-free biosensing platform that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated detection on a microfluidic paper-based analytical device (μPAD) for colorimetric, visual readout of double-stranded DNA (dsDNA). The μPAD features seven functional zones, including lyophilized RPA and CRISPR reagents, and immobilized streptavidin and anti-FAM antibodies for signal generation. Upon target recognition, Cas12a's trans-cleavage activity releases biotinylated-FAM-labeled reporters that form a sandwich complex with gold nanoparticle (AuNP)-conjugated anti-FAM antibodies, producing a visible red signal at the test zone. The gray value of the colorimetric signal correlates linearly with target concentration, enabling the quantitative detection of HPV16 E7 dsDNA down to 100 pM within 60 min. The assay demonstrated high accuracy and reproducibility in spiked samples. By combining isothermal amplification, CRISPR specificity, and paper-based microfluidics, this platform offers a rapid, low-cost, and user-friendly solution for point-of-care HPV screening in resource-limited settings. This work advances the integration of CRISPR diagnostics with μPAD, paving the way for scalable point-of-care molecular diagnostics beyond HPV.},
}
@article {pmid41590269,
year = {2025},
author = {Liang, Z and Zhang, J and Zhang, S},
title = {Engineering a CRISPR-Mediated Dual Signal Amplification-Based Biosensor for miRNA Determination.},
journal = {Biosensors},
volume = {16},
number = {1},
pages = {},
doi = {10.3390/bios16010017},
pmid = {41590269},
issn = {2079-6374},
support = {2023A1515110638//Guangdong Basic and Applied Basic Research Foundation/ ; 2025A1515011683//Guangdong Basic and Applied Basic Research Foundation/ ; 2025A04J4037//Guangzhou Science and Technology Planning Project/ ; 2022GDASZH-2022010110//GDAS' Project of Science and Technology Development/ ; },
mesh = {*MicroRNAs/analysis ; *Biosensing Techniques/methods ; Humans ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electrochemical Techniques ; },
abstract = {MicroRNAs, pivotal regulators of gene expression and physiology, serve as reliable biomarkers for early cancer diagnosis and therapy. As one of the earliest discovered miRNAs in the human genome, miRNA-21 provides critical information for early cancer diagnosis, drug therapy, and prognosis. In this work, we harness CRISPR as a bridge to integrate target-induced self-priming hairpin isothermal amplification (SIAM) with terminal transferase (TdT) polymerization labeling, constructing a facile, straightforward electrochemical biosensor for sensitive miRNA-21 detection. Unlike conventional single-strand template-based exponential amplification (EXPAR), the SIAM hairpin undergoes target triggered intramolecular conformational change, initiating extension and strand displacement reactions that suppress nonspecific dimer formation and lower background current. Notably, the assay requires only a single probe, enabling unidirectional signal amplification while nonspecific reactions caused by system complexity. The generated SIAM products activate the Cas12a/crRNA complex to trans-cleave PO4[3-] modified single-stranded DNAs (ssDNAs); the resulting 3' hydroxyl ssDNAs are subsequently labeled by TdT, with the assistance of SA-HRP catalyzing hydrogen peroxide, achieving robust signal amplification. Under optimized conditions, the cathodic current exhibits a logarithmic relationship with miRNA concentrations from 20 fM to 5.0 × 10[8] fM, with a detection limit of 9.2 fM. The biosensor successfully quantified miRNA-21 in commercial serum samples and biological lysates, demonstrating its potential for cancer diagnostics and therapy.},
}
@article {pmid41588195,
year = {2026},
author = {Taveneau, C and Chai, HX and D'Silva, J and Bamert, RS and Chen, H and Hayes, BK and Calvert, RW and Purcell, J and Curwen, DJ and Munder, F and Martin, LL and Barr, JJ and Rosenbluh, J and Fareh, M and Grinter, R and Knott, GJ},
title = {De novo design of potent CRISPR-Cas13 inhibitors.},
journal = {Nature chemical biology},
volume = {},
number = {},
pages = {},
pmid = {41588195},
issn = {1552-4469},
abstract = {CRISPR-Cas systems are transformative tools for gene editing that can be tuned or controlled by anti-CRISPRs (Acrs)-phage-derived inhibitors that regulate CRISPR-Cas activity. However, Acrs that can inhibit biotechnologically relevant CRISPR systems are relatively rare and challenging to discover. To overcome this limitation, we describe a highly successful and rapid approach that leverages de novo protein design to develop new-to-nature proteins for controlling CRISPR-Cas activity. Here, using Leptotrichia buccalis CRISPR-Cas13a as a representative example, we demonstrate that Acrs designed using artificial intelligence (AIcrs) are capable of highly potent and specific inhibition of CRISPR-Cas13a nuclease activity. We present a comprehensive workflow for design validation and demonstrate AIcr functionality in controlling CRISPR-Cas13 activity in bacterial and human cells. The ability to design bespoke inhibitors of Cas effectors will contribute to the ongoing development of CRISPR-Cas tools in diverse applications across research, medicine, agriculture and microbiology.},
}
@article {pmid41588051,
year = {2026},
author = {Molina, MC and Quiroga, C},
title = {Functional characterization of a type I-F1 CRISPR-cas system from the clinical isolate Shewanella xiamenensis Sh95 reveals constitutive activity and plasmid-curing capability.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-025-34486-2},
pmid = {41588051},
issn = {2045-2322},
support = {ANPCyT 2020-03222//Agencia Nacional de Promoci&#xf3;n de la Investigaci&#xf3;n, el Desarrollo Tecnol&#xf3;gico y la Innovaci&#xf3;n/ ; IP-PUE 0085//Consejo Nacional de Investigaciones Cient&#xed;ficas y T&#xe9;cnicas/ ; PIDAE-UBA 2025 EX-2024-04115022-UBA-DME#REC//Universidad de Buenos Aires/ ; },
}
@article {pmid41587008,
year = {2026},
author = {Cai, M and Song, K and Yao, C and Wang, S and Wang, R and Wang, Q and Chen, H and Wang, H},
title = {Global spread and evolution of KPC-2 and NDM-1-producing Gram-negative bacteria.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
pmid = {41587008},
issn = {1869-1889},
abstract = {The co-occurrence of KPC and NDM carbapenemases in Gram-negative bacteria presents a serious and expanding global health threat. This study characterized 338 KPC-2/NDM-1 dual-positive isolates from 23 countries, including 41 clinical strains sequenced through hybrid second- and third-generation platforms from China's national surveillance network. These isolates spanned six genera, 58 species, and 138 sequence types, reflecting substantial taxonomic and geographic diversity. Molecular analysis identified IncFII(p14) plasmids as the principal vectors for cross-genus dissemination of KPC-2, while IncX3, IncN, and IncFIB(pB171)/IncFII(Yp) plasmids were dominant carriers of NDM-1 among the studied strains. Codon usage analysis indicated stronger bias in KPC-2 plasmids (effective codon number: 39.17, optimal codons: 17) compared to NDM-1 plasmids (effective codon number: 41.25, optimal codons: 12), indicating differential evolutionary pressures. Dual-positive strains exhibited significantly higher virulence scores and broader resistance profiles than reference strains (P<0.001). Notably, only 14.6% of isolates harbored Type I-E CRISPR-Cas systems, all of which encoded the anti-CRISPR protein AcrIE10. Furthermore, Type II methyltransferase numbers were significantly enriched in dual-positive strains (P<0.005), suggesting a potential role in modulating host defense evasion. We propose that in Klebsiella spp., KPC-2 plasmids are typically acquired prior to NDM-1 plasmids and can form hybrid plasmids. In non-Klebsiella genera, dual resistance is primarily driven by independent acquisition of high-risk plasmids such as IncFII(p14) and IncX3, without a fixed temporal order. These findings highlight the convergence of global plasmid-mediated resistance, host-pathogen immune interplay, and pan-resistance evolution. Targeting high-risk plasmid lineages and host defense-modulating elements may be key to forecasting resistance emergence and guiding early interventions against dual-carbapenemase-producing pathogens.},
}
@article {pmid41586841,
year = {2026},
author = {Graça, M and Virgolini, N and Correia, R and Escandell, J and Roldão, A},
title = {An improved CRISPR-Cas9 protein-based method for knocking out insect Sf9 cell genes.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {42},
pmid = {41586841},
issn = {1432-0614},
mesh = {Animals ; *CRISPR-Cas Systems ; Sf9 Cells ; *Gene Knockout Techniques/methods ; *Gene Editing/methods ; Apoptosis ; Baculoviridae/genetics ; Spodoptera/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Insect cells are one of the uprising expression systems in the biopharmaceutical industry to produce vaccines and gene therapy vectors, but cell line development has been limited by the lack of established genetic engineering tools and genomic characterization. CRISPR-Cas9 has arisen as a powerful tool for gene editing but has seen little application in insect cells. In this work, a gene editing pipeline for the delivery of a ribonucleoprotein (RNP) complex comprised of a guide RNA and the enzyme Cas9 to insect Sf9 cells was implemented and then applied to knockout caspase initiator Sf-Dronc, aiming at alleviating cell apoptosis during an infection process. The resulting engineered cell lines were characterized as per their phenotype and production of three different product modalities. Utilizing the established workflow, a knockout rate of 68% was achieved with the implemented protocol (vs. the 12% presumed efficiency of a previously reported system) when targeting the fdl gene. When applied to Sf-Dronc, mutants containing deletions in several alleles of the host genome were identified and confirmed by next-generation sequencing. Generated clones exhibited higher apoptosis resistance and delayed onset of cell viability drop following infection with baculovirus. While Sf-Dronc deletion was shown to have negligible impact on the production of rAAV and PfRipr5, production of iVLPS showed an > twofold increase over wild-type Sf9. Overall, this study showcases the successful implementation of an efficient CRISPR-Cas9 pipeline, further leveraging the usage of genetic engineering in insect Sf9 cells towards the development of enhanced cell hosts for biopharmaceutical production. KEY POINTS: • Implementation of an efficient CRISPR-Cas9 RNP complex delivery strategy to insect cells. • Establishment of the genome editing pipeline demonstrated through Sf-Dronc knockout, resulting in increased apoptosis resistance and delayed loss of viability upon baculovirus infection. • Sf-Dronc deletion led to over a twofold increase in the production of influenza VLPs compared to wild-type Sf9 cells.},
}
@article {pmid41586305,
year = {2025},
author = {Pérez-Rodríguez, M and Serrano-Pertierra, E and Blanco-López, MC},
title = {Advances in biosensor technologies for the detection of antimicrobial resistance in Staphylococcus aureus.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1741845},
pmid = {41586305},
issn = {2235-2988},
mesh = {*Biosensing Techniques/methods ; Humans ; *Staphylococcal Infections/diagnosis/microbiology ; *Methicillin-Resistant Staphylococcus aureus/drug effects/genetics/isolation &amp; purification ; *Staphylococcus aureus/drug effects/genetics/isolation &amp; purification ; CRISPR-Cas Systems ; *Drug Resistance, Bacterial ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests/methods ; },
abstract = {The rise of methicillin-resistant Staphylococcus aureus (MRSA) underscores the urgent need for rapid, sensitive, and portable diagnostics. In this paper, we have critically reviewed recent advances in biosensor technologies, integrating nanomaterials, aptamers, CRISPR/Cas systems, and microfluidic lab-on-a-chip platforms, that enable sub-hour and ultrasensitive detection of S. aureus and its resistance genes. These innovations offer powerful alternatives to conventional culture and PCR assays, forming the way for real-time, point-of-care antimicrobial resistance testing. Remaining challenges include matrix interference, lack of standardization, and limited clinical validation, yet continued integration with artificial intelligence and digital systems promises transformative diagnostic capabilities.},
}
@article {pmid41584446,
year = {2026},
author = {Yang, F and Ran, Q and Chen, J and Bao, G and Xian, Y and Zhang, C},
title = {Spatiotemporally regulated mitochondrial genome editing via enzyme and NIR-activated CRISPR/Cas9 nanoplatform.},
journal = {Chemical science},
volume = {},
number = {},
pages = {},
pmid = {41584446},
issn = {2041-6520},
abstract = {Mitochondrial DNA (mtDNA) mutations play critical roles in tumor progression and metabolic reprogramming. Controllable gene editing within tumor cell mitochondria remains a challenge due to the double-membrane barrier and the lack of tumor-selective activation. Herein, we report a dual-responsive CRISPR/Cas delivery platform (UCRP-TPP) that enables spatiotemporally regulated mtDNA editing for targeted tumor therapy. This nanoplatform integrates near infrared light-responsive upconversion nanoparticle (UCNP), an apurinic endonuclease 1 (APE-1)-responsive DNA complex, and a mitochondrial-targeting ligand (TPP), ensuring selective activation and mitochondrial release of Cas9/sgRNA complexes. Upon activation by endogenous APE-1 enzyme and exogenous NIR light, UCRP-TPP induces mtDNA editing by CRISPR/Cas, which leads to mtDNA copy number reduction, mitochondrial membrane depolarization, reactive oxygen species generation, and tumor cell apoptosis. In vivo studies further confirm the robust antitumor efficacy of the UCRP-TPP-based nanoplatform. This work presents a versatile and controllable mitochondrial gene-editing strategy.},
}
@article {pmid41581986,
year = {2026},
author = {Dhariwal, R and Jain, M},
title = {Cell-free systems for low-cost diagnostics.},
journal = {Progress in molecular biology and translational science},
volume = {218},
number = {},
pages = {157-185},
doi = {10.1016/bs.pmbts.2025.08.005},
pmid = {41581986},
issn = {1878-0814},
mesh = {Humans ; Biosensing Techniques ; *Cell-Free System ; Point-of-Care Systems ; Diagnosis ; },
abstract = {Cell-free systems have also become a revolutionary platform for low-cost diagnostics, providing fast, flexible, and scalable solutions to the conventional cell-based assays. Such systems, which utilize the fundamental biochemical machinery of cells without the intricacies of living organisms, have been of great use in point-of-care (POC) diagnostics, particularly in resource-poor environments. This chapter offers a broad overview of the basic principles, design approaches, and technological breakthroughs behind cell-free diagnostic development. It discusses the biochemical underpinnings of cell-free expression, such as ribosomal function, transcriptional control, and energy regeneration, with emphases on the leading platforms including E. coli lysates, wheat germ extracts, and PURE systems. The application of synthetic biology in the form of gene circuits, CRISPR-Cas tools, and RNA aptamers is presented here in the framework of improving the sensitivity and specificity of diagnostics. The chapter further discusses recent innovations in paper-based assays, microfluidic biosensors, and wearable biosensors, which are capable of offering real-time and field-deployable diagnostics. Major challenges in the form of reagent stability, scalability, and regulatory implications are analyzed carefully along with recent trends such as AI-based system design and personalization of diagnostics. In extensive case studies, the chapter highlights the promise of cell-free systems in filling diagnostic gaps, enhancing access to healthcare, and revolutionizing global health. This book strives to offer an encyclopedic sourcebook for researchers, clinicians, and innovators interested in bringing cell-free diagnostics forward.},
}
@article {pmid41578173,
year = {2026},
author = {Rahimian, M and Aghazadeh-Soltan-Ahmadi, M and Panahi, B},
title = {Genomic landscape of biosynthetic gene clusters in Iranian extremophiles reveals prolific metabolite potential, prophage associations, and integrated defensive-metabolic islands.},
journal = {BMC microbiology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12866-025-04690-1},
pmid = {41578173},
issn = {1471-2180},
abstract = {The extreme and underexplored ecosystems of Iran represent a significant reservoir of microbial diversity with profound biosynthetic potential. To systematically investigate this resource, we employed a comprehensive genome mining approach on 16 bacterial isolates from hypersaline, desert, and petroleum-contaminated soils. Our analysis revealed an extraordinary density and complexity of biosynthetic gene clusters (BGCs), identifying 229 BGCs in total. A substantial majority (56.8%) showed no significant similarity to known clusters, underscoring the extensive novelty encoded within these extremophiles. Notably, we discovered highly intricate "trio" and "quartet" hybrid BGCs, which encode the machinery for three or four distinct classes of secondary metabolites, pushing the boundaries of known biosynthetic complexity. Parallel analysis identified six novel, high-quality prophages, largely uncharacterized in public databases. These prophages were found to carry a putative bacteriocin cluster (UviB) indicating a direct role in enhancing host fitness. Furthermore, we uncovered a dynamic co-evolutionary arms race, with bacterial genomes fortified by diverse defense systems, including abundant CRISPR-Cas arrays, and prophages encoding a repertoire of counter-defense anti-CRISPR proteins. Genomic architecture analysis revealed widespread co-localization of BGCs, prophages, and defense systems into functional genomic islands, suggesting a synergistic linkage between secondary metabolism and phage resistance. This study illuminates the remarkable biosynthetic and defensive landscape of Iranian extremophiles, highlighting them as a premier resource for discovering novel natural products and understanding virus-host evolutionary dynamics.},
}
@article {pmid41578091,
year = {2026},
author = {Torres-Higuera, LD and Rojas-Tapias, DF and Jiménez-Velásquez, S and Renjifo-Ibáñez, C},
title = {Comprehensive genotyping and taxonomic analysis uncovers extensive distribution of intermediate Leptospira species in Colombia.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {2},
pages = {57},
pmid = {41578091},
issn = {1573-0972},
mesh = {Colombia/epidemiology ; *Leptospira/genetics/classification/isolation &amp; purification ; Phylogeny ; *Leptospirosis/microbiology/epidemiology/veterinary ; Humans ; Animals ; RNA, Ribosomal, 16S/genetics ; Genotype ; Genetic Variation ; DNA, Bacterial/genetics ; Multilocus Sequence Typing ; Whole Genome Sequencing ; Genome, Bacterial ; Ribotyping ; DNA-Directed RNA Polymerases/genetics ; Virulence Factors/genetics ; Minisatellite Repeats ; },
abstract = {Leptospirosis, a globally prevalent zoonosis caused by pathogenic and intermediate Leptospira species, poses significant threats to public health and livestock industries. Despite its substantial impact, knowledge gaps persist regarding the prevalence and genetic diversity of Leptospira strains in many regions, including South America. This study aimed to characterize a diverse collection of Leptospira strains isolated from various sources in Colombia to enhance our understanding of the genetic diversity within this genus. Using a tiered approach combining conventional and genomic methods, we genotyped 55 isolates from various sources using 16S rRNA and rpoB gene sequencing, DNA ribotyping, and Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA). Most isolates were classified into phylogenetic groups containing pathogenic and intermediate strains of L. interrogans and L. wolffii, respectively, which was corroborated by ribotyping and MLVA. Whole-genome sequencing of selected strains revealed distinct genomic characteristics compared to related strains. Pan-genome analysis identified strain-specific genes, primarily hypothetical, while virulence factor analysis distinguished species-specific patterns. Furthermore, CRISPR-Cas system analysis uncovered genetic variations among the isolates. This study provides a framework for understanding Leptospira genetic diversity in Colombia and its potential implications on human and animal health. Our findings highlight the need for improved diagnostic methods and surveillance strategies that encompass both pathogenic and intermediate Leptospira species, which could significantly impact public health policies and veterinary practices in the region.},
}
@article {pmid41578087,
year = {2026},
author = {Gautam, B and Jarvis, BA and Esfahanian, M and McGinn, M and Williams, D and Liu, S and Phippen, ME and Heller, NJ and Wesley, TL and Phippen, WB and Ulmasov, T and Marks, MD and Chopra, R and Sedbrook, JC},
title = {Creating a new oilseed crop, pennycress, by combining key domestication traits using CRISPR genome editing.},
journal = {Nature plants},
volume = {12},
number = {1},
pages = {74-87},
pmid = {41578087},
issn = {2055-0278},
support = {2018-67009-27374//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2018-67009-27374//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2018-67009-27374//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2018-67009-27374//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; },
mesh = {*Gene Editing ; *Domestication ; *Crops, Agricultural/genetics ; CRISPR-Cas Systems ; Seeds/genetics ; *Brassica/genetics ; },
abstract = {Considerable off-season farmland lies fallow because few crops can profitably fit between primary crops. As a remedy, we performed de novo domestication of the freeze-tolerant, rapid-cycling wild brassica Thlaspi arvense L. (field pennycress), identifying and stacking CRISPR-Cas9-induced mutations that have minimal impacts on seed yields. High-yielding varieties were created with seed compositions such as 'double-low' canola (low erucic acid and reduced glucosinolate) and reduced seed fibre content. Seed glucosinolate content was reduced by 75% by combining mutations in R2R3-MYB (MYB28/HAG1) and basic helix-loop-helix MYC (MYC3) transcription factors. Pennycress weediness was greatly reduced by knockout of the basic helix-loop-helix transcription factor TRANSPARENT TESTA8 (TT8), which lowered seed dormancy and seed coat protections, thereby mitigating re-emergence in fields. Domesticated pennycress offers farmers a low-carbon-intensity intermediate crop that fits between two full-season summer crops, resulting in three cash crops in 2 years, conferring cover-crop-like ecosystem benefits while producing grain for renewable fuels and enhanced food security.},
}
@article {pmid41577144,
year = {2026},
author = {Mocchetti, A and Steelant, P and Hosseinkhani, M and De Rouck, S and Khajehali, J and Van Leeuwen, T},
title = {Knockout of nAChR subunits in spider mites and their phytoseiid predators confers spinosyn cross-resistance and reveals a conserved mode of action in mites.},
journal = {Insect biochemistry and molecular biology},
volume = {},
number = {},
pages = {104498},
doi = {10.1016/j.ibmb.2026.104498},
pmid = {41577144},
issn = {1879-0240},
abstract = {Spinosyns are allosteric modulators of nicotinic acetylcholine receptors (nAChRs) which in insects specifically target subunit α6. However, their mode of action in mites and compatibility with phytoseiid predators remain unclear. We combined phylogenetics with CRISPR/Cas-based reverse genetics to test whether α6-like subunits mediate spinosyn toxicity in mites and to assess prospects for resistance breeding in phytoseiids. The phylogenetic analysis identified seven α and three β subunits in multiple phytoseiids and in Tetranychus urticae. A single phytoseiid subunit clustered within the insect α6/α7 clade, whereas T. urticae possessed three (Tuα5/α6/α7) without strict one-to-one insect orthology. Using SYNCAS maternal delivery of CRISPR RNPs, we disrupted the putative α6 ortholog in Amblyseius swirskii (Asα6) and each of the three α6/α7-clade genes in T. urticae. In A. swirskii, all survivors of a discriminating spinosad dose carried Asα6 indels, and three independently edited lines exhibited insensitivity to both spinosad and spinetoram (no significant mortality at 10,000 mg a.i./L), whereas the wild type showed LC50 = 163 mg/L (spinosad) and 54 mg/L (spinetoram). In T. urticae, Tuα6 knockouts conferred high cross-resistance to both compounds, while Tuα5 knockouts slightly increased susceptibility and Tuα7 knockouts produced modest resistance. Our data demonstrate that α6-mediated spinosyn action is conserved in mites, with α6 loss conferring strong cross-resistance in a key phytoseiid predator and in a model tetranychid. These findings enable marker-assisted editing/selection of spinosyn-resistant phytoseiid strains to improve pesticide-biocontrol compatibility and establish α6 as a practical universal marker gene for genome editing in acarine systems.},
}
@article {pmid41576918,
year = {2026},
author = {Pinglay, S and Atwater, JT and Brosh, R and Shendure, J and Maurano, MT and Boeke, JD},
title = {Mammalian genome writing: Unlocking new length scales for genome engineering.},
journal = {Cell},
volume = {189},
number = {2},
pages = {356-374},
pmid = {41576918},
issn = {1097-4172},
support = {DP5 OD036167/OD/NIH HHS/United States ; R01 HG012743/HG/NHGRI NIH HHS/United States ; RM1 HG009491/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; Animals ; *Gene Editing/methods ; *Genome ; *Genetic Engineering/methods ; *Mammals/genetics ; CRISPR-Cas Systems ; },
abstract = {The ability to design and engineer mammalian genomes across arbitrary length scales would transform biology and medicine. Such capabilities would enable the systematic dissection of mechanisms governing gene regulation and the influence of complex haplotypes on human traits and disease. They would also facilitate the engineering of disease models that more faithfully recapitulate human physiology and of next-generation cell therapies harboring sophisticated genetic circuits. Over the past decade, advances in genome editing have made small, targeted modifications at single sites routine. However, achieving multiple coordinated alterations across long sequence windows (>10 kb) or installing large synthetic DNA segments in mammalian cells remains a major challenge. Recent advances in mammalian genome writing-the bottom-up design, assembly, and targeted integration of large custom DNA sequences, independent of any natural template-offer a potential solution. Here, we review key technological developments, highlight emerging applications, and discuss current bottlenecks and strategies for overcoming them.},
}
@article {pmid41574295,
year = {2025},
author = {Zhang, R and Zheng, Y and Ding, C and Wu, J and Zhu, W and Zhu, X and Xu, G and Chen, L},
title = {OxyR contributes to the oxidative stress capacity and virulence of hypervirulent Klebsiella pneumoniae ATCC 43816.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1661384},
pmid = {41574295},
issn = {2235-2988},
mesh = {*Oxidative Stress ; *Klebsiella pneumoniae/pathogenicity/genetics/growth &amp; development/drug effects ; Animals ; Virulence ; Klebsiella Infections/microbiology/pathology ; Biofilms/growth &amp; development ; Mice ; Hydrogen Peroxide/toxicity ; Disease Models, Animal ; Gene Expression Regulation, Bacterial ; *Bacterial Proteins/genetics/metabolism ; Gene Deletion ; Moths/microbiology ; *Repressor Proteins/genetics/metabolism ; Genetic Complementation Test ; Virulence Factors/genetics ; Gene Expression Profiling ; CRISPR-Cas Systems ; Lepidoptera/microbiology ; },
abstract = {INTRODUCTION: Hypervirulent Klebsiella pneumoniae (hvKP) is an emerging pathogen associated with severe invasive infections and high mortality, in which resistance to host-derived reactive oxygen species (ROS) is critical for immune evasion and persistence. However, the mechanisms underlying oxidative stress resistance in hvKP remain poorly understood, and the role of the global regulator OxyR in this species has not been fully elucidated.<br><br>METHODS: In this study, VK055_RS16305 was first identified as an OxyR homologue in K. pneumoniae ATCC 43816 by sequence alignment. The oxyR deletion mutant was generated using a CRISPR/Cas9-based genome editing system, whereas the complemented strain was obtained using the pSTV28 plasmid carrying oxyR. We then compared their growth characteristics, susceptibility to H&#x2082;O&#x2082;, biofilm formation, and virulence in Galleria mellonella and mouse infection models, and performed RNA sequencing followed by qRT-PCR to characterize the OxyR regulon under oxidative stress.<br><br>RESULTS: Deletion of oxyR did not alter bacterial growth or colony morphology under non-stress conditions, but markedly increased susceptibility to H&#x2082;O&#x2082; and impaired biofilm formation. In vivo, the oxyR mutant exhibited attenuated virulence, with improved survival of Galleria mellonella and mice and significantly reduced bacterial burdens in blood, liver, lung, and spleen, all of which were restored by genetic complementation. Transcriptomic analysis revealed that OxyR positively regulates multiple oxidative stress-associated genes, including hemH, grxA, gsk, katG, and ahpC, in response to H&#x2082;O&#x2082;.<br><br>DISCUSSION: Together, these findings demonstrate that OxyR is a key regulator of oxidative stress defense, biofilm formation, and systemic virulence in hvKP, providing new insight into OxyR-mediated pathogenic mechanisms in K. pneumoniae.},
}
@article {pmid41570748,
year = {2026},
author = {Gholami, S and Aghbash, PS and Ravanlo, ZZ and Rahimi, SB and Baghi, HB},
title = {Therapeutic targeting of the HPV E7 oncoprotein: Current advances and emerging strategies.},
journal = {International immunopharmacology},
volume = {172},
number = {},
pages = {116193},
doi = {10.1016/j.intimp.2026.116193},
pmid = {41570748},
issn = {1878-1705},
abstract = {Cervical cancer is one of the most common malignancies among women, with persistent infection by high-risk human papillomavirus (HPV) types, particularly HPV16 and HPV18, being the primary etiological factor. The viral oncoproteins E6 and E7 play pivotal roles in carcinogenesis by inactivating the tumor suppressor proteins p53 and pRb, respectively. E7 has emerged as a promising therapeutic target due to its continuous expression in transformed cells and its essential role in maintaining the malignant phenotype. Recent advances in molecular biology and nanotechnology have led to the development of novel therapeutic strategies aimed at silencing or inhibiting E7, such as immunotherapy, RNA interference (RNAi), CRISPR/Cas9-based genome editing, and the use of natural bioactive compounds. Immunotherapeutic approaches aim to elicit specific cytotoxic T-cell responses against E7, whereas RNAi and CRISPR/Cas systems enable precise suppression or disruption of the E7 oncogene. As a result, it leads to the reactivate of p53 and pRb pathways, cell cycle arrest, and apoptosis. Additionally, the design of innovative delivery systems, such as liposomal nanoparticles, polymeric carriers, and viral vectors, has improved the efficiency and safety of therapeutic gene delivery. Collectively, these targeted approaches offer promising prospects for the treatment of HPV-related cancers. However, further optimization of delivery platforms and minimization of off-target effects are essential for the successful clinical translation of E7-targeted therapies in cervical cancer.},
}
@article {pmid41570230,
year = {2025},
author = {Demidova, NA and Klimova, RR and Kushch, AA and Karpov, DS},
title = {CRISPR-Cas genome editing system in the diagnosis and therapy of infection caused by herpes simplex virus type 1 (Orthoherpesviridae: Alphaherpesviridae: Simplexvirus: Simplexvirus humanalpha1).},
journal = {Voprosy virusologii},
volume = {70},
number = {6},
pages = {493-507},
doi = {10.36233/0507-4088-307},
pmid = {41570230},
issn = {2411-2097},
mesh = {Humans ; *Herpesvirus 1, Human/genetics/pathogenicity ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Herpes Simplex/therapy/diagnosis/genetics/virology ; Animals ; Genome, Viral ; *Genetic Therapy/methods ; },
abstract = {Herpes simplex virus type 1 (HSV-1), newly named as Simplexvirus humanalpha1 is one of the most common pathogens in the human population, which can cause severe disease, often with fatal outcomes. Diagnostic methods currently in use are specific and sensitive, but time-consuming, require expensive laboratory equipment and highly qualified personnel. Existing therapeutic agents have a number of significant drawbacks. To successfully treat and prevent the spread of the infection, new rapid, easy-to-use, and highly sensitive diagnostic tools and effective therapeutic agents are required. One approach to achieve this goal is CRISPR-based technology. This review analyzes information obtained from a literature search in the Scopus, Web of Science and MedLine databases on the topics «HSV-1, structure, distribution, life cycle», «new methods for molecular diagnosis of HSV-1-infection», «classification of CRISPR-Cas systems», «nucleic acid amplification methods», «CRISPR-Cas effector proteins», «application of CRISPR-Cas systems in molecular diagnostics of HSV-1-infection», «application of CRISPR-Cas systems in therapy of HSV-1-infection». New approaches of CRISPR using effector proteins Cas12 and Cas13 in the diagnosis of HSV-1 infections are reviewed. The article discusses the progress in the development of CRISPR-Cas-based therapies against HSV-1-infection in vitro and in vivo. CRISPR gene therapy in vivo has a great clinical potential, but its safety and efficacy require further investigation. An analysis of the available data suggests that CRISPR-based technologies offer promising prospects for expanding the arsenal of diagnostic tools and antiviral drugs in the context of current and future outbreaks of viral diseases.},
}
@article {pmid41569521,
year = {2026},
author = {Yang, X and Wu, H and Zeng, Z and Chen, WN and Luan, GX and Zhang, QL and Chen, JM},
title = {Recent advances in highly sensitive and specific functional nucleic acid sensors for environmental pollutant detection: from mechanism to application.},
journal = {The Analyst},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5an01139f},
pmid = {41569521},
issn = {1364-5528},
abstract = {Functional nucleic acids (FNAs) have emerged as a cutting-edge tool in environmental pollutant detection, attributed to their exceptional stability, robust specificity, and remarkable capacity for signal transduction and amplification. This review elaborates comprehensively on four pivotal categories of FNAs-aptamers, RNA-cleaving DNAzymes, G-quadruplex/hemin DNAzymes, and gRNAs-alongside their applications in monitoring a spectrum of pollutants. These encompass organic contaminants (e.g., pesticides and bisphenols), heavy metals (such as Pb[2+] and Hg[2+]), biotoxins, and pathogenic microorganisms. It also underscores the integration of FNAs with sophisticated technologies like nanomaterials and CRISPR/Cas systems to augment detection sensitivity and efficacy. Despite prevailing challenges, including susceptibility to environmental variables (pH and temperature) and intricate synthesis procedures, FNAs hold immense potential for advancing environmental monitoring and pollution control.},
}
@article {pmid41569163,
year = {2026},
author = {Luo, Y and Jiang, Q and Qu, Y and Li, W and Liu, R and Zhu, Y and Xie, Y and Jiang, C and Chen, C and Cong, L and Han, F and Bao, J and Wang, C},
title = {Compact bacterial recombination complexes drive efficient kilobase-scale knock-in in mammalian cells.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41569163},
issn = {1362-4962},
support = {2024YFC3408100//National Key Research and Development Program of China/ ; JSSCTD202450//Jiangsu Shuangchuang Project/ ; TJ-2023-005//Jiangsu Science and Technology Association Youth Science and Technology/ ; QYPY20230032//Center for Advanced Interdisciplinary Science and Biomedicine of IHM/ ; BK20240529//Natural Science Foundation of Jiangsu Province/ ; //Nanjing Medical University/ ; 82403421//National Natural Science Foundation of China/ ; 2408085J016//Anhui Provincial Natural Science Foundation/ ; },
mesh = {Animals ; Humans ; Mice ; *Gene Knock-In Techniques/methods ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Recombinational DNA Repair ; Escherichia coli/genetics/enzymology ; *Escherichia coli Proteins/genetics/metabolism ; HEK293 Cells ; Neurons/metabolism ; *Rec A Recombinases/genetics/metabolism ; },
abstract = {Efficient homologous recombination, homology-directed repair (HDR), remains a major hurdle for precise genome editing in mammalian cells, particularly for kilobase-scale insertions. Bacterial recombineering proteins, such as RecE and RecT, offer potential solutions, but their activity in eukaryotic systems has been largely uncharacterized. Here, we identify Escherichia coli RecE (EcRecE) as a potent enhancer of HDR in mammalian cells. Targeted recruitment of EcRecE via CRISPR/Cas9 significantly increased HDR efficiency at multiple genomic loci across different cellular contexts, including human embryonic stem cells, achieving a 3-6-fold enhancement in the integration efficiency of kilobase-scale sequences. Furthermore, in combination with RecT and a catalytically inactive Cas9 (dCas9), applying functional domain engineering, we developed a dCas9-miniRecTE editor that enhances large-fragment integration without introducing double-strand breaks in human cells and primary mouse neurons, achieving ∼20% kilobase-scale knock-in efficiency. These results establish EcRecE as a versatile tool for improving precision genome engineering, with potential applications in therapeutic gene editing.},
}
@article {pmid41569151,
year = {2026},
author = {Zhang, W and Kong, J and Zeng, Y and Su, Y and Zhang, S and Li, Y and Hu, C and Chen, Q and Xiao, Y and Lu, M},
title = {Structural plasticity enables broad cAn binding and dual activation of CRISPR-associated ribonuclease Cdn1.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41569151},
issn = {1362-4962},
support = {2023YFC3402300//National Key Research and Development Program of China/ ; 2021ZD0203400//STI2030-Major Projects/ ; 31970547//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Protein Binding ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Models, Molecular ; Adenine Nucleotides/metabolism/chemistry ; Catalytic Domain ; *Ribonucleases/metabolism/chemistry/genetics ; Oligoribonucleotides ; },
abstract = {Prokaryotes have naturally evolved diverse RNA-guided defense systems against viral infections, with the type III CRISPR-Cas systems representing the most intricate. These systems feature accessory proteins activated by cyclic oligoadenylates (cOAs) produced upon target RNA recognition, synergizing with the CRISPR-Cas machinery to defend against exogenous invaders. Typically, each accessory protein is activated by only one specific cOA type. Here, we characterize Cdn1, a type III-B CRISPR accessory protein from Psychrobacter lutiphocae, which binds to cA3, cA4, and cA6, but activated by cA4 and cA6 with different efficacies to catalyze ssRNA cleavage. Combined structural and biochemical analyses reveal that cOA binding triggers dramatic conformational reorganization, including the formation of a dimerization interface of nuclease domains, the emergence of substrate binding cleft, and the reconstruction of a metal-dependent catalytic center essential for RNA cleavage. This dual activation mechanism illustrates evolutionary innovation within CRISPR-associated Rossman-fold nucleases. We propose that such structural plasticity evolved to maximize defensive resilience during microbial competition and horizontal gene transfer, while preserving broad-spectrum antiviral ability. These findings not only elucidate the activation mechanisms of Cdn1 within the type III systems but also underscore the functional complexity and adaptability of CRISPR-Cas ancillary proteins.},
}
@article {pmid41568169,
year = {2026},
author = {Zhang, W and Wang, H and Liu, D and Mao, X and Zhang, Y and Yang, Y and Liu, Z and Pan, T and Liu, Y and Zhang, Q},
title = {Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {1},
pages = {102811},
pmid = {41568169},
issn = {2162-2531},
abstract = {The CRISPR-Cas13 system exhibits potent RNA cleavage activity and has been widely utilized for RNA-targeting applications. However, its collateral cleavage of bystander RNAs limits in vivo therapeutic applications. In this study, we generated a series of LwaCas13a mutants through structure-based design and site-directed mutagenesis strategies. A triple mutant enCas13a (Q521R/E796A/E810A) was obtained with significantly enhanced target RNA cleavage activity along with only slightly increased collateral activity. To reduce the collateral activity, we optimized crRNA terminal extensions and obtained M1crRNA and M3crRNA variants that, in combination with enCas13a, maintained or reduced collateral activity while preserving enhanced targeted cleavage activity. Thus, by optimizing the Cas protein and crRNA, we have created an improved CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity. This system demonstrated superior performance in targeting endogenous genes and antiviral applications. Mechanistic studies revealed that enhanced protein-crRNA interactions and altered complex conformations underlie the improved cleavage activity. This engineering approach provides a generalizable strategy for developing CRISPR-Cas systems with enhanced therapeutic potential.},
}
@article {pmid41564866,
year = {2026},
author = {Watterson, A and Picco, G and Veninga, V and Samarakoon, Y and Cattaneo, CM and Vieira, SF and Karakoc, E and Bhosle, S and Battaglia, TW and Consonni, S and Halim, TYF and Voest, EE and Garnett, MJ and Coelho, MA},
title = {CRISPR screens in the context of immune selection identify CHD1 and MAP3K7 as mediators of cancer immunotherapy resistance.},
journal = {Cell reports. Medicine},
volume = {7},
number = {1},
pages = {102565},
doi = {10.1016/j.xcrm.2025.102565},
pmid = {41564866},
issn = {2666-3791},
mesh = {Humans ; Animals ; *Immunotherapy/methods ; Mice ; *MAP Kinase Kinase Kinases/genetics/metabolism ; *DNA Helicases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *DNA-Binding Proteins/genetics/metabolism ; Cell Line, Tumor ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; CD8-Positive T-Lymphocytes/immunology ; *Drug Resistance, Neoplasm/genetics ; *Neoplasms/immunology/therapy/genetics ; Interferon-gamma ; Melanoma/immunology/genetics ; Mice, Inbred C57BL ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cancer immunotherapy is only effective in a subset of patients, highlighting the need for effective biomarkers and combination therapies. Here, we systematically identify genetic determinants of cancer cell sensitivity to anti-tumor immunity by performing whole-genome CRISPR-Cas9 knockout screens in autologous tumoroid-T cell co-cultures, isogenic cancer cell models deficient in interferon signaling, and in the context of four cytokines. We discover that loss of CHD1 and MAP3K7 (encoding TAK1) potentiates the transcriptional response to IFN-γ, thereby creating an acquired vulnerability by sensitizing cancer cells to tumor-reactive T cells. Immune checkpoint blockade is more effective in a syngeneic mouse model of melanoma deficient in Chd1 and Map3k7 and is associated with elevated intra-tumoral CD8[+] T cell numbers and activation. CHD1 and MAP3K7 are recurrently mutated in cancer, and reduced expression in tumors correlates with response to immune checkpoint inhibitors in patients, nominating these genes as potential biomarkers of immunotherapy response.},
}
@article {pmid41564857,
year = {2026},
author = {Balke-Want, H and Keerthi, V and Del Carmen Arenas, M and Chen, Y and Malipatlolla, M and Klysz, DD and Xu, P and Ho, K and Asano, K and Stahl, D and Huang, J and Retherford, A and Patel, S and Fowler, C and Maas, L and Gkitsas-Long, N and Jiang, Q and Liu, X and Ullrich, R and George, J and Heitzeneder, S and Tunuguntla, R and Sage, J and Sotillo, E and Mackall, CL and Feldman, SA},
title = {c-JUN enhances CRISPR knockin anti-B7-H3 CAR T cell function in small cell lung cancer and thoracic SMARCA4-deficient undifferentiated tumors.},
journal = {Cell reports. Medicine},
volume = {7},
number = {1},
pages = {102549},
doi = {10.1016/j.xcrm.2025.102549},
pmid = {41564857},
issn = {2666-3791},
mesh = {Humans ; *DNA Helicases/deficiency/genetics/metabolism ; *Lung Neoplasms/immunology/pathology/genetics/therapy/metabolism ; *Nuclear Proteins/deficiency/genetics/metabolism ; *Transcription Factors/deficiency/genetics/metabolism ; *Small Cell Lung Carcinoma/immunology/pathology/therapy/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *T-Lymphocytes/immunology/metabolism ; Cell Line, Tumor ; *Proto-Oncogene Proteins c-jun/metabolism/genetics ; *Receptors, Chimeric Antigen/metabolism/immunology ; Immunotherapy, Adoptive/methods ; Animals ; },
abstract = {Small cell lung cancer (SCLC), a highly lethal disease, limits T cell responses by downregulating major histocompatibility (MHC) class I molecules. Because chimeric antigen receptor (CAR) T cells are not MHC restricted, they may provide a powerful strategy against SCLC. However, few CAR targets for SCLC are known. Here, we show that B7-H3/CD276 is expressed in SCLC and thoracic SMARCA4-deficient undifferentiated tumors (UTs) that can clinicopathologically mimic SCLC. Thoracic SMARCA4-deficient UTs limit killing by B7-H3 CAR T cells via secretion of transforming growth factor β1 (TGF-β1). To overcome tumor-driven CAR T cell suppression, we knock in c-JUN alongside a B7-H3 CAR into the TRAC locus of primary human T cells utilizing CRISPR-Cas9. Non-viral c-JUN+B7-H3 CAR T cells show enhanced killing of both SCLC cells with low antigen density and thoracic SMARCA4-deficient UTs, providing a platform to address these highly aggressive entities. We also provide evidence that good manufacturing practice (GMP) clinical-scale manufacturing is feasible for c-JUN+B7-H3 CAR T cells.},
}
@article {pmid41535129,
year = {2026},
author = {Du, J and Pu, X and Yuan, T and Peng, F and Hu, J and Li, H and Chen, B and Luo, J and Li, S and Teng, Y and Zhu, X and Chen, W and Xie, Q and Jiang, L and Xiong, E and Yang, R},
title = {Plug-and-Play Photo-Initiated CRISPR-Cas12a One-Pot Nucleic Acid Detection via Universal Repeat RNA Acylation Strategy.},
journal = {Analytical chemistry},
volume = {98},
number = {3},
pages = {2136-2145},
doi = {10.1021/acs.analchem.5c05769},
pmid = {41535129},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Acylation ; *RNA/genetics/analysis/chemistry ; Humans ; Gene Editing ; CRISPR-Associated Proteins/metabolism ; Photochemical Processes ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Precise spatiotemporal control of CRISPR activity is central to both accurate gene editing and sensitive molecular diagnostics. However, current regulatory strategies are often sequence-specific, labor-intensive, and difficult to generalize. Here, we report a minimalist plug-and-play tactic: acylation of the repeat region (rRNA) of a split crRNA with photolabile groups. Because the modification is introduced post-synthesis and is independent of the spacer region (sRNA), every rRNA, regardless of its target sequence, can be activated by light irradiation alone, entirely eliminating the need for redesign or reoptimization. Integrating the photo-initiated CRISPR-Cas12a system with recombinase polymerase amplification into a one-pot format yields an upgraded platform, named POIROTv2 (PhotO-Initiated CRISPR-Cas12a system for Robust One-pot Testing, version 2). POIROTv2 achieves a 100-fold sensitivity gain over conventional always-on Cas12a-based one-pot assays and matches the analytical performance of a two-step assay while remaining a more streamlined and potentially faster detection process and avoiding the risk of aerosol contamination. In clinical validation with HCMV- and EBV-suspected samples, POIROTv2 delivered diagnostic accuracy statistically indistinguishable from that of gold-standard qPCR, highlighting its potential for robust and sensitive molecular diagnostics. Overall, the strategy opens up exciting possibilities for applications in infectious virus diagnostics and has broad prospects in the field of spatiotemporally controllable gene editing.},
}
@article {pmid41533833,
year = {2026},
author = {Chen, X and Mao, C and Gao, Y and Shi, C and Wang, Y and Jin, Z and Xia, B and Zhou, Y},
title = {Ultrasensitive Detection of Cardiac Troponin I via CRISPR/Cas12a-Mediated Liposomal Amplification Coupled with Electrospray Ionization Mass Spectrometry.},
journal = {Analytical chemistry},
volume = {98},
number = {3},
pages = {2183-2190},
doi = {10.1021/acs.analchem.5c05804},
pmid = {41533833},
issn = {1520-6882},
mesh = {*Troponin I/blood/analysis ; Humans ; *Spectrometry, Mass, Electrospray Ionization/methods ; *CRISPR-Cas Systems ; *Liposomes/chemistry ; Limit of Detection ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Direct quantitative analysis of low-abundance protein biomarkers by electrospray ionization mass spectrometry (ESI-MS) remains challenging due to poor ionization efficiency and matrix interferences. Herein, we report an ultrasensitive analytical platform, termed CRISPR/Cas12a-mediated liposomal amplification coupled with electrospray ionization mass spectrometry (CMLA-MS), that overcomes this limitation by integrating CRISPR/Cas12a-mediated dual-cascade signal amplification with an ESI-MS readout. The strategy converts the detection of poorly ionizable protein molecules into the quantification of numerous, highly ionizable small-molecule reporters: proteins trigger Cas12a trans-cleavage (first amplification), which subsequently cleaves single-stranded DNA (ssDNA) probes anchored to signal-loaded liposomes, causing the burst release of thousands of MS-detectable reporters (second, physical amplification). This dual-amplification strategy enabled an exceptionally low limit of detection (LOD) of 10.8 fg/mL, and the method successfully quantified cardiac troponin I (cTnI) in clinical serum samples with high recoveries (90.3-101.6%).},
}
@article {pmid41512333,
year = {2026},
author = {Zhao, J and Sui, Z and Chen, B and Wu, R and Xu, J and Dong, H},
title = {Customizable NAND Logic-Gate Biosensing System Enabled by an Engineered Methylation-CRISPR/Cas12a Consensus Sequence for Ultrasensitive DNA Methyltransferase Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {3},
pages = {2368-2378},
doi = {10.1021/acs.analchem.5c06772},
pmid = {41512333},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism/analysis ; Limit of Detection ; Humans ; Logic ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {DNA methyltransferases (MTases) play crucial roles in epigenetic regulation, and their abnormal activity is closely associated with various human diseases. Here, we report a customizable NAND logic-gate biosensing platform for highly sensitive and intelligent detection of DNA adenine methyltransferase (Dam MTase). An engineered methylation-CRISPR/Cas12a consensus sequence (MCCS, 5'-TTTGATC-3') was rationally designed to integrate the Cas12a PAM site, Dam methylation site, and DpnI recognition sequence into a unified functional motif. Coupled with a primer-triggered hybridization chain reaction (HCR), multiple tandem MCCS units were generated to amplify the fluorescence signal output. In this logic circuit, Dam, SAM, and DpnI serve as three biochemical inputs, and their combined presence ("1,1,1") yields a low-fluorescence "OFF" output according to the NAND logic rule. The system exhibited a broad linear detection range with an ultralow detection limit of 0.00032 U mL[-1], outstanding selectivity toward nontarget MTases, and satisfactory recoveries (98.16-100.03%) in human serum samples. Furthermore, it enabled quantitative evaluation of Dam inhibitors, revealing IC50 values of 1.75 μM for 5-fluorouracil and 11.9 μM for penicillin G. This strategy provides a universal molecular computation-driven biosensing framework for enzyme activity analysis and inhibitor screening in complex biological systems.},
}
@article {pmid41511442,
year = {2026},
author = {Li, Y and Li, X and Chen, Y and Wang, Y and Zuo, Z},
title = {Differential Allosteric Modulation of Cas9 Specificity.},
journal = {Journal of chemical theory and computation},
volume = {22},
number = {2},
pages = {806-817},
doi = {10.1021/acs.jctc.5c01919},
pmid = {41511442},
issn = {1549-9626},
mesh = {Allosteric Regulation ; Molecular Dynamics Simulation ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; Mutation ; },
abstract = {Both RNA- and protein-based strategies have been developed to mitigate off-target cleavage by CRISPR-Cas9, yielding noncanonical guide RNAs (gRNAs) and Cas9 variants with enhanced gene-editing precision. However, the molecular mechanisms by which such PAM-distal alterations─remote from the nuclease centers─modulate Cas9 activity and specificity remain incompletely understood. Here, we performed near-millisecond all-atom molecular dynamics simulations to elucidate how diverse PAM-distal perturbations─including gRNA truncation, base mismatching, and evolved mutations─reshape the conformational dynamics and allosteric regulation of Cas9. Despite their distinct origins, all perturbations ultimately modulate Cas9 function by altering HNH dynamics that impede the transition from the checkpoint to the catalytically active state, yet they do so through distinct allosteric routes. The 16-nt gRNA induces a pronounced REC3 reorientation toward the L2 linker and HNH domain, while PAM-distal mismatches with the 18-nt gRNA promote engagement of the unwound target DNA strand with L2─both effectively restraining HNH rotation. In contrast, evolved mutations remodel the global motional modes so that REC2 swivels inward, constraining the HNH flexibility. These perturbations delineate multiple structural paths converging on a shared allosteric outcome─HNH immobilization and catalytic suppression─thereby unifying RNA-, DNA-, and protein-level effects within a single dynamic framework linking distal structural perturbations to activity control. This work provides mechanistic insight into the regulation of Cas9 fidelity and offers principles for the design of next-generation genome editors.},
}
@article {pmid41475353,
year = {2026},
author = {Marks, D and Garcia, E and Kumar, S and Tyson, K and Koch, C and Ivanov, AP and Edel, JB and Mirza, HB and Flanagan, W and Dunsby, C and French, PMW and McNeish, IA},
title = {Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor.},
journal = {Cell reports methods},
volume = {6},
number = {1},
pages = {101270},
doi = {10.1016/j.crmeth.2025.101270},
pmid = {41475353},
issn = {2667-2375},
mesh = {Female ; Humans ; *Ovarian Neoplasms/drug therapy/pathology/metabolism/genetics ; *Fluorescence Resonance Energy Transfer/methods ; *Biosensing Techniques/methods ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Animals ; Mice ; *Poly(ADP-ribose) Polymerases/metabolism ; *Poly (ADP-Ribose) Polymerase-1/metabolism/genetics ; },
abstract = {Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionized the treatment of ovarian high-grade serous carcinoma (HGSC), particularly in homologous recombination-deficient tumors. However, the emergence of resistance poses a critical challenge, as over 50% of patients relapse within 3 years. The mechanisms underlying changes in PARP trapping, a central aspect of PARPi efficacy, are not well understood, as current experimental methodologies lack resolution and throughput. To address this, we develop an intramolecular fluorescence resonance energy transfer (FRET)-based biosensor by CRISPR-Cas9 dual labeling of endogenous PARP1 with EGFP and mCherryFP in OVCAR4 cells. This biosensor enables real-time, single-cell analysis of PARP trapping dynamics. Using fluorescence lifetime imaging microscopy (FLIM), we reveal dose-dependent PARP trapping, differentiate the trapping efficiencies of four clinically approved PARPi, and observe reduced trapping in PARPi-resistant models in vitro and in vivo. This biosensor provides critical insights into PARPi resistance mechanisms, with implications for developing more effective therapies and advancing personalized treatment for ovarian cancer patients.},
}
@article {pmid41436498,
year = {2025},
author = {Jiang, J and Jiang, Z and Luo, Q and Chen, X and Zhuang, J and Chen, J and Mu, Q and Qiu, J and Li, Y and Chen, S and Zhang, P and Yu, K and Chen, S and Liu, GS and Zhuang, J},
title = {Loss of ELF2 drives topotecan resistance in retinoblastoma revealed by genome-wide CRISPR-Cas9 screening.},
journal = {Cell death &amp; disease},
volume = {17},
number = {1},
pages = {128},
pmid = {41436498},
issn = {2041-4889},
support = {82472143//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82372131//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024A1515012562//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; GNT2029648//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {*Topotecan/pharmacology/therapeutic use ; Humans ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Retinoblastoma/genetics/drug therapy/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; Animals ; Mice ; Cell Line, Tumor ; Topoisomerase I Inhibitors/pharmacology ; Xenograft Model Antitumor Assays ; Apoptosis/drug effects ; *Retinal Neoplasms/genetics/drug therapy/pathology ; Female ; },
abstract = {The topoisomerase I inhibitor topotecan is an effective chemotherapeutic agent for retinoblastoma; however, treatment resistance remains a major clinical challenge, and its mechanisms remain elusive. Using genome-wide CRISPR-Cas9 knockout screening, we identified ELF2 as a key gene involved in topotecan resistance. Here, we show that surviving retinoblastoma cells exposed to topotecan showed progressively decreased ELF2 expression, accompanied by reduced apoptosis. In a mouse xenograft model, ELF2 disruption diminished the antitumor efficacy of topotecan, with ELF2-knockout cells exhibiting reduced topotecan-induced apoptosis. RNA sequencing further revealed that the MT-CYB pathway, associated with ATP synthesis, contributes to ELF2-mediated resistance. Importantly, clinical analysis demonstrated a correlation between ELF2 expression and tumor volume in retinoblastoma patients treated with topotecan. Together, these findings interrogate the mechanisms underlying topotecan resistance in retinoblastoma and suggest ELF2 as a potential therapeutic target to overcome drug resistance.},
}
@article {pmid41427723,
year = {2026},
author = {Ortiz-Severin, J and Geoffroy, P and Aravena, P and Hodar, C and Palma, DE and González, M and Cambiazo, V},
title = {Mobile-CRISPRi as a tool for genetic manipulation in the intracellular pathogen Piscirickettsia salmonis.},
journal = {Applied and environmental microbiology},
volume = {92},
number = {1},
pages = {e0156025},
pmid = {41427723},
issn = {1098-5336},
support = {1211893//ANID-Fondecyt/ ; 2024T2DID//Doctoral Fellowship/ ; ICN2021_044//Millennium Science Initiative Program/ ; },
mesh = {*Piscirickettsia/genetics ; Fish Diseases/microbiology ; Piscirickettsiaceae Infections/microbiology/veterinary ; Animals ; *CRISPR-Cas Systems ; Bacterial Proteins/genetics ; Gene Silencing ; },
abstract = {UNLABELLED: Piscirickettsia salmonis is the causative agent of salmonid rickettsial septicemia (SRS), the main bacterial disease affecting the salmon industry in Chile. In this work, we implemented a Mobile-CRISPRi system to generate gene silencing using a catalytically inactive dCas9 protein and an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible single-guide RNA (sgRNA). We demonstrate the efficacy of the CRISPRi system in P. salmonis by silencing an exogenous reporter (sfGFP) and an endogenous regulator (Fur) that controls intracellular iron homeostasis in bacteria. The inducible expression of dCas9 and the sfGFP-directed sgRNA caused a 98.7% decrease in fluorescence in the knockdown strain. This silencing system was effective in seven P. salmonis strains from both genogroups. Furthermore, the same system was used to construct fur knockdown strains. A 50-fold decrease in fur expression level was determined in these strains when the expression of the fur gRNA was induced with IPTG. By RNA-seq, we detected a significant increase in the expression of genes encoding the Fe[2+] and Fe[3+] acquisition systems and iron mobilization in the fur1 knockdown after IPTG induction. All the genes with over 2-fold increased expression in the RNA-seq presented the Fur box consensus sequence in their regulatory region. The implementation of the Mobile-CRISPRi system in P. salmonis has been demonstrated to be effective, thus providing a tool with potential application for the analysis of gene function in this pathogen. It is anticipated that these analyses will be valuable in identifying genes involved in the mechanisms of pathogenesis of P. salmonis.<br><br>IMPORTANCE: Salmonid rickettsial septicemia (SRS) is an infectious disease caused by the marine bacterium Piscirickettsia salmonis. This Gamma-proteobacteria is a fastidious and facultative intracellular pathogen that has a nearly worldwide distribution, particularly impacting Chilean salmonid aquaculture. Its fastidious nature has made it hard to grow in labs, hindering research into its virulence and treatment, especially because of the lack of molecular techniques to study gene function. We show here the successful implementation of the Mobile-CRISPRi system for gene silencing. Significantly, we have adapted this technique for use with the marine pathogen P. salmonis, inserting exogenous genes into the bacterium's chromosome to ensure their constitutive and inducible expression and silencing both exogenous and endogenous gene expression. The Mobile-CRISPRi system was also used to study the iron regulator Fur, confirming Fur's relevance to the iron metabolism in the pathogen.},
}
@article {pmid41423621,
year = {2025},
author = {Goswami, SG and Gupta, P and Arvinden, VR and Bhargava, N and Iyer, AR and Saravanakumar, V and Yadav, P and Jha, SK and Singh, S and Kumar, A and Singh, P and Gunda, P and Jain, S and Mehta, P and Nakamura, Y and Kurita, R and Bajaj, A and Ramalingam, S},
title = {CRISPR editing of HPFH3 genotype induces γ-globin expression and reverses sickle cell disease and β-thalassemia phenotypes.},
journal = {Stem cell research &amp; therapy},
volume = {17},
number = {1},
pages = {46},
pmid = {41423621},
issn = {1757-6512},
mesh = {*beta-Thalassemia/genetics/therapy/pathology/metabolism ; *Anemia, Sickle Cell/genetics/therapy/pathology/metabolism ; Humans ; *gamma-Globins/genetics/metabolism ; Animals ; *Gene Editing/methods ; *Fetal Hemoglobin/genetics/metabolism ; CRISPR-Cas Systems ; Genotype ; Mice ; Phenotype ; Hematopoietic Stem Cells/metabolism ; },
abstract = {BACKGROUND: Hereditary persistence of Fetal Hemoglobin (HPFH) is a benign condition known to mitigate symptoms in individuals with co-inherited β-hemoglobinopathies, such as β-thalassemia (BT) and sickle cell disease (SCD), through the reactivation of fetal hemoglobin (HbF). HPFH typically arises from deletions of varying sizes affecting the β-globin gene cluster or point mutations in the promoters of the γ-globin genes. While the therapeutic benefits of point mutations have been extensively studied, the potential of deletional forms of HPFH remains underexplored in preclinical settings.<br><br>METHOD: In this study, we generated benign deletional HPFH3 genotype in SCD and BT patient-derived HSPCs using CRISPR/Cas9 and showed that therapeutically relevant levels of HbF reactivation result in the alleviation of the pathological phenotypes.<br><br>RESULTS: In edited cells derived from SCD patients, we observed reduced sickling and oxidative stress, while in edited from BT cells, restoration of the &#x3b1;-globin/&#x3b2;-globin ratio improved erythroid lineage maturation and reduced ROS levels. Importantly, HPFH3-edited HSPCs retained their genome integrity and showed no detrimental effect on their regeneration or differentiation into erythroid, myeloid, T, and B cell lineages in immunodeficient NBSGW mice post-xenotransplantation. Additionally, we showed a reduced interaction between the LCR and HBB, suggesting that the HPFH3 deletion specifically promoted LCR interactions with HBG1/2, likely due to the absence of the HBB locus.<br><br>CONCLUSIONS: Collectively, our preclinical findings suggest that the generation of the HPFH3 genotype has the potential to significantly enhance HbF levels, offering a promising universal therapeutic strategy for treating both SCD and &#x3b2;-thalassemia.},
}
@article {pmid41344324,
year = {2026},
author = {Djamshidi, M and Hill, A and Heshmatzad, K and Langley, J and Krowicki, H and Ali, M and Yang, Y and Tanida, R and Abdul-Careem, MF and Billon, P and Riabowol, K},
title = {FAME-CRISPR improves CRISPR-Cas9 genome editing via HDAC inhibition and engineered virus-like particle delivery.},
journal = {Cell reports methods},
volume = {6},
number = {1},
pages = {101248},
doi = {10.1016/j.crmeth.2025.101248},
pmid = {41344324},
issn = {2667-2375},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Histone Deacetylase Inhibitors/pharmacology ; *Virion/genetics ; DNA Breaks, Double-Stranded ; HEK293 Cells ; },
abstract = {CRISPR-mediated gene editing using engineered virus-like particles (eVLPs) can achieve high efficiency, but performance varies with reduced effectiveness often seen in primary cells or when generating polyclonal models at scale. We developed a faster, accurate and 4-fold more efficient CRISPR-Cas9 (FAME-CRISPR) method using pan-histone deacetylase inhibitors with eVLP transduction compared to previous reports using other histone deacetylase inhibitors. Combined optimization of pan-HDACi treatment with eVLP enhanced double-strand break (DSB)-mediated CRISPR and base editing gave significantly edited populations within 2- to 3-cell mean population doublings, reducing the need for post-editing selection in immortalized cancer cells and in primary diploid fibroblasts that have limited replicative lifespans.},
}
@article {pmid41208252,
year = {2026},
author = {Patterson, FM and Nguyen Tran, MT and Guinan, T and Mohd Khalid, MKN and Kc, R and Fairfax, KA and Liu, GS and Cook, AL and Hung, SS and Hewitt, AW},
title = {Generalizable features of pegRNA design for prime editing of inherited retinal diseases.},
journal = {Ophthalmic genetics},
volume = {47},
number = {1},
pages = {59-66},
doi = {10.1080/13816810.2025.2576786},
pmid = {41208252},
issn = {1744-5094},
mesh = {*Gene Editing/methods ; Humans ; *Retinal Diseases/genetics/therapy ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND AND OBJECTIVES: The variety of ocular cell types involved in inherited retinal disease (IRD) necessitates the use of gene editing therapeutics which have generalizable components. In our study, we investigate the generalizable characteristics of non-engineered pegRNA design (PE2) for efficient, proof-in-principle gene correction of over 21 genes implicated in IRDs and associated syndromes. We use a single-transgene oligopool approach, comprising approximately 12,000 uniquely barcoded pegRNAs that target a synthetically integrated, 50 bp sequence motif, which faithfully recapitulate the disease context of their various counterpart IRDs. Using this approach, we perform a high throughput, pooled analysis of pegRNA characteristics across non- and ocular cell types to propose a cell-line agnostic set of pegRNA design guidelines.<br><br>RESULTS: Briefly, we find that non-engineered pegRNA 3' extensions should mediate substitution-type edits and that the desired edit should be placed within five nucleotides upstream of the nick site induced by the Cas-endonuclease. Further, PBS and RTT lengths of at least 12 and 14 nucleotides, respectively, should be used and each non-engineered pegRNA 3' extension should obviate an initial templating cytosine nucleotide.<br><br>CONCLUSION: We establish a set of recommendations for the generalizable design of the non-engineered pegRNA 3' extension for the correction of several IRDs, enabling overall simplification of design parameters for PE2-based systems.},
}
@article {pmid41084330,
year = {2026},
author = {Huppertz, F and Caturelli, MS and Lehmann, LS and Kurth, F and Maier, AG and Matuschewski, K},
title = {Plasmodium falciparum gametogenesis essential protein 1 (GEP1) is a transmission-blocking target.},
journal = {FEBS letters},
volume = {600},
number = {2},
pages = {239-250},
pmid = {41084330},
issn = {1873-3468},
support = {IRTG2290//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Plasmodium falciparum/genetics/drug effects/metabolism/growth &amp; development ; Animals ; *Protozoan Proteins/genetics/metabolism ; *Gametogenesis/genetics/drug effects ; Mice ; Humans ; *Malaria, Falciparum/transmission/parasitology ; Female ; Male ; Xanthurenates/pharmacology ; Anopheles/parasitology ; CRISPR-Cas Systems ; Polymorphism, Single Nucleotide ; },
abstract = {Transmission of Plasmodium parasites to Anopheles mosquitoes relies on rapid activation of mature gametocytes in the midgut, triggered by a temperature drop and xanthurenic acid. In Plasmodium yoelii, the gametogenesis essential protein 1 (GEP1) was linked to xanthurenic acid (XA)-dependent gamete activation. We characterized GEP1 in Plasmodium falciparum using CRISPR-Cas9 to create PfGEP1 loss-of-function lines. These lines failed to undergo male or female gametogenesis, even when stimulated by XA or a temperature drop. The defect persisted despite treatment with the phosphodiesterase inhibitor Zaprinast. Analysis of field samples revealed two GEP1 single-nucleotide polymorphisms (V241L and S263P) in 12% and 20% of 49 cases. Our findings confirm GEP1's essential role in gamete activation, highlight an XA-independent function, and support its potential as a transmission-blocking target. Impact statement For sustainable malaria control, transmission-blocking drug targets are urgently needed. Work in murine models showed that GEP1 is a candidate. We show complete block of life cycle progression of the human malarial parasite Plasmodium falciparum when GEP1 is deleted, warranting targeted drug development to achieve gamete-free mosquito blood meals.},
}
@article {pmid41039747,
year = {2026},
author = {Chen, L and Huang, Q and Liu, Y and Chen, K and Yang, Q and Tang, H and Wang, D and Tang, Z},
title = {A Galactose-Engineered Dual-Responsive Nanocarrier for ASO/CRISPR-Cas9 Delivery to Inhibit HBV Replication.},
journal = {Advanced healthcare materials},
volume = {15},
number = {4},
pages = {e02835},
doi = {10.1002/adhm.202502835},
pmid = {41039747},
issn = {2192-2659},
support = {2023MD734126//China Postdoctoral Science Foundation/ ; BJRC202426//Chongqing Medical University Top Talent Cultivation Program/ ; CSTB2024NSCQ-MSX0202//Natural Science Foundation of Chongqing/ ; 2022COBSHTB3003//Chongqing Municipal Key Research and Development Program of China/ ; },
mesh = {*Hepatitis B virus/physiology/drug effects ; Animals ; *CRISPR-Cas Systems/genetics ; *Virus Replication/drug effects ; Humans ; Mice ; *Oligonucleotides, Antisense/pharmacology/chemistry ; *Galactose/chemistry ; Hepatitis B/virology ; Gene Editing ; *Nanoparticles/chemistry ; *Drug Carriers/chemistry ; Hep G2 Cells ; },
abstract = {Complete hepatitis B virus (HBV) cure is hindered primarily by the stable persistence of covalently closed circular DNA (cccDNA). Gene editing approaches to eradicate HBV by targeting cccDNA face challenges and limitations due to suboptimal editing efficiency and substantial off-target effects. Herein, a combinatorial therapeutic strategy is developed that integrates CRISPR/Cas9-mediated cccDNA disruption with an antisense oligonucleotide (ASO)-targeted degradation of pregenomic RNA (pgRNA). To overcome delivery challenges, a hepatocyte-targeting nanocarrier (UACPG) is engineered, featuring low immunogenicity, high payload capacity, and dual-stimuli responsiveness. The UACPG platform enabled liver-specific delivery through surface-conjugated targeting ligands, followed by on-demand release of Cas9 ribonucleoprotein complexes and ASO via RNase H-dependent degradation and near-infrared (NIR) light activation. The results demonstrated that UACPG can effectively reduce HBV replication and viral antigen levels, while significantly lowering cccDNA in hydrodynamic HBV-infected mouse models, with no significant off-target effects observed. This nanocarrier achieved the spatiotemporally controlled release of gene-editing systems in vitro and in vivo, significantly inhibiting the replication of HBV, thereby establishing an innovative technological platform for developing curative HBV therapies.},
}
@article {pmid40372972,
year = {2025},
author = {Sobral, LM and Walker, FM and Madhavan, K and Janko, E and Donthula, S and Danis, E and Bompada, P and Balakrishnan, I and Wang, D and Pierce, A and Haag, MM and Carstens, BJ and Serkova, NJ and Foreman, NK and Venkataraman, S and Veo, B and Vibhakar, R and Dahl, NA},
title = {Targeting processive transcription for Myc-driven circuitry in medulloblastoma.},
journal = {Neuro-oncology},
volume = {27},
number = {10},
pages = {2697-2710},
doi = {10.1093/neuonc/noaf121},
pmid = {40372972},
issn = {1523-5866},
support = {K08 NS121592/NS/NINDS NIH HHS/United States ; P30 CA046934/CA/NCI NIH HHS/United States ; R01 NS091219/NS/NINDS NIH HHS/United States ; S10 OD023485/OD/NIH HHS/United States ; K08NS121592/NS/NINDS NIH HHS/United States ; R01NS091219/NS/NINDS NIH HHS/United States ; MUJP.2021.003//University of Colorado Cancer Center/Molecular, Cellular, and Developmental Biology/ ; P30 CA046934/CA/NCI NIH HHS/United States ; S10 OD023485/CD/ODCDC CDC HHS/United States ; },
mesh = {*Medulloblastoma/genetics/pathology/drug therapy ; Humans ; Animals ; Mice ; *Proto-Oncogene Proteins c-myc/genetics ; *Cerebellar Neoplasms/genetics/pathology/drug therapy ; CRISPR-Cas Systems ; Xenograft Model Antitumor Assays ; *Gene Expression Regulation, Neoplastic/drug effects ; *Transcription, Genetic ; Tumor Cells, Cultured ; Cyclin-Dependent Kinase 9/antagonists &amp; inhibitors ; Cell Proliferation ; },
abstract = {BACKGROUND: Medulloblastoma is the most common malignant brain tumor of childhood. The highest-risk tumors are driven by recurrent Myc amplifications (Myc-MB) and experience poorer outcomes despite intensive multimodal therapy. The Myc transcription factor defines core regulatory circuitry for these tumors and acts to broadly amplify downstream pro-survival transcriptional programs. Therapeutic targeting of Myc directly has proven elusive, but inhibiting transcriptional cofactors may present an indirect means of drugging the oncogenic transcriptional circuitry sustaining Myc-MB.<br><br>METHODS: Independent CRISPR-Cas9 screens were pooled to identify conserved dependencies in Myc-MB. We performed chromatin conformation capture (Hi-C) from primary patient Myc-MB samples to map enhancer-promoter interactions. We then treated in vitro and xenograft models with CDK9/7 inhibitors to evaluate the effect on Myc-driven programs and tumor growth.<br><br>RESULTS: Eight CRISPR-Cas9 screens performed across 3 independent labs identify CDK9 as a conserved dependency in Myc-MB. Myc-MB cells are susceptible to CDK9 inhibition, which is synergistic with concurrent inhibition of CDK7. Inhibition of transcriptional CDKs disrupts enhancer-promoter activity in Myc-MB and downregulates Myc-driven transcriptional programs, exerting a potent antitumor effect.<br><br>CONCLUSIONS: Our findings identify CDK9 inhibition as a translationally promising strategy for the treatment of Myc-MB.},
}
@article {pmid41564380,
year = {2026},
author = {Myojin, Y and Kodama, T and Takahashi, R and Nagasawa, H and Kondo, Y and Yusa, K and Yoshida-Hashidate, T and Shindou, H and Furuta, K and Murai, K and Saito, Y and Hikita, H and Takehara, T},
title = {Genome-wide CRISPR screen identifies ACSL3 as a regulator of lipotoxicity and progression of MASLD.},
journal = {Hepatology communications},
volume = {10},
number = {2},
pages = {},
pmid = {41564380},
issn = {2471-254X},
mesh = {Humans ; *Coenzyme A Ligases/genetics/metabolism ; Long-Chain-Fatty-Acid-CoA Ligase ; Hepatocytes/metabolism ; Disease Progression ; Endoplasmic Reticulum Stress/genetics ; Apoptosis/genetics ; *Fatty Liver/genetics/metabolism/pathology ; CRISPR-Cas Systems ; Animals ; Oxidative Stress ; Mice ; Male ; Liver/pathology/metabolism ; Lipid Metabolism ; Lipogenesis ; },
abstract = {BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD) and its progressive form, metabolic dysfunction-associated steatohepatitis, are highly prevalent and lack effective pharmacotherapies. Hepatocellular lipotoxicity-driven by the accumulation of saturated fatty acids (eg, palmitate)-promotes disease progression; however, the determinants of hepatocyte susceptibility remain incompletely defined.<br><br>METHODS: We performed a genome-wide CRISPR-Cas9 loss-of-function screening to identify the regulators of palmitate-induced lipotoxicity. The top candidates were validated using genetic perturbation and pharmacological inhibition. Lipid handling, endoplasmic reticulum/oxidative stress, apoptosis, and lipogenic transcriptional programs were also quantified. Human MASLD liver tissues were analyzed for ACSL3 expression in relation to histology and aminotransferases. Single-cell and spatial transcriptomics were used to localize ACSL3 expression and the associated pathway signatures in metabolic dysfunction-associated steatohepatitis.<br><br>RESULTS: The screen recovered established mediators (CASPASE-8, AGPAT9, RNF213) and identified ACSL3 as a novel determinant of hepatocyte survival under lipotoxic stress. Genetic deletion or pharmacological inhibition of ACSL3 renders hepatocytes resistant to palmitate-induced apoptosis and endoplasmic reticulum stress, accompanied by reduced lipid-droplet accumulation, decreased incorporation of saturated fatty acids into neutral lipids and phospholipids, and blunted induction of lipogenic programs. In human MASLD, hepatic ACSL3 expression positively correlated with histological severity and aminotransferase levels. Single-cell transcriptomics localized ACSL3 predominantly to hepatocytes in advanced metabolic dysfunction-associated steatohepatitis displaying oxidative and endoplasmic reticulum stress signatures, whereas spatial transcriptomics showed ACSL3-high hepatocyte regions enriched for apoptotic and inflammatory pathways and spatially coupled to macrophage-rich and plasma cell-rich niches.<br><br>CONCLUSIONS: ACSL3 is a central regulator of lipotoxic hepatocyte injury and MASLD progression, mechanistically linking lipid-droplet biogenesis to apoptosis and inflammatory niche formation. These data suggest that ACSL3 is a promising therapeutic target and support further translational studies to evaluate ACSL3 modulation in steatotic liver disease.},
}
@article {pmid41563484,
year = {2026},
author = {Pardy, F and Reblova, K and Svozilova, H and Tichy, B and Pospisilova, S and Kotaskova, J and Navrkalova, V},
title = {Assessment of long-read strategies for the enrichment of clinically relevant breakpoints in lymphomas: towards a diagnostic implementation.},
journal = {Annals of hematology},
volume = {105},
number = {2},
pages = {47},
pmid = {41563484},
issn = {1432-0584},
mesh = {Humans ; *Translocation, Genetic ; *Lymphoma/genetics/diagnosis ; *Chromosome Breakpoints ; Cell Line, Tumor ; High-Throughput Nucleotide Sequencing/methods ; CRISPR-Cas Systems ; },
abstract = {Recurrent chromosomal translocations are hallmarks of many hematological malignancies, including lymphomas and leukemias. Accurate breakpoint detection is essential for diagnostics, treatment optimization, and disease monitoring. Long-read sequencing (Oxford Nanopore Technologies) enables unambiguous mapping and translocation identification. We designed a Cas9-based enrichment panel targeting common translocations in lymphoid malignancies. To accommodate both well-defined and promiscuous translocation partners, we employed single-side and dual-side sequencing strategies. Using well-established lymphoid cell lines, we benchmarked three enrichment approaches: (i) Cas9 read-out, (ii) Cas9 excision with multiplexing, and (iii) adaptive sampling. Cas9-mediated enrichment achieved superior on-target coverage, particularly in densely targeted regions (such as the IGH locus), while single-probe targets showed lower coverage depth. Adaptive sampling offered higher throughput, flexibility, and better pore occupancy, however with limited breakpoint detection. Cas9 excision has been demonstrated as a fast and reliable method to detect canonical translocation partners in clinical lymphoma samples. Our findings indicate that long-read enrichment strategies are suitable for targeting breakpoint hotspots, although the choice of approach depends heavily on the laboratory's specific goal. We propose a decision algorithm for selecting the optimal method based on experimental and clinical needs: Cas9-mediated enrichment suits focused diagnostic intent, while adaptive sampling is preferable for broader research use.},
}
@article {pmid41491310,
year = {2026},
author = {Arena, KA and Kearns, CA and Ahmed, M and O'Rourke, R and Sagerström, CG and Franco, SJ and Appel, B},
title = {Gsx2 regulates oligodendrocyte precursor formation in the zebrafish spinal cord.},
journal = {Developmental biology},
volume = {531},
number = {},
pages = {30-44},
doi = {10.1016/j.ydbio.2026.01.001},
pmid = {41491310},
issn = {1095-564X},
mesh = {Animals ; *Zebrafish/embryology/genetics/metabolism ; *Spinal Cord/embryology/cytology/metabolism ; *Zebrafish Proteins/metabolism/genetics ; *Oligodendroglia/metabolism/cytology ; Cell Differentiation/genetics ; Gene Expression Regulation, Developmental ; *Oligodendrocyte Precursor Cells/metabolism/cytology ; Neural Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; *Homeodomain Proteins/genetics/metabolism ; Gene Regulatory Networks ; Cell Lineage ; },
abstract = {Nervous system development relies on sequential and coordinated formation of diverse neurons and glia from neural progenitor cells (NPCs). In the spinal cord, NPCs of the pMN domain produce neurons early in development followed by oligodendrocyte precursor cells (OPCs), which subsequently differentiate as oligodendrocytes (OLs), the myelinating glia of the central nervous system. The mechanisms that specify neural progenitor cells to the OL lineage are not yet well understood. Using zebrafish as an experimental model system, we generated single-cell RNA sequencing and single-nuclei ATAC sequencing data that identified a subpopulation of NPCs, called pre-OPCs, that appeared fated to produce OPCs. pre-OPCs uniquely express several genes that encode transcription factors specific to the OL lineage, including Gsx2, which regulates OPC formation in the mouse forebrain. To investigate Gsx2 function in zebrafish OPC specification, we used CRISPR/Cas9 genome editing to create gsx2 loss-of-function alleles. gsx2 homozygous mutant embryos initiated OPC formation prematurely and produced excess OPCs without altering OL differentiation. Using our single-nuclei multi-omics dataset, we predicted a gene regulatory network centered around gsx2 and identified genes that might be transcriptionally regulated by Gsx2. Taken together, our studies suggest that Gsx2 expression in pre-OPCs contributes to the timing of OPC specification.},
}
@article {pmid41443566,
year = {2026},
author = {Ghosh, P and Wadsworth, BC and Terry, L and Evans, TA},
title = {Evolutionary conservation of midline axon guidance activity between Drosophila and Tribolium Frazzled.},
journal = {Developmental biology},
volume = {531},
number = {},
pages = {1-9},
doi = {10.1016/j.ydbio.2025.12.015},
pmid = {41443566},
issn = {1095-564X},
mesh = {Animals ; *Axon Guidance/physiology/genetics ; *Tribolium/embryology/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Drosophila melanogaster/embryology/genetics/metabolism ; Axons/metabolism ; Signal Transduction ; Netrin Receptors/genetics/metabolism ; Gene Expression Regulation, Developmental ; Biological Evolution ; CRISPR-Cas Systems ; },
abstract = {The regulation of midline crossing of axons is of fundamental importance for the proper development of nervous system connectivity in bilaterian animals. A number of conserved axon guidance signaling pathways coordinate to attract or repel axons at the nervous system midline to ensure the proper regulation of midline crossing. The attractive Netrin-Frazzled/DCC (Net-Fra) signaling pathway is widely conserved among bilaterians, but it is not clear whether the mechanisms by which Net and Fra promote midline crossing are also conserved. In Drosophila, Fra can promote midline crossing via Netrin-dependent and Netrin-independent mechanisms, by acting as a canonical midline attractive receptor and also through a non-canonical pathway to inhibit midline repulsion via transcriptional regulation. To examine the conservation of Fra-dependent axon guidance mechanisms among insects, in this paper we compare the midline attractive roles of the Frazzled receptor in the fruit fly (Drosophila melanogaster) and flour beetle (Tribolium castaneum) using CRISPR/Cas9-mediated gene editing. We replace the Drosophila fra gene with sequences encoding Drosophila Fra (DmFra) or Tribolium Fra (TcFra) and examine midline crossing of axons in the ventral nerve cord of embryos carrying these modified alleles. We show that Tribolium Fra can fully substitute for Drosophila Fra to promote midline crossing of axons in the embryonic nervous system, suggesting that the mechanisms by which Frazzled regulates midline axon guidance are evolutionarily conserved within insects.},
}
@article {pmid41435996,
year = {2026},
author = {Gopalakrishnan, R and Kannan, K and Gunasekaran, R and Ramachandran, P and Ganapathy, D and Pitchiah, S},
title = {A comparative review of vector insertion techniques in Saccharomyces cerevisiae.},
journal = {Journal of microbiological methods},
volume = {241},
number = {},
pages = {107378},
doi = {10.1016/j.mimet.2025.107378},
pmid = {41435996},
issn = {1872-8359},
mesh = {*Saccharomyces cerevisiae/genetics ; *Genetic Vectors/genetics ; CRISPR-Cas Systems ; Homologous Recombination ; *Genetic Engineering/methods ; *Mutagenesis, Insertional/methods ; Synthetic Biology/methods ; Gene Editing/methods ; Genome, Fungal ; },
abstract = {Saccharomyces cerevisiae, a model organism in genetics and molecular biology has been extensively engineered using various vector insertion techniques. This review compares and contrasts three prominent techniques: In vivo homologous recombination (HR), Cre-lox recombination and CRISPR/Cas9. In vivo HR leverages the organism's innate DNA repair machinery for easy vector integration and targeted genome modifications. Cre-lox recombination offers high specificity and efficiency at loxP sites, making it ideal for targeted gene excision or integration. CRISPR/Cas9 has revolutionized genome engineering with its precision and ability to target multiple loci simultaneously. Each technique has its strengths and limitations, including site dependency, off-target effects, and strain-specific variability. This review provides a comprehensive overview of these vector insertion techniques, highlighting their applications, advantages, and limitations in S. cerevisiae genome engineering and synthetic biology.},
}
@article {pmid41420208,
year = {2025},
author = {Holmlund, H and Yamauchi, Y and Tekayev, M and Jakobs, S and Robin, A and Fujii, W and Ward, MA},
title = {CRISPR/Cas9-mediated knock-in of the murine Y chromosomal genes Zfy1 and Zfy2.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {88},
pmid = {41420208},
issn = {1471-2164},
support = {NIH F31HD111279/GF/NIH HHS/United States ; HD114645/GF/NIH HHS/United States ; 17CON-86294//Hawai'i Community Foundation/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Male ; Mice ; *Gene Knock-In Techniques ; Spermatogenesis/genetics ; *Y Chromosome/genetics ; Testis/metabolism ; Gene Editing ; Zinc Fingers/genetics ; },
abstract = {BACKGROUND: The Y-linked mouse zinc finger genes Zfy1 and Zfy2 are critical fertility factors in mice but the mechanisms by which they regulate spermatogenesis remain unclear. We recently produced Zfy1/2 double knock-out mice and observed a complete loss in fertility. However, the biochemical mechanism by which Zfy regulates spermatogenesis is unknown, and ZFY expression has not yet been confirmed at the protein level. As both Zfy homologues share ~ 95% sequence similarity, it is difficult to produce an anti-ZFY antibody specific to either homologue.<br><br>RESULTS: To overcome this technical challenge, we used CRISPR/Cas9 genome editing to develop tagged Zfy1 knock-in (XY[Zfy1-HA]), Zfy2 knock-in (XY[Zfy2-FLAG], XY[Zfy2-3xFLAG], and XY[Zfy2-HA]), and Zfy1/2 double knock-in (XY[Zfy1-HA,Zfy2-MYC]) mice. Successful targeting was confirmed by genotyping and sequencing. The knock-in lines were fertile with normal sperm parameters. Using Western blot on testes, knock-in specific bands were detected matching the predicted ZFY expression patterns. Using immunofluorescence on testis sections from knock-in males, ZFY1 and ZFY2 expression was detected in zygotene spermatocytes, and ZFY2 expression was also detected in spermatids step 7-8 and 9.<br><br>CONCLUSIONS: These novel knock-in mice can be used in future investigations to determine how ZFY controls spermatogenesis.},
}
@article {pmid41160201,
year = {2026},
author = {Wang, YY and Lin, YH and Ke, CC and Lai, TH and Yu, IS and Au, CF and Singh, R and Lin, YH},
title = {Human teratozoospermia-related AGTPBP1 R791H mutation is associated with sperm head and tail defects in a CRISPR-engineered murine model.},
journal = {Journal of assisted reproduction and genetics},
volume = {43},
number = {1},
pages = {133-142},
pmid = {41160201},
issn = {1573-7330},
support = {NSTC 111-2320-B-030 -007-MY3//National Science and Technology Council/ ; CTH-109A-2215//Cardinal Tien Hospital/ ; },
mesh = {Animals ; Male ; *Teratozoospermia/genetics/pathology ; Mice ; *Sperm Head/pathology ; Humans ; *Sperm Tail/pathology ; Disease Models, Animal ; Spermatozoa/pathology ; CRISPR-Cas Systems/genetics ; Mutation ; *Infertility, Male/genetics/pathology ; *Poly-ADP-Ribose Binding Proteins/genetics ; },
abstract = {BACKGROUND: Infertility is a pervasive global health concern affecting millions of couples worldwide. Approximately 7% of the male population is infertile. Teratozoospermia, defined by > 96% abnormal sperm morphology, is a major cause of infertility often linked to genetic defects. In our previous study, we identified three AGTPBP1 mutations (p.Glu423Asp, p.Pro631Leu, and p.Arg811His) in teratozoospermia cases. AGTPBP1 is a key enzyme involved in regulating tubulin polyglutamylation and generating Δ-2 tubulin, a major structural component of the sperm tail and an essential structure for sperm head differentiation. However, functional proof of the impact of AGTPBP1 Arg811His on sperm head and tail impairment remained unestablished.<br><br>METHODS: Knock-in mice carrying the equivalent mutation, Arg791His (R791H) corresponding to the human mutation (R811H), in the Agtpbp1 gene were generated and analyzed for sperm morphological abnormalities.<br><br>RESULTS: Sperm morphological evaluation revealed a significant increase in the proportion of morphologically abnormal sperm in the Agtpbp1[R791H/R791H] mice. Detailed morphological analysis revealed a significantly higher incidence of sperm head abnormalities and abnormal attachment of the head to the midpiece in the Agtpbp1[R791H/R791H] mice relative to wild-type controls. Further, sperm with head defects from Agtpbp1[R791H/R791H] mice exhibited abnormal accumulation of polyglutamylated tubulin within the sperm head. The mutant mice showed exactly the same morphological defects as seen in human patients and those displayed by mice lacking the complete carboxypeptidase A domain of AGTPBP1 but at a relatively lesser frequency.<br><br>CONCLUSIONS: We conclude that the R791H mutation in the Agtpbp1 gene impairs sperm head and tail differentiation, resulting in sperm morphological defects.},
}
@article {pmid41128695,
year = {2026},
author = {Plugge, SF and Ma, H and van der Vaart, JY and Sprangers, J and Morsink, FHM and Xanthakis, D and Jamieson, C and Stroot, KBW and Keijzer, AR and Margaritis, T and Candelli, T and Straver, R and de Ridder, J and Holstege, FCP and de Leng, WWJ and Offerhaus, GJA and Merenda, A and Maurice, MM},
title = {Intestinal LKB1 Loss Drives a Premalignant Program Along the Serrated Cancer Pathway.},
journal = {Gastroenterology},
volume = {170},
number = {2},
pages = {298-314},
doi = {10.1053/j.gastro.2025.07.041},
pmid = {41128695},
issn = {1528-0012},
mesh = {Humans ; *Protein Serine-Threonine Kinases/genetics/deficiency/metabolism ; Animals ; AMP-Activated Protein Kinase Kinases ; *Peutz-Jeghers Syndrome/genetics/pathology/enzymology ; Organoids/pathology ; *Colorectal Neoplasms/genetics/pathology/enzymology ; Mice ; *Precancerous Conditions/genetics/pathology/enzymology ; Signal Transduction ; ErbB Receptors/metabolism ; Mice, Knockout ; Colon/pathology/enzymology ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Disease Models, Animal ; Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *Cell Transformation, Neoplastic/genetics/pathology ; Intestinal Mucosa/pathology ; CRISPR-Cas Systems ; AMP-Activated Protein Kinases ; },
abstract = {BACKGROUND &amp; AIMS: Heterozygous inactivating mutations of Serine Threonine Kinase 11/Liver Kinase B1 (LKB1) are causative to the Peutz-Jeghers syndrome (PJS), a hereditary disease characterized by gastrointestinal hamartomatous polyposis and increased cancer susceptibility. Although LKB1 loss-induced polyp formation has been ascribed to nonepithelial tissues, how LKB1 deficiency increases cancer risk of patients by altering the phenotypical landscape and hierarchical organization of epithelial tissues remains poorly understood.<br><br>METHODS: Using CRISPR/Cas9, we generated heterozygous and homozygous Lkb1-deficient mouse small intestinal and human colon organoids. These organoids were characterized by an integrated approach that combines imaging, bulk and single-cell RNA sequencing, and growth factor dependency assays. Our findings were validated in human PJS-derived tissues using immunohistochemistry and linked to colorectal cancer profiles using the Cancer Genome Atlas (TCGA) cancer database.<br><br>RESULTS: Our results reveal that heterozygous Lkb1 loss is sufficient to push intestinal cells into a premalignant transcriptional program associated with serrated colorectal cancer, which is further amplified by loss of heterozygosity. This altered epithelial growth state associates with persistent features of regeneration and enhanced EGFR ligand and receptor expression, conferring niche-independent growth properties to Lkb1-deficient organoids. Moreover, our newly generated LKB1-mutant signature is enriched in sporadic serrated colorectal cancer, and synergistic cooperation of Lkb1 deficiency with mutant Kras was experimentally confirmed by assessing organoid growth properties and transcriptomes.<br><br>CONCLUSIONS: Heterozygous loss of LKB1 pushes intestinal cells into a chronic regenerative state, which is amplified on loss of heterozygosity. Lkb1 deficiency thereby generates fertile ground for serrated colorectal cancer formation in the intestine, potentially explaining the increased cancer risk observed in PJS.},
}
@article {pmid39894889,
year = {2026},
author = {Shahid, A and Zahra, A and Aslam, S and Shamim, A and Ali, WR and Aslam, B and Khan, SH and Arshad, MI},
title = {Appraisal of CRISPR Technology as an Innovative Screening to Therapeutic Toolkit for Genetic Disorders.},
journal = {Molecular biotechnology},
volume = {68},
number = {1},
pages = {71-94},
pmid = {39894889},
issn = {1559-0305},
support = {DDWP-2021//Ministry of Science and Technology, Pakistan/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Diseases, Inborn/diagnosis/therapy/genetics ; *Genetic Therapy/methods ; },
abstract = {The high frequency of genetic diseases compels the development of refined diagnostic and therapeutic systems. CRISPR is a precise genome editing tool that offers detection of genetic mutation with high sensitivity, specificity and flexibility for point-of-care testing in low resource environment. Advancements in CRISPR ushered new hope for the detection of genetic diseases. This review aims to explore the recent advances in CRISPR for the detection and treatment of genetic disorders. It delves into the advances like next-generation CRISPR diagnostics like nano-biosensors, digitalized CRISPR, and omics-integrated CRISPR technologies to enhance the detection limits and to facilitate the "lab-on-chip" technologies. Additionally, therapeutic potential of CRISPR technologies is reviewed to evaluate the implementation potential of CRISPR technologies for the treatment of hematological diseases, (sickle cell anemia and β-thalassemia), HIV, cancer, cardiovascular diseases, and neurological disorders, etc. Emerging CRISPR therapeutic approaches such as base/epigenetic editing and stem cells for the development of foreseen CRIPSR drugs are explored for the development of point-of-care testing. A combination of predictive models of artificial intelligence and machine learning with growing knowledge of genetic disorders has also been discussed to understand their role in acceleration of genetic detection. Ethical consideration are briefly discussed towards to end of review. This review provides the comprehensive insights into advances in the CRISPR diagnostics/therapeutics which are believed to pave the way for reliable, effective, and low-cost genetic testing.},
}
@article {pmid41562469,
year = {2026},
author = {Wan, Z and Xu, C and Wang, Y and Song, L and Yuan, W and Chen, M and Gong, R and Zhang, XE},
title = {An AND-Logic Gate-Based Biosensor for Simultaneous Detection of SARS-CoV-2 Nucleic Acids and Nucleocapsid Proteins.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c07321},
pmid = {41562469},
issn = {1520-6882},
abstract = {Nucleic acids and proteins are recognized as gold standard biomarkers for disease diagnosis and pathogen detection. However, conventional single-analyte detection methods remain susceptible to false positives caused by manual operational errors or sample contamination, thereby undermining diagnostic reliability and increasing the burden on healthcare systems. To address this limitation, we developed a one-pot isothermal amplification and CRISPR-Cas cooperative system (OIACS) that functions as an AND-logic gate biosensor for the simultaneous detection of SARS-CoV-2 RNA and nucleocapsid protein. Unlike conventional methods relying solely on CRISPR RNA (crRNA) recognition, the OIACS employs antibody-mediated target binding with blocker release for target recognition, offering increased flexibility in assay design for different targets. A universal Cas12a-targetable DNA barcode is generated via strand displacement isothermal amplification, enabling signal amplification upon dual-target recognition. The OIACS assay exhibited practical utility by reliably detecting SARS-CoV-2 transcription- and replication-competent virus-like-particles at 5000 copies/mL, and the limit of detection was determined to be as low as 1698 copies/mL, highlighting its robustness and potential for clinical diagnosis.},
}
@article {pmid41562237,
year = {2026},
author = {Mattivi, C and Wang, S and Ji, L and Xiao, Q and Cao, J},
title = {HLA-Knockout: Enabling Allele-Specific Knockout of HLA Class I Genes for Immunogenic Engineering.},
journal = {HLA},
volume = {107},
number = {1},
pages = {e70548},
pmid = {41562237},
issn = {2059-2310},
support = {P30CA072720//Rutgers Cancer Institute of New Jersey/ ; //Nanjing University/ ; //Science Fund Program for Distinguished Young Scholars (Overseas)/ ; 82473308//National Natural Science Foundation of China Grant/ ; 2025300355//Fundamental Research Funds for the Central Universities/ ; },
mesh = {Humans ; *Alleles ; *Gene Knockout Techniques/methods ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Histocompatibility Antigens Class I/genetics/immunology ; Receptors, Antigen, T-Cell/genetics/immunology ; },
abstract = {The interaction between T-cell receptors (TCRs) and antigenic peptides presented by HLA molecules is fundamental to adaptive immunity. However, the extreme polymorphism of HLA genes poses major challenges for transplantation, antigen discovery, immunotherapy and studies of allele-specific function. Although CRISPR/Cas9 has transformed gene editing, existing sgRNA design tools are not optimised for knockout of HLA Class I genes due to their high rates of polymorphism. To address this, we developed HLA-Knockout (https://hlaknockout.rutgers.edu), a novel web-based tool that enables precise, allele-specific targeting of HLA Class I genes. HLA-Knockout retrieves user-defined HLA sequences from the IPD-IMGT/HLA database and applies stringent design criteria, including mismatch filtering and PAM disruption analysis, to ensure high specificity and minimal off-target effects on non-target HLA Class I alleles. Using HLA-Knockout, we achieved efficient single- and double-allele HLA Class I knockouts in human cells without disrupting non-target HLA Class I alleles. Functional assays confirmed allele-specific loss of antigen-specific TCR activation, validating the platform's utility. HLA-Knockout provides a unique resource for dissecting HLA-restricted immune interactions and has broad applications in transplantation biology, autoimmunity and cancer immunotherapy.},
}
@article {pmid41562083,
year = {2025},
author = {Liu, J and Hong, W and Sun, Z and Zhang, S and Xue, C and Dong, N},
title = {The gut-lung axis: effects and mechanisms of gut microbiota on pulmonary diseases.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1693964},
pmid = {41562083},
issn = {1664-3224},
mesh = {Humans ; *Gastrointestinal Microbiome/immunology ; Animals ; *Lung/immunology/metabolism/microbiology ; Dysbiosis/immunology ; *Lung Diseases/microbiology/immunology/metabolism/therapy/etiology ; Fatty Acids, Volatile/metabolism ; Fecal Microbiota Transplantation ; },
abstract = {The proposal of the gut-lung axis has profoundly reshaped our understanding of the mechanisms underlying respiratory diseases. As a crucial component of this axis, the gut microbiota plays a central role in pulmonary immune regulation through inter-organ communication mediated by metabolic products. However, a systematic integration of mechanisms explaining how gut microbes achieve precise cross-organ immune regulation remains elusive. Existing research predominantly focuses on descriptive observations, such as the association between early-life microbiota dysbiosis and an increased risk of asthma and chronic obstructive pulmonary disease (COPD), as well as the frequent occurrence of acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF), often accompanied by gut microbiome disruption. This paper focuses on three key gut microbial metabolites-short-chain fatty acids (SCFAs), tryptophan metabolites, and polyamines (PAs)-to examine their roles in immune regulation, maintenance of barrier function, and modulation of metabolic signaling networks. Based on the latest experimental and clinical evidence, this study systematically elucidates how dysbiosis of the gut microbiota, a key component of the gut-lung axis, crosses physiological barriers to exacerbate pulmonary inflammation. Regarding intervention strategies, probiotics, fecal microbiota transplantation (FMT), and CRISPR-Cas systems have demonstrated significant therapeutic potential in restoring gut microbial balance. Finally, this paper outlines future research directions, emphasizing the need to further explore non-invasive microbial sampling techniques, molecular interaction mechanisms of the gut-lung axis, and personalized microbiome-based diagnostic and therapeutic strategies to provide new insights for the prevention and treatment of respiratory diseases involving gut microbiota.},
}
@article {pmid41561392,
year = {2026},
author = {Choudhary, DK and Turgeman-Grott, I and Robinzon, S and Gophna, U},
title = {CRISPR-Cas targeting in Haloferax volcanii promotes within-species gene exchange by triggering homologous recombination.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqaf047},
pmid = {41561392},
issn = {2633-6693},
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated genes) systems provide adaptive immunity in bacteria and archaea against mobile genetic elements, but the role they play in gene exchange and speciation remains unclear. Here, we investigated how CRISPR-Cas targeting affects mating and gene exchange in the halophilic archaeon Haloferax volcanii. Surprisingly, we found that CRISPR-Cas targeting significantly increased mating efficiency between members of the same species, in contrast to its previously documented role in reducing interspecies mating. This enhanced mating efficiency was dependent on the Cas3 nuclease/helicase and extended beyond the targeted genomic regions. Further analysis revealed that CRISPR-Cas targeting promoted biased recombination in favor of the targeting strain (the strain containing the CRISPR-Cas system) during mating, resulting in an increased proportion of recombinant progeny that are positive for CRISPR-Cas. To test whether an increase in recombination is sufficient to increase mating efficiency, we tested whether strains lacking the Mre11-Rad50 complex, which are known to have elevated recombination activity, also exhibited higher mating success. Indeed, these strains showed higher mating, as did cells that were exposed to DNA damage using methyl methanesulfonate. These findings suggest that CRISPR-Cas systems in archaea play roles beyond their canonical immune function. They may contribute to speciation by facilitating within-species gene exchange while limiting between-species genetic transfer, thereby maintaining species boundaries.},
}
@article {pmid41561390,
year = {2026},
author = {Fehrenbach, A and Mitrofanov, A and Backofen, R and Baumdicker, F},
title = {The complexity of multiple CRISPR arrays in strains with (co-occurring) CRISPR systems.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqaf042},
pmid = {41561390},
issn = {2633-6693},
abstract = {CRISPR and their associated Cas proteins provide adaptive immunity in prokaryotes, protecting against invading genetic elements. These systems are categorized into types and are highly diverse. Genomes often harbor multiple CRISPR arrays varying in length and distance from Cas loci. However, the ecological roles of multiple CRISPR arrays and their interactions with multiple Cas loci remain poorly understood. We present a comprehensive analysis of CRISPR systems that uncovers variation between diverse Cas types regarding the occurrence of multiple arrays, the distribution of their lengths and positions relative to Cas loci, and the diversity of their repeat sequences. Some types tend to occur as the sole Cas locus present in the genome, but typically have two or more associated arrays, especially for types I-E and I-F. Multiple Cas types are also common, with some systems showing a preference for specific co-occurrence. Distinct array distributions and orientations around Cas loci indicate substantial differences in functionality and transcriptional behavior among Cas types. Our analysis suggests that arrays with identical repeats in the same genome acquire new spacers at comparable rates, irrespective of their proximity to the Cas locus. Furthermore, repeat similarities indicate that arrays of systems that often co-occur with other systems tend to have more diverse repeats than those mostly appearing alongside solitary systems. Our results indicate that co-occurring Cas-type pairs might not only collaborate in spacer acquisition but also maintain independent and complementary functions and that CRISPR systems distribute their defensive spacer repertoire equally across multiple CRISPR arrays.},
}
@article {pmid41560876,
year = {2026},
author = {More, R and Yadav, V and Vadakedath, N},
title = {Calyptranema fuscum gen. sp. nov.: a novel cyanobacterial genus within Oculatellaceae based on polyphasic and genomic characterization.},
journal = {Current research in microbial sciences},
volume = {10},
number = {},
pages = {100542},
pmid = {41560876},
issn = {2666-5174},
abstract = {This study presents a comprehensive characterization and classification of a novel cyanobacterial isolate, strain S582, proposed as Calyptranema fuscum gen. sp. nov. within the family Oculatellaceae using an integrated polyphasic approach. Strain S582 was isolated from a lake in the Botanical Garden, Sarangpur, Chandigarh, India. Initial molecular characterization with the 16S rRNA gene revealed ≤ 94.90% of similarity with related genera and showed unique 16S-23S ITS secondary structures, indicating its delineation as a novel genus. Morphological assessment highlighted the presence of a cap-like structure called calyptra at the terminal cells, further distinguishing it from related genera. Furthermore, whole genome sequencing yielded an assembly of 7962,515 bp with GC content of 48.27%. Genome-based analysis encompassing average nucleotide identity (ANI), average amino acid identity (AAI), percentage of conserved proteins (POCP) was subsequently performed. The observed values for ANI (71.15% to 73.00%) and AAI (63.30% to 69.62%) were below the established genus-level thresholds. Phylogenetic analysis using maximum-likelihood and Bayesian inference along with phylogenomic reconstruction based on 1434 single copy core genes supported its taxonomic novelty. Functional classification revealed the presence of biosynthetic gene clusters (BGCs), tRNAs, insertion elements, CRISPR/Cas systems, and genes associated with metabolism, carbon fixation and photosynthesis. Additionally, the pangenome was constructed to study the genomic diversity of the studied isolate and related genera among the Oculatellaceae family and identified core, accessory, and singleton gene clusters. Collectively, these findings establish Calyptranema fuscum gen. sp. nov. as a novel genus within Oculatellaceae while expanding our understanding of cyanobacterial diversity and genomic potential.},
}
@article {pmid41556308,
year = {2026},
author = {Xie, Z and Jin, YS and Miller, MJ},
title = {Exploiting the Endogenous Type II-A CRISPR-Cas System for Functional Engineering of Probiotic Lacticaseibacillus rhamnosus GG.},
journal = {Microbial biotechnology},
volume = {19},
number = {1},
pages = {e70303},
pmid = {41556308},
issn = {1751-7915},
support = {//University of Illinois at Urbana-Champaign/ ; },
mesh = {*CRISPR-Cas Systems ; *Lacticaseibacillus rhamnosus/genetics ; *Gene Editing/methods ; *Probiotics ; Glucuronidase/genetics/metabolism ; },
abstract = {Lacticaseibacillus rhamnosus GG (LGG) is one of the most extensively studied probiotic strains, widely used in food and health applications. However, the absence of efficient, precise genome editing methods has limited its broader potential and functional versatility. Here, we present an endogenous type II-A CRISPR-Cas genome editing workflow for LGG designed for functional strain construction. Using a plasmid interference assay together with single-nucleotide substitutions, we confirm the precise PAM requirement as 5'-NGAAA-3'. We pair a synthetic sgRNA cassette with homology-directed repair donors to enable targeted deletions and insertions across multiple loci, achieving modest but practically relevant editing efficiencies (11.1-25.0% of recovered transformants) that support routine strain construction. Using this optimised and precise genome engineering method, we generated a β-glucuronidase (GUS)-expressing LGG strain for robust strain tracking within complex microbial communities. This work removes barriers to LGG engineering, expands the probiotic CRISPR toolkit, and provides broadly applicable strategies for designing next-generation probiotics with applications in food biotechnology and microbial therapeutics.},
}
@article {pmid41556260,
year = {2026},
author = {Neherin, K and Holloway, K and Song, Y and Houston, A and Chen, F and Ding, L and Zhang, H},
title = {Introducing Cellular Senescence in Human Induced Pluripotent Stem Cells and Differentiated Neural Lineage for Modeling of Age-Associated Diseases.},
journal = {Advanced biology},
volume = {10},
number = {1},
pages = {e00468},
doi = {10.1002/adbi.202500468},
pmid = {41556260},
issn = {2701-0198},
support = {RF1AG056302/AG/NIA NIH HHS/United States ; R21AG086894/AG/NIA NIH HHS/United States ; U54AG075934//NIH Common Fund/ ; },
mesh = {Humans ; *Cellular Senescence/genetics/physiology ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Cell Differentiation ; *Neural Stem Cells/cytology/metabolism ; *Aging/genetics ; CRISPR-Cas Systems ; Cell Lineage ; },
abstract = {Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenetic landscapes that mark the aging clock, and consequently cells differentiated from iPSCs resemble fetal cells rather than adult or aged cells. The lack of proper cellular aging in cells differentiated from iPSCs presents a unique challenge in iPSC-based modeling of age-associated diseases such as neurodegeneration. To address this challenge, we seek to introduce cellular senescence, a hallmark of aging, into iPSC-based models in a robust and temporally controlled manner. An inducible CRISPR interference (CRISPRi) is used to suppress the expression of TERF2, a key component of the telomere protecting Shelterin complex. We demonstrate that suppression of TERF2 robustly activates the DNA damage response, p53/p21 signaling, and cellular senescence in iPSCs in a highly homogeneous and synchronous manner. Applying this inducible CRISPRi-TERF2 system to differentiation of iPSCs to neural progenitor cells (NPCs), we show efficient activation of senescence-associated phenotypes in NPCs. This inducible cell model allows isogenic comparisons of the same cell populations over the course of differentiation with or without the activation of cellular senescence in a synchronous and homogeneous manner, and has broad applications in investigating the role of cellular senescence in the progression of age-related diseases.},
}
@article {pmid41555514,
year = {2026},
author = {Xiao, B and Zhang, J},
title = {A Simple Programmable Cas12a/crRNA Induced Walking System for Sensitive Methicillin-Resistant Staphylococcus aureus Detection via Integrated cis- and trans-Cleavage Activity.},
journal = {Journal of microbiology and biotechnology},
volume = {36},
number = {},
pages = {e2511026},
doi = {10.4014/jmb.2511.11026},
pmid = {41555514},
issn = {1738-8872},
mesh = {*Methicillin-Resistant Staphylococcus aureus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics/metabolism ; Aptamers, Nucleotide/genetics ; *Staphylococcal Infections/diagnosis/microbiology ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Biosensing Techniques/methods ; Humans ; },
abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) represents a serious threat to public health due to its strong antibiotic resistance, wide dissemination, and high infection rates. Rapid identification of MRSA strains is essential for accurate diagnosis and timely treatment of related infections. In this study, we propose an analytical method for MRSA that employs a hairpin-structured locker-probe to directly regulate the trans-cleavage activity of Cas12a. This designed locker-probe connects a target-specific aptamer to an inhibitory aptamer of the CRISPR/Cas12a system. Upon binding to the specific target, the probe undergoes a conformational change that abolishes its inhibitory effect on Cas12a. As a result, the structure-switchable probe modulates Cas12a activity in a target-dependent manner. Additionally, the sensing substrate combines a "cis-cleavage trigger" and a "trans-cleavage trigger" to integrate both cis- and trans-cleavage activities of Cas12a/crRNA within a single probe. This design significantly simplifies the probe architecture while maintaining high signal amplification efficiency. The proposed method was successfully applied to detect MRSA, achieving a detection limit as low as 2.5 CFU/ml with high specificity. By exploiting the inhibitory aptamer of Cas12a as a regulatory element for MRSA analysis, this work expands the toolbox of CRISPR/Cas12a-based methodologies and offers a promising strategy for bacterial detection.},
}
@article {pmid41555077,
year = {2026},
author = {Yu, L and Yin, M and Zhu, Y and Lu, Z and Xiao, B and Zhou, F and Yu, Y and Huang, Z},
title = {An anti-CRISPR targets the sgRNA to block Cas9 and guides the design of enhanced genome editors.},
journal = {Nature structural &amp; molecular biology},
volume = {},
number = {},
pages = {},
pmid = {41555077},
issn = {1545-9985},
abstract = {Bacteriophages have evolved anti-CRISPR (Acr) proteins to combat the adaptive immunity provided by bacterial CRISPR-Cas systems. Here, we report the cryo-electron microscopy structure of an anti-Cas9 protein AcrIIA27 bound to SpyCas9-sgRNA (single guide RNA) complex. Our structure reveals that AcrIIA27 binds the solvent-exposed phosphate backbone of the sgRNA, acting as a potent inhibitor of diverse Cas9 orthologs. AcrIIA27 in the structure is positioned near the protospacer-adjacent motif DNA-binding pocket on SpyCas9, causing steric hindrance that prevents substrate DNA recognition. This mechanism suggests solvent-exposed regions of sgRNAs (PTP RNAs), prone to nonspecific binding of positively charged components, may compromise CRISPR-Cas genome-editing efficiency. Indeed, truncations of the PTP RNAs in different editing systems significantly enhance genome-editing efficiency in human cells. Overall, our findings reveal a previously uncharacterized inhibition mechanism of an anti-Cas protein and offers a general strategy for developing more efficient genome-editing tools.},
}
@article {pmid41554027,
year = {2025},
author = {Ireri, SW and Cao, M},
title = {CRISPR-Cas9-based Mutagenesis in the Entomopathogenic Nematode Steinernema hermaphroditum and the Maintenance of Mutant Lines.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {226},
pages = {},
doi = {10.3791/68932},
pmid = {41554027},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Mutagenesis ; *Rhabditida/genetics ; Microinjections/methods ; Gene Editing/methods ; },
abstract = {Entomopathogenic nematodes (EPNs) in the genus Steinernema and Heterorhabditis maintain mutualistic interactions with Xenorhabdus and Photorhabdus symbiotic bacteria, respectively. Together, these nematode-bacterium pairs infect and kill insect hosts that are primarily larvae from the orders of Lepidoptera and Coleoptera, forming a tractable tripartite system for dissecting the molecular basis of mutualism and parasitism. A key step towards fully utilizing this model is the development of stable and transgenerational genetic tools in EPNs. Here, we demonstrate a reliable CRISPR-Cas9 genome editing platform in the emerging model Steinernema hermaphroditum, a species that is readily maintained in vivo and in vitro, and is highly amenable to gonadal microinjection. Importantly, its hermaphroditic reproduction greatly streamlines the generation and maintenance of homozygous mutant lines. We provide a detailed protocol for efficient, targeted gene disruption using microinjection-based delivery of Cas9 ribonucleoprotein complexes. As a proof of concept, we modified the conserved muscle-associated gene unc-22, generating a characteristic twitching phenotype that validates targeted mutagenesis in this system. This CRISPR-Cas9 platform opens the door to stable genetic manipulation in S. hermaphroditum, such as transgene expression, and provides a framework that can be extended to additional EPN species of agricultural and ecological importance.},
}
@article {pmid41540063,
year = {2026},
author = {Port, F and Buhmann, MA and Zhou, J and Stricker, M and Vaughan-Brown, A and Michalsen, AC and Roßmanith, E and Pöltl, A and Großkurth, L and Huber, J and Menendez Kury, LB and Weberbauer, B and Hübl, M and Puscher, E and Heigwer, F and Boutros, M},
title = {Improved in vivo gene knockout with high specificity using multiplexed Cas12a sgRNAs.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {877},
pmid = {41540063},
issn = {2041-1723},
support = {ERC-DECODE//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; SFB1324//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Gene Editing/methods ; Drosophila melanogaster/genetics ; Gene Targeting/methods ; *Bacterial Proteins/genetics/metabolism ; Drosophila/genetics ; },
abstract = {CRISPR nuclease-mediated gene knock-out is limited by suboptimal sgRNAs, inaccessible target sites, and undesired repair outcomes. Here, we present a Cas12a-based system in Drosophila that targets each gene with four sgRNAs to overcome these limitations. Multiplexed sgRNAs act through redundancy and synergism, frequently creating deletions between target sites and increasing the fraction of loss-of-function mutations. We show that multiplexed gene targeting is well tolerated and does not cause widespread proximity effects. To visualize CRISPR-nuclease activity in living animals, we developed a screening assay and used it to assess Cas12a activity across 33% of the Drosophila genome in combination with over 2000 sgRNAs. This revealed remarkably high on-target (>99%) and very low (<1%) off-target activity of multiplexed Cas12a sgRNA arrays. Quantitative side-by-side comparisons with current Cas9-based systems targeting over 100 genes in parallel demonstrate that multiplexed Cas12a gene targeting achieves superior performance and reveals phenotypes missed by established methods. The system described here provides a framework for reliable gene knock-out in multicellular systems.},
}
@article {pmid41506457,
year = {2026},
author = {Jiménez Lancho, V and Leitner, K and Agarwal, K and Krishnakumar, A and Khetan, A and Borth, N and Marx, N},
title = {CRISPR-based precise methylation of specific FUT8 promoter regions allows isolation of CHO cells with a fine-tuned glycoprofile.},
journal = {Journal of biotechnology},
volume = {410},
number = {},
pages = {341-352},
doi = {10.1016/j.jbiotec.2026.01.001},
pmid = {41506457},
issn = {1873-4863},
mesh = {CHO Cells ; Cricetulus ; Animals ; *DNA Methylation/genetics ; *Promoter Regions, Genetic/genetics ; *Fucosyltransferases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Glycosylation ; Gene Editing/methods ; },
abstract = {A major advantage of producing therapeutic proteins in mammalian cells is their ability to tailor proteins with human-like posttranslational modifications such as glycosylation, which ultimately defines aspects like stability, protein folding or immunogenicity. However, producing therapeutic proteins with a consistent and reproducible glycoprofile remains a major challenge for the biopharmaceutical industry, especially with biosimilar production. While the enzymes responsible for glycosylation of proteins have been the subject of various cell engineering approaches, tuning their gene expression to precise levels is still difficult to achieve. While CRISPR/Cas9 enabled the genetic engineering of cells to drastically overexpress or remove a target gene, CRISPR/dCas9-based epigenetic editing by targeted DNA methylation promises to stably change the expression pattern of target genes after transient transfection of the CRISPR-tool. Application of targeted DNA methylation so far has mostly been used to completely silence gene expression by fully methylating the corresponding promoter regions. Here, we aim to tune expression of the associated gene by DNA methylation of confined promoter regions and to apply this technique as a new glycoengineering approach. By coupling CRISPR-based targeted DNA methylation with lectin-FACS assisted sorting we obtained CHO cell lines with a fine-tuned phenotype. First, dCas9-DNMT3A3L in combination with one single gRNA is targeted to the FUT8 promoter to induce confined DNA methylation, resulting in a phenotypically diversified population. Next, a window sorting strategy based on lectin-stained cells using five different sorting gates spanning from low to high FUT8 expression was applied to isolate single clones with a defined phenotype. Isolated clones were phenotypically assessed and re-sorted to obtain a homogenous expression profile. The resulting clonal cell lines showed either tuned or knock-down phenotypes with varying gene expression levels. Two out of seven clones that showed tuned FUT8 gene expression were phenotypically stable over 60 days. Gene expression levels, on the other hand, showed a steady decline over time that in part, however, can be explained by the general variation of FUT8 expression in different growth phases. Importantly, glycan analysis of recombinant EpoFc produced in the tuned clonal cell lines showed ranges of 35-70 % fucosylation, demonstrating that isolated clones can produce recombinant proteins with a distinct glycosylation profile. To understand why some clones showed tuned FUT8 gene expression levels while others were completely knocked-down, we analyzed the DNA methylation status of their respective FUT8 promoter. Critical areas within the FUT8 promoter were identified, with some associated with general repression and others with the tuning of FUT8 gene expression when affected by DNA methylation. Additionally, a combination of histone marks associated with active and repressed promoters was found to potentially define clones with a fine-tuned expression. Combined, the data demonstrates that using targeted DNA methylation in a manner confined to specific promoter regions opens new engineering strategies to fine-tune gene expression in mammalian cells.},
}
@article {pmid41496287,
year = {2026},
author = {Chatrousse, L and Poullion, T and Mahiou, H and El-Kassar, L and Giraud-Triboult, K and Boissart, C and Benchoua, A},
title = {Knock-out of specific DMD gene isoforms in the parental hESC line SA001 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103899},
doi = {10.1016/j.scr.2025.103899},
pmid = {41496287},
issn = {1876-7753},
mesh = {Humans ; *Dystrophin/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Protein Isoforms/genetics/metabolism ; Cell Line ; Male ; Muscular Dystrophy, Duchenne/genetics ; *Gene Knockout Techniques ; },
abstract = {The DMD gene, which encodes the protein dystrophin, is involved in a group of diseases known as dystrophinopathies, which includes Duchenne Muscular Dystrophy (DMD). DMD is a progressive and lethal muscular disorder mainly affecting boys that results from the loss of function of the longer dystrophin isoform DP427 in skeletal muscles. Dystrophinopathies are also associated with poorly understood neurocognitive and neurodevelopmental disorders. To investigate the role of dystrophin isoforms in neural development, we specifically disrupted three dystrophin isoforms expressed in the brain, namely DP427, DP140 and DP71, in the male human embryonic stem cell line SA001 using the CRISPR/Cas9 system. (100 / 100 words).},
}
@article {pmid41496285,
year = {2026},
author = {Wang, J and Gao, Y and Jin, K and Jin, ZB and Xu, J},
title = {Generation of RB1 knockout human embryonic stem cell lines derived from H9 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103897},
doi = {10.1016/j.scr.2025.103897},
pmid = {41496285},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Knockout Techniques ; *Retinoblastoma Binding Proteins/genetics/metabolism/deficiency ; Cell Line ; *Ubiquitin-Protein Ligases/genetics/deficiency/metabolism ; Cell Differentiation ; Animals ; Mice ; },
abstract = {RB1 is a tumor suppressor gene which plays a crucial role in cell cycle and cellular differentiation. Mutations or loss of RB1 are associated with retinoblastoma and a variety of other cancers. We generated RB1 knockout human embryonic stem cell (hESC) lines (H9) using CRISPR/Cas9-mediated gene targeting. These RB1-deficient cell lines maintain typical stem cell morphology, normal karyotype and expression of pluripotent marker genes. Furthermore, they retain their in vivo differentiation capacity, enabling the generation of multiple cell lineages. These RB1 knockout hESC lines provide valuable models for investigating the role of RB1 in tumorigenesis, neurodevelopment, and cell cycle regulation.},
}
@article {pmid41496284,
year = {2026},
author = {Fan, M and Zhao, M and Su, W and Tang, Z and Sun, W and Zhou, T and Liu, P},
title = {Generation of a PLIN2-GFP2-P2A-Puro human induced pluripotent stem cell line (SEUi001-A) via CRISPR/Cas9-mediated gene editing technology.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103896},
doi = {10.1016/j.scr.2025.103896},
pmid = {41496284},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Perilipin-2/genetics/metabolism ; Cell Line ; Green Fluorescent Proteins/metabolism/genetics ; Cell Differentiation ; },
abstract = {Perilipin 2 (PLIN2) dysregulation drives metabolic pathologies including non-alcoholic fatty liver disease (NAFLD). To enable real-time tracking of PLIN2 dynamics, we established a human induced pluripotent stem cell (hiPSC) line with endogenous GFP2 knock-in at the PLIN2 locus via CRISPR/Cas9-mediated non-homologous end joining (NHEJ). This PLIN2-GFP2 reporter line demonstrated synchronous fluorescence and transcriptional expression validated by flow cytometry. Genomic integrity was confirmed by normal diploid karyotype (46, XY). Pluripotency markers (POU5F1, SOX2, NANOG) were stably expressed. Furthermore, the cells possessed the ability to differentiate into three germ layers. As the first reported endogenous PLIN2 reporter in human stem cells, this model overcomes limitations of antibody-based detection and transgenic overexpression systems, preserving native regulatory mechanisms. The model provides a physiologically relevant platform for: (1) live monitoring of LD-mitochondria interactions, (2) high-throughput compound screening for metabolic disorders, and (3) modeling NAFLD pathogenesis in vitro, advancing precision therapeutics and mechanistic disease modeling.},
}
@article {pmid41496283,
year = {2026},
author = {Low, YC and McKnight, CL and Stojanovski, D and Thorburn, DR and Frazier, AE},
title = {Generation of a pluripotent human AGK knockout embryonic stem cell model (WAe009-A-3C) of Sengers syndrome.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103895},
doi = {10.1016/j.scr.2025.103895},
pmid = {41496283},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *Phosphotransferases (Alcohol Group Acceptor)/genetics/metabolism/deficiency ; Cell Line ; CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism ; Gene Knockout Techniques ; *Mitochondrial Diseases/genetics/pathology ; Gene Editing ; Cell Differentiation ; },
abstract = {Sengers syndrome is a rare mitochondrial disorder caused by the loss of a nuclear encoded mitochondrial protein, acylglycerol kinase (AGK). Here, we describe the generation of a novel in vitro stem cell model of Sengers syndrome (AGK[KO] C10) using CRISPR/Cas9 gene editing. This cell line displayed normal characteristics of pluripotent stem cells, including colony morphology, expression of pluripotency markers, trilineage potential, and no karyotypic abnormalities. Together with the parental H9 hESC control line, the AGK[KO] C10 line can ultimately be used for investigation of disease mechanisms and drug testing.},
}
@article {pmid41496278,
year = {2026},
author = {Bozaoglu, K and Massie, S and Irion, FE and Davies, KC and Kantor, I and Raabus, M and Haebich, KM and Vlahos, K and Howden, SE and Wright, J and Payne, JM and Lockhart, PJ},
title = {Simultaneous reprogramming and gene correction to generate six iPSC lines and isogenic controls from individuals with neurofibromatosis type 1.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103904},
doi = {10.1016/j.scr.2025.103904},
pmid = {41496278},
issn = {1876-7753},
mesh = {Humans ; *Neurofibromatosis 1/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cellular Reprogramming ; *Gene Editing ; CRISPR-Cas Systems ; Cell Line ; Male ; Female ; },
abstract = {Neurofibromatosis type 1 (NF1) is a neurodevelopmental disorder that affects ∼ 1: 2700 individuals (Lee et al., 2023) however the underlying pathogenic mechanisms are poorly understood. In this study, we performed simultaneous reprogramming and CRISPR-Cas9 genome editing to generate pluripotent stem cell (iPSCs) lines and their respective isogenic controls from six individuals with different pathogenic NF1 variants. All iPSC lines had a normal karyotype, were pluripotent and able to differentiate into the three embryonic germ layers. These iPSC lines are valuable pre-clinical models to investigate the pathomechanisms of NF1 and can be used for future screening to identify new therapeutic treatments for NF1.},
}
@article {pmid41483428,
year = {2026},
author = {Deng, X and Gao, Q and Shen, K and Mu, W and Ge, T and Gu, J and Yang, X and Cheng, J and Wang, J and Zhang, W and Li, D and Zhou, J and Xiao, M},
title = {TNFRSF13B Variant-Induced TACI Dysregulation Underlies CAEBV Pathogenesis.},
journal = {Journal of clinical immunology},
volume = {46},
number = {1},
pages = {12},
pmid = {41483428},
issn = {1573-2592},
support = {82200259//National Natural Science Foundation of China/ ; 81830008//National Natural Science Foundation of China/ ; 82270203//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Transmembrane Activator and CAML Interactor Protein/genetics/metabolism ; *Epstein-Barr Virus Infections/genetics/immunology/metabolism ; *Herpesvirus 4, Human/physiology/immunology ; Frameshift Mutation ; Signal Transduction ; Exons/genetics ; CRISPR-Cas Systems ; },
abstract = {The tumor necrosis factor (TNF) receptor superfamily member, transmembrane activator and CAML interactor (TACI) encoded by TNFRSF13B, are extensively involved in immune responses. In our previous work, TNFRSF13B exon 2 variants were recurrently identified in chronic active Epstein-Barr virus disease (CAEBV). Here we aim to reveal the roles of TNFRSF13B variants in CAEBV, and investigate the feasibility of targeting TNFRSF13B/TACI as a new approach to control EBV infection. The lymphoblastoid cell lines (LCL) models carrying homozygous TNFRSF13B exon 2 frameshift mutations were constructed using CRISPR/Cas9. Immunological assays, transcriptomic analysis, and gene silencing experiments were performed on LCL models to measure the effect of TNFRSF13B exon 2 variants and explore the underlying mechanisms. TACI ligands and a TLR9 agonist were applied to modulate TACI signaling and EBV activities. Frameshift mutations in exon 2 of TNFRSF13B significantly up-regulated the short isoforms of TACI (TACI-S) at the expense of its long isoforms (TACI-L) in LCLs. The up-regulated TACI-S induced more intense activation of NF-κB, MAPK, and Rho signaling pathways, leading to the switch of EBV activities to lytic reactivation. The subsequent increased viral load and viral IL-10 provide a rational for the susceptibility of variant carriers to CAEBV. The BAFF trimer, an indirect TACI-signaling inhibitor, also significantly suppressed the EBV lytic program. Gene silencing experiments indicated that XBP-1 might be involved in the TACI-mediated regulation of EBV lytic activities in EBV-immortalized B cells. This study underscores the impact of TNFRSF13B variants on EBV infection and host immune responses, offering insights into CAEBV pathogenesis and potential therapeutic strategies.},
}
@article {pmid41478438,
year = {2026},
author = {Ma, Q and Wang, N and Qiao, K and Luo, K and Zhao, C and Yan, J and Fan, S and Rong, J and Ma, Q},
title = {The R2R3-MYB transcription factor GhMYB35 governs anther development and pollen viability in upland cotton.},
journal = {Gene},
volume = {984},
number = {},
pages = {149990},
doi = {10.1016/j.gene.2025.149990},
pmid = {41478438},
issn = {1879-0038},
mesh = {*Gossypium/genetics/growth &amp; development/metabolism ; *Pollen/genetics/growth &amp; development ; *Transcription Factors/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Flowers/genetics/growth &amp; development ; Plant Infertility/genetics ; CRISPR-Cas Systems ; },
abstract = {Male reproductive development is fundamental to the life cycle of flowering plants, culminating in seed production. Aberrations in anther development frequently lead to male sterility, yet the underlying molecular mechanisms in upland cotton (Gossypium hirsutum) remain largely uncharacterized. The R2R3-MYB family of transcription factors are known key regulators of diverse developmental processes, including male fertility in several model species. Here, we identify and functionally characterize GhMYB35, an R2R3-MYB transcription factor that plays an essential role in cotton anther development. CRISPR/Cas9-mediated knockout of GhMYB35 resulted in complete male sterility, with mutants (ghmyb35) exhibiting indehiscent anthers, shorter filaments, and a total absence of viable pollen. Expression analyses reveal that GhMYB35 is predominantly expressed in anthers, with peak expression of its A- and D-subgenome homoeologs occurring at developmental stage 7. Subcellular localization results show that bothGhMYB35_AandGhMYB35_Dare nuclear-localized transcription factors. Furthermore, the total absence of GhMYB35 leads to pollen abortion and subsequent anther collapse without dehiscence. Collectively, our findings establish GhMYB35 as a critical regulator of anther maturation, thereby elucidating a key component of the molecular network governing male fertility in cotton.},
}
@article {pmid41476041,
year = {2025},
author = {Xi, W and Xu, Y and Bao, W and Ding, Z and Xiao, H and Yang, J and Yan, X and Ping, Y},
title = {In vivo chemogenetic RNA editing of macrophages by bioengineered viruses for sepsis treatment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {927},
pmid = {41476041},
issn = {2041-1723},
support = {82425055//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32261143727//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82504685//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024M762890//China Postdoctoral Science Foundation/ ; 2025T180980//China Postdoctoral Science Foundation/ ; GZB20240672//China Postdoctoral Science Foundation/ ; LMS25H300002//Natural Science Foundation of Zhejiang Province (Zhejiang Provincial Natural Science Foundation)/ ; },
mesh = {*Sepsis/therapy/genetics/immunology ; Animals ; *Macrophages/metabolism ; Mice ; *RNA Editing/genetics ; *Lentivirus/genetics ; Disease Models, Animal ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics/metabolism ; Mice, Inbred C57BL ; Genetic Vectors/genetics ; Humans ; Genetic Therapy/methods ; Bioengineering ; CRISPR-Cas Systems ; Male ; Chemogenetics ; },
abstract = {Sepsis, a life-threatening condition arising from a dysregulated host response to infection, remains a significant clinical challenge with limited therapeutic options. RNA editing presents a promising avenue for modulating gene expression to attenuate the inflammatory cascade characteristic of sepsis. Here, we introduce an approach utilizing chemogenetic activation of CasRx-based RNA editing via bioengineered lentiviruses for the treatment of sepsis. Our strategy involves the targeted delivery of biomineralized lentiviral vectors encoding RNA-editing enzymes and chemogenetic switches to M1 macrophage populations implicated in sepsis pathogenesis. Upon the administration of a small molecule ligand, the chemogenetic switches activate the RNA-editing tool, CasRx, thereby enabling precise and repeated downregulation of NLRP3 mRNA. We demonstrate the efficacy and repeatability of this viral-based approach in mouse models of sepsis, highlighting its potential as a versatile therapeutic strategy for mitigating sepsis-induced inflammation. This study underscores the utility of chemogenetic technologies in harnessing the power of RNA editing for the treatment of sepsis and other inflammatory disorders.},
}
@article {pmid41422102,
year = {2025},
author = {Zhong, C and Yu, L and Zhao, T and Shen, X and Li, Z and Zhu, W and Hu, Z and Tian, R and Han, Z and He, D},
title = {A Plug-in system for reprogramming the editing patterns of base editors.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {910},
pmid = {41422102},
issn = {2041-1723},
support = {32171465//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32500460//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023M744121//China Postdoctoral Science Foundation/ ; 2023M734090//China Postdoctoral Science Foundation/ ; 2023M734091//China Postdoctoral Science Foundation/ ; },
mesh = {*Gene Editing/methods ; Zebrafish/genetics/embryology ; Animals ; Humans ; CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; DNA/genetics/metabolism ; HEK293 Cells ; },
abstract = {DNA base editors are transformative genome editing tools that enable nucleotide conversions without inducing double-stranded DNA breaks, making them promising for correcting genetic mutations. Current base editors, however, are limited by fixed editing windows and constrained location of deaminases. To address these constraints, we develop a modular system termed Plug-in Base Editor (Plug-in BE), which dynamically programs deaminase positioning via integrating various epitopes and antibody-fused deaminases. This system expands the editing capabilities of base editors by optimizing deaminase's spatial interaction with DNA, leading to improvements in efficiencies, window restrictions, and safety profiles. We validate Plug-in BE's versatility and high fidelity in cancer gene therapy and zebrafish embryo editing, demonstrating its potential as a powerful and adaptable tool for basic research and therapeutic applications. This innovation can generate a series of base editors without extensive protein evolution, positioning Plug-in BE as a significant advancement in the field of genome editing.},
}
@article {pmid41421670,
year = {2026},
author = {Fan, X and Li, B and Xu, X and Long, B and Jia, Z and Wang, R and Gao, J and Chen, Y and Peng, M and Zhou, M},
title = {Deciphering the regulatory role of the pfs gene on biofilm formation in Lactobacillus plantarum R: Insights from transcriptome and metabolome.},
journal = {Bioresource technology},
volume = {443},
number = {},
pages = {133833},
doi = {10.1016/j.biortech.2025.133833},
pmid = {41421670},
issn = {1873-2976},
mesh = {*Lactiplantibacillus plantarum/genetics/physiology/metabolism ; *Biofilms/growth &amp; development ; *Transcriptome/genetics ; *Metabolome/genetics ; Gene Expression Regulation, Bacterial ; *Bacterial Proteins/genetics/metabolism ; Quorum Sensing/genetics ; *Genes, Bacterial/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Lactobacillus plantarum is a widely recognized probiotic that forms biofilms to enhance environmental tolerance and probiotic properties, but the mechanisms regulating its biofilm formation remain unclear. This study successfully used CRISPR-Cas9 to delete the pfs gene in the high biofilm-producing strain L. plantarum R, and first investigated its role by integrated transcriptomic and metabolomic analyses. The pfs gene participates in the activated methyl cycle and AI-2 synthesis, which is involved in quorum sensing and biofilm formation. Deletion of pfs increased biofilm biomass by 91% and markedly enhanced matrix accumulation, including exopolysaccharides, extracellular proteins and extracellular DNA (eDNA). Transcriptomic analysis revealed significant perturbation of cysteine and methionine metabolism and altered expression of key genes involved in exopolysaccharide synthesis. Metabolomic profiling identified 223 differentially expressed metabolites, primarily associated with carbon flux and EPS precursor pathways. In summary, pfs deletion enhances biofilm formation via metabolic reprogramming rather than classical AI-2 dependent QS pathways. This study provides new insights into the pfs gene-mediated regulation of biofilm formation in L. plantarum and establishes a foundation for future strategies to manipulate biofilm formation in industrial applications.},
}
@article {pmid41419745,
year = {2025},
author = {Ying, Q and Chen, Y and Shen, L and Xu, Y and Tian, R},
title = {SPLiCR-seq: A CRISPR-Based Screening Platform for RNA splicing Identifies Novel Regulators of IRE1α-XBP1 Signaling Under ER Stress.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {911},
pmid = {41419745},
issn = {2041-1723},
support = {82171416//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*X-Box Binding Protein 1/metabolism/genetics ; *Endoribonucleases/metabolism/genetics ; *Protein Serine-Threonine Kinases/metabolism/genetics ; *RNA Splicing/genetics ; Humans ; Signal Transduction/genetics ; Animals ; *Endoplasmic Reticulum Stress/genetics ; Protein Phosphatase 1/metabolism/genetics/antagonists &amp; inhibitors ; Unfolded Protein Response/genetics ; Mice ; CRISPR-Cas Systems ; HEK293 Cells ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {RNA splicing is fundamental to cellular function, yet systematic investigation of its complex regulation has been limited by existing methods. Here, we present SPLiCR-seq (SPLicing regulator identification through CRISPR screening), a high-throughput CRISPR screening platform that enables direct measurement of RNA splicing outcomes for pooled genetic perturbations, overcoming limitations of traditional fluorescence-based approaches. Applying SPLiCR-seq to investigate XBP1 splicing during the unfolded protein response (UPR), we conduct targeted and genome-wide screens across diverse cellular contexts, revealing both common and cell-type specific regulators. Notably, we identify GADD34 (PPP1R15A) as a novel modulator of IRE1α-XBP1 signaling, demonstrating that it directly interacts with IRE1α and functions independently of its canonical role in eIF2α dephosphorylation. Pharmacological inhibition of GADD34 using Sephin1 effectively suppressed XBP1 splicing and alleviated CAR-T cell exhaustion in an ex vivo model, leading to enhanced tumor-killing capacity across multiple cancer models. This work not only establishes a powerful new tool for systematically studying RNA splicing regulation but also uncovers a promising therapeutic strategy for improving CAR-T cell immunotherapy through modulation of the IRE1α-XBP1 pathway.},
}
@article {pmid41418729,
year = {2026},
author = {Wang, Y and Zheng, W and Qiu, B and Chen, Q and Yang, T and Zhou, S and Liu, J and Yang, B},
title = {Generation of a human embryonic stem cell line (SMUDHe010-A-3F) with Cas9 expression cassette integrated at the AAVS1 locus via CRISPR/Cas9-mediated homologous recombination.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103882},
doi = {10.1016/j.scr.2025.103882},
pmid = {41418729},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Homologous Recombination/genetics ; Cell Line ; Gene Editing ; Genetic Loci ; Cell Differentiation ; CRISPR-Associated Protein 9/metabolism ; },
abstract = {Cas9, an RNA-guided nuclease, enables precise genome editing by recognizing sgRNA-complementary sequences and cleaving target DNA. In this study, we used CRISPR/Cas9-mediated homologous recombination to integrate a loxP-flanked STOP cassette-controlled Cas9 expression framework (LSL-Cas9) into the AAVS1 safe-harbor locus of human embryonic stem cells. The resulting cell line, SMUDHe010-A-3F, allows Cre-dependent activation of Cas9 but remains inactive in the absence of Cre recombinase. Karyotype and tri-lineage differentiation confirmed genomic stability and pluripotency. This line provides a valuable platform for organoid gene editing and studies of human development and disease.},
}
@article {pmid41406514,
year = {2026},
author = {Ghodrat, R and Ramachandran, H and Hildebrandt, B and Binder, S and Rossi, A and Reichert, AS},
title = {CRISPR/Cpf1-mediated editing of PINK1 in induced pluripotent stem cells.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103887},
doi = {10.1016/j.scr.2025.103887},
pmid = {41406514},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Protein Kinases/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; },
abstract = {The PTEN induced kinase 1 (PINK1) gene is crucial for mitophagy and mitochondrial quality control. Mutations in the PINK1 gene are associated with several neurological disorders. To decipher the role of PINK1-mediated mitophagy in human induced pluripotent stem cells (hiPSCs) and in their differentiated counterparts, we used CRISPR/Cpf1 and generated a human iPSC line with homozygous out-of-frame deletions by targeting exon 6 of the PINK1 gene. The generated homozygous PINK1 mutant cell line showed normal cell morphology, genomic stability, and expression of classical stem cell markers. Furthermore, the cells can be differentiated efficiently into the three germ layers.},
}
@article {pmid41398410,
year = {2025},
author = {Chi, H and Hoikkala, V and McMahon, S and Graham, S and Gloster, T and White, MF},
title = {Structure and mechanism of the broad spectrum CRISPR-associated ring nuclease Crn4.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {889},
pmid = {41398410},
issn = {2041-1723},
support = {101018608//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {Crystallography, X-Ray ; *CRISPR-Cas Systems ; Adenine Nucleotides/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Models, Molecular ; *Endonucleases/metabolism/chemistry/genetics ; Bacteriophages/genetics ; Nucleotides, Cyclic/metabolism ; Oligoribonucleotides/metabolism ; },
abstract = {Type III CRISPR systems detect the presence of RNA from mobile genetic elements (MGE) in prokaryotes, providing antiviral immunity. On activation, the catalytic Cas10 subunit conjugates ATP to form cyclic oligoadenylate (cOA) signalling molecules that activate ancillary effectors, providing an immune response. Cellular ring nucleases degrade cOA to reset the system. Here, we describe the structure and mechanism of a new family of ring nucleases, Crn4, associated with type III-D CRISPR systems. The crystal structure of Crn4 reveals a small homodimeric protein with a fold unrelated to any known ring nuclease or, indeed, any known protein structure. Crn4 degrades a wide range of cOA species to linear oligoadenylates in vitro and ameliorates type III CRISPR immunity in vivo. Phage and plasmids also encode Crn4 orthologues that may function as anti-CRISPRs. These observations expand our understanding of ring nucleases and reveal a new protein fold for cyclic nucleotide recognition.},
}
@article {pmid41385899,
year = {2026},
author = {Fathy, K and Bharti, J and Khan Sony, S and Nehra, M and Kaul, R and Rawat, B and Sopory, SK and Agrawal, PK and Prakash, A and Kaul, T},
title = {Triumphing over hidden hunger: Redesigning rice (Oryza sativa L.) for enhanced nutraceutical grain composition utilizing multiplexed genome editing.},
journal = {Journal of plant physiology},
volume = {316},
number = {},
pages = {154667},
doi = {10.1016/j.jplph.2025.154667},
pmid = {41385899},
issn = {1618-1328},
mesh = {*Oryza/genetics/metabolism/chemistry ; *Gene Editing/methods ; *Edible Grain/chemistry/genetics ; Cadmium/metabolism/analysis ; *Dietary Supplements ; Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; Plant Breeding ; Zinc/metabolism ; Iron/metabolism ; },
abstract = {Rice, a staple food crop, is consumed by most of the world's population. Micronutrient malnutrition is a severe health issue, leading to diseases such as cancer, anemia, diabetes, heart disease, and disorders in physical and psychological development. We aimed to create rice with low cadmium in the grain but having high cadmium in shoots, safe biofortified protein, high iron, and zinc using CRISPR/Cas9 and breeding technologies instead of adding drugs. The triple gene Knockout rice lines for two iron sensors and one negative regulator gene for cadmium were created to offer high Fe/Zn and low Cd content for breeders. Multiplexed gene editing mediated biolistic transformation of rice callus, and genotyping was used to check the genetic stability of the edited rice lines. Rice lines were found to have enhanced iron, zinc, and protein content, with concentrations varying based on growth conditions. These lines can be used as phytoremediators for cadmium by storing Cd on plant shoots. The rice-edited plants possessed excellent agro-morphological traits, photosynthetic, and physiological performance. The developed edited indica rice lines have crucial agronomic traits with more nutritional value. Compared to the other lines and the wild wildtype, the genome-edited free Cas9 line 2 showed better traits: 13.48 μg/g (iron), 22.9 μg/g (zinc), and a high protein content, which depends on how bioavailable metals and nutrients are in the soil. The line also had 20.60 g of seeds per 1000 g of plant, a total plant yield of 102.76 g, and 101 days of 50 % flowering. This work offers efficient and precise multiple gene-editing in rice with an effective, sustainable strategy for multi-trait enhancement. The developed lines could be used in breeding programs for sustainable solutions for malnutrition worldwide. The experimental results can provide reference and support for the safe use of edited crops as a diet.},
}
@article {pmid41285723,
year = {2025},
author = {Morshedi Rad, D and Richards, C and Zhand, S and de Alwis, N and Hannan, NJ and Faiz, A and McClements, L and Ebrahimi Warkiani, M},
title = {CRISPR/Cas9-mediated gene editing in trophoblast cells via mechanoporation for preeclampsia insight.},
journal = {Cell death &amp; disease},
volume = {17},
number = {1},
pages = {61},
pmid = {41285723},
issn = {2041-4889},
support = {106628//National Heart Foundation of Australia (Heart Foundation)/ ; DP200101860//Department of Education and Training | Australian Research Council (ARC)/ ; 2021CDF1148//Cancer Institute NSW (Cancer Institute New South Wales)/ ; },
mesh = {*Trophoblasts/metabolism/pathology ; Female ; *Pre-Eclampsia/genetics/pathology/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Pregnancy ; Cell Movement/genetics ; Cell Line ; },
abstract = {Preeclampsia is a severe pregnancy complication marked by impaired trophoblast function and abnormal placental development, leading to significant maternal and fetal morbidity. FK506-binding protein-like (FKBPL) has been identified as a potential biomarker as it is significantly downregulated in early pregnancy stages of women who progress to develop preeclampsia. However, editing the Fkbpl gene in trophoblast cells to create a model of preeclampsia using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is challenging due to inefficient delivery, leading to low editing efficiency and reduced cell viability. To address these challenges, we developed a cost-effective and minimally invasive mechanoporation system using micro-engineered filters to deliver CRISPR/Cas9 plasmid DNA (pDNA) targeting the Fkbpl gene into trophoblast cells. This approach successfully generated cell lines with a 38% knockout (K/O) of Fkbpl expression, significantly reducing cell migration (wildtype (WT): 28.77% ± 4.7 vs. 38% K/O: 4.95% ± 0.8, wound closure, **p < 0.01) and proliferation (WT: 1.26 ± 0.06 vs. 38% K/O: 0.81 ± 0.01, ****p < 0.0001). Lower Fkbpl-K/O efficiency of 17% showed a similar reduction in cell proliferation as the 38% K/O clone. Although a full Fkbpl-K/O in the ACH-3P first-trimester trophoblast cell line was not achieved, the partial K/O provided valuable insights into Fkbpl's role in trophoblast function relevant to preeclampsia pathogenesis. Moreover, treatment with mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) or MSC-sEVs did not restore migratory capacity in Fkbpl-deficient cells (p = 0.14). MSC-sEVs increased proliferation in WT ACH-3P cells at 1 µg (p < 0.05) and 2 µg (p < 0.01) doses, however, were not effective in either 17% or 38% Fkbpl-K/O clones, suggesting that FKBPL is an important mechanism of MSC-sEV-mediated therapeutic effect in trophoblasts in the context of preeclampsia. This study advances gene-editing techniques in placental biology and proposes new therapeutic strategies and mechanisms for pregnancy-related complications. A Schematic overview of CRISPR/Cas9 plasmid delivery using microfiltroporation compared to gold standard electroporation and lipofection technologies in trophoblast cells. A CRISPR/Cas9 plasmid targeting Fkbpl was delivered to the first trimester trophoblast cell line, ACH-3P. Cells were sorted according to green fluorescence protein (GFP) expression, expanded and assessed for changes in cell function using proliferation and migration assays. B Actual images of the isopore silicon nitride (SiN) microfilters used in this study and diagram of cell membrane dynamics in response to mechanoporation. This figure was created with Biorender.com. CRISPR clustered regularly interspaced short palindromic repeats, EP electroporation, MFP microfiltroporation.},
}
@article {pmid41194485,
year = {2026},
author = {Yang, TT and Zhang, JR and Xie, ZH and Ren, ZL and Zhao, MY and Hu, WJ and Yan, JW and Ni, M},
title = {CRISPR-Cas9-Targeted Nanopore Sequencing for STR Typing.},
journal = {Electrophoresis},
volume = {47},
number = {1},
pages = {57-67},
doi = {10.1002/elps.70051},
pmid = {41194485},
issn = {1522-2683},
support = {2021QY2004//We thank the National Key Research and Development Program of China/ ; 82030058//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Nanopore Sequencing/methods ; *Microsatellite Repeats/genetics ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *DNA Fingerprinting/methods ; Pilot Projects ; *Sequence Analysis, DNA/methods ; DNA/genetics ; },
abstract = {CRISPR-Cas9-targeted sequencing can enrich DNA regions of interest by directing the Cas9 protein to bind and cleave specific DNA sequences via single-guide RNA (sgRNA). It is interesting to explore the efficacy of using CRISPR-Cas9-targeted nanopore sequencing (referred to as Cas9-seq), a polymerase chain reaction (PCR)-free workflow, for forensic short tandem repeats (STR) profiling, and to compare it with the amplification-based approach. In this pilot study, we constructed a Cas9-seq method for profiling seven STR loci, including D18S51, FGA, TPOX, D16S539, vWA, CSF1PO, and TH01. With 3 µg DNA inputs from human NA12878 and 293T cell lines, we achieved 643.45- and 468.34-fold enrichment ratios of the sgRNA-targeted regions by using Cas9-seq, respectively. Compared to nanopore sequencing of PCR amplicon products (amplicon-seq) of the ForenSeq DNA Signature Prep kit, the Cas9-seq reads had an ultralow strand bias. However, surprisingly, Cas9-seq did not show advantages in allele balance and had higher noise in the reads. At the seven STR loci for the two samples, both Cas9-seq and amplicon-seq had three genotyping errors. Additionally, there were no false-positive single-nucleotide polymorphisms (SNPs) introduced by Cas9-seq, whereas amplicon-seq produced three. In sum, we conclude that the PCR-free Cas9-seq might not be favorable for forensic STR genotyping.},
}
@article {pmid40522172,
year = {2025},
author = {Xue, B and Zhang, MX and Bi, XC and Lai, SP and Bie, XT and Dong, Y and Li, JF and Gao, F and Zhang, X and Wang, Y},
title = {An Amygdala-hippocampus Circuit for Endocannabinoid Modulation of Anxiety Avoidance.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {34},
pages = {e05121},
pmid = {40522172},
issn = {2198-3844},
support = {2021ZD0203000 (2021ZD0203002//STI2030-Major/ ; 82090033//National Natural Science Foundation of China/ ; 81830035//National Natural Science Foundation of China/ ; 82003729//National Natural Science Foundation of China/ ; ZR2019ZD35//Natural Science Foundation of Shandong Province/ ; ZR2020QH357//Natural Science Foundation of Shandong Province/ ; 202205AF150068//Zhang Xia Expert Workstation of the Yunnan Province/ ; ZYGD23034//West China Hospital, Sichuan University/ ; },
mesh = {*Amygdala/physiology ; *Hippocampus/physiology ; *Endocannabinoids/metabolism ; *Anxiety/metabolism/physiopathology ; Male ; Animals ; Mice ; Receptor, Cannabinoid, CB1/metabolism ; Mice, Inbred C57BL ; Basolateral Nuclear Complex/physiology ; Neurons/physiology ; Avoidance Learning/physiology ; CRISPR-Cas Systems ; Brain/physiology ; },
abstract = {Recent studies indicate a therapeutic potential of increased brain endocannabinoids (eCBs) in anxiety disorders, but the underlying brain circuits are still elusive. Here, it is observed that optogenetic inhibition and activation of anterior basolateral amygdala (aBLA) - ventral hippocampus (vHPC) glutamatergic projections respectively decrease and increase anxiety avoidance behaviors. Then, the contributions of eCBs in aBLA-vHPC projections to anxiety avoidance are investigated by employing three newly developed synapse- and circuit-specific eCB-targeted viral strategies to achieve real-time monitoring of eCB release, in vivo optogenetic activation of CB1 receptors, and CRISPR-Cas9 gene knockdown of eCB biosynthesis enzymes. Prominent eCB release are surprisingly found at aBLA-vHPC glutamatergic synapses during anxiety avoidance, suggesting inhibitory effects of increased eCBs in aBLA-vHPC projections on anxiety avoidance. This idea is further supported by findings that specific activation of CB1 receptors at aBLA-vHPC synapses inhibit presynaptic glutamate release and reduce anxiety avoidance. In contrast, specific knockdown of eCB biosynthesis enzymes at aBLA-vHPC synapses reduce eCB levels at aBLA-vHPC glutamatergic synapses and increase anxiety avoidance. Additionally, inhibition of aBLA-innervated vHPC glutamatergic neurons alleviates anxiety avoidance. Together, these findings reveal counteracting effects of increased eCB signaling in aBLA-vHPC circuits on anxiety avoidance.},
}
@article {pmid40434188,
year = {2026},
author = {Wu, F and Li, N and Xiao, Y and Palanki, R and Yamagata, H and Mitchell, MJ and Han, X},
title = {Lipid Nanoparticles for Delivery of CRISPR Gene Editing Components.},
journal = {Small methods},
volume = {10},
number = {2},
pages = {e2401632},
pmid = {40434188},
issn = {2366-9608},
support = {XDB0570000//Strategic Priority Research Program of the Chinese Academy of Science/ ; 32471401//National Natural Science Foundation of China/ ; ssIII-2024B01//Shanghai Sci-Tech Inno Center for Infection &amp; Immunity/ ; },
mesh = {*Gene Editing/methods ; *Nanoparticles/chemistry ; Humans ; *Lipids/chemistry ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Liposomes ; },
abstract = {Gene editing has emerged as a promising therapeutic option for treating genetic diseases. However, a central challenge in the field is the safe and efficient delivery of these large editing tools, especially in vivo. Lipid nanoparticles (LNPs) are attractive nonviral vectors due to their low immunogenicity and high delivery efficiency. To maximize editing efficiency, LNPs should efficiently protect gene editing components against multiple biological barriers and release them into the cytoplasm of target cells. In this review, the widely used CRISPR gene editing systems are first overviewed. Then, each component of LNPs, as well as their effects on delivery, are systematically discussed. Following this, the current LNP engineering strategies to achieve non-liver targeting are summarized. Finally, preclinical and clinical applications of LNPs for in vivo genome editing are highlighted, and perspectives for the future development of LNPs are provided.},
}
@article {pmid41553913,
year = {2026},
author = {Garnica, M and San Martin-Uriz, P and Rodriguez-Marquez, P and Calleja-Cervantes, ME and Rodriguez-Diaz, S and Martinez-Turrillas, R and Hernaez, M and Prosper, F and Rodriguez-Madoz, JR},
title = {Improving CRISPR-Cas9 Screens in CAR T Cells: A Refined Method for Library Preparation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {227},
pages = {},
doi = {10.3791/69721},
pmid = {41553913},
issn = {1940-087X},
mesh = {Humans ; *CRISPR-Cas Systems ; *T-Lymphocytes/immunology ; *Receptors, Chimeric Antigen/genetics/immunology ; *Gene Library ; Immunotherapy, Adoptive/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Chimeric antigen receptor (CAR) T cell therapies have demonstrated remarkable efficacy in several hematological malignancies, yet their success has not been fully replicated in solid tumors. Moreover, even in hematological cancers, relapse after CAR T cell infusion continues to compromise long-term outcomes. These challenges highlight the urgent need to develop strategies that enhance CAR T cell efficacy, persistence, overcoming tumor and microenvironment-mediated resistance. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based screening platforms provide a powerful approach to systematically identify genes that regulate CAR T cell function. By linking genetic perturbations to phenotypic outcomes, these assays enable the discovery of pathways controlling activation, proliferation, memory formation, and cytotoxicity. Standard workflows involve transduction of substantial numbers of cells with a single guide RNA (sgRNA) library, Cas9-mediated editing, selection of edited cells, and PCR amplification of sgRNA cassettes from genomic DNA (gDNA) prior to sequencing. However, PCR amplification using large amounts of gDNA poses significant challenges and often fails to selectively amplify and retrieve sgRNAs. Here, we describe an optimized CRISPR-Cas9 knockout screening protocol, which we have tested on primary human CAR T cells. The method here incorporates an intermediate step during sgRNA library preparation that reduces gDNA carryover through enzymatic digestion and selective pulldown of the sgRNA cassette, thereby increasing the efficiency of the first PCR amplification. This modification allowed us to retrieve sgRNA information across our CAR T cell screens, which had remained elusive in our previous attempts using traditional 1 and 2-step PCR amplification protocols. In conclusion, this optimized workflow facilitates CRISPR screening library preparation in challenging samples and enables the identification of key genetic determinants that can be targeted to improve therapeutic efficacy.},
}
@article {pmid41551929,
year = {2026},
author = {Mir-Pedrol, J and Kuhlburger, L and Sanvicente-García, M and Yazar, M and Ryan, CJ and Krakau, S and Gabernet, G and Güell, M and Bonfanti, M and Nf-Core Community, and Nahnsen, S},
title = {nf-core/crisprseq: a versatile pipeline for comprehensive analysis of CRISPR gene editing and screening assays.},
journal = {NAR genomics and bioinformatics},
volume = {8},
number = {1},
pages = {lqaf214},
pmid = {41551929},
issn = {2631-9268},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Software ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {In recent years, CRISPR technology has become widely applied in scientific research, being simpler, cheaper, and more precise than previous gene-editing techniques. This editing technology can be used for various applications, such as gene knockout, gene knock-in, CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), CRISPR screens, base editing, and prime editing. The share of pipelines to analyze the variety of CRISPR editing methods is low, and until now, none of them caters to both gene editing and CRISPR-based functional genomics. Here, we introduce nf-core/crisprseq, a Nextflow DSL2 pipeline for the assessment of CRISPR gene editing and screening assays. The workflow is written in a modularized fashion to allow the easy incorporation of new steps. nf-core/crisprseq is the first generic pipeline enabling the analysis of the broad spectrum of CRISPR designs. We show the performance and usability of the software using publicly available datasets.},
}
@article {pmid41550651,
year = {2025},
author = {Yan, W and Weng, X},
title = {Targeted RNA base editing for therapeutic: mechanisms and advances.},
journal = {Pharmaceutical science advances},
volume = {3},
number = {},
pages = {100089},
pmid = {41550651},
issn = {2773-2169},
abstract = {RNA base editing, which enables RNA base modification through effector proteins guided by targeting systems, is a powerful technology to correct disease-associated point mutations. Although overshadowed by CRISPR-based genome editing, RNA editing has seen rapid development in recent years, with significant improvements in efficiency and precision. In this review, we summarize the core components of RNA base editing systems (RNA-targeting systems and effector proteins) and describe major RNA editing types, including A-to-I, C-to-U, A-to-m[6]A/m[6]A-to-A, and U-to-Ψ base editors, along with their research progress. In addition, we systematically summarize the delivery methods of the developed RNA editors and their initial exploration in treating diseases caused by nonsense mutations. Finally, combining the current development status of the RNA editing related field, we reflect on the problems encountered in the current development of the RNA editing field and offer our own insights on the future development direction.},
}
@article {pmid41550334,
year = {2025},
author = {Lin, S and Li, H and Bai, S and Han, X},
title = {Mobile RNAs as systemic signaling beyond boundaries for plant stress resistance.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1712714},
pmid = {41550334},
issn = {1664-462X},
abstract = {Plant mobile RNAs-including small RNAs (miRNAs, siRNAs), mRNAs, lncRNAs, and tRNA fragments-function as systemic signaling molecules that traverse cellular, tissue, and species boundaries to coordinate plant adaptation to environmental stresses. Here, we summarize the critical roles of mobile RNAs in mediating systemic adaptation to abiotic challenges and biotic interactions. Crucially, we highlight the diverse transport mechanisms enabling their movement and discuss the emerging functional versatility of mobile RNAs, which extends beyond transcriptional regulation to encompass epigenetic modifications, resource allocation, and cross-species communication. These fundamental insights are driving transformative applications: Mobile RNAs provide the foundation for developing systemic RNAi-based biopesticides and are being integrated with CRISPR-Cas technologies to overcome delivery barriers and enable heritable, transgene-free genome editing in crops. Understanding and harnessing mobile RNA networks offers unprecedented potential for engineering resilient crops and implementing precise, sustainable crop protection strategies to address global food security challenges.},
}
@article {pmid41549062,
year = {2026},
author = {Mukherjee, P and Benicky, J and Panigrahi, A and Ailles, L and Goldman, R},
title = {SULF1 in Cancer Associated Fibroblasts Promotes Invasion in Head and Neck Cancer Cell Lines.},
journal = {Cancer medicine},
volume = {15},
number = {1},
pages = {e71540},
pmid = {41549062},
issn = {2045-7634},
support = {R01CA238455/GF/NIH HHS/United States ; 2P30CA051008/CA/NCI NIH HHS/United States ; /NH/NIH HHS/United States ; 1S10OD028623-01A1//NIH Office of the Director/ ; S10OD032420//NIH Office of the Director/ ; //George Washington University/ ; //School of Medicine and Health Sciences/ ; },
mesh = {Humans ; *Cancer-Associated Fibroblasts/metabolism/pathology ; *Head and Neck Neoplasms/pathology/genetics/metabolism ; *Sulfotransferases/metabolism/genetics ; Cell Movement ; Neoplasm Invasiveness ; Cell Line, Tumor ; *Squamous Cell Carcinoma of Head and Neck/pathology/genetics/metabolism ; Cell Proliferation ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Gene Expression Regulation, Neoplastic ; },
abstract = {BACKGROUND: Cancer-associated fibroblasts (CAFs) significantly influence tumor behavior in head and neck squamous cell carcinoma (HNSCC) and other malignancies. We identified the extracellular sulfatase SULF1 as a key stromal factor highly expressed in CAFs and associated with poor prognosis.<br><br>METHODS AND RESULTS: Using CRISPR/Cas9-edited SULF1-knockout primary HNSCC CAFs, we demonstrate that loss of SULF1 reduces fibroblast proliferation and markedly impairs cancer cell migration and invasion in&#xa0;vitro. Two-photon microscopy in 3D spheroid cocultures revealed that SULF1-deficient CAFs fail to support invasiveness of Cal33 cells, resulting in spheroids with fewer invasive projections and altered morphology. Proteomic analysis confirmed the absence of SULF1 in the knockout cell cultures and revealed that SULF2, expressed in tumor cells, does not compensate for its loss.<br><br>CONCLUSION: These findings highlight the importance of CAF-derived SULF1 in regulating tumor invasion and suggest that SULF1 is a promising therapeutic target in HNSCC.},
}
@article {pmid41548978,
year = {2026},
author = {Zheng, SH and Liu, Y and Xia, XX and Liu, YM},
title = {Advances in base editing technology and the construction of precise zebrafish disease models.},
journal = {Yi chuan = Hereditas},
volume = {48},
number = {1},
pages = {46-60},
doi = {10.16288/j.yczz.25-157},
pmid = {41548978},
issn = {0253-9772},
mesh = {Animals ; *Zebrafish/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Disease Models, Animal ; Humans ; },
abstract = {Single nucleotide variants (SNVs) are among the primary pathogenic factors of human genetic diseases, accounting for a significant proportion of all mutation types. Conducting in-depth research on the pathogenic significance of these mutations in animal models is essential for understanding disease mechanisms and developing therapeutic strategies. The progress of such research largely depends on the continuous innovation and advancement of gene editing technologies. In recent years, base editing technology based on the CRISPR/Cas9 system has emerged, enabling precise conversion of individual nucleotides. Owing to its efficiency and convenience, base editing has been widely applied in gene therapy, the construction of animal models, and molecular breeding, bringing new breakthroughs and opportunities to life sciences and medical research. Zebrafish, with their advantages of small size, high fecundity, transparent embryos, and external development, have become an ideal model organism for studying disease mechanisms and drug screening. In this review, we summarize the development of CRISPR/Cas9-based base editing technologies, highlight the emergence of novel editing tools, and explore the application and progress of base editing in constructing precise zebrafish disease models.},
}
@article {pmid41546796,
year = {2026},
author = {Borgohain, T and Suma, R and Muttappagol, M and Saikia, B and Keithellakpam, A and Laskar, A and Hiremath, SS and Basu, U and Velmurugan, N and Palakolanu, SR and Chikkaputtaiah, C},
title = {Precision breeding in a changing climate: unlocking resilience through omics and gene editing.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {26},
pmid = {41546796},
issn = {1438-7948},
support = {31/0025 (15358) 2022-EMR-I//Human Resource Development Centre, Council of Scientific And Industrial Research/ ; DST/WISE-PDF/LS-112/2024(G)//Department of Science and Technology, Ministry of Science and Technology, India/ ; MMP025301//Council of Scientific and Industrial Research, India/ ; },
mesh = {*Gene Editing/methods ; *Plant Breeding/methods ; *Climate Change ; *Crops, Agricultural/genetics/growth &amp; development ; *Genomics ; Genome-Wide Association Study ; },
abstract = {Climate change, rising global food demand, and shrinking resources require transformative innovations in crop breeding. This review outlines recent advances in new breeding technologies (NBTs), including molecular markers, genome-wide association studies (GWAS), genomic selection (GS), next-generation sequencing (NGS), and gene editing (GE) tools such as the clustered regularly interspaced short palindromic repeat (CRISPR/Cas), base editing, and prime editing. These methods enable the accurate improvement of traits, thereby accelerating the development of crops resistant to both abiotic and biotic stresses. The integration of multi-omics platforms, including genomics, transcriptomics, proteomics, metabolomics, and phenomics, provides a comprehensive framework for deciphering and manipulating complex trait architectures. Artificial intelligence (AI) and machine learning (ML) enhance precision breeding by providing data-driven insights and enabling the forecasting of traits. Emphasis is also placed on combining gene editing with other strategies, such as speed breeding, to accelerate the development of traits. This review underscores the importance of an integrated systems biology approach that combines multi-omics, gene editing, AI, and speed breeding to accelerate the development of climate-resilient, high-yielding, and nutritionally enhanced crops. The integration of these innovative technologies holds great promise for addressing global food security, environmental sustainability, and agricultural resilience in the face of climate change. A strategic framework for the future of plant breeding is outlined, emphasizing the importance of interdisciplinary collaboration in building a sustainable agricultural future.},
}
@article {pmid41543903,
year = {2026},
author = {Yin, S and Jarosz, DF and Ting, AY},
title = {Towards CRISPR-based editing of the mitochondrial genome in yeast.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {3},
pages = {e2505894123},
pmid = {41543903},
issn = {1091-6490},
support = {NA//Stanford University (SU)/ ; NA//Chan Zuckerberg Initiative (CZI)/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Gene Editing/methods ; *Genome, Mitochondrial/genetics ; *CRISPR-Cas Systems/genetics ; Mitochondria/genetics/metabolism ; DNA, Mitochondrial/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Mitochondria, which evolved from symbiotic bacteria, possess their own genomes (mtDNA) and support independent transcription and translation within the organelle. Given the essential role of mtDNA in energy production, metabolism, as well as cellular homeostasis, and the high density of confirmed pathogenic mutations that map to mtDNA, there is a pressing need for versatile methods to study and manipulate this genome. Although CRISPR technology has revolutionized the editing of nuclear genomes, it has not been successfully extended to mtDNA, primarily due to the challenge of delivering single guide RNAs (sgRNAs) across both outer and inner mitochondrial membranes. Here we develop a survival-based reporter in Saccharomyces cerevisiae to screen for potential RNA import motifs. We identify a 40-nucleotide aptamer (IM83) that facilitates sgRNA entry into the mitochondrial matrix, enabling CRISPR editing by a mitochondrially-localized adenine base editor. We show that mitochondrial import of IM83 is ATP-dependent and enhanced by the tRNA synthetase Msk1. Further investigations identify barriers to efficient CRISPR editing of mtDNA, including loss of membrane potential associated with mitochondrial targeting of the base editor. These insights lay the groundwork for future improvements in CRISPR-based editing of mtDNA in eukaryotes.},
}
@article {pmid41543528,
year = {2026},
author = {O'Connor-Moneley, J and Lange, T and Flanagan, PR and Brunke, S and Fischer, L and Sharma, R and Puri, S and Hube, B and Sullivan, DJ and Moran, GP},
title = {Depletion of the Candida albicans TLO gene family reveals a requirement for alpha TLO genes for wild-type virulence.},
journal = {Microbiology (Reading, England)},
volume = {172},
number = {1},
pages = {},
doi = {10.1099/mic.0.001654},
pmid = {41543528},
issn = {1465-2080},
mesh = {*Candida albicans/genetics/pathogenicity/growth &amp; development ; Animals ; Virulence/genetics ; Mice ; *Fungal Proteins/genetics/metabolism ; Hyphae/growth &amp; development/genetics ; Disease Models, Animal ; Candidiasis, Oral/microbiology ; Humans ; Macrophages/microbiology ; Female ; Gene Expression Regulation, Fungal ; CRISPR-Cas Systems ; *Mediator Complex/genetics/metabolism ; Multigene Family ; },
abstract = {Candida albicans uniquely possesses an expanded family of genes (the TLO gene family) that encodes 10-15 paralogues of the Med2 component of the transcriptional regulator Mediator. Previous studies have shown that TLO null mutants are unable to form hyphae and are hypersensitive to environmental stress. However, the reason for the TLO gene expansion remains unclear, and the current study aimed to determine if reduction in the TLO family copy number affected virulence. In order to investigate this, we used CRISPR-Cas9 mutagenesis to generate two TLO-depleted mutants: one mutant retaining only TLOβ2 (CaTLO2) and the second mutant containing only TLOγ5 (CaTLO5). Both TLO-depleted mutants exhibited increased filamentous growth, increased susceptibility to specific stresses and reduced virulence in a murine model of oropharyngeal candidiasis (OPC). In vitro, the CaTLO5 mutant also exhibited impaired hyphal escape from macrophages and reduced hyphal invasion of oral keratinocytes. We then investigated if complementation with TLOα1, a gene previously shown to restore wild-type growth in a Δtlo null mutant, could restore virulence. In vitro infection models showed that TLOα1 could restore true hypha formation, epithelial invasion and hyphal escape from macrophages in the CaTLO5 background. The murine OPC model showed that TLOα1 could restore wild-type virulence in both CaTLO2 and CaTLO5 strains, suggesting an essential role for α-TLO in oral mucosal infection. Together, these findings highlight the functional specialization between the α, β and γ TLO gene groups and establish α-TLO as a major regulator of virulence in C. albicans.},
}
@article {pmid41543272,
year = {2026},
author = {Lummerstorfer, M and Lächelt, U},
title = {Non-Viral CRISPR carriers: transient delivery with lasting effects.},
journal = {Drug delivery},
volume = {33},
number = {1},
pages = {2614125},
pmid = {41543272},
issn = {1521-0464},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Gene Transfer Techniques ; Animals ; Genetic Vectors ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR-Cas9 has revolutionized the field of genome editing. While conventional gene supplementation therapies and the market of related gene therapy products are dominated by viral vectors, non-viral delivery strategies are increasingly being explored for in vivo CRISPR applications. Given the permanent nature of genome editing, prolonged expression of the CRISPR machinery is not required, and transient delivery nevertheless can achieve lasting therapeutic effects. In contrast, short-term availability of genome editing components is rather considered advantageous to reduce the risk of off-target effects in a 'hit-and-run' fashion. In this article, we provide a systematic survey of the current clinical trial landscape with focus on in vivo CRISPR therapies and discuss utilized delivery strategies. As of December 2025, 136 CRISPR trials are ongoing, including 36 based on in vivo delivery of CRISPR components which show a clear shift towards non-viral vectors. The article describes the clinically employed CRISPR technologies and non-viral delivery platforms, highlighting both the present opportunities and key challenges associated with CRISPR delivery in the future.},
}
@article {pmid41414648,
year = {2026},
author = {Wang, LR and Zhu, ST and Liao, ZH and Wu, N and Nie, ZK and Ye, C and Shi, TQ},
title = {Establishing a CRISPR/Cas9 Genome Editing System Combined with URA3-Blaster in Botrytis cinerea for Enhanced Abscisic Acid Production.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {2},
pages = {2207-2217},
doi = {10.1021/acs.jafc.5c14153},
pmid = {41414648},
issn = {1520-5118},
mesh = {*Botrytis/genetics/metabolism ; *Abscisic Acid/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Metabolic Engineering ; *Fungal Proteins/genetics/metabolism ; },
abstract = {Abscisic acid (ABA) is a key plant growth regulator widely used in agriculture and ecological restoration. Although metabolic engineering of the fungus Botrytis cinerea can enhance ABA production, it has been hindered by inefficient genetic tools. In this study, we first established a recyclable selection marker system in B. cinerea based on orotidine-5'-phosphate decarboxylase. Subsequently, the CRISPR/Cas9 system was optimized, achieving up to 100% editing efficiency, far surpassing traditional homologous recombination. Based on this platform, multiple metabolic engineering strategies were systematically explored to enhance ABA biosynthesis. Increasing acetyl-CoA supply, inhibiting squalene synthesis, and knocking out key secondary metabolism genes Bcpks12 and Bcphs1 all significantly promoted ABA accumulation. Notably, co-overexpression of Bcacly1 and Bcacly2 combined with 1 g/L citrate increased ABA production to 1.36 g/L, representing a 38.66% improvement. Overall, this study provides an efficient genetic toolkit and a solid foundation for the industrial-scale production of ABA via engineered B. cinerea.},
}
@article {pmid41397984,
year = {2025},
author = {Chen, J and Hu, L and Vernuccio, R and Shi, N and Tian, J and Zhang, Y and Tian, S and Cao, X and Ha, Z and Lu, J and Battini, L and Raynal, B and Haouz, A and Xue, J and Cai, Q and Zhao, Y and Lu, Y and Smith, GL and Xie, Y and Lu, H and Guardado-Calvo, P and Zhang, P and Zhang, R},
title = {Development of a replication-defective mpox virus platform for fundamental and therapeutic research.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {798},
pmid = {41397984},
issn = {2041-1723},
mesh = {*Virus Replication/drug effects/genetics ; Humans ; *Antiviral Agents/pharmacology/chemistry ; Animals ; CRISPR-Cas Systems ; Chromosomes, Artificial, Bacterial/genetics ; Genome, Viral ; *Orthopoxvirus/genetics/drug effects/physiology ; Virion/genetics/drug effects ; *Defective Viruses/genetics/drug effects ; Virus Assembly/drug effects/genetics ; },
abstract = {The recent global outbreaks of mpox highlight the urgent need for both fundamental research and antiviral development. However, studying the mpox virus (MPXV), with its large and complex genome, remains challenging due to the requirement for high-containment facilities. Here, we describe a strategy for de novo assembly of MPXV clade IIb genomes in bacterial artificial chromosomes using transformation-associated recombination cloning. Leveraging CRISPR-Cas9 and Lambda Red recombination, we engineer replication-defective MPXV particles with dual deletions of OPG96 (M2R) and OPG158 (A32.5 L)-genes essential for virion assembly, that are capable of recapitulating key stages of the viral life cycle. We apply this system to screen a compound library and identify G243-1720, a potent anti-poxvirus inhibitor with broad activity in vitro and in vivo. G243-1720 blocks the formation of extracellular enveloped virions and cell-cell spread. Resistance mutation selection, crystallographic analysis, analytical ultracentrifugation, and mass photometry reveal that, despite its distinct chemical structure, G243-1720 shares a mode of action with tecovirimat, both functioning by affecting dimerization of protein OPG57 (F13). Our findings underscore the potential of G243-1720 as a promising broad-spectrum anti-poxvirus lead compound and demonstrate the utility of replication-defective MPXV particles as a reliable platform for viral biology studies and antiviral development.},
}
@article {pmid41365122,
year = {2026},
author = {Kim, WN and Kim, HU},
title = {Precise DGAT1 base editing and in-frame deletion reveal motif-specific regulation of seed oil biosynthesis in Arabidopsis.},
journal = {Plant physiology and biochemistry : PPB},
volume = {230},
number = {},
pages = {110861},
doi = {10.1016/j.plaphy.2025.110861},
pmid = {41365122},
issn = {1873-2690},
mesh = {*Arabidopsis/genetics/metabolism ; *Diacylglycerol O-Acyltransferase/genetics/metabolism ; *Seeds/metabolism/genetics ; *Arabidopsis Proteins/genetics/metabolism ; *Gene Editing ; *Plant Oils/metabolism ; Triglycerides/biosynthesis ; *Sequence Deletion ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in triacylglycerol (TAG) biosynthesis and is a key determinant of seed oil content and composition. To dissect the functional contribution of the conserved DGAT1 domains, we employed adenine and cytosine base editors and CRISPR/Cas9-mediated in-frame deletion to generate targeted alleles in Arabidopsis thaliana. A total of 25 single guide RNAs were designed to introduce precise nucleotide substitutions across functional domains, and the edited lines were screened using seed fluorescence and Sanger sequencing. Five base-edited (BE) DGAT1 mutants affecting acyl-CoA/CoA allosteric binding site (S124F, S123R/S124L), thiolase acyl-enzyme intermediate signature motif (L229P), diacylglycerol (DAG)-binding motif (W416C/R419Q, V418I), and an in-frame deletion in the intrinsically disordered N-terminal region (Δ49-76H) were characterized. Amino acid substitutions in the conserved domains led to distinct shifts in seed fatty acid profiles. Loss-of-function-like mutants (S123R/S124L, L229P, W416C/R419Q) reduced 18:1 and 20:1 levels and increased 18:3 levels, whereas putative gain-of-function mutants (S124F, V418I) enhanced 20:1 incorporation and elevated total oil content. Overexpression of DGAT1[S124F] and DGAT1[V418I] in the dgat1 mutant background further increased seed oil accumulation beyond that achieved with wild-type DGAT1. Structural modeling of DGAT1 proteins revealed the location of substituted amino acids and their interactions with surrounding residues, as well as the absence of putative N-terminal regulatory segment. These results demonstrate that precise base editing can modulate DGAT1 activity and TAG composition by targeting functional motifs, providing insights into the structure-function relationships of this key enzyme and offering strategies for metabolic engineering of seed oils.},
}
@article {pmid41344457,
year = {2026},
author = {Liu, L and Huang, X and Wan, S and Gao, Z and Liu, H},
title = {Ectodysplasin A regulates the development of scale and intermuscular bone in teleosts.},
journal = {International journal of biological macromolecules},
volume = {337},
number = {Pt 2},
pages = {149465},
doi = {10.1016/j.ijbiomac.2025.149465},
pmid = {41344457},
issn = {1879-0003},
mesh = {Animals ; Zebrafish/genetics/growth &amp; development ; *Ectodysplasins/genetics/metabolism ; *Fishes/genetics/growth &amp; development ; *Bone and Bones/metabolism ; Phylogeny ; *Animal Scales/growth &amp; development/metabolism ; Signal Transduction ; Gene Expression Regulation, Developmental ; *Fish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Most bony fish are extensively covered by scales, which play crucial roles in locomotion, balance, and sensory perception. However, the molecular mechanisms underlying fish scales development remain poorly understood. In this study, comparative genomic analyses were performed between scaled and scaleless fish species, leading to the identification of key genes (eda, wnt3a, gsk3bb, etc.) involved in scale formation. Phylogenetic and sequence analyses of the eda gene revealed that the Eda protein in Monopterus albus lacks a transmembrane domain, disrupting Eda/Edar binding and potentially driving scale degeneration. Using CRISPR/Cas9 technology, we generated zebrafish eda[-/-] mutants, which exhibited a complete absence of scales, fin rays, pharyngeal teeth, and gill rakers. Notably, the intermuscular bones in these mutants showed significantly reduced length, and simplified morphology, indicating impaired growth. Furthermore, quantitative PCR (qPCR) analysis demonstrated that eda deficiency disrupts the Eda/Edar/NF-κB signaling pathway. Our findings provide significant insights into the molecular regulatory mechanisms underlying the development of skin appendages (e.g., scales) and intermuscular bones.},
}
@article {pmid41298443,
year = {2025},
author = {Wei, T and Yang, X and Jiang, C and Liao, K and Ye, H and Luo, HY and Liu, YR and Wang, PY and Meng, F and Dou, SX and Rong, Z and Li, H},
title = {Osmotic pressure regulates DNA labelling and transcription with dCas9-SunTag system in live cells.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {773},
pmid = {41298443},
issn = {2041-1723},
support = {12122402, 12074043//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Osmotic Pressure/physiology ; *DNA/metabolism/genetics ; Humans ; *Transcription, Genetic ; Cell Nucleus/metabolism/genetics ; CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/genetics ; Telomere/metabolism/genetics ; HeLa Cells ; },
abstract = {Extracellular osmotic pressure is a key modulator of intracellular biophysical properties and cellular functions. However, its impact on the cell nucleus remains elusive, largely due to the challenges in real-time measurement of local environmental properties and reaction kinetics at specific loci within the nucleus. Here, we employ the dCas9-SunTag system to investigate the biophysical response at target DNA loci to osmotic pressure alterations. We reveal that variations in extracellular osmotic pressure modulate the efficiency of dCas9-SunTag-mediated fluorescent labelling rapidly and reversibly, with hypoosmotic condition increasing and hyperosmotic condition decreasing the number and fluorescence intensity of foci for telomeres and genes. Strikingly, osmotic pressure also regulates gene transcription with the dCas9-SunTag system, mirroring its effects on fluorescent labelling, as evidenced by changes in mRNA burst frequency. The underlying mechanism is that osmotic pressure shifts the binding-unbinding equilibrium of specific proteins to dCas9-SunTag complex by altering intranuclear crowding. These findings not only highlight the role of mechanical cues in modulating DNA-related processes within the nucleus, but also establish the dCas9-SunTag system as a sensitive probe for intranuclear crowding in response to extracellular cues, notably osmotic pressure.},
}
@article {pmid41543170,
year = {2026},
author = {Romanowski, JS and Myles, KM and Adelman, ZN},
title = {Microhomology-mediated end joining is the predominant form of DNA repair in the mosquito Aedes aegypti with implications for gene editing, gene drive, and transgene removal.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41543170},
issn = {1362-4962},
support = {//National Institute of Allergies and Infectious Diseases/ ; AI148787/NH/NIH HHS/United States ; //NIAID/ ; },
mesh = {Animals ; *Aedes/genetics ; *Gene Editing/methods ; *DNA End-Joining Repair ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Transgenes ; Gene Drive Technology ; DNA Repair ; },
abstract = {Programmable site-specific nucleases have revolutionized the field of genetics, and in the field of mosquito vector control, gene editing by these tools has inspired a new wave of population control approaches that aim to prevent disease transmission. Little is known of how DNA repair is prioritized in mosquitoes, which diverged from the nearest model system (Drosophila) by >200 million years, despite site-specific gene editing now being commonplace. Here, we report a scalable, high-throughput platform for studying DNA double-stranded DNA break (DSB) repair in mosquitoes by delivering CRISPR/Cas9, I-SceI, or other nucleases to Aedes aegypti embryos, capable of measuring single-strand annealing (SSA), non-homologous end joining, and microhomology-mediated end-joining (MMEJ) repair outcomes. We find CRISPR/Cas9 can induce deletions of up to 8.6 kb through SSA repair and is tolerant of resection distances of 3.5 kb. Indel events were insensitive to lig4 knockouts, and across 20 synthetic guide RNAs (sgRNAs) representing 5 locations in 2 transgenic strains were almost exclusively attributed to MMEJ repair, establishing MMEJ as the dominant form of repair in A. aegypti at CRISPR/Cas9 DSBs. This information is critical to our understanding of how DNA repair shapes processes required for genetic control strategies involving gene drive action/resistance as well as transgene stability.},
}
@article {pmid41542957,
year = {2026},
author = {Lee, SY and Park, HH},
title = {Structural insights into promoter recognition by Aca7.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.70405},
pmid = {41542957},
issn = {1742-4658},
support = {RS-2025-02316334//NRF/ ; RS-2025-16065724//NRF korea/ ; },
abstract = {CRISPR-Cas systems provide adaptive immunity to bacteria, although bacteriophages counter these defenses with anti-CRISPR (Acr) proteins. Acr expression is frequently regulated by anti-CRISPR associated (Aca) proteins, which repress transcription by binding inverted repeat (IR) sequences in operon promoters. Here, we report the first identification of an IR motif within the AcrIF11-Aca7 operon promoter from Halomonas caseinilytica and present the crystal structure of Aca7 bound to this IR DNA. Biochemical assays demonstrated that Aca7 specifically recognizes the IR element, and structural analysis revealed a symmetric Aca7 dimer engaging both major grooves via helix-turn-helix motifs while stabilizing DNA bending through minor groove contacts. Residue-level interactions, including those mediated by R38, Q42, K46, and K49, establish a detailed basis for sequence-specific recognition. Comparison with Aca2 highlights distinct dimer architectures and DNA deformation strategies among Aca proteins. Our findings uncover the molecular mechanism by which Aca7 represses AcrIF11 expression and broaden the understanding of Aca-mediated transcriptional regulation.},
}
@article {pmid41541975,
year = {2025},
author = {Hummel, L and Carr, C and Biow, S and Asher, K and Sauer, T and Maylin, M},
title = {Re-examining the Diagnostic Criteria for Wilson's Disease: A Case Report and Literature Review.},
journal = {Cureus},
volume = {17},
number = {12},
pages = {e99271},
pmid = {41541975},
issn = {2168-8184},
abstract = {Wilson's disease (WD) is a heterogeneous genetic disorder for which diagnosis is challenging. We present the case of a 54-year-old woman with a complicated medical history, including hepatitis C, cirrhosis, hepatic encephalopathy, and extensive psychiatric disease, who was transferred to our hospital for management of a spinal epidural abscess. Further findings suggested undiagnosed WD, including bilateral rings around Descemet's membrane and modestly low ceruloplasmin but normal urinary copper excretion. Many algorithms have been proposed for Wilson disease diagnosis, including clinical, laboratory, imaging, and genetic findings; however, no single test is diagnostic. The European Association for the Study of the Liver (EASL) guidelines are the most commonly employed algorithm. Updated 2022 guidelines by both the American Association for the Study of Liver Diseases (AASLD) and the British Association for the Study of the Liver (BASL) build upon this algorithm. Our patient scored 5 points on the EASL scale, which is sufficient for diagnosis. Nevertheless, we believe that she met the diagnostic criteria without having the actual disease. Newer guidelines by AASLD and BASL do not provide additional conclusivity. Our case demonstrates the need for re-evaluation of the diagnostic criteria of WD, where uncertainty can mean permanent hepatic and brain damage. Diagnostic guidelines should incorporate new biomarkers, ophthalmological techniques, and advanced technologies such as next-generation sequencing or CRISPR-Cas-based tools.},
}
@article {pmid41499124,
year = {2026},
author = {Wang, K and Wang, W and Hu, Y and Li, X and Yu, H and Xia, X and Kai, T and Wen, M},
title = {A Relay CRISPR/Cas12a System for Extraction-Free and Ultrasensitive Detection of Staphylococcus aureus in Complex Food Samples.},
journal = {Analytical chemistry},
volume = {98},
number = {2},
pages = {1657-1666},
doi = {10.1021/acs.analchem.5c06345},
pmid = {41499124},
issn = {1520-6882},
mesh = {*Staphylococcus aureus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Food Microbiology ; Electrochemical Techniques ; DNA, Bacterial/genetics/analysis ; Limit of Detection ; RNA, Ribosomal, 16S/genetics ; },
abstract = {Staphylococcus aureus (SA) is a predominant foodborne pathogen, frequently associated with nutrient-rich matrices. However, rapid and ultrasensitive detection of SA in complex food samples remains a formidable challenge. In this work, we developed a relay biosensing system for detecting SA-16S rDNA that combines a magnetic bead-anchored APE1 DNA walker with an asymmetric CRISPR/Cas12a system. The magnetic bead-anchored DNA walker enables efficient separation of SA-targets from complex food matrices, while generating abundant activators through APE1-mediated catalytic cleavage. Employing spatially decoupled reactions with differential crRNAs, the asymmetric Cas12a system achieves programmable cascade amplification. Dual-mode fluorescence/electrochemical readout supports both laboratory analysis and on-site detection. The system demonstrates exceptional sensitivity, capable of detecting trace amounts of SA-16S rDNA (7.5 aM by fluorescence, 1.0 fM by electrochemistry) and SA (4 CFU/mL by fluorescence, 34 CFU/mL by electrochemistry). Overall, this work provides a modular and programmable biosensing strategy for bacterial identification, thereby facilitating advanced nucleic acid analysis within complex biological matrices.},
}
@article {pmid41439322,
year = {2026},
author = {Wang, Z and Yang, F and Zeng, S and Sun, R and Hu, Q and Du, Y},
title = {An integrated valved microfluidic platform for rapid and simultaneous nucleic acid detection.},
journal = {Lab on a chip},
volume = {26},
number = {2},
pages = {507-514},
doi = {10.1039/d5lc01096a},
pmid = {41439322},
issn = {1473-0189},
mesh = {Humans ; *Lab-On-A-Chip Devices ; *Nucleic Acid Amplification Techniques/instrumentation ; Human papillomavirus 16/genetics/isolation &amp; purification ; Human papillomavirus 18/genetics/isolation &amp; purification ; *DNA, Viral/analysis/genetics ; *Nucleic Acids/analysis ; *Microfluidic Analytical Techniques/instrumentation ; CRISPR-Cas Systems ; },
abstract = {Applying CRISPR-based diagnostics to point-of-care pathogen detection remains challenging because of the multi-step and time-consuming sample preparation process. This study presents a low-cost, integrated valved microfluidic device that combines recombinase polymerase amplification (RPA), CRISPR signal amplification, and lateral flow readout for simultaneous nucleic acid detection. The core advantage of the platform lies in its ability to sequentially control the entire multi-step assay through simple valve operation, significantly minimizing user intervention. All key reagents, including the RPA mix, Cas12a/crRNA complex, and proteinase K lysis buffer, are pre-lyophilized, ensuring stability and ready-to-use functionality. The platform demonstrates a sensitivity of 20 copies/reaction for HPV16/18 plasmids and accurately genotypes HPV in lysates of cervical cancer cells within one hour, showing complete concordance with quantitative PCR results. This integrated device, achieving a user-friendly protocol and visual readout, provides a powerful tool for nucleic acid-based point-of-care testing and self-testing in resource-limited settings.},
}
@article {pmid41422144,
year = {2025},
author = {Felício, D and Osório, H and Pereira, C and Brandão, AF and Freixo, JP and Carvalho, I and Sousa, AP and Castro-Caldas, M and Sequeiros, J and Lemos, C and Santos, M},
title = {Missense variant in TTBK2 kinase domain causes loss of function and impaired protein phosphorylation.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {2501},
pmid = {41422144},
issn = {2045-2322},
support = {UI/BD/154402/2023//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; UIDB/00215/2020, UIDP/00215/2020, LA/P/0064/2020 , UID/215/2025//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; DL 57/2016 - Norma Transit&#xf3;ria//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125//Rede Nacional de Espectrometria de Massa/ ; ZGRACA//Ataxia UK/ ; },
mesh = {Humans ; Phosphorylation ; *Mutation, Missense ; *Protein Serine-Threonine Kinases/genetics/metabolism/chemistry ; Protein Domains ; CRISPR-Cas Systems ; *Loss of Function Mutation ; HEK293 Cells ; },
abstract = {Tau tubulin kinase 2 (TTBK2) is a ubiquitous serine-threonine protein kinase implicated in diverse cellular processes, including microtubule regulation, ciliogenesis, synaptic signaling, and the phosphorylation of key proteins like TDP-43. Despite its relevance, many aspects of TTBK2 function in both physiological and pathological conditions remain poorly understood. Truncating variants in TTBK2 gene cause spinocerebellar ataxia type 11 (SCA11), a rare form of autosomal dominant cerebellar ataxia. However, the functional consequences and pathogenic potential of missense variants have yet to be elucidated. In this study, we developed a CRISPR/Cas9 knock-in cell model harboring a missense variant in TTBK2 kinase domain (NM_173500.4:c.625 C > T; p.Leu209Phe) to evaluate its impact on TTBK2 expression, associated protein levels, and phosphoproteomic profiles. TTBK2 missense variant (TTBK2-L209F) was associated with reduced TTBK2 protein levels, altered levels of cytoskeleton-related proteins, and impaired kinase activity, namely toward TDP-43. Phosphoproteomic analyses identified dysregulation in pathways linked to gene regulation, protein degradation, cytoskeletal organization, and TGF-β signaling. These findings provide valuable insights into the biological roles of TTBK2 in cellular signaling. Moreover, this study underscores the importance of functional studies to better understand the consequences of TTBK2 missense variants, particularly those affecting the kinase domain, and their potential contribution to disease.},
}
@article {pmid41388295,
year = {2025},
author = {Ji, T and Zhang, Y and Wang, Y and Yuan, K and Wang, M and Ye, J and Zhang, H and Zhang, N and Zhang, H},
title = {AND logic-gated CRISPR/Cas9 and hybridization chain reaction system for precise ctDNA detection.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {43},
pmid = {41388295},
issn = {1477-3155},
support = {2022YFB3808200//National Key Research and Development Program of China/ ; },
mesh = {*Circulating Tumor DNA/genetics/blood/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; Polymorphism, Single Nucleotide ; Nucleic Acid Amplification Techniques/methods ; Biomarkers, Tumor/genetics/blood ; Nucleic Acid Hybridization ; Mutation ; Proto-Oncogene Proteins p21(ras)/genetics ; Limit of Detection ; },
abstract = {Circulating tumor DNA (ctDNA) is a critical biomarker for liquid biopsies, enabling the non-invasive acquisition of cancer-related information from blood samples. Precise detection of ctDNA, particularly the identification of single-nucleotide variations (SNVs), is crucial for early cancer diagnosis, therapeutic monitoring, and prognostic evaluation. However, current ctDNA detection methods often encounter challenges such as complex procedures, difficult data analysis, and false-positive signals during pre-amplification. In this study, we introduce a novel detection method based on AND logic-gated integration of interspaced short palindromic repeats and associated proteins (CRISPR/Cas9) system with hybridization chain reaction (HCR) isothermal amplification. This strategy enhances the specific and sensitive detection of ctDNA. The incorporation of the AND logic gate effectively minimizes the off-target effects of Cas9 and enables the differentiation of single-nucleotide mutations, such as KRAS G12D, even in complex serum environments. Our system exhibits high sensitivity and specificity, achieving a limit of detection as low as 1 fM and capable of identifying SNVs mutations with allele fractions as low as 0.1% among wild-type sequences. Furthermore, we validated the specificity of our approach by successfully detecting various mutations, including KRAS G12C, KRAS G12D, EGFR T790M and TP53 R273H, in simulated clinical samples. These findings highlight a reliable method for precise ctDNA detection, offering high specificity, selectivity, and accuracy, thus paving the way for potential cancer diagnostic application.},
}
@article {pmid41387738,
year = {2025},
author = {Zhang, X and Zhu, T and Zhang, W and Zhang, Y and Zhang, J and Yang, J and Xia, C and Zhao, H and Yu, Y and Wen, C},
title = {Ethylene promotes branch formation but inhibits tendril development in cucumber.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {745},
pmid = {41387738},
issn = {2041-1723},
support = {JKZX202207//Beijing Academy of Agricultural and Forestry Sciences (BAAFS)/ ; },
mesh = {*Ethylenes/pharmacology/metabolism ; *Cucumis sativus/growth &amp; development/genetics/metabolism/drug effects ; Gene Expression Regulation, Plant/drug effects ; Plant Proteins/genetics/metabolism ; Signal Transduction/drug effects ; CRISPR-Cas Systems ; *Plant Growth Regulators/pharmacology/metabolism ; Mutation ; Promoter Regions, Genetic ; },
abstract = {Ethylene coordinates numerous plant growth processes, particularly in cucurbit crops, yet its role in vegetative growth regulation remains largely unexplored. Here, we report the function of ethylene in controlling branch and tendril development in cucumber. We find that ethylene promotes branches formation but inhibits tendrils development in a dose-dependent manner. CRISPR-Cas9-generated gene-edited Csein2 and Csein3/Cseil1 mutants exhibit few branches and more tendrils. Exogenous ethylene can recover the branch/tendril defective phenotypes of the Csein3 and Cseil1 mutants but not those of the Csein2 mutant or the Csein3/Cseil1 double mutant. Transcriptomic and metabolic analyses reveal that CsCYP707A4 and CsTL are the key downstream targets of ethylene signaling. We show that CsEIN3 can bind to its promoters to activate the expression of CsCYP707A4 but inhibit the expression of CsTL, which leads to the opposite effect on branch and tendril development. The study sets the foundation for designing ideal plant architecture to increase production efficiency.},
}
@article {pmid41382257,
year = {2025},
author = {Chen, W and Wu, P and Champer, J},
title = {Strategies to improve the efficiency of homing gene drives with multiplexed gRNAs.},
journal = {BMC biology},
volume = {24},
number = {1},
pages = {12},
pmid = {41382257},
issn = {1741-7007},
support = {32270672//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics ; *Gene Drive Technology/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; },
abstract = {BACKGROUND: CRISPR homing gene drive holds great potential for pest control, but its success is challenged by the generation of resistance alleles through end-joining repair. Using multiple gRNAs to target adjacent sites within a conserved gene can prevent functional resistance by allowing repeated cleavage events, but poor homology during DNA repair may compromise efficiency.<br><br>RESULTS: We first assessed the efficiency of single gRNA drives with truncated homology arms in Drosophila melanogaster mimicking a multiplexed system in which only one site is cleaved. Integrating results into a detailed gRNA multiplexing model, we found that efficiency loss was greater than expected. To mitigate this, we evaluated two new strategies: (1) extended homology arms to span all target sites (with mutations in the PAMs to prevent self-cleavage) and (2) a population-level gRNA multiplexing system involving two or more drives, each carrying two gRNAs. Extended homology arms did not result in notable improvement in conversion efficiency, and the extended region could be lost during drive conversion. The population-level multiplexing gRNAs strategy was more promising, though the intentionally mutated PAM also could not be consistently inherited. Simulations of homing suppression drives applying population-level multiplexed gRNAs increased the success rate of population elimination and reduced the time required for suppression.<br><br>CONCLUSIONS: Future drive designs requiring a larger number of gRNAs could potentially be improved. The design relying on extended homology arms may not represent an optimal strategy. However, population-level multiplexing gRNAs could serve as a promising alternative, enhancing efficiency while maintaining tolerance to functional resistance.},
}
@article {pmid41370122,
year = {2026},
author = {Pfisterer, L and Boyle, C and Cole, A and Mitchell, I and Flanagan, M and Gromley, Z and Gromley, A},
title = {Disruption of the centriolin/Cep110 gene (CNTRL) with CRISPR/Cas9 leads to cell cycle arrest and cell death of rhabdomyosarcoma cells in vitro.},
journal = {Molecular biology of the cell},
volume = {37},
number = {2},
pages = {br4},
doi = {10.1091/mbc.E25-08-0365},
pmid = {41370122},
issn = {1939-4586},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; *Rhabdomyosarcoma/genetics/metabolism/pathology ; Cell Line, Tumor ; *Cell Cycle Checkpoints/genetics ; *Cell Cycle Proteins/genetics/metabolism ; Gene Editing/methods ; Centrosome/metabolism ; Cell Death/genetics ; *Microtubule-Associated Proteins/genetics/metabolism ; Cilia/metabolism ; Cell Survival/genetics ; Cell Proliferation/genetics ; Apoptosis/genetics ; },
abstract = {Rhabdomyosarcoma is the most common pediatric soft tissue cancer, thought to arise from primitive mesenchymal cells that differentiate into skeletal muscle. Previous studies suggest that primary cilia may play a role in the development of rhabdomyosarcoma. Primary cilia are cellular structures that arise from the centrosome and serve important functions in sensory signaling, cell migration, and developmental processes. However, most rhabdomyosarcoma cell lines do not have primary cilia. Because primary cilia are derived from centrosomes, the development of rhabdomyosarcoma may, in fact, be due to the function of centrosome proteins rather than the primary cilia itself. Therefore, this study sought to determine if the centrosomal protein centriolin/Cep110, which is localized to both centrosomes and primary cilia, plays a role in rhabdomyosarcoma biology. The gene editing tool CRISPR/Cas9 was used to disrupt the centriolin/Cep110 gene in the rhabdomyosarcoma cell line CCL-136, and the effects on cell viability and cell cycle progression were assayed. Our results show that loss of centriolin/Cep110 leads to cell cycle arrest and apoptotic cell death in rhabdomyosarcoma cells. These findings suggest that centriolin/Cep110 plays a key role in rhabdomyosarcoma cell proliferation and viability and that this centrosome protein may represent a potential target for future rhabdomyosarcoma therapies.},
}
@article {pmid41540035,
year = {2026},
author = {Zhang, H and Shang, R and Zhang, Z and Zhou, M and Bigot, A and Cai, Y and Zhao, Y and Wang, Y and Deshmukh, A and Kudryashova, E and Kudryashov, DS and He, C and Mouly, V and Bi, P},
title = {Development of a split-toxin CRISPR screening platform to systematically identify regulators of human myoblast fusion.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {547},
pmid = {41540035},
issn = {2041-1723},
support = {GM147209//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; AR080330//U.S. Department of Health &amp; Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; },
mesh = {Humans ; *Myoblasts/metabolism/cytology ; *Muscle Development/genetics ; Cell Differentiation/genetics ; Cell Fusion ; *CRISPR-Cas Systems/genetics ; Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Muscle, Skeletal/metabolism ; High-Throughput Screening Assays/methods ; Single-Cell Analysis ; Cell Line ; },
abstract = {Muscle defects are common in human developmental disorders and often cause severe functional impairment. These defects arise from intricate tissue crosstalk and rare genetic mutations, underscoring the need to systematically identify cell-autonomous mechanisms regulating human myogenesis. Here we show a rationally designed, high-throughput genetic screening platform that integrates human myoblast models, customized CRISPR libraries, and a split-toxin strategy that enables quantitative selection of fusion-defective myocytes. Leveraging this platform, our initial screen uncovers a large group of hits essential for human myoblast fusion. The majority of these hits converge into 23 protein complexes. Notably, mutations in 41 screen hits are associated with human diseases marked by abnormal skeletal-muscle morphology. Applying a new single-cell CRISPR &amp; RNA-seq approach, we show that majority of these hits control human myoblast fusion as well as influence early-stage myogenic differentiation. This work establishes a scalable approach to identify cell-autonomous regulators of human muscle differentiation and fusion.},
}
@article {pmid41538317,
year = {2026},
author = {Ham, DT and Browne, TS and Zhang, CQ and Foo, GW and Uruthirapathy, AS and Gloor, GB and Edgell, DR},
title = {Machine learning reveals sequence and methylation determinants of SaCas9-PAM interactions in bacteria.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41538317},
issn = {1362-4962},
support = {PJT 159708/CAPMC/CIHR/Canada ; PJT 191939/CAPMC/CIHR/Canada ; },
mesh = {*Machine Learning ; Staphylococcus aureus/genetics/enzymology ; *CRISPR-Associated Protein 9/metabolism/genetics ; Escherichia coli/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Cas Systems ; Nucleotide Motifs ; DNA Methylation ; Methylation ; Gene Editing/methods ; },
abstract = {Cas9 nucleases defend bacteria against invading DNA and can be used with single guide RNAs (sgRNAs) as antimicrobials and genome-editing tools. However, bacterial applications are limited by incomplete knowledge of Cas9-target interactions. Here, we generated large-scale Staphylococcus aureus Cas9 (SaCas9)/sgRNA activity datasets in bacteria and trained a machine learning model (crispr macHine trAnsfer Learning) to predict SaCas9 activity. Incorporating downstream sequences flanking the canonical NNGRRN protospacer adjacent motif (PAM) at positions [+1] and [+2] improved predictive performance, with T-rich dinucleotides at these positions correlating with higher in vivo activity. Crucially, SaCas9 showed $\sim$10-fold reduced activity at sites containing a 5$^{\prime}
$-NNGGAT[C]-3$^\prime$ PAM [+1] sequence in pooled sgRNA experiments in Escherichia coli and Citrobacter rodentium. Plasmid cleavage assays in DNA adenine methyltransferase (DAM)-deficient E. coli confirmed that adenine methylation at GATC motifs inhibited SaCas9 activity. Removal of a DAM site within a PAM sequence enhanced cleavage, while introduction of a site reduced activity, directly linking adenine methylation to SaCas9 activity. These findings demonstrate that machine learning can uncover biologically relevant determinants of Cas9 activity. Avoidance of methylated PAMs may reflect an evolutionary adaptation by SaCas9 to discriminate self from nonself or to counter methylation as a phage and plasmid antirestriction strategy.},
}
@article {pmid41538311,
year = {2026},
author = {Xiao, Z and Sun, Y},
title = {Illuminating the genome: emerging approaches in CRISPR-Cas live-cell imaging.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41538311},
issn = {1362-4962},
support = {2022YFA1303103//National Key Research and Development Program of China/ ; 2022YFA3401100//National Key Research and Development Program of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Genome ; Gene Editing/methods ; Animals ; },
abstract = {CRISPR-Cas-based live-cell imaging has rapidly become a central technology for studying genome dynamics with high specificity and flexibility. By coupling nuclease-deactivated Cas (dCas) with programmable guide RNAs, genomic loci can be tracked in living cells, providing direct insights into nuclear organization and chromatin behavior. While repetitive regions such as telomeres and centromeres are readily visualized, labeling non-repetitive loci remains more challenging due to weak signals and high background. Recent advances, including multicolor labeling strategies, innovative amplification systems based on dCas9 and single-guide RNA (sgRNA) engineering, and integration with novel fluorescent reporters, have markedly expanded the applicability of CRISPR imaging across the genome. These developments have expanded the multiplexing capacity of CRISPR imaging, improved signal-to-background ratios, and even enabled the visualization of non-repetitive genomic loci. Nonetheless, key challenges remain, including cellular toxicity, replication stress, and genomic instability associated with prolonged CRISPR expression. In this review, we summarize recent advances in CRISPR live-cell imaging and highlight key design trade-offs and biological constraints.},
}
@article {pmid41536960,
year = {2026},
author = {van der Wilt, CN and Veltrop, RJA and Janssens, MH and Bakker, I and Stillitano, F and Sluijter, JPG and van Laake, LW and van der Velden, J and Villard, E and Montag, J and Denning, C and van Tintelen, JP and Te Riele, ASJM and van der Harst, P and Schurgers, LJ and van Steenbeek, FG and Harakalova, M},
title = {Setting the stage for cardiomyopathy gene editing trials: a systematic review of isogenic pair use in human induced pluripotent stem cell-derived cardiomyocyte research.},
journal = {European heart journal open},
volume = {6},
number = {1},
pages = {oeaf161},
pmid = {41536960},
issn = {2752-4191},
abstract = {In vitro gene editing using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has demonstrated the feasibility of introducing or correcting specific pathogenic variants. These successes represent a key first step towards therapeutic genome editing for cardiomyopathies, showing that precise, variant-specific interventions are achievable. To translate in vitro findings to the clinic, it is essential to develop robust disease models that yield meaningful, translatable data. The next challenge is systematically identifying disease-causing variants amenable to gene editing with strong pre-clinical support. Therefore, we conducted a systematic search of published studies on isogenic hiPSC-CM pairs in cardiomyopathy research with specific criteria, including (likely) pathogenic variants causing cardiomyopathy, correction and/or introduction of variants, differentiation into CMs, and functional follow-up. We systematically assessed 785 papers and highlighted 101 studies meeting our inclusion criteria reporting 69 patients carrying 56 unique variants across 31 genes, most commonly MYH7, MYBPC3, and DMD. This expanded to 91 variants across 38 genes upon inclusion of the introduced variants in a donor line. However, reported clinical data were often incomplete, underscoring the need for standardized phenotypic documentation. We reveal a lack of patient details, which creates an incomplete picture of underlying disease variables that hinder the design of targeted personalized treatments. Omitted key clinical data can lead to misinterpretations or overlooked variables that impact treatment outcomes. This systematic review integrates current evidence from successful in vitro studies using isogenic hiPSC-CM models and proposes a reporting framework for variant prioritization and the rigorous application of isogenic controls in cardiomyopathy research.},
}
@article {pmid41536809,
year = {2026},
author = {Liou, RH and Urrutia-Cabrera, D and Liu, CF and Wu, S and Westin, IM and Golovleva, I and Liu, GS and Kumar, S and McLenachan, S and Chen, FK and Hsu, FT and Huang, CL and Edwards, T and Martin, KR and Cheng, AW and Wong, RCB},
title = {Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to target ABCA4 splice variants in Stargardt disease.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {1},
pages = {102789},
pmid = {41536809},
issn = {2162-2531},
abstract = {Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in the ABCA4 gene. The splicing variants c.5461-10T>C and c.4773+3A>G in ABCA4 cause the skipping of exon 39-40 and exon 33-34, respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate these ABCA4 splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C, and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containing ABCA4 variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-spliced ABCA4 c.5461-10T>C variants and up to 78% reduction of the ABCA4 c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced ABCA4 c.4773+3A>G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts for ABCA4, providing an important step to advance the development of gene therapy to treat STGD1.},
}
@article {pmid41535388,
year = {2026},
author = {Rolando, JC and Thieme, A and Weckman, NE and Kim, N and de Puig, H and Tan, X and Cotnoir, E and Chaturvedi, V and Collins, JJ and Walt, DR},
title = {Digital CRISPR-based diagnostics for quantification of Candida auris and resistance mutations.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {41535388},
issn = {2157-846X},
support = {WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; },
abstract = {Candida auris, an increasingly prevalent fungal pathogen, requires both rapid identification and antifungal susceptibility testing to enable proper treatment. This study introduces digital SHERLOCK (dSHERLOCK), a platform that combines CRISPR/Cas nucleic acid detection, single-template quantification and real-time kinetics monitoring. Assays implemented on this platform display excellent sensitivity to C. auris from major clades 1-4, while maintaining specificity when challenged with common environmental and pathogenic fungi. dSHERLOCK detects C. auris within 20 min in minimally processed swab samples and achieves sensitive quantification (1 c.f.u. µl[-1]) within 40 min. To address antifungal susceptibility testing, we develop assays that detect mutations that are commonly associated with azole and echinocandin multidrug resistance. We use machine learning and real-time monitoring of reaction kinetics to achieve highly accurate simultaneous quantification of mutant and wild-type FKS1 SNP alleles in fungal populations with mixed antifungal susceptibility, which would be misdiagnosed as completely susceptible or resistant under standard reaction conditions. Our platform's use of commercially available materials and common laboratory equipment makes C. auris diagnostics widely deployable in global healthcare settings.},
}
@article {pmid41526513,
year = {2026},
author = {Ngo, W and Wu, JLY and Wasko, KM and Doudna, JA},
title = {Targeted delivery of genome editors in vivo.},
journal = {Nature biotechnology},
volume = {44},
number = {1},
pages = {49-59},
pmid = {41526513},
issn = {1546-1696},
support = {DE-AC52-07NA27344//DOE | LDRD | Lawrence Livermore National Laboratory (LLNL)/ ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; Animals ; *Genetic Therapy/methods ; *Gene Transfer Techniques ; *Drug Delivery Systems ; },
abstract = {Genome editing has revolutionized the treatment of genetic diseases, yet the difficulty of tissue-specific delivery currently limits applications of editing technology. In this Review, we discuss preclinical and clinical advances in delivering genome editors with both established and emerging delivery mechanisms. Targeted delivery promises to considerably expand the therapeutic applicability of genome editing, moving closer to the ideal of a precise 'magic bullet' that safely and effectively treats diverse genetic disorders.},
}
@article {pmid41468873,
year = {2026},
author = {Zhang, J and Zhang, X and Xie, X and Han, B and Zhao, F and Yang, X and Zhang, W and Jiang, Y and Zhang, X},
title = {CRISPR/Cas12a-mediated marker-free fluorescent biosensing platform based on a lightful copper nanocluster for highly sensitive detection of mycotoxin.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129326},
doi = {10.1016/j.talanta.2025.129326},
pmid = {41468873},
issn = {1873-3573},
mesh = {*Copper/chemistry ; *Biosensing Techniques/methods ; *Aflatoxin B1/analysis ; *CRISPR-Cas Systems ; *Metal Nanoparticles/chemistry ; Limit of Detection ; Food Contamination/analysis ; Fluorescence ; *Mycotoxins/analysis ; },
abstract = {The problem of mycotoxin contamination in foodstuffs has attracted widespread attention and posed a great threat to human health. Therefore, the sensitive and effective detection of mycotoxins is of great importance for preserving public health worldwide. In this study, a CRISPR/Cas12a-mediated marker-free fluorescent biosensing platform was constructed for highly sensitive and fast detection of aflatoxin B1. The copper nanoclusters were synthesized with marker-free DNA single strands within 5 min, showing the outstanding fluorescence properties. With the existence of aflatoxin B1, the released complementary DNA (cDNA) triggered multiple isothermal amplification reaction. Subsequently, the obtained amplification products triggered the trans-cleavage activity of CRISPR/Cas12a system, which degraded the DNA single strands for synthesis of copper nanoclusters, leading to a decreased fluorescent signal. Benefiting from good fluorescence properties of copper nanoclusters, the established biosensing platform for aflatoxin B1 detection had a high specificity and a limit of detection of 47.51 pg/mL in the linear range of 0.05-10 ng/mL. The proposed platform provided a new insight for the detection of non-nucleic acid targets.},
}
@article {pmid41454886,
year = {2026},
author = {Yang, H and Gao, X and Jin, ZC and Zhang, R and Ning, B and Yan, X},
title = {Simple and Versatile Toolkit for Genetic Manipulation of Bacillus licheniformis.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {262-270},
doi = {10.1021/acssynbio.5c00699},
pmid = {41454886},
issn = {2161-5063},
mesh = {*Bacillus licheniformis/genetics ; Plasmids/genetics ; *Genetic Engineering/methods ; DNA Transposable Elements/genetics ; Mutagenesis ; CRISPR-Cas Systems/genetics ; },
abstract = {Bacillus licheniformis is a spore-forming bacterium with probiotic, environmental, and industrial applications. Many wild strains with diverse functions have been described in recent years. Nevertheless, the lack of efficient and universal genetic manipulation tools hinders the study and engineering of these strains. Here, a versatile and simple genetic manipulation toolkit is established for B. licheniformis. The cornerstone of this toolkit is a conjugative DNA transfer system. This system could effectively transfer temperature-sensitive plasmid pTSMK into all ten tested B. licheniformis strains, with efficiencies ranging from 10[-5] to 10[-3]. Based on this DNA transfer system, the tools for maker-free knockout and knock-in, CRISPRi, as well as transposon mutagenesis, were built. A transposition frequency of 7.68 × 10[-3] was observed. The toolkit developed in this study fulfills most tasks in the engineering of this species and will promote the basic and applied research of B. licheniformis.},
}
@article {pmid41418786,
year = {2026},
author = {Sherman, A and Benvenisty, N},
title = {Genetic screening of long non-coding RNAs in human embryonic stem cells reveals novel regulators of pluripotency.},
journal = {Stem cell reports},
volume = {21},
number = {1},
pages = {102743},
doi = {10.1016/j.stemcr.2025.102743},
pmid = {41418786},
issn = {2213-6711},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Differentiation/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; Octamer Transcription Factor-3/genetics/metabolism ; *Genetic Testing ; Cell Line ; CRISPR-Cas Systems ; Apoptosis/genetics ; },
abstract = {The human genome encodes thousands of long non-coding RNAs (lncRNAs), transcripts of over 200 nucleotides that lack protein-coding potential. lncRNAs are emerging as key players in diverse cellular processes, particularly in tissue-specific contexts, yet their functionality remained poorly understood. Here, we performed a CRISPR interference (CRISPRi) screen in human embryonic stem cells (hESCs), identifying over 100 essential and about 150 growth-restricting lncRNAs. We show that growth-modifying lncRNAs display distinctive properties, including unique expression signatures, genomic structure, evolutionary conservation, chromosomal distribution, and potential involvement in teratoma formation. Notably, we uncovered two primate-conserved, uncharacterized, essential lncRNAs that regulate neighboring pluripotency transcription factors: lncOCT4, which positively regulates OCT4 and induces p53-mediated apoptosis upon knockdown, and lncVRTN, which acts as a putative negative regulator of VRTN, affecting cell fate determination. These findings shed light on the contribution of lncRNAs to the human-specific pluripotency network and provide insights into lncRNA-mediated regulation of hESC growth and differentiation.},
}
@article {pmid41391726,
year = {2026},
author = {Liao, XR and Han, D and Qi, LJ and Huang, QY and Gao, QY and He, XY and Guo, T and Lei, JJ and Cheng, SX},
title = {Aptamer-functionalized nanoparticles for CRISPR-Cas9 delivery to circulating malignant cells for therapeutic efficacy evaluation.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {390},
number = {},
pages = {114542},
doi = {10.1016/j.jconrel.2025.114542},
pmid = {41391726},
issn = {1873-4995},
mesh = {Humans ; *CRISPR-Cas Systems ; *Nanoparticles/chemistry/administration &amp; dosage ; *Aptamers, Nucleotide/administration &amp; dosage/chemistry ; Cell Line, Tumor ; Hyaluronic Acid/chemistry ; *Neoplastic Cells, Circulating/metabolism ; Gene Editing/methods ; *Neoplasms/therapy/genetics ; Hyaluronan Receptors/metabolism ; Epithelial Cell Adhesion Molecule/genetics ; Proto-Oncogene Proteins c-met/genetics ; *Gene Transfer Techniques ; ErbB Receptors/genetics ; Plasmids ; },
abstract = {Genome editing therapies targeting oncogenic pathways represent a promising alternative to small-molecule inhibitors, enabling durable therapeutic responses without inducing drug resistance. However, their success hinges on overcoming tumor heterogeneity, as malignant cells of cancer patients exhibit significant phenotypic variability. To advance personalized research on genome editing efficacy, tailored delivery systems capable of precisely targeting heterogeneous cancer cell populations are essential. Herein, we developed a facile modification strategy to construct a multiplexed surface-functionalized gene delivery system targeting heterogeneous cancer cells for personalized therapeutic studies. The system integrates the EGFR-targeting TuTu22 aptamer with SYL3C-conjugated hyaluronic acid (SYL3C-HA) for EpCAM and CD44 recognition. This triple-targeting platform enables efficient delivery of genome editing plasmid for c-Met knockout in both cancer cell lines and circulating malignant cells (CMCs) from cancer patients. The c-Met knockout not only reduces tumor malignancy but also reverses immune suppression, evidenced by PD-L1 downregulation and restored immune surveillance. By combining gene delivery with an ex vivo patient-derived evaluation platform, this system provides a robust tool for personalized research on the therapeutic strategies for tumor progression inhibition and immunity restoration.},
}
@article {pmid41387457,
year = {2025},
author = {Cheng, F and Soleimani Samarkhazan, H and Khazaei, Y},
title = {CRISPR-engineered microbiome: living therapeutics revolutionize blood cancer immunotherapy.},
journal = {NPJ biofilms and microbiomes},
volume = {12},
number = {1},
pages = {17},
pmid = {41387457},
issn = {2055-5008},
mesh = {Humans ; *Immunotherapy/methods ; Animals ; *Hematologic Neoplasms/therapy/immunology/microbiology ; *CRISPR-Cas Systems ; *Gastrointestinal Microbiome/genetics ; *Microbiota ; },
abstract = {Blood cancers such as leukemia, lymphoma, and myeloma remain refractory in many patients due to immune escape, antigen heterogeneity, and therapy‑related toxicities. To address these challenges, we review recent strategies that harness CRISPR‑engineered gut commensals as precision "living therapeutics" to modulate host immunity and directly target malignant clones. We frame this review around three principal themes: (1) mechanistic strategies whereby CRISPR-engineered commensals modulate host immunity and directly antagonize malignant clones; (2) the enabling technologies and delivery/containment platforms, CRISPR variants, phage/LNP delivery, genetic circuits and biocontainment, that make living therapeutics feasible; and (3) translational progress, outstanding technical and safety barriers, and ethical/regulatory challenges that must be addressed for clinical deployment. To illustrate these themes, we discuss three concrete therapeutic modalities: engineered microbial secretion of immunomodulators, targeted delivery of tumor-lytic payloads, and engineered production of anticancer metabolites, and how these are enabled by contemporary CRISPR and synthetic-biology toolkits. Selected preclinical models report substantial antitumor effects, often >60% tumor reduction in rodent studies, and restoration of CAR-T cell function in controlled settings; however, effect sizes vary across models, and human translation remains unproven. We also analyze key technical barriers, strain stability, biocontainment, off‑target effects, and propose solutions, including auxotrophic kill-switches and AI‑guided strain optimization. Finally, we outline future directions, from in situ phage delivery to multi‑omics-driven patient stratification. CRISPR‑microbiome editing represents a paradigm shift in hematologic oncology, offering localized, sustained therapy with reduced systemic toxicity.},
}
@article {pmid41319963,
year = {2026},
author = {Kong, H and Wang, S and Zhuo, C and Zhong, Q and Xu, Y and Lao, YH and Lv, S and Xie, X and Yuan, Q and Li, K and Tao, Y and Li, M},
title = {Nanovesicles integrating PD-1-mediated targeting and CRISPR/Cas9-based CD47 editing for dual immune checkpoint blockade.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {390},
number = {},
pages = {114480},
doi = {10.1016/j.jconrel.2025.114480},
pmid = {41319963},
issn = {1873-4995},
mesh = {*CD47 Antigen/genetics ; CRISPR-Cas Systems ; Animals ; Gene Editing ; *Immune Checkpoint Inhibitors/administration &amp; dosage ; *Programmed Cell Death 1 Receptor/genetics/immunology ; Humans ; B7-H1 Antigen/immunology ; Mice ; Cell Line, Tumor ; *Neoplasms/therapy/immunology/genetics ; Mice, Inbred C57BL ; Female ; Immunotherapy/methods ; },
abstract = {Immunotherapy with immune checkpoint inhibitors has revolutionized cancer treatment, yet many tumors evade immune surveillance through multiple suppressive mechanisms. In particular, the adaptive immune checkpoint programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) and the innate "don't eat me" signal CD47/signal-regulatory protein alpha (SIRPα) represent two distinct pathways that cancers exploit to avoid T-cell attack and macrophage phagocytosis, respectively. Herein, we present BITE (Biomimetic Immune Targeting and Editing), a genetically engineered biomimetic nanoplatform designed to concurrently blockade both pathways by combining PD-1-mediated tumor targeting with CRISPR/Cas9 gene editing of CD47. BITE nanovesicles display PD-1 on their surface, enabling selective binding to PD-L1-expressing tumor cells and local disruption of PD-1/PD-L1 signaling. Simultaneously, they deliver a CRISPR/Cas9 payload that knocks out the CD47 gene in tumor cells, abolishing the anti-phagocytic signal and thus activating innate immune clearance. We demonstrate that BITE efficiently homes to PD-L1-positive tumors in vitro and in vivo, achieves significant CD47 gene disruption in tumor cells, and triggers robust phagocytosis by macrophages. In a mouse tumor model, dual checkpoint blockade by BITE reshapes the tumor microenvironment, yielding increased infiltration of CD4[+] T cells, CD8[+] T cells, and M1 macrophages; treatment with BITE induces pronounced tumor regression and extended survival, outperforming single-target controls. Our results establish a proof-of-concept for this dual-function nanovesicle approach, highlighting its potential to engage both adaptive and innate immunity synergistically. The BITE platform offers a versatile and targeted strategy to overcome immune resistance in cancer, representing a promising therapeutic avenue in biomedical engineering and nanomedicine.},
}
@article {pmid41147788,
year = {2026},
author = {Saika, H and Hara, N and Yasumoto, S and Muranaka, T and Yoshimi, K and Mashimo, T and Toki, S},
title = {Versatile genome editing using Type I-E CRISPR-Cas3 in rice.},
journal = {Plant &amp; cell physiology},
volume = {67},
number = {1},
pages = {82-92},
doi = {10.1093/pcp/pcaf138},
pmid = {41147788},
issn = {1471-9053},
support = {JPJ008000//Ministry of Agriculture, Fisheries and Food/ ; //Cross-ministerial Strategic Innovation Promotion Program/ ; JPJ012287//Bio-oriented Technology Research Advancement Institution/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; Plants, Genetically Modified ; },
abstract = {The Type I-E CRISPR-Cas3 derived from Escherichia coli (Eco CRISPR-Cas3) can introduce large deletions in target sites and is available for mammalian genome editing. The use of Eco CRISPR-Cas3 in plants is challenging because seven CRISPR-Cas3 components (six Cas proteins and CRISPR RNA) must be expressed simultaneously in plant cells. To date, application has been limited to maize protoplasts, and no mutant plants have been produced. In this study, we developed a genome editing system in rice using Eco CRISPR-Cas3 via Agrobacterium-mediated transformation. Deletions in the target gene were detected in 39%-71% of transformed calli by polymerase chain reaction (PCR) analysis, and the frequency of alleles lacking a region 7.0 kb upstream of the protospacer adjacent motif sequence was estimated as 21%-61% by quantifying copy number by droplet digital PCR, suggesting that mutant plants could be obtained with reasonably high frequency. Deletions were determined in plants regenerated from transformed calli, and stably inherited to the progenies. Sequencing analysis showed that deletions of 0.1-7.2 kb were obtained, as reported previously in mammals. Interestingly, deletions separated by intervening fragments or with short insertion and inversion were also determined, suggesting the creation of novel alleles. Moreover, we demonstrated C to T base editing based on Type I-E CRISPR-Cas3 in rice, whereas base editing based on Type I-C and Type I-F2 CRISPR-Cas3 has been reported previously only in human cells. Overall, Eco CRISPR-Cas3 could be a promising genome editing tool for gene knockout, gene deletion, base editing, and genome rearrangement in plants.},
}
@article {pmid40971496,
year = {2026},
author = {V Pillai, V and Koganti, PP and Gurung, S and Cheong, SH and Selvaraj, V},
title = {Transformed bovine trophoblast stem cell lines, characterization, gene editing and secretion†.},
journal = {Biology of reproduction},
volume = {114},
number = {1},
pages = {273-286},
doi = {10.1093/biolre/ioaf212},
pmid = {40971496},
issn = {1529-7268},
support = {USDA-NIFA 2023-08329//United States Department of Agriculture/ ; NE-2227//USDA-Multistate Program/ ; },
mesh = {Animals ; *Trophoblasts/cytology/physiology/metabolism ; Cattle ; *Gene Editing/veterinary ; Female ; *Stem Cells/cytology/physiology/metabolism ; Pregnancy ; Cell Differentiation ; Cell Line, Transformed ; CRISPR-Cas Systems ; },
abstract = {Trophoblast stem cells (TSCs) serve as a critical model for understanding placental development, early embryo-maternal interactions, and pregnancy establishment in mammals. In cattle, the developing trophectoderm plays an essential role in conceptus elongation and secretion of factors necessary for maternal recognition of pregnancy. Building on previous work identifying signaling pathways regulating bovine TSC self-renewal and differentiation, we report the generation and characterization of transformed bovine TSC (bTSC) lines derived from blastocysts via lentiviral transduction of simian vacuolating virus 40 large T antigen. These rapidly proliferating TSC cell lines, maintained in the presence of Rho-associated protein kinase (ROCK) inhibition, retain key morphological and transcriptional characteristics of bovine TSCs. Upon transforming growth factor β-induced differentiation, they exhibit morphological and molecular changes consistent with trophoblast maturation. To evaluate their utility for functional studies, we demonstrated stable gene introduction of tdTomato and EGFP using lentiviral vectors and employed CRISPR/Cas9-mediated gene editing to target lentiviral EGFP integration sites, confirming efficient gene deletion. Additionally, proteomic analysis of conditioned medium identified secreted proteins with potential roles in embryo-uterine interactions, aligning with factors previously reported in bovine conceptus secretomes. These findings establish transformed bTSC lines as a valuable model for investigating bovine trophoblast biology, functional gene studies, and trophoblast-endometrial signaling. By providing a renewable in vitro system with stable proliferative capacity, these cell lines enable further exploration of the molecular mechanisms governing early pregnancy in cattle.},
}
@article {pmid41535270,
year = {2026},
author = {Taguchi, J and Kikuchi, M and Jeon, H and Shimizu, R and Mori, H and Ikawa, M and Yamada, Y and Sato, K and Ikeda, T and Yamazaki, S and Ozawa, M},
title = {A scalable two-step genome editing strategy for generating full-length gene-humanized mice at diverse genomic loci.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {356},
pmid = {41535270},
issn = {2041-1723},
support = {23K27084//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 25K18393//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21H05033//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21H05033//Japan Society for the Promotion of Science London (JSPS London)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Humans ; Mice ; CRISPR-Cas Systems/genetics ; Gene Knock-In Techniques/methods ; Male ; Mice, Transgenic ; Alleles ; Homologous Recombination ; Embryonic Stem Cells/metabolism ; Genetic Loci ; Hematopoiesis/genetics ; Female ; },
abstract = {Full-length gene-humanized mice engineered by completely replacing mouse loci with human counterparts, including untranslated and regulatory regions, provide a robust in vivo platform for human gene function studies. However, reliably humanizing large genomic regions remains challenging due to limited DNA insert sizes, complex protocols, and specialized material requirements. This study introduces a streamlined approach that enables full-length gene humanization through two sequential CRISPR-assisted homologous recombination steps in embryonic stem cells. This method supports targeted knock-in of genomic fragments (> 200 kbp) and is applicable across multiple mouse strains. Humanized alleles generated using the developed method recapitulate human-like splicing isoforms and organ-specific gene expression while restoring essential functions in hematopoiesis, spermatogenesis, and survival. Furthermore, disease-associated mutations can be engineered into humanized alleles to model human genetic disorders in vivo. This versatile platform enables the creation of physiologically relevant, fully gene-humanized mouse models for broad applications in biomedical research.},
}
@article {pmid41533983,
year = {2026},
author = {Zhang, RR and Wang, YH and Peng, XF and Sun, YJ and Xu, ZS and Liu, H and Xiong, AS},
title = {Expansion protein DcEXP22 regulates taproot enlargement via mediating root cell extension in carrot.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {1},
pages = {e70677},
doi = {10.1111/tpj.70677},
pmid = {41533983},
issn = {1365-313X},
support = {KYLH2025002//Fundamental Research Funds for the Central Universities/ ; 2023-SYS-02//Open Research Project of Key Laboratory of Biotechnology of Qinghai-Tibet Plateau Biotechnology of the Ministry of Education/ ; //Priority Academic Program Development of Jiangsu Higher Education Institutions Project PAPD/ ; //Bioinformatics Center of Nanjing Agricultural University/ ; },
mesh = {*Daucus carota/genetics/growth &amp; development/metabolism ; *Plant Roots/growth &amp; development/genetics/cytology/metabolism ; *Plant Proteins/genetics/metabolism/physiology ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; CRISPR-Cas Systems ; },
abstract = {Carrot (Daucus carota L.) is an important root vegetable crop of the Daucus genus in the Apiaceae. As the main product organ of carrot, the taproot has high nutritional and economic value. Expansins, a class of proteins involved in plant cell wall relaxation and cell extension, are mainly found in growing tissues and organs. Expansins play an important role in plant root development. Here, the DcEXP22 gene with a length of 789 bp was cloned from the carrot cultivar 'Kurodagosun'. Based on the stable genetic transformation system and CRISPR/Cas9 gene-editing technology, the DcEXP22 gene was overexpressed and knocked out in carrots. The results indicated that overexpression of the DcEXP22 gene increased carrot root fresh weight, root diameter, and root-shoot ratio, and enlarged the perimeter and area of taproot phloem cells. In contrast, knockout of the DcEXP22 gene inhibited the development of carrot taproots and the extension of phloem cells, suggesting that the DcEXP22 gene might promote the enlargement of carrot taproots by regulating the size of phloem cells. RNA-seq analysis identified several genes that were co-expressed with DcEXP22, including DcCYP734A1, DcERF1, DcMAP2K1, and DcSAD9. It was hypothesized that the DcEXP22 gene might influence the enlargement of carrot taproot by participating in the signal transduction of phytohormones such as brassinosteroids, cell wall synthesis and modification, and fatty acid metabolisms. These findings will advance our knowledge of the molecular mechanisms of carrot taproot enlargement.},
}
@article {pmid41500123,
year = {2026},
author = {Zang, Z and Chen, J and Dong, Y and Chen, L and Yang, M and Mu, M and Zhou, L and Zhang, W and Zou, G and Liu, C},
title = {CRISPR-Cas12a biosensing technology advances and applications in precision diagnostics and cancer research.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129298},
doi = {10.1016/j.talanta.2025.129298},
pmid = {41500123},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/diagnosis/genetics ; *Precision Medicine/methods ; Biomarkers, Tumor/genetics ; Gene Editing ; },
abstract = {CRISPR-Cas12a has become a versatile biotechnology platform with important applications in biosensing, diagnostics, and precision genome editing. This system is activated by a single crRNA, exhibits target-responsive trans-cleavage activity, and recognizes programmable PAM sequences. These features provide a robust basis for accurate detection of diverse biomarkers. Its detection capabilities include nucleic acid targets such as viral RNA and cancer mutations, as well as non-nucleic acid molecules like exosomes and proteins. Recent advancements have shown significant advantages, including multi-temperature adaptability, rapid kinetics, and compatibility with both DNA and RNA targets. Technical improvements include machine learning-assisted crRNA design for enhanced prediction accuracy and engineered EnAsCas12a variants that overcome conventional PAM restrictions. Notable achievements involve entropy-driven circuits that achieve attomolar-level sensitivity, smartphone-compatible four-channel quantitative detection systems, and streamlined integrated workflows completed within 30 min. Advances in sensor design, such as metal-organic framework encapsulation and high-performance aptamer-based sensors, have further expanded detection capabilities. In oncology research, CRISPR-Cas12a technology provides powerful tools to comprehensively analyze complex molecular networks within the tumor microenvironment (TME) and facilitate ultrasensitive detection of early-stage cancer biomarkers. Additionally, in genome editing, CRISPR-Cas12a enables precise genomic modifications due to distinct repair pathways, versatile delivery methods, and efficient creation of transgenic models. Thus, it expands its functional scope beyond diagnostics. With ongoing development, this technology is expected to evolve into an integrated platform combining TME research, point-of-care cancer diagnostics, and programmable genome engineering, offering innovative solutions for both biomedical research and clinical translation.},
}
@article {pmid41478231,
year = {2026},
author = {Liu, Y and Mei, H and Gao, C and Yang, Y},
title = {Rapid detection of respiratory syncytial virus using RT-LAMP-CRISPR/Cas12b on a gravity-driven microfluidic chip.},
journal = {Diagnostic microbiology and infectious disease},
volume = {114},
number = {3},
pages = {117244},
doi = {10.1016/j.diagmicrobio.2025.117244},
pmid = {41478231},
issn = {1879-0070},
mesh = {Humans ; *Respiratory Syncytial Virus Infections/diagnosis/virology ; *Molecular Diagnostic Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; *Respiratory Syncytial Virus, Human/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Lab-On-A-Chip Devices ; RNA, Viral/genetics ; Limit of Detection ; Point-of-Care Systems ; },
abstract = {AIMS: Respiratory syncytial virus (RSV) is the most significant pathogen causing acute lower respiratory tract infections in children. Early detection of RSV can help control disease progression and reduce complications. However, RT-qPCR-based detection methods cannot provide accurate results within one hour and are not suitable for use in resource-limited settings. Therefore, there is a pressing need to develop a rapid and precise bedside RSV detection assay to meet clinical demands.<br><br>METHODS AND RESULTS: Firstly, we developed a RT-LAMP-assisted CRISPR/Cas12b method for detecting RSV M gene, capable of identifying target RNA with a limit of detection as low as 100 copies/&#x3bc;L within 40 min. Secondly, we created and tested a sampling lysis reagent, demonstrating its effectiveness in enabling direct detection without the need for nucleic acid extraction, thus improving bedside detection efficiency. Lastly, to facilitate use in resource-limited areas, we designed and developed a gravity-driven microfluidic chip that simplifies the stepwise process of RT-LAMP amplification and CRISPR/Cas12b detection. This chip allows for visual recognition of results without the need for an external power source when used in point-of-care (POC) settings. This assay showed 99% agreement with RT-qPCR, highlighting its potential for practical application. Additionally, no cross-reactivity was observed with other respiratory pathogens infection, demonstrating good clinical specificity.<br><br>CONCLUSIONS: In summary, the platform we developed is faster and more user-friendly than RT-qPCR, while achieving comparable sensitivity.<br><br>IMPACT STATEMENT: Our finding fills the gap in the inability to detect RSV in POC setting, safeguarding the health of children, and offers new insights for the innovation of CRISPR diagnostics.},
}
@article {pmid41476394,
year = {2026},
author = {Zhuang, M and Song, J and Hu, X and Wang, X},
title = {Metabolic Engineering of Escherichia coli Nissle 1917 for Efficient Production of p-Coumaric Acid.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {210-222},
doi = {10.1021/acssynbio.5c00639},
pmid = {41476394},
issn = {2161-5063},
mesh = {*Escherichia coli/metabolism/genetics ; *Metabolic Engineering/methods ; *Coumaric Acids/metabolism ; *Propionates/metabolism ; Ammonia-Lyases/genetics/metabolism ; Tyrosine/biosynthesis/metabolism ; CRISPR-Cas Systems/genetics ; Plasmids/genetics/metabolism ; Escherichia coli Proteins/genetics/metabolism ; Quorum Sensing/genetics ; Phenylalanine/biosynthesis ; },
abstract = {p-Coumaric acid is a valuable phytochemical with significant roles in anticancer cell proliferation, antianxiety, and neuroprotection and as a key precursor for various flavonoids. However, the production of p-coumaric acid in microorganisms is often limited by enzyme compatibility and its antimicrobial effects. In this study, a p-coumaric acid producing Escherichia coli strain was constructed. First, the cryptic plasmids pMUT1 and pMUT2 were eliminated from E. coli Nissle 1917 by using the CRISPR/Cas9 method to mitigate their interference with heterologous gene expression, and the resulting strain WEN01 was used to screen for the genes encoding for tyrosine ammonia-lyase with superior host compatibility. Next, the gene tyrR encoding a global regulator was knocked out to alleviate the repression of l-tyrosine production. The key genes pheL and pheA involved in phenylalanine biosynthesis were knocked out to reduce byproduct formation, resulting in the strain WEN06. Finally, the quorum sensing system was used to overexpress the key genes aroG[fbr] and tyrA[fbr] in the l-tyrosine biosynthetic pathway, and the resulting strain WEN06/pWT101-AT, pWT104F could produce 462.6 mg/L p-coumaric acid in shake flask fermentation. In fed-batch fermentation, the engineered strain WEN06/pWT101-AT, pWT104F could produce 10.3 g/L p-coumaric acid with a glucose conversion yield of 0.13 g/g and a productivity of 0.14 g/L/h. This work provides a novel strategy for the efficient production of p-coumaric acid and lays a foundation for the efficient production of antimicrobial natural products in bacteria.},
}
@article {pmid41455263,
year = {2026},
author = {Liu, F and Cheng, AX and Zhang, C and Li, J and Huang, YF and Zhang, YP and Li, CP and Zhao, H},
title = {Amplification-free, CRISPR-Cas12a-mediated fluorescence biosensor using mesoporous silica nanomaterials for ultrasensitive detection of nucleic acid biomarkers.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129289},
doi = {10.1016/j.talanta.2025.129289},
pmid = {41455263},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *Silicon Dioxide/chemistry ; *CRISPR-Cas Systems ; Humans ; *Nanostructures/chemistry ; Porosity ; Limit of Detection ; Fluorescent Dyes/chemistry ; Rhodamines/chemistry ; Fluorescence ; ErbB Receptors/genetics ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Sensitive detection of nucleic acid biomarkers is crucial in many fields, including biomedical diagnosis, veterinary medicine, and food safety. Thus, developing an accurate and cost-effective detection method for nucleic acid biomarkers is essential. Here, we developed a sensitive CRISPR-Cas12a-based fluorescence biosensor using mesoporous silica nanomaterials (MSNs). A large quantity of rhodamine B (RB) was enriched on the MSNs to synthesize RB@MSN nanocomposites, which served as fluorescent probe materials, and Au NPs acted as fluorescence quenching materials. Combined with the high specific recognition capability of the CRISPR-Cas12a system, we detected three important nucleic acids without requiring amplification: the EGFR exon 19 deletion mutation (EGFR 19Del, found in circulating tumor DNA), African swine fever virus (ASFV), and human papilloma virus (HPV). Under optimal conditions and using quantitative analysis, there were strong linear correlations between the concentrations of the targets and their respective fluorescence intensities. The lowest detection limits were 55 aM for EGFR 19Del, 51 aM for ASFV, and 24 aM for HPV. By enriching and encapsulating MSNs with RB, our method avoided the problems of fluorescence modifications in typical CRISPR-Cas12a systems, such as professional outsourcing requirements and easily quenched fluorescence. Moreover, the results exhibited good repeatability and stability. This method provides a novel approach to nucleic acid fluorescence detection using the CRISPR-Cas12a system.},
}
@article {pmid41448060,
year = {2026},
author = {Long, Y and Sun, S and Mei, H and Zhou, D and Zhou, H and Fang, Z and Li, X and Li, N and Zhuang, T and Guo, C},
title = {RT-LAMP-CRISPR/Cas12b-based hand-pressure-actuated microfluidic chip for rapid and portable detection of severe fever with thrombocytopenia syndrome virus.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129277},
doi = {10.1016/j.talanta.2025.129277},
pmid = {41448060},
issn = {1873-3573},
mesh = {*Phlebovirus/genetics/isolation &amp; purification ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology ; *Nucleic Acid Amplification Techniques/instrumentation/methods ; Humans ; *Lab-On-A-Chip Devices ; *Molecular Diagnostic Techniques/instrumentation/methods ; *CRISPR-Cas Systems ; Pressure ; RNA, Viral/genetics/blood ; },
abstract = {Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, highly pathogenic tick-borne virus causing severe viral hemorrhagic fever, posing a significant public health threat. Rapid and accurate detection of SFTSV in resource-limited settings is critical for early diagnosis and effective control of severe fever with thrombocytopenia syndrome (SFTS). Here, we developed the RT-LAMP-CRISPR/Cas12b-based Hand-Pressure-Actuated Microfluidic Chip for Rapid and Portable Detection of Severe Fever with Thrombocytopenia Syndrome Virus (HARD). The hand-pressure-actuated microfluidic chip integrates reverse transcription loop-mediated isothermal amplification (RT-LAMP), clustered regularly interspaced short palindromic repeats (CRISPR) and its associated proteins (CRISPR associated proteins, Cas) 12b in order to achieve rapid, low-cost, and contamination-free point-of-care testing. The HARD system achieves a detection limit of 5 copies per reaction, utilizing direct RNA lysis from blood samples and a hand warmer as a heat source, enabling electricity-free operation. Clinical validation with blood samples from vector-borne infectious diseases demonstrated high concordance with laboratory RT-qPCR, with 88.9 % sensitivity, 100 % specificity, and 95 % accuracy. Thus, the HARD platform offers a rapid, portable, and efficient solution for the early diagnosis of SFTSV in resource-limited settings, with potential for broader application in POCT for infectious diseases.},
}
@article {pmid41439415,
year = {2026},
author = {Zhao, R and Wan, P and Huang, H and Li, Q and Zeng, Z and Xiong, W},
title = {Harnessing CRISPR-Cas9 and CRISPRi systems to reverse antibiotic resistance in a clinical multidrug-resistant Escherichia coli isolate.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {81},
number = {1},
pages = {},
doi = {10.1093/jac/dkaf442},
pmid = {41439415},
issn = {1460-2091},
support = {2022YFD1800400//National Key R&amp;D Program/ ; },
mesh = {*Escherichia coli/drug effects/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Drug Resistance, Multiple, Bacterial/genetics ; Microbial Sensitivity Tests ; *Anti-Bacterial Agents/pharmacology ; Escherichia coli Proteins/genetics ; Escherichia coli Infections/microbiology ; Humans ; Multidrug Resistance-Associated Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Deletion ; },
abstract = {OBJECTIVES: To evaluate the CRISPR-Cas9 and CRISPR interference (CRISPRi) systems as an antibiotic re-sensitization strategy for reversing multidrug resistance in a clinical Escherichia coli isolate.<br><br>MATERIALS AND METHODS: The CRISPR-Cas9 system was applied for precise deletion of the acrB gene from clinical E. coli isolate GP53 and homologous recombination (HR) was provided for accurate repairs of double-strand breaks. An arabinose-inducible CRISPRi system was developed and optimized using fluorescent reporter strain GH01. Multiple guide RNAs (gRNAs) targeting acrB were designed, and the most effective gRNA was selected based on its transcriptional suppression of gene acrB. The minimum inhibitory concentrations (MICs) of selected antibiotics in GP53&#x394;acrB, CRISPRi strains, WT and WT combined with efflux pump inhibitor PA&#x3b2;N were evaluated.<br><br>RESULTS: The CRISPR-Cas9 system precisely deleted the acrB gene in clinical E. coli isolate GP53 with 11.46% knockout efficiency. The constructed arabinose-inducible CRISPRi system effectively repressed fluorescent protein expression in strain GH01. Although dCas9 expression increased with L-arabinose concentration, the transcriptional repression efficiency of the target gene under 1&#x2005;mM induction reached a significant inhibitory level. The CRISPRi system targeting gene acrB exhibited 44.9%, 5.4% and 23.5% inhibition rates on the transcriptional levels with 1&#x2005;mM L-arabinose for three distinct gRNAs. Both the knockout and CRISPRi strains successfully restored susceptibility of the multidrug-resistant E. coli GP53 to quinolones and tetracyclines, outperforming the effect of PA&#x3b2;N combination therapy.<br><br>CONCLUSIONS: In this study, CRISPR-based systems effectively reversed multidrug resistance in a clinical E. coli isolate, advancing the applications of CRISPR systems in controlling bacterial multidrug resistance.},
}
@article {pmid41433963,
year = {2026},
author = {Schneider, O and Zehl, M and Miele, M and Pace, V and Brungs, C and Cheng, JF and Hummelbrunner, S and Dirsch, VM and Zotchev, SB},
title = {Heterologous Expression and CRISPR/Cas9-Assisted Manipulation of the Hybrid Gene Cluster Specifying the Biosynthesis of Meroterpenoids and Phenazines.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {137-148},
doi = {10.1021/acssynbio.5c00531},
pmid = {41433963},
issn = {2161-5063},
mesh = {*Multigene Family/genetics ; *CRISPR-Cas Systems/genetics ; *Phenazines/metabolism/chemistry ; *Terpenes/metabolism/chemistry ; Streptomyces/genetics/metabolism ; Streptomyces coelicolor/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Anti-Bacterial Agents/biosynthesis ; },
abstract = {A hybrid gene cluster, mfq, predicted to govern the biosynthesis of both meroterpenoids and phenaziterpenes, was cloned from the genome of Streptomyces sp. S4.7 and introduced into the heterologous host Streptomyces coelicolor M1154. The biosynthesis of the meroterpenoids marfuraquinocins C and D, previously isolated from Streptomyces niveus SCSIO 3406, as well as a new congener, marfuraquinocin E, which exhibited antibacterial activity, was activated upon overexpression of the regulatory protein MfqF. However, production of neither phenaziterpenes nor phenazines was detected. The structure of marfuraquinocin E was elucidated, revealing the attachment of a terpene moiety at C-2, in contrast to C-6 as seen in the known congeners A-D. Using the CRISPR/Cas9 system, several genes in the mfq cluster were inactivated, confirming the role of MfqW as a prenyltransferase specific to the meroterpenoid pathway. Both gene overexpression and further knockouts provided the first insights into the complex regulation of this hybrid gene cluster. To restore the presumably deficient phenazine biosynthetic pathway, a gene encoding a PhzF homologue from another gene cluster in S4.7 was heterologously expressed alongside the mfq cluster, leading to the production of 1,6-phenazine dicarboxylic acid upon MfqF overexpression. This work lays the foundation for elucidating the complete biosynthetic pathway of marfuraquinocins and its potential coregulation with that of phenazines.},
}
@article {pmid41396964,
year = {2026},
author = {Jiang, C and Liu, Y and Han, W and Zou, D and Chen, K and Jiang, X and Ma, A and Wei, X},
title = {Regulation of Single and Multiple Genes in Bacillus amyloliquefaciens by an Evolution System In Vivo.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {88-98},
doi = {10.1021/acssynbio.5c00480},
pmid = {41396964},
issn = {2161-5063},
mesh = {*Bacillus amyloliquefaciens/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Directed Molecular Evolution/methods ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Cytidine Deaminase/genetics/metabolism ; Mutation ; },
abstract = {With the development of synthetic biology, an evolution system in vivo has been applied to accelerate the construction of cell factories. In this study, an efficient in vivo evolution system was developed for regulation of single and multiple genes in Bacillus amyloliquefaciens. First, the CRISPR/Cas9n-AID base editor was constructed through integration expression of the fused Cas9n protein and activation-induced cytidine deaminase (AID), and the base conversion efficiency from C to T was as high as 90% in single-gene editing. Subsequently, the evolution template (XP43) with an editable RBS sequence (GGGGGGGG) was designed for in vivo evolution through two strategies. By next-generation sequencing of RBS mutation libraries, the extended sgRNA strategy was confirmed to be the optimal evolution scheme. Using the alkaline protease gene (aprE) as the single gene target, the evolution program was initiated to successfully obtain a series of mutant strains with gradient AprE activities. Furthermore, multiple key genes (dhemA, SAM2, and hemEHY) were evolved simultaneously to balance the heme metabolic network, and the optimal mutant strain (HZHA-C2) produced 14.02 mg/L heme, 93% higher than the control strain. Finally, the overexpression of the hemH gene further increased the heme titer by 49%. By a fed-batch fermentation strategy, the heme titer of the optimal engineered strain (HZHA2/pHY-hemH) was improved by 64%, achieving 32.61 mg/L.},
}
@article {pmid41352420,
year = {2026},
author = {Tang, M and Liang, R and Wu, Z and Chen, C and He, B and Zhou, N and Wang, S and Xiao, X and Li, G and Jiang, Y and Gong, G and Zhou, Y},
title = {Deciphering OCT4A-dose-dependent transcriptional profiles associated with tumorigenic potential in somatic cancer cells.},
journal = {SLAS technology},
volume = {36},
number = {},
pages = {100381},
doi = {10.1016/j.slast.2025.100381},
pmid = {41352420},
issn = {2472-6311},
mesh = {*Octamer Transcription Factor-3/genetics/metabolism ; Humans ; *Neoplasms/genetics/pathology ; Cell Line, Tumor ; *Carcinogenesis/genetics ; Gene Expression Regulation, Neoplastic ; Gene Expression Profiling ; *Transcriptome ; CRISPR-Cas Systems ; Gene Regulatory Networks ; Prognosis ; },
abstract = {AIMS: The transcription factor OCT4A, a well-established master pluripotency factor, exerts regulatory effects on cell fate determination that are closely associated with its protein levels. This study aims to uncover the downstream gene profile features relevant to tumorigenic potential mediated by OCT4A under varying protein abundance in somatic cancer cells (SCCs).<br><br>MATERIALS AND METHODS: CRISPR-Cas9-mediated knockout and doxycycline-inducible OCT4A expression systems were established in cervical (HeLa) and hepatocellular (HepG2, Huh7) cancer cells. Single-cell sequencing, spatial transcriptomic and survival analysis data were used to elucidate the expression pattern of OCT4 in somatic cancer tissues and its prognostic relevance. The plate colony formation assay was performed to assess the tumorigenic capacity of SCCs, and Bulk RNA sequencing coupled with weighted gene co-expression network analysis (WGCNA) identified dose-relevant downstream pathways. Functional enrichment, survival modeling, and RT-qPCR validation were used to construct OCT4A-dose-dependent transcriptional regulatory networks.<br><br>KEY FINDINGS: OCT4 transcript, is heterogeneously present and confined to a small subset of tumor cells within somatic cancer tissues, with a significantly higher proportion of OCT4-positive cells in tumor tissues compared to paired paraneoplastic tissues and is significantly correlated with poor prognosis in SCCs. Endogenous low-level OCT4A positively regulates tumorigenic capacity predominantly through targeting non-coding genes, whereas high-level OCT4A suppresses tumorigenic capacity primarily via protein-coding genes in SCCs. A prognostic model based on high-level OCT4A-regulated protein-coding genes was associated with favorable clinical outcomes, aligning with in vitro phenotypic results.<br><br>SIGNIFICANCE: Our findings further confirm in SCCs that the functional pleiotropy of OCT4A is closely linked to its protein abundance, and further systematically elucidate the molecular signatures of OCT4A-regulated downstream gene networks associated with tumorigenic phenotypes at differential protein levels, providing novel insights for its translational exploitation in both oncological intervention and regenerative medicine strategies.},
}
@article {pmid41313840,
year = {2026},
author = {Mohammad, SI and Kareem, AK and Vasudevan, A and Rekha, MM and Jabir, MS and Nayak, P and AlKhafaje, Z and Arora, V and Kadhum, W and Chennakesavulu, K},
title = {Genome editing of immune checkpoints: CRISPR-mediated PD-1 inhibition in cancer.},
journal = {Seminars in oncology},
volume = {53},
number = {1},
pages = {152438},
doi = {10.1016/j.seminoncol.2025.152438},
pmid = {41313840},
issn = {1532-8708},
mesh = {Humans ; *Gene Editing/methods ; *Neoplasms/genetics/therapy/immunology ; *Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors/genetics ; Animals ; *CRISPR-Cas Systems ; *Immune Checkpoint Inhibitors/therapeutic use/pharmacology ; B7-H1 Antigen/antagonists &amp; inhibitors/genetics ; Immunotherapy, Adoptive/methods ; },
abstract = {The programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint is a primary mechanism by which tumors evade immune surveillance, limiting the efficacy of cytotoxic T lymphocytes (CTLs) and tumor-infiltrating lymphocytes (TILs). Although immune checkpoint blockade therapies have revolutionized cancer treatment, their efficacy is restricted by acquired resistance, T-cell exhaustion, and tumor heterogeneity. The advent of CRISPR-Cas9 genome editing provides a precise and versatile approach to disrupt PD-1 or PD-L1, directly enhancing anti-tumor immune responses. Preclinical studies demonstrate that ex vivo PD-1 knockout in primary human T cells or TILs enhances proliferation, cytokine production, and cytotoxicity, resulting in improved tumor clearance in xenograft and humanized mouse models. In chimeric antigen receptor (CAR) T cell therapy, CRISPR-mediated disruption of PD-1 improves effector function, persistence, and resistance to exhaustion, with universal and allogeneic CAR-T platforms benefiting from multiplex genome editing. Direct PD-L1 knockout in tumor cells, often facilitated via nanoparticle- or biomaterial-assisted delivery, reshapes the immunosuppressive tumor microenvironment, promotes T cell infiltration, and enhances the efficacy of adoptive cellular therapy. Combination approaches integrating PD-1 editing with viral antigen targeting, long noncoding RNA (lncRNA) modulation, or conventional checkpoint blockade demonstrate synergistic anti-tumor effects. Clinically, early-phase trials in non-small cell lung cancer, mesothelin-positive solid tumors, and hematological malignancies establish the feasibility, safety, and preliminary efficacy of PD-1-deficient T cells. Despite these promising outcomes, challenges such as off-target effects, delivery efficiency, immunogenicity, long-term persistence, and regulatory considerations remain. This review aims to comprehensively evaluate preclinical and clinical studies investigating CRISPR-mediated PD-1/PD-L1 inhibition across various cancers, summarize mechanistic insights, and highlight translational opportunities and challenges for clinical implementation.},
}
@article {pmid41286106,
year = {2026},
author = {Angelini Stewart, A and Ahrens-Nicklas, RC and Tsai, SQ and Musunuru, K and Giannikopoulos, P and Clelland, CD},
title = {Measurement and clinical interpretation of CRISPR off-targets.},
journal = {Nature genetics},
volume = {58},
number = {1},
pages = {20-27},
pmid = {41286106},
issn = {1546-1718},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods/adverse effects ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; },
abstract = {CRISPR genetic therapies are revolutionizing the landscape of preclinical research and clinical studies, providing new potential routes for curative intervention for a range of previously untreatable diseases. As with any therapy, the therapeutic benefits and risks must be weighed against consideration of the disease threat. Genome-related adverse events are an inherent risk of CRISPR genetic therapies, including off-target edits. The perception that CRISPR therapies ought to have near-zero off-targets belies clinical medicine, therapy development and biology, which demonstrate that 'perfect' therapeutics do not exist. Given that not all genomic off-target events are equal, we provide a practical framework to evaluate and assess off-target safety based on the tools available today and ones that will be developed in the future. With the comprehensive information and assessment gathered using these guidelines, we aim to streamline the transition of CRISPR therapeutics from bench to bedside.},
}
@article {pmid41270550,
year = {2026},
author = {Hu, Q and Liu, J and Zhang, W and Liao, X and Guo, Y and Lu, Q and Zhang, R and Yang, B and Zhang, T and Zhai, X and Luo, Q},
title = {Rapid nucleic acid detection of Mycoplasma synoviae using dual-mode RAA-CRISPR/Cas12a system.},
journal = {Poultry science},
volume = {105},
number = {1},
pages = {106126},
pmid = {41270550},
issn = {1525-3171},
mesh = {Animals ; *Mycoplasma synoviae/isolation &amp; purification ; *Mycoplasma Infections/veterinary/diagnosis/microbiology ; *Poultry Diseases/diagnosis/microbiology ; *Chickens ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; Ducks ; Columbidae ; },
abstract = {Mycoplasma synoviae is a significant avian pathogen implicated in diverse clinical manifestations, including air sacculitis, infectious synovitis with arthritis, and eggshell apex abnormalities, which significantly threaten the poultry industry. To enable the rapid and accurate detection of M. synoviae, we developed a dual-mode diagnostic platform that combines recombinant enzyme-assisted amplification (RAA) with CRISPR/Cas12a-based fluorescence and lateral flow dipsticks (LFD). The Qsep100 automatic nucleic acid analysis system was used to screen for RAA primers by evaluating the amplicon size and concentration. Fluorescence monitoring revealed that the optimal reaction time for the RAA-CRISPR/Cas12a system was 28 min. After optimizing the concentrations of LbaCas12a, crRNA, and the ssDNA reporter, the assay achieved a sensitivity of 5.2 copies/µL. Cross-reactivity testing with seven common avian pathogens confirmed the high specificity of the established method for detecting M. synoviae. In the clinical validation, the method perfectly matched the results of quantitative real time polymerase chain reaction (qPCR). Furthermore, an epidemiological investigation revealed that chickens had the highest positivity rate for M. synoviae among the chickens, ducks, and pigeons. In summary, we developed a rapid, accurate, and portable diagnostic platform for M. synoviae detection, which provides a valuable tool for disease prevention and control in resource-limited settings.},
}
@article {pmid41265135,
year = {2026},
author = {Wright, M and Choi, W and Deng, Z and Kim, D and Lee, K},
title = {CRISPR/Cas9 Gene Editing of Turkey Cells Using Adenoviral Delivery Running Head: RESEARCH NOTE.},
journal = {Poultry science},
volume = {105},
number = {1},
pages = {106096},
pmid = {41265135},
issn = {1525-3171},
mesh = {Animals ; *Gene Editing/veterinary/methods ; *CRISPR-Cas Systems ; *Turkeys/genetics ; *Adenoviridae/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Line ; Receptors, Leptin/genetics ; Genetic Vectors ; },
abstract = {Poultry, including birds such as chicken, duck, turkey, geese, and other game birds is the most widely consumed animal protein globally, with per capita poultry consumption steadily rising in the United States over the past several decades. To meet the demand for poultry, many avenues of improving poultry production have been explored including genetic modification solutions. While genetic modification in many poultry species has been well explored, gene editing in turkey has no research into its application. This study investigated the feasibility of using CRISPR/Cas9 mediated gene editing using adenoviral delivery in vitro, a method that has been proven successful for other species of poultry but never turkey. Three guide RNAs (gRNAs) were designed, targeting the leptin receptor (LEPR) gene matching the gene sequences of both chicken and turkey. Following the assessment of each gRNA's editing efficiency in turkey embryonic fibroblast (TEF) cells and commercially available chicken DF-1 cells via liposome-mediated transfection of the CRISPR vector, the most effective gRNA was chosen for recombinant adenovirus type 5 production. From there, both DF-1 and TEF cells were transduced using the adenovirus and the genome editing efficiency was assessed. The results show that turkey cells can be genetically modified in vitro with indel frequencies comparable to those observed in chicken cells, using both CRISPR plasmids and adenoviral vectors. These findings can lay the groundwork for future experiments and adaptation to in vivo modification of turkey in the future.},
}
@article {pmid41236017,
year = {2026},
author = {Zhang, C and Goldsmith, SR and Htut, M and Rhee, JW and Vyas, V and Clark, MC and Armenian, SH and Forman, SJ and Wang, X},
title = {CRISPR Knockdown of CHIP Genes in Macrophages Drives Increased Production of Inflammatory Cytokines in CAR-T Cell Therapy.},
journal = {Transplantation and cellular therapy},
volume = {32},
number = {1},
pages = {10.e1-10.e5},
doi = {10.1016/j.jtct.2025.08.022},
pmid = {41236017},
issn = {2666-6367},
mesh = {Humans ; *Macrophages/metabolism/immunology ; *Cytokines/metabolism ; DNA Methyltransferase 3A ; *Immunotherapy, Adoptive/methods/adverse effects ; *CRISPR-Cas Systems ; Dioxygenases/genetics ; Receptors, Chimeric Antigen ; Gene Knockdown Techniques ; Proto-Oncogene Proteins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Binding Proteins/genetics ; Repressor Proteins/genetics ; DNA (Cytosine-5-)-Methyltransferases/genetics ; Gene Editing ; },
abstract = {Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by somatic mutations in leukemogenic genes (e.g., DNMT3A, TET2, and ASXL1) in hematopoietic stem cells and is linked to age-related clonal expansion and inflammation. We previously showed that patients with CHIP receiving chimeric antigen receptor (CAR)-T cell therapy have a higher risk of developing clinically significant cytokine release syndrome (CRS) compared to those without CHIP. Here, we investigated the mechanisms through which CHIP mutations can contribute to CAR-T-related toxicities. To model the interaction of CAR-T cell-mediated inflammatory response and macrophages harboring CHIP gene mutations, we employed CRISPR-based gene editing to knock down three key CHIP-associated genes (DNMT3A, TET2, and ASXL1) in human macrophages, using multiple guide RNAs for each. We co-cultured gene-edited macrophages with autologous CS1 and BCMA CAR-T cells and multiple myeloma (MM) tumor cells. Compared to nonedited macrophages, DNMT3A-, TET2-, and ASXL1-edited macrophages secreted significantly higher levels of pro-inflammatory cytokines characteristic of CRS, including IL-6, MCP-1, and IL-1β (P < .05 to .01). These results suggest that the presence of CHIP mutations in human macrophages may exacerbate the inflammatory response during CAR-T therapy. This study highlights CHIP mutations as potential biomarkers for identifying patients at high risk of developing CRS and for guiding personalized prophylactic interventions in MM CAR-T therapy.},
}
@article {pmid41150843,
year = {2026},
author = {Butt, H and Sathish, S and London, E and Le, A and Li, Q and Gudmundsdottir, B and Lee, DY and Burke, EV and Yates, BP and Liu, DR and Hsieh, M and Leonard, A and Eaton, WA and Uchida, N and Pierciey, FJ and Newby, GA and Tisdale, JF and Demirci, S},
title = {Comparative analysis of CRISPR-Cas9, lentiviral transduction, and base editing for sickle cell disease in a murine model.},
journal = {Blood advances},
volume = {10},
number = {2},
pages = {289-300},
doi = {10.1182/bloodadvances.2025017321},
pmid = {41150843},
issn = {2473-9537},
mesh = {Animals ; *Anemia, Sickle Cell/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; *Lentivirus/genetics ; Disease Models, Animal ; Humans ; *Transduction, Genetic ; Hematopoietic Stem Cells/metabolism ; Hematopoietic Stem Cell Transplantation ; },
abstract = {Sickle cell disease (SCD) is a red blood cell disorder caused by a mutation in the β-globin gene, leading to sickle hemoglobin polymerization under low oxygen conditions. Both CRISPR-Cas9 editing and lentiviral transduction have shown promising clinical outcomes, but it remains unclear which approach is superior. Alternatively, new editing tools such as base editing may also be promising and reduce risks of genotoxicity. To compare these approaches, we studied them in an immunocompromised mouse model. We optimized ex vivo conditions in CD34+ hematopoietic stem and progenitor cells (HSPC) and infused edited SCD HSPC into busulfan-conditioned nonirradiated NOD,B6.SCID Il2rγ-/-KitW41/W41 (NBSGW) mice. Ex vivo analysis confirmed successful editing and transduction. At 16 weeks, bone marrow analysis showed similar human CD45+ cell engraftment across all groups (75%-90%). In the competitive transplantation group, there was a lower amount of B-cell lymphoma/leukemia 11A enhancer editing than base editing and lentiviral transduction. A secondary transplantation model yielded similar results. An antisickling assay showed significantly higher red blood cell sickling reduction in the base editing, transduction, and competitive transplantation groups compared to CRISPR-Cas9. In conclusion, although all methods showed therapeutic potential, base editing and lentiviral transduction provided superior outcomes over CRISPR-Cas9-mediated editing in a competitive murine transplantation model.},
}
@article {pmid40971839,
year = {2026},
author = {Lv, X and Wei, Q and Zhi, Q and Liu, X and Li, F and Niu, Y and Sun, H and Jin, K and Chen, GH and Li, B and Zuo, Q},
title = {CRISPR/Cas9-mediated TOP1 knockout in chicken DF-1 cells reveals its critical role in apoptosis regulation and genomic stability.},
journal = {Journal of animal science},
volume = {104},
number = {},
pages = {},
doi = {10.1093/jas/skaf315},
pmid = {40971839},
issn = {1525-3163},
mesh = {Animals ; *Apoptosis/genetics/physiology ; *Chickens/genetics ; *CRISPR-Cas Systems ; *Genomic Instability/genetics ; *DNA Topoisomerases, Type I/genetics/metabolism ; Cell Line ; Gene Knockout Techniques/veterinary ; DNA Damage ; },
abstract = {The role of topoisomerase I (encoded by TOP1) in avian cell survival and apoptosis regulation remains unclear, limiting its potential application in poultry biotechnology. This study aimed to establish a CRISPR/Cas9-mediated TOP1 knockout platform in chicken DF-1 cells and evaluate its functional impact on apoptosis. Three sgRNAs targeting TOP1 were designed and delivered via liposome vectors, achieving knockout efficiencies up to 50% as confirmed by T7 Endonuclease I (T7E1) assay and Sanger sequencing, with no detectable off-target effects. Functional analysis revealed that TOP1 knockout significantly increased apoptosis rates and upregulated DNA damage markers (γH2AX) and apoptotic genes (Caspase 8 and BRCA1). These results demonstrate that TOP1 is essential for maintaining genomic stability in avian somatic cells, and its depletion triggers apoptosis through DNA damage accumulation. Although synthetic lethality-based sex control was not directly tested here, our findings provide critical evidence that TOP1 dysfunction could theoretically enable selective elimination of specific cell populations (e.g., primary germ cells) via CRISPR editing. Notably, developing targeted delivery systems for PGCs-a focus of future research not addressed in this study-will be essential to achieve such selectivity in vivo, highlighting a significant technological hurdle to overcome.},
}
@article {pmid39962280,
year = {2026},
author = {Zheng, C and Zhang, G and Dean, LJ and Sontheimer, EJ and Xue, W},
title = {The reverse transcriptase domain of prime editors contributes to DNA repair in mammalian cells.},
journal = {Nature biotechnology},
volume = {44},
number = {1},
pages = {146-153},
pmid = {39962280},
issn = {1546-1696},
support = {R01 CA275945/CA/NCI NIH HHS/United States ; UH3HL147367//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; P01HL158506//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 GM150273/GM/NIGMS NIH HHS/United States ; UH3 HL147367/HL/NHLBI NIH HHS/United States ; R01GM150279//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {Humans ; *DNA Repair/genetics ; *RNA-Directed DNA Polymerase/metabolism/genetics ; *Gene Editing/methods ; HEK293 Cells ; Animals ; DNA Damage ; Protein Domains ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {Reverse transcriptase (RT) has been shown to play a role in double-strand break repair in bacteria, yet the impact of the RT component of prime editors (PEs) on normal mammalian cellular functions is unclear. Here we show that overexpressed RT or PE increases short insertions and diminishes homology-directed repair following Cas9 cleavage at multiple loci in multiple cell lines. Live-cell imaging shows that RT and PEs are rapidly recruited to laser-induced DNA damage sites and promote endogenous repair, independent of known DNA damage sensors. Interestingly, RT-mCherry partially impairs green fluorescent protein-PARP1 recruitment. A compact PE without an RNase H domain shows reduced DNA repair activity and may therefore be more suitable for clinical application. These data reveal a role for untethered RT or the RT domain of PEs in the repair of chromosomal breaks, calling for evaluation of the long-term effect of PEs and retroviral RT in mammalian cells.},
}
@article {pmid39934271,
year = {2026},
author = {Hart, SK and Müller, S and Wessels, HH and Méndez-Mancilla, A and Drabavicius, G and Choi, O and Sanjana, NE},
title = {Precise RNA targeting with CRISPR-Cas13d.},
journal = {Nature biotechnology},
volume = {44},
number = {1},
pages = {64-69},
pmid = {39934271},
issn = {1546-1696},
support = {DP2HG010099//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA/genetics ; *Gene Editing/methods ; HEK293 Cells ; Transcriptome/genetics ; },
abstract = {The possibility of collateral RNA degradation poses a concern for transcriptome perturbations and therapeutic applications using CRISPR-Cas13. We show that collateral activity only occurs with high RfxCas13d expression. Using low-copy RfxCas13d in transcriptome-scale and combinatorial pooled screens, we achieve high on-target knockdown without extensive collateral activity. Furthermore, analysis of a high-fidelity Cas13 variant suggests that its reduced collateral activity may be due to overall diminished nuclease capability.},
}
@article {pmid41533919,
year = {2026},
author = {Sun, X and Liang, Y and Liang, Y and Li, W and Chen, H and Ding, C and Duan, C and Zhou, Y and Zhang, R and Xu, H and Chen, J},
title = {PRPF8 Mutation-Induced Defects in Human iPSC-Derived RPE Are Rescued by Adenine Base Editing.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {67},
number = {1},
pages = {21},
pmid = {41533919},
issn = {1552-5783},
mesh = {Humans ; *Retinal Pigment Epithelium/metabolism/ultrastructure/pathology ; *Induced Pluripotent Stem Cells/metabolism ; *Mutation ; *Gene Editing/methods ; *Retinitis Pigmentosa/genetics/pathology/metabolism ; *Adenine ; Cell Differentiation ; Cells, Cultured ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {PURPOSE: The pathological effects of pre-mRNA processing factor 8 (PRPF8) mutations on the retinal pigment epithelium (RPE) are not fully understood. We aimed to identify disease-specific cellular and molecular phenotypes in PRPF8 retinitis pigmentosa (RP) patient-derived induced pluripotent stem cell (iPSC)-RPE and to test whether adenine base editing (ABE), which corrects the PRPF8 mutation in iPSCs, can reverse abnormal RPE phenotypes.<br><br>METHODS: We obtained patient-derived iPSCs with the heterozygous PRPF8 (c.5792C>T) mutation and created an induced mutation iPSC line by introducing the same mutation into wild-type iPSCs using CRISPR/Cas9. These cells were differentiated into RPE cells. We measured PRPF8 expression, barrier integrity, and apicobasal polarity. Electron microscopy examined apical microvilli and pigment granules. RNA sequencing quantified splicing events and affected pathways. ABE corrected the PRPF8 mutation in patient iPSCs, and the corrected clones were re-differentiated into RPE cells for evaluation.<br><br>RESULTS: PRPF8-mutant RPE cells exhibited decreased PRPF8 mRNA and protein levels, weakened barrier function, and disrupted cell polarity. Ultrastructural analysis showed loss of apical microvilli and pigment granules. Transcriptomic analysis identified abnormal splicing events, with enrichment in cilium assembly and melanosome pathways. ABE correction restored PRPF8 expression, normalized barrier integrity, apicobasal polarity, and rescued the defects in apical microvilli and pigment granules.<br><br>CONCLUSIONS: PRPF8 mutations in patient-derived iPSC RPE cause functional and ultrastructural defects driven by splicing abnormalities. ABE correction of the PRPF8 mutation in iPSCs can restore PRPF8 expression and alleviate cellular and molecular defects in RPE and highlights the therapeutic potential of precise gene editing correction strategies for RP.},
}
@article {pmid41287270,
year = {2026},
author = {Yao, W and Xu, X and Zhai, X and Ji, T and Zhang, R and Xu, S and Luo, X},
title = {Autocatalytic Circular DNA Powered Plasmonic CRISPR/Cas12a Platform for Ultrasensitive Non-Nucleic Acid Target Sensing.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {65},
number = {3},
pages = {e16838},
doi = {10.1002/anie.202516838},
pmid = {41287270},
issn = {1521-3773},
support = {21505081//National Natural Science Foundation of China/ ; 22374085//National Natural Science Foundation of China/ ; ZR2023MB110//Natural Science Foundation of Shandong Province/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA, Circular/chemistry/metabolism/genetics ; *Biosensing Techniques/methods ; Nucleic Acid Amplification Techniques ; Catalysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a-based detection of non-nucleic acid targets faces two major challenges: 1) limited sensitivity due to the inherent inability to pre-amplify non-nucleic acid targets, and 2) suboptimal performance of traditional reporters caused by photobleaching of fluorescent dyes, rapid degradation, and slow reaction kinetics resulting from random molecular collisions. To overcome these limitations, we developed an innovative plasmonic CRISPR/Cas12a platform featuring positive-feedback autocatalytic circular DNA (cir-DNA) amplification. This system synergistically combines spatial confinement effects with plasmon-enhanced fluorescence (PEF) to achieve ultrasensitive detection of non-nucleic acid targets. The engineered cir-DNA enables continuous Cas12a regeneration for autocatalytic signal amplification, while the designed plasmonic spherical nucleic acids significantly accelerate reaction kinetics while enhancing fluorescence signals. This integrated approach reduced the required reaction time to 15 min while improving the detection limit by approximately 52-fold compared to conventional methods. Furthermore, by leveraging a convolutional neural network (CNN) machine learning model, not only the assessment of the risk level of perfluorooctanoic acid (PFOA) based on threshold-positive and threshold-negative serum concentrations but also highly accurate blind testing were both achieved, highlighting its potential for clinical applications such as pregnancy risk assessment.},
}
@article {pmid40759200,
year = {2026},
author = {He, J and Zhang, M and Shi, Y and Ji, Z and Lu, Y and Yu, W and Wang, F and Guan, Q and Yan, H and Chen, G and Zhang, E and Lian, S and Pu, X and Zhang, J and Ge, S and Zhou, R and Li, J and Wang, P and Sun, B and Zhang, Z},
title = {Targeting xanthine oxidoreductase reverses resistance to EGFR tyrosine kinase inhibitors in intrahepatic cholangiocarcinoma.},
journal = {Journal of hepatology},
volume = {84},
number = {2},
pages = {355-369},
doi = {10.1016/j.jhep.2025.07.016},
pmid = {40759200},
issn = {1600-0641},
mesh = {*Cholangiocarcinoma/drug therapy/genetics/pathology ; Animals ; Mice ; *Drug Resistance, Neoplasm/drug effects/genetics ; Humans ; ErbB Receptors/antagonists &amp; inhibitors/metabolism ; *Bile Duct Neoplasms/drug therapy/pathology/genetics ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; *Xanthine Dehydrogenase/antagonists &amp; inhibitors/genetics/metabolism ; Cell Line, Tumor ; CRISPR-Cas Systems ; Male ; Tyrosine Kinase Inhibitors ; },
abstract = {BACKGROUND &amp; AIMS: Despite the overexpression and aberrant activation of epidermal growth factor receptor (EGFR) in intrahepatic cholangiocarcinoma (iCCA), the disease remains refractory to EGFR tyrosine kinase inhibitors (TKIs). Multiple clinical trials involving EGFR-targeting agents have been conducted; however, none have demonstrated clinically significant efficacy. The aim of this study was to elucidate the mechanisms underlying EGFR TKI resistance in iCCA.<br><br>METHODS: Genome-scale CRISPR-Cas9 chemical-genetic screens were conducted, identifying xanthine oxidoreductase (XOR) as a critical modulator. XOR expression was evaluated in surgical samples from patients with iCCA and matched peritumor tissue via immunohistochemistry, western blotting, and quantitative reverse-transcription PCR. iCCA models were established in Xdh[f/f] and Xdh[hep-/-] mice, as well as in adeno-associated virus-treated mice via hydrodynamic tail vein injection of the oncogenic driver myr-AKT combined with either Yap[S127A] or NICD1 using the Sleeping Beauty system.<br><br>RESULTS: CRISPR-Cas9 screening revealed that XOR, which is upregulated in iCCA compared to peritumor tissue and bile ducts, is required for EGFR TKI resistance. XOR knockdown inhibited iCCA cell proliferation, increased DNA damage, reduced tumor progression in iCCA models, and significantly enhanced the anti-tumor efficacy of gefitinib both in vitro and in vivo. CEBP&#x3b2; was found to regulate XOR transcription. Mechanistically, XOR promoted EGFR stability via USP8-mediated deubiquitination and MUC1 upregulation, thereby enhancing signaling cascades and DNA damage repair - collectively driving resistance to TKIs.<br><br>CONCLUSIONS: Targeting XOR-mediated EGFR stabilization and MUC1 upregulation represents a promising therapeutic strategy to inhibit iCCA progression and enhance sensitivity to EGFR TKIs in patients with iCCA.<br><br>IMPACT AND IMPLICATIONS: This study identifies XOR as a key driver of resistance to EGFR tyrosine kinase inhibitors (TKIs) in intrahepatic cholangiocarcinoma (iCCA), addressing a major limitation of current targeted therapies. By promoting EGFR stability through USP8-mediated deubiquitination and MUC1 upregulation, XOR enhances DNA repair and sustains oncogenic signaling. Inhibition of XOR sensitizes iCCA cells to EGFR TKIs, suppresses tumor progression, and exacerbates DNA damage. These findings suggest that targeting XOR could overcome TKI resistance and offer a novel therapeutic strategy for iCCA, potentially improving outcomes in a patient population with limited effective treatment options.},
}
@article {pmid41533584,
year = {2026},
author = {Zhang, J and He, X and Huang, J and Cheng, C and He, G and Xia, R and Yang, J and Chen, J and Guo, L and Xiang, D and Li, F and Shi, J and Li, P},
title = {Steric regulation of CRISPR/Cas12a trans-cleavage kinetics via split-activator extensions.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41533584},
issn = {1362-4962},
support = {2025ZDXM001//Chongqing Municipal Health Commission/ ; KJZD-K202400103//Chongqing Municipal Education Commission/ ; CSTB2024TIAD-CYKJCXX0031//Scientific and Technological Innovation Cooperation Program/ ; YXGD2025029//Chongqing Young and Middle-aged Medical High-level Talent Project/ ; YXQN2025049//Chongqing Youth Outstanding Medical Talent Project/ ; 2026MSXM018//Chongqing Science and Technology Bureau/ ; 2024YCXM010//2024 Hospital-level Cultivation Project of Chongqing University Jiangjin Hospital/ ; 2025qdjfxm001//Chongqing University Jiangjin Hospital/ ; 2025qdjfxm002//Chongqing University Jiangjin Hospital/ ; PS202511//2025 Key Research Project for Enhancing Medical Service Capabilities of County-level Medical Institutions/ ; CSTB2024NSCQ-KJFZMSX0018//Chongqing Science and Technology Development Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Kinetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism/chemistry ; MicroRNAs/genetics/analysis ; DNA/genetics/chemistry/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a holds substantial promise for molecular diagnostics, yet its rapid and uncontrolled activation often results in background leakage and disrupts the coordination of upstream reaction modules. Here, we established a steric-regulation framework that enables predictable tuning of Cas12a trans-cleavage kinetics through rationally engineered extensions on split activators. Systematic analysis of extension orientation, length, and hybridization state revealed quantitative and direction-dependent rules governing steric control of activator assembly and Cas12a activation. Guided by these insights, we integrated the sterically regulated split activator into an entropy-driven DNA circuit to construct a fully one-pot cascaded detection system. The engineered steric barriers effectively suppressed premature activation and established precise kinetic matching between the DNA circuit and Cas12a. The resulting platform achieved a detection limit of 1.24 pM for microRNA-21 and demonstrated high fidelity. This work defines a predictable steric-gating mechanism for Cas12a activation and delivers a nucleic-acid-only regulatory module that can be incorporated into diverse CRISPR architectures, supporting the development of robust, leakage-resistant one-pot diagnostic systems.},
}
@article {pmid41533581,
year = {2026},
author = {Sugimoto, Y and Kachi, T and Watanabe, Y and Kubokawa, M and Ogami, K and Kawamata, M and Yoshino, S and Suzuki, HI},
title = {Optimized CRISPR-Cas9 system for efficient engineering of ecDNA in cancer cells.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41533581},
issn = {1362-4962},
support = {JP24H00614//Japan Society for the Promotion of Science/ ; JP22K06925//Japan Society for the Promotion of Science/ ; JP25K10464//Japan Society for the Promotion of Science/ ; 19K24694//Japan Society for the Promotion of Science/ ; JP22ama221111//Japan Agency for Medical Research and Development/ ; JP23ck0106791//Japan Agency for Medical Research and Development/ ; JP23tk0124003//Japan Agency for Medical Research and Development/ ; JP24ck0106875//Japan Agency for Medical Research and Development/ ; JP25ck0106019//Japan Agency for Medical Research and Development/ ; JP25ak0101291//Japan Agency for Medical Research and Development/ ; JP23kk0305026//Japan Agency for Medical Research and Development/ ; JP25kk0305028//Japan Agency for Medical Research and Development/ ; //Takeda Science Foundation/ ; 22-6304//oray Science Foundation/ ; //Inamori Research Institute for Science/ ; 24KJ1238//JSPS/ ; //Nagoya University/ ; //JST-SPRING/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Neoplasms/genetics ; Cell Line, Tumor ; Gene Knock-In Techniques ; *DNA/genetics ; },
abstract = {Extrachromosomal DNA (ecDNA) amplification represents an emerging mechanism underlying oncogene amplification, tumor heterogeneity, and drug resistance in cancer. However, the biology of ecDNA remains poorly understood because tools to engineer ecDNAs and precisely monitor their dynamics are limited. In particular, genome engineering strategies have not been established for ecDNA, which exists in tens to hundreds of copies within a single cell. Here, we report a systematic validation of ecDNA editing using standard CRISPR-Cas9 system and optimized CRISPR-Cas9 system with safeguard single-guide RNAs (sgRNAs), in which the addition of cytosine extensions finely reduces excessive Cas9 activity. The conventional CRISPR-Cas9 system induced severe cytotoxicity and markedly reduced ecDNA copy number, together with frequent micronucleus formation. Knock-in efficiency was remarkably low, highlighting an intrinsic difficulty in editing ecDNA. In contrast, the safeguard sgRNA strategy not only alleviated cytotoxicity and ecDNA loss in a cytosine-length-dependent manner but also enabled efficient knock-in into multiple ecDNA per cell. Computational simulations suggested that the degree and temporal patterns of multiple DNA cleavage events shape cell death, micronucleus formation, and rapid expansion of knock-in ecDNA. Collectively, optimization of Cas9 activity using safeguard sgRNAs enables efficient and nondisruptive ecDNA engineering, providing a powerful tool to study ecDNA biology.},
}
@article {pmid41533573,
year = {2026},
author = {Kammerdiener, EK and Garcia, SK and Bales, MK and Klingeman, DM and Guss, AM and Giannone, RJ and Hettich, RL and Eckert, CA and Alexander, WG},
title = {Multilayered regulation by RNA thermometers enables precise control of Cas9 expression in E. coli.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41533573},
issn = {1362-4962},
support = {ERKP886//U.S. Department of Energy/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Promoter Regions, Genetic ; Temperature ; Escherichia coli Proteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics ; Gene Editing/methods ; Heat-Shock Proteins/genetics/metabolism ; },
abstract = {Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We also show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs in Escherichia coli. This strategy should be applicable to all bacterial Cas9-based methods and technologies.},
}
@article {pmid41533202,
year = {2026},
author = {Kumar, P and Yadav, H and Mahakalkar, B and Mandlik, R and Vats, S and Thakral, V and Kumar, V and Nishad, SK and Nichal, S and Deshmukh, R and Sharma, TR and Sonah, H},
title = {Challenges and Opportunities with CRISPR-Based Genome Editing in Legume Crops.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {24},
pmid = {41533202},
issn = {1438-7948},
support = {HSCSIT/R&amp;D/2024/511//Haryana State Council for Science and Technology/ ; HSCSIT/R&amp;D/2024/511//Haryana State Council for Science and Technology/ ; Agril. Edn / 27/05/NP(VP)-2023-HRD//Indian Council of Agricultural Research/ ; BT/PR38279/GET/119/351/2020//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; *Fabaceae/genetics ; *Genome, Plant ; Plants, Genetically Modified/genetics ; Plant Breeding ; },
abstract = {Over the last couple of decades, tremendous progress has been made in legume genomics. Genomics information generated for legume crops is being explored through molecular breeding and transgenic approaches. However, the gap between knowledge generation and its utilization is increasing. In this regard, recent developments in genome editing techniques provide an excellent opportunity to utilize the available knowledge for the improvement of legume crops. This review highlights recent developments with Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based genome-editing approaches, including Cas variants/orthologs and Protospacer adjacent motif-less (PAMless) Genome Editing, multiplex genome editing, base editing, prime editing, transcriptional regulation, methylome editing, and DNA-free editing methods. Furthermore, the applications of non-homologous end joining (NHEJ) and homology-directed repair (HDR)- based editing, are addressed which enable targeted and precise genomic modifications. Moreover, virus-mediated genome editing, in planta transformation, and mobile guide RNAs are increasingly being leveraged to enhance the efficiency and heritability of genome editing. Additionally, the role of artificial intelligence in guide RNA design, off-target prediction, and the development of novel Cas variants is also discussed, which can speed up the legume improvement. This article highlights the successful examples of efforts utilizing CRISPR/Cas9 for the development of legume crops with biotic and abiotic stress tolerance, desirable plant architecture, improved nutrient uptake, and enhanced yield and quality. The biggest limitation in the genome editing of legume crops is their recalcitrance to both transformation and tissue culture. This article discusses how this particular limitation can be addressed in the context of genome editing of legume crops. Finally, the possibilities of integrating these recently developed tools with translational breeding have also been discussed, which will facilitate the legume production for sustainable agriculture and food security.},
}
@article {pmid41533153,
year = {2026},
author = {Ricci, ML and Fillo, S and Giordani, F and Ciammaruconi, A and Girolamo, A and Anselmo, A and Monte, A and Mentasti, M and Cusimano, V and Caporali, MG and Rota, MC and Petzold, M and Afshar, B and Lista, F and Luck, C and Scaturro, M},
title = {Genomic characterization of Legionella pneumophila serogroup 1 ST901 isolates responsible for recurrent travel-associated Legionnaires' disease cases and clusters.},
journal = {Pathogens and global health},
volume = {},
number = {},
pages = {1-12},
doi = {10.1080/20477724.2025.2610657},
pmid = {41533153},
issn = {2047-7732},
abstract = {Cases of travel-associated Legionnaires' disease (TALD) are frequently reported in Italy. From 1987 to 2021, 61 cases of TALD were linked to 22 hotels in a municipality in northern Italy. Legionella pneumophila serogroup 1 (Lp1) strains isolated from both patients and hotel water systems were identified as sequence type (ST) 901, a genotype rarely associated with travel-related infections in Italy or elsewhere. Whole-genome sequencing was used to analyze 41 isolates, and phylogenetic relationships were inferred by core genome multilocus sequence typing (cgMLST), single nucleotide polymorphisms (SNP) and pangenome analyses. The Lp ST901 isolates were found to form a clade characterized by some accessory genomic islands (AGI) already described in other epidemic strains, such as Alcoy, Corby, Paris and Philadelphia; other islands, containing either transposase/recombinase or transcriptional regulator factors or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-cas systems, were specific to Lp ST901. Lp ST901 also appears to have undergone possible recombination with other strains, such as Lp ST47 (Lorraine strain). Additionally, CRISPR-Cas systems may have contributed to the protection of Lp ST901 from external dangers, while the colonized hotel water systems may have provided an ideal environmental protective niche. Our findings highlight that Lp ST901 has public health significance and deserves attention in Legionnaires' disease surveillance and risk assessment.},
}
@article {pmid41532605,
year = {2026},
author = {Chen, Q and Gou, H and Xu, C and Wang, S and Zhang, H and Song, M and Wang, M and Ji, X and Wei, X and Tan, Y and Quan, H and Luo, P and Shou, H and Liu, P and Liang, Y and Zhu, JK},
title = {Structure-Guided Engineering of a Cas12i Nuclease Unlocks Near-PAMless Genome Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e16670},
doi = {10.1002/advs.202516670},
pmid = {41532605},
issn = {2198-3844},
support = {KJZD20240903102703005//Shenzhen Science and Technology Program/ ; JCYJ20241202125332043//Shenzhen Science and Technology Program/ ; 2024B1111130001//Guangdong S&amp;T Program/ ; },
abstract = {The therapeutic and research applications of CRISPR-Cas nucleases are constrained by their reliance on specific Protospacer Adjacent Motifs (PAMs), which limit the accessible sites in the genome. To overcome this critical barrier, we performed structure-guided engineering of SF01, a compact Cas12i nuclease. Using AlphaFold-predicted structural models, we identified and systematically mutagenized 38 residues at the PAM-interacting interface. This iterative engineering process yielded three superior variants-KR, IKRR, and STKRR-that exhibit dramatically relaxed PAM specificity, enabling efficient editing at a broad spectrum of 5'-NNTN-3' sites. Importantly, while the most broad-spectrum variant (STKRR) shows a trade-off at canonical sites, the IKRR variant retains high activity at canonical 5'-NTTN-3' PAMs while simultaneously enabling efficient editing at 5'-NNTN-3' sites. This near-PAMless activity expands the targetable portion of the genome to over 25%, a four-fold increase over the parental nuclease. Furthermore, adenine base editors (ABEs) constructed with these variants achieve high-efficiency editing (∼80%) at endogenous loci with expanded targeting scope. Comprehensive off-target analysis using GUIDE-tag and Digenome-seq revealed that the enhanced on-target activity of the SF01 variants is not accompanied by a loss of specificity. These engineered nucleases represent a powerful and versatile expansion of the genome editing toolkit, enabling applications previously inaccessible due to PAM constraints.},
}
@article {pmid41528700,
year = {2026},
author = {Wei, W and Yang, Y and Shih, J},
title = {Rapid and Sensitive Detection of Phytoplasma Diseases Using a CRISPR/Cas12a DETECTR Assay Combined with Isothermal Recombinase Polymerase Amplification.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3008},
number = {},
pages = {63-74},
pmid = {41528700},
issn = {1940-6029},
mesh = {*Phytoplasma/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism ; DNA, Bacterial/genetics ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases/genetics ; Bacterial Proteins/genetics ; },
abstract = {Our protocol outlines a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay, which combines CRISPR/Cas12a technology with isothermal Recombinase Polymerase Amplification (RPA) for the rapid and specific detection of phytoplasma diseases in plants. This isothermal method utilizes RPA to amplify the target DNA fragment from the genomic DNA of phytoplasmas, followed by incubation with Cas12a nuclease and CRISPR RNAs (crRNAs) specifically designed to target unique phytoplasma DNA sequences. Upon initial cleavage of the amplified target DNA, Cas12a gains enzymatic activity to indiscriminately cleave single-stranded fluorescent oligonucleotide reporters, generating a fluorescent signal for highly sensitive detection of the pathogen. The protocol provides detailed instructions on: (i) sample collection and preparation; (ii) assay reaction setup, including RPA and Cas12a detection steps; (iii) reaction and detection conditions; and (iv) guidelines for accurately interpreting fluorescence data to detect phytoplasma DNA. This protocol is designed for researchers and agricultural professionals to effectively adopt and implement this advanced diagnostic technique.},
}
@article {pmid41505468,
year = {2026},
author = {Ma, J and Zhang, Y and Li, Z and Liu, L and A, J and Liang, R and Cao, C and Zhou, J and Cheng, P and Li, Y and Li, Z and Ma, L and Jiang, L and A, X},
title = {CRISPR/Cas12a-RCA enables ultrasensitive detection of circulating free DNA for noninvasive diagnosis of echinococcosis.},
journal = {PLoS neglected tropical diseases},
volume = {20},
number = {1},
pages = {e0013069},
doi = {10.1371/journal.pntd.0013069},
pmid = {41505468},
issn = {1935-2735},
mesh = {Humans ; *CRISPR-Cas Systems ; *Echinococcosis/diagnosis/blood/parasitology ; *Echinococcus/genetics/isolation &amp; purification ; *Cell-Free Nucleic Acids/blood/genetics ; Sensitivity and Specificity ; *DNA, Helminth/blood/genetics ; *Molecular Diagnostic Techniques/methods ; Animals ; Male ; Female ; Limit of Detection ; Middle Aged ; High-Throughput Nucleotide Sequencing ; Adult ; },
abstract = {OBJECTIVE: To develop a novel non-invasive CRISPR/Cas12a-RCA assay for the primary screening of human echinococcosis via detection of circulating Echinococcus cell-free DNA (cfDNA) in peripheral blood.<br><br>METHODS: Plasma cfDNA from 20 AE patients was analyzed via high-throughput sequencing to identify conserved repetitive Echinococcus fragments.A one-pot RCA system coupled with CRISPR/Cas12a was optimized for Echinococcus-cfDNA detection. The limit of detection (LOD) was determined using serially diluted synthetic standards, while specificity was validated through mismatch probes and cross-reactivity testing. Clinical validation included 50 AE cases, 22 cystic echinococcosis (CE) cases, 43 non-Echinococcus hepatic disease (HD) cases, and 53 healthy controls (CON).<br><br>RESULTS: A conserved repetitive 28S rDNA fragment (pan-Echinococcus-28S) was identified as a biomarker. The CRISPR/Cas12a-RCA assay achieved amplification within 30 minutes at 37 &#xb0;C, with a linear range of 1 aM to 100 pM and an LOD of 1.41 aM. Visual detection limits were 10 aM (UV light) and 1 aM (blue light). The assay demonstrated high sensitivity (87.5%) and specificity (96.9%, AUC&#x2009;=&#x2009;0.933) in distinguishing Echinococcus infection (AE/CE) from HD and CON.<br><br>CONCLUSION: The optimized one-pot CRISPR/Cas12a-RCA system enables rapid and ultrasensitive detection of pan-Echinococcus cfDNA, providing a cost-effective and highly accurate solution for the primary screening of echinococcosis.},
}
@article {pmid41494531,
year = {2026},
author = {Wan, W and Ji, X and Song, H and Zhang, Z and Kwok, CK and Fang, X and Li, X},
title = {Fluorogenic CRISPR for DNA imaging in live mammalian cells.},
journal = {Cell chemical biology},
volume = {33},
number = {1},
pages = {33-44},
doi = {10.1016/j.chembiol.2025.10.013},
pmid = {41494531},
issn = {2451-9448},
mesh = {Humans ; *DNA/genetics/analysis/metabolism ; *Fluorescent Dyes/chemistry ; Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Optical Imaging ; },
abstract = {Spatiotemporal imaging of genomic DNA dynamics in live mammalian cells is essential for elucidating eukaryotic organization and processes relevant to health and disease. CRISPR systems greatly facilitate the development of live cell DNA imaging tools. However, conventional CRISPR imaging tools typically utilize constitutively fluorescent proteins, resulting in high background noise, nonspecific nucleolar signals, and low signal-to-noise ratios. To address this, fluorogenic CRISPR-based imaging tools have been developed. These tools remain non-fluorescent until they bind to the target DNA, thus significantly reducing the background and enhancing the sensitivity. This review summarizes four fluorogenic CRISPR strategies, each utilizing different fluorogenic reporters, including fluorogenic proteins, fluorogenic RNA aptamers, split fluorescent proteins, and molecular beacons. These fluorogenic CRISPR approaches successfully monitored the subnucleus gene loci localization, dynamics, and DNA breaks and repairs. We anticipate that this review can inspire researchers to expand the fluorogenic CRISPR for cellular DNA imaging and diverse bioapplications.},
}
@article {pmid41444344,
year = {2025},
author = {Li, X and Guo, J and Yang, H and Wu, Y and Xie, Z and Li, D},
title = {A CRISPR-assisted passive microfluidic chip for rapid, visual detection of multiple respiratory viruses.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {2033},
pmid = {41444344},
issn = {2045-2322},
support = {GJHZ20220913143207014//Science and Technology Innovation Commission of Shenzhen/ ; 12274197//China National Natural Science Fund/ ; 2022B1515020093//Guangdong Scientific and Technological Project/ ; },
mesh = {Humans ; SARS-CoV-2/genetics/isolation &amp; purification ; *Lab-On-A-Chip Devices ; *CRISPR-Cas Systems ; COVID-19/diagnosis/virology ; *Respiratory Tract Infections/virology/diagnosis ; Coinfection/diagnosis/virology ; Nucleic Acid Amplification Techniques ; Influenza B virus/genetics/isolation &amp; purification ; Influenza A virus/genetics/isolation &amp; purification ; },
abstract = {In recent years, viral co-infections, particularly with respiratory viruses, have resulted in more complex symptoms, a greater disease burden, and increased challenges in clinical decision-making. These complexities underscore the urgent need for improved diagnostic tools in the managing acute respiratory infections. To address the limitations of conventional qPCR and current POCT methodologies, we developed a passively driven microfluidic chip capable of rapidly screening multiple respiratory viruses. This platform is particularly suited for the point-of-care diagnosis of viral co-infections. Our device integrates nucleic acid amplification and CRISPR-based detection within a single, passively operated system. By utilizing a rapid, 10-minute sample preparation protocol and a 35-minute on-chip assay, this platform enables the multiplex detection of influenza A/B, human parainfluenza virus, and SARS-CoV-2. The total assay time from sample to answer is approximately 45 min, with equipment requirements minimized to a heating block. The assay demonstrated a detection sensitivity of about 10 copies/µL for viral RNA in dilution series experiments. The sensitivity of the assay was 98.44% (95% CI: 91.6%-99.96%), and the specificity was 100% (95% CI: 79.4%-100%). The system combines CRISPR-Cas12a-mediated sensing with reverse transcription recombinase polymerase amplification (RPA) for highly specific nucleic acid detection. The chip design utilizes capillary action and gravity-driven flow for autonomous fluid control, while lyophilized reagent preloading ensures storage stability and minimizes user intervention.},
}
@article {pmid41435708,
year = {2026},
author = {Fang, J and Chen, Q and Ran, M and Chen, R and Chen, W and Cui, J and Wang, J and Zhong, K and Shi, L and Lu, C and Jiang, H},
title = {RPA-CRISPR/Cas12a-coupled microfluidic biosensor enabling on-site, sensitive quantification of Vibrio parahaemolyticus.},
journal = {Biosensors &amp; bioelectronics},
volume = {296},
number = {},
pages = {118327},
doi = {10.1016/j.bios.2025.118327},
pmid = {41435708},
issn = {1873-4235},
mesh = {*Vibrio parahaemolyticus/isolation &amp; purification/genetics/pathogenicity ; *Biosensing Techniques/instrumentation ; *Nucleic Acid Amplification Techniques/instrumentation ; Limit of Detection ; *CRISPR-Cas Systems/genetics ; Animals ; Seafood/microbiology ; Lab-On-A-Chip Devices ; *Vibrio Infections/microbiology/diagnosis ; Equipment Design ; },
abstract = {Vibrio parahaemolyticus is a major cause of seafood-associated gastroenteritis and aquatic animal diseases, posing persistent threats to public health and aquaculture. Rapid and accurate on-site quantitative detection is essential for risk assessment and early intervention. Although qPCR and digital PCR provide reliable quantification, their reliance on complex instrumentation limits field deployment. RPA-CRISPR-based isothermal assays offer a low-equipment alternative; however, existing approaches lack robust methodological strategies to achieve standard-curve-based quantitative reliability under field-deployable conditions. Here, we propose a methodological framework that enables in-run calibration and variance control for quantitative isothermal amplification by integrating reaction-volume locking and simultaneous standard-curve generation, implemented here within a closed centrifugal microfluidic system. A multi-unit microfluidic platform preloaded with gradient concentration standard plasmids allows concurrent construction of standard curves and sample analysis in a single run, reducing the impact of environmental and batch-to-batch variability. Signal generation was achieved using a one-pot RPA-CRISPR/Cas12a assay, in which balanced amplification and cleavage kinetics were obtained by screening crRNAs targeting suboptimal PAM sites and optimizing reaction conditions. The platform achieved a detection limit of 6.08 copies/μL and a linear quantitative range of 10[0]-10[4] copies/μL (R[2] > 0.96), with performance comparable to qPCR (AUC = 0.984), and acceptable intra- and inter-assay variability under the tested conditions, with relative standard deviations of 2.63-6.07 %, at a cost of approximately $3.30 per test. Validation using spiked and real seafood samples demonstrated reliable on-site quantification. This work establishes a transferable quantitative methodology for RPA-CRISPR-based isothermal assays, advancing field-deployable pathogen detection in aquaculture and food safety.},
}
@article {pmid41414712,
year = {2026},
author = {Congdon, ST and Bennett, J and Opinya, R and Agosto, AR and Dossias, O and Kokko, C and Levesque, AA and Koob, AO and Silver, AC and Thomas-Charles, CA},
title = {Investigating and correcting a rare pathogenic mutation in GDF11.},
journal = {HGG advances},
volume = {7},
number = {1},
pages = {100559},
doi = {10.1016/j.xhgg.2025.100559},
pmid = {41414712},
issn = {2666-2477},
mesh = {Humans ; HEK293 Cells ; *Growth Differentiation Factors/genetics/metabolism ; *Gene Editing/methods ; *Bone Morphogenetic Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Mutation ; Golgi Apparatus/metabolism ; Codon, Nonsense ; },
abstract = {Single-nucleotide variants (SNVs) and small insertions or deletions (indels) underlie most rare monogenic disorders, yet therapeutic strategies to precisely correct these mutations remain limited. Prime editing enables the repair of such pathogenic variants without introducing double-stranded breaks. Here, we applied CRISPR prime editing to model and correct a de novo GDF11 nonsense mutation (Tyr336∗) identified in a participant from the Undiagnosed Diseases Network with growth delay and multisystem abnormalities. Using HEK293T cells, we generated heterozygous (HET) GDF11 Tyr336∗ clones, which exhibited reduced GDF11 protein levels due to post-translational degradation likely mediated by endoplasmic reticulum- and Golgi-associated quality control pathways. These cells displayed marked Golgi abnormalities, including an increased number of compact, irregularly shaped Golgi structures, findings consistent with Golgi fragmentation and stress. Transcriptomic profiling of HET cells revealed a broad dysregulation of gene networks, including downregulation of metabolic and Golgi-linked biosynthetic genes, and upregulation of cell-adhesion and extracellular matrix genes. These transcriptional shifts paralleled the participant's developmental, neural, and cardiovascular phenotypes. To correct the mutation, we tested multiple bespoke prime editing strategies and identified PE7, in combination with a prime editing guide RNA designed by Pridict, as the most effective ribonucleoprotein complex for rescue. Editing efficiency was further enhanced by introducing an additional silent protospacer-adjacent motif-disrupting mutation, likely preventing both Cas9 re-binding and mismatch repair. Together, these findings support a haploinsufficiency mechanism for the GDF11 Tyr336∗ allele and establish a generalizable framework for disease modeling and allele-specific correction of pathogenic variants in human cells.},
}
@article {pmid41401738,
year = {2026},
author = {Feng, J and Lin, X and Kang, L and Duan, M and Duan, N and Wang, Z and Wu, S},
title = {Integrating a microfluidic chip@LFA biosensor enabled by Pt3Sn@MGO nanocomposites for RAA/CRISPR-Cas12b mediated food adulteration monitoring.},
journal = {Biosensors &amp; bioelectronics},
volume = {296},
number = {},
pages = {118315},
doi = {10.1016/j.bios.2025.118315},
pmid = {41401738},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; Animals ; *Food Contamination/analysis ; *Nanocomposites/chemistry ; Salmon/genetics ; CRISPR-Cas Systems/genetics ; Limit of Detection ; Lab-On-A-Chip Devices ; Nucleic Acid Amplification Techniques ; Food Analysis ; Recombinases/chemistry ; Myoglobin/genetics ; },
abstract = {Traditional methods for food species genetic authentication typically involve time-consuming laboratory procedures and inconsistent operations, easily resulting in gene damage and inaccurate diagnostics. Here, by fully integrating reagent flow and reactions through micromachining technology, a standardized and lab-free operational PMMA-based microfluidic chip@ lateral flow assay (LFA) biosensor was developed for salmon adulteration detection. The primers recognizing the myoglobin nuclear gene of salmon were designed and optimized, enabling efficient recombinase-aided amplification (RAA) of target gene and subsequent activation of the CRISPR-Cas12b system. A multifunctional Pt3Sn@MGO nanocomposite was synthesized with enhanced FRET efficiency and photothermal properties, then employed as a signal probe in LFA test strip, achieving fluorescent, photothermal, and colorimetric quantitative detection of salmon contents in mixed samples, with detection limits of 0.007 %, 0.092 %, and 0.153 %, respectively, salmon contents in commercially products were evaluated to verify the practicality. This work presents an integrated, portable, and automation-enabled platform for standardized genetic authentication of food adulteration, which would be utilized as a universal lab-free method for on-site adulteration monitoring and species gene diagnostics by matching conserved genes and primer designs.},
}
@article {pmid41372199,
year = {2025},
author = {Nan, AX and Chickering, M and Bartolome, CL and Shadija, N and Li, D and Estes, BJG and Stetina, JV and Li, W and Andresen, J and Molugu, K and Amunugama, R and Fang, M and Bai, C and Wang, J and Norouzi, D and Cochrane, JC and Gatlin, JT and Dunyak, MT and Kumar, S and Chavez, L and Seth, A and Halperin, S and Finn, JD and Xie, J},
title = {Ligase-mediated programmable genomic integration (L-PGI).},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {563},
pmid = {41372199},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mice ; Humans ; Genomics/methods ; *Ligases/metabolism/genetics ; HEK293 Cells ; },
abstract = {Since their discovery, CRISPR systems have been repurposed for programmable targeted genomic editing, leading to applications for gene disruption, single base editing, insertion, deletion, and manipulation of short genomic sequences. Pairing Cas9 nickase with reverse transcriptase allows applications for insertion, substitution, and deletion of short genomic sequences from an RNA template without generating double stranded breaks however this technology typically shows reduced efficacy in post mitotic cells, limiting its translatability in vivo. Here we present a novel, ligase-based method that addresses these limiations. We introduce edits through delivery and ligation of a synthetic DNA donor to genomic nicks created with Cas9 nickase and report editing activity in cell lines, primary cell cultures, and adult mice via nonviral delivery. With favorable on target outcomes compared to transcription-based editing in key cell types, good tolerability, and deliverability, ligation-mediated gene editing has the potential to further advance genomic medicine.},
}
@article {pmid41353404,
year = {2025},
author = {Burgold, T and Karakoc, E and Gonçalves, E and Barrio-Hernandez, I and Dwane, L and Silva, R and Souster, E and Sharma, M and Beck, A and Koh, GCC and Zalmas, LP and Garnett, MJ and Bassett, AR},
title = {A next-generation dual guide CRISPR system for genetic interaction library screening.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {561},
pmid = {41353404},
issn = {2041-1723},
support = {220540/Z/20/A//Wellcome Trust (Wellcome)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Library ; Cell Line, Tumor ; Streptococcus pyogenes/genetics ; Colorectal Neoplasms/genetics ; CRISPR-Associated Protein 9/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Transfer/genetics ; },
abstract = {Pairwise perturbation of gene function using the CRISPR/Cas9 system has potential in screening for genetic interactions and synthetic lethal gene pairs to identify combination therapies for cancer. However, existing dual guide expression systems are cumbersome to clone, often result in a large proportion of undesired guide pairs and have an imbalance of guide expression from the two positions. Here, we demonstrate a next-generation system for dual guide delivery based around a tRNA spacer that allows a single-step cloning strategy, as little as 2% of undesired guide pairs, and highly balanced expression of the two guides. This system allows efficient library-scale screening for hundreds of thousands of genetic interactions using the well-understood Streptococcus pyogenes Cas9 (SpCas9) system. We use this to screen a 100,136 guide pair library in colorectal cancer cells and successfully identify synthetic lethal genetic interactions between paralogs or other known interacting genes, establishing our method for performing efficient large-scale genetic interaction screens. This system is versatile and could be used with most guide RNA vector systems, and for other uses of paired guide delivery, such as improving single gene knockout efficiency or improving guide detection in single cell or optical CRISPR screens.},
}
@article {pmid41338219,
year = {2026},
author = {Yang, Z and Zhang, L and Jiang, X and Yang, X and Ma, K and Yoo, D and Lu, Y and Zhang, S and Chen, J and Nie, Y and Bian, X and Han, J and Fu, L and Zhang, J and Ventura, M and Zhang, G and Sun, Q and Eichler, EE and Mao, Y},
title = {Incomplete lineage sorting of segmental duplications defines the human chromosome 2 fusion site early during African great ape speciation.},
journal = {Cell genomics},
volume = {6},
number = {1},
pages = {101079},
doi = {10.1016/j.xgen.2025.101079},
pmid = {41338219},
issn = {2666-979X},
mesh = {Animals ; Humans ; *Hominidae/genetics ; *Segmental Duplications, Genomic/genetics ; *Chromosomes, Human, Pair 2/genetics ; *Genetic Speciation ; Chromosome Inversion/genetics ; Evolution, Molecular ; CRISPR-Cas Systems ; },
abstract = {All great apes differ karyotypically from humans due to the fusion of chromosomes 2a and 2b, resulting in human chromosome 2. Here, we show that the fusion was associated with multiple pericentric inversions, segmental duplications (SDs), and the turnover of subterminal repetitive DNA. We characterized the fusion site at the single-base-pair resolution and identified three distinct SDs that originated more than 5 million years ago. These three distinct SDs were differentially distributed among African great apes as a result of incomplete lineage sorting (ILS) and lineage-specific duplication. One of these SDs shares homology to a hypomethylated SD spacer sequence present in the subterminal heterochromatin of Pan but is completely absent subtelomerically in both humans and orangutans. CRISPR-Cas9-mediated depletion of the fusion site in human neural progenitor cells alters the expression of genes, indicating a potential regulatory consequence to this human-specific karyotypic change. Overall, this study offers insights into how complex regions subject to ILS may contribute to speciation.},
}
@article {pmid40795361,
year = {2026},
author = {Prillo, S and Ravoor, A and Yosef, N and Song, YS},
title = {ConvexML: Fast and Accurate Branch Length Estimation under Irreversible Mutation Models, Illustrated through Applications to CRISPR/Cas9-Based Lineage Tracing.},
journal = {Systematic biology},
volume = {75},
number = {1},
pages = {115-134},
pmid = {40795361},
issn = {1076-836X},
support = {R56-HG013117/NH/NIH HHS/United States ; R01-HG013117/NH/NIH HHS/United States ; 101089213//ERC/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Mutation ; *Classification/methods ; *Phylogeny ; *Models, Genetic ; Software ; Cell Lineage/genetics ; },
abstract = {Branch length estimation is a fundamental problem in statistical phylogenetics and a core component of tree reconstruction algorithms. Traditionally, general time-reversible mutation models are employed, and many software tools exist for this scenario. With the advent of CRISPR/Cas9 lineage tracing technologies, there has been significant interest in the study of branch length estimation under irreversible mutation models. Under the CRISPR/Cas9 mutation model, irreversible mutations-in the form of DNA insertions or deletions-are accrued during the experiment, which are then read out at the single-cell level to reconstruct the cell lineage tree. However, most of the analyses of CRISPR/Cas9 lineage tracing data have so far been limited to the reconstruction of single-cell tree topologies, which depict lineage relationships between cells, but not the amount of time that has passed between ancestral cell states and the present. Time-resolved trees, known as chronograms, would allow one to study the evolutionary dynamics of cell populations at an unprecedented level of resolution. Indeed, time-resolved trees would reveal the timing of events on the tree, the relative fitness of subclones, and the dynamics underlying phenotypic changes in the cell population-among other important applications. In this work, we introduce the first scalable and accurate method to refine any given single-cell tree topology into a single-cell chronogram by estimating its branch lengths. To do this, we perform regularized maximum likelihood estimation (MLE) under a general irreversible mutation model, paired with a conservative version of maximum parsimony that reconstructs only the ancestral states that we are confident about. To deal with the particularities of CRISPR/Cas9 lineage tracing data-such as double-resection events, which affect runs of consecutive sites-we avoid making our model more complex and instead opt for using a simple but effective data encoding scheme. Similarly, we avoid explicitly modeling the missing data mechanisms-such as heritable missing data-by instead assuming that they are missing completely at random. We stabilize estimates in low-information regimes by using a simple penalized version of MLE using a minimum branch length constraint and pseudocounts. All this leads to a convex MLE problem that can be readily solved in seconds with off-the-shelf convex optimization solvers. We benchmark our method using both simulations and real lineage tracing data, and show that it performs well on several tasks, matching or outperforming competing methods such as TiDeTree and LAML (Lineage Analysis via Maximum Likelihood) in terms of accuracy, while being $10\sim 100\times$ faster. Notably, our statistical model is simpler and more general, as we do not explicitly model the intricacies of CRISPR/Cas9 lineage tracing data. In this sense, our contribution is 2-fold: (1) a fast and robust method for branch length estimation under a general irreversible mutation model and (2) a data encoding scheme specific to CRISPR/Cas9 lineage tracing data, which makes it amenable to the general model. Our branch length estimation method, which we call "ConvexML," should be broadly applicable to any evolutionary model with irreversible mutations (ideally, with high diversity) and an approximately ignorable missing data mechanism. "ConvexML" is available through the convexml open-source Python package.},
}
@article {pmid41528123,
year = {2025},
author = {Dong, Q and Luo, C},
title = {A recombinase polymerase amplification-coupled Cas12a for detection of Salmonella Typhi - a preliminary report.},
journal = {Folia histochemica et cytobiologica},
volume = {63},
number = {4},
pages = {185-192},
doi = {10.5603/fhc.108406},
pmid = {41528123},
issn = {1897-5631},
mesh = {*Salmonella typhi/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems/genetics ; *Typhoid Fever/diagnosis/microbiology ; *Recombinases/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *Bacterial Proteins/genetics/metabolism ; DNA, Bacterial/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; CRISPR-Associated Proteins ; },
abstract = {INTRODUCTION: . Typhoid fever, a disease resulting from an infection with Salmonella Typhi (S. Typhi) remains widespread in economically disadvantaged regions, where it continues to be a critical public health concern. As the symptoms and signs are non-specific, they are difficult to diagnose directly based on the clinical picture. Therefore, laboratory examinations are essential for diagnosis.<br><br>MATERIAL AND METHODS: . This research introduces a fast and equipment-independent approach for detecting S. Typhi by employing CRISPR/Cas12a-based technology. The optimized CRISPR/Cas12a system achieved a detection limit of 103 copies/&#x3bc;L of S. Typhi DNA per reaction, with the entire assay completed within 60 min.<br><br>RESULTS: . Four clinical isolates cultured from patients with typhoid fever were collected and evaluated using our CRISPR/Cas12a-based detection system. The assay results demonstrated that all four samples were accurately identified as positive.<br><br>CONCLUSIONS: . We showed that the developed CRISPR/Cas12a-based detection method provides a promising alternative for the on-site and simple detection of S. Typhi.},
}
@article {pmid41527500,
year = {2026},
author = {Atceken, N and Kahya, A and Yigci, D and Tasoglu, S},
title = {CRISPR-on-Chip for Point-of-Care Diagnostics.},
journal = {ACS nano},
volume = {},
number = {},
pages = {},
doi = {10.1021/acsnano.5c19771},
pmid = {41527500},
issn = {1936-086X},
abstract = {CRISPR-based diagnostic platforms have gained significant momentum in recent years, enabling highly sensitive and specific detection of pathogens and diseases. Due to their practical benefits, these platforms have become widely adopted in point-of-care (PoC) applications. CRISPR-on-chip technology integrates CRISPR-Cas platforms with diverse microfluidic systems, allowing scalability and portable, real-time, and precise biomolecule detection. This approach enhances diagnostic accuracy, reduces processing times, and minimizes the need for complex laboratory infrastructures, unlike in conventional diagnostics. Using CRISPR-Cas enzymes in microfluidic systems, CRISPR-on-chip platforms offer key advantages such as single-molecule sensitivity, multiplex detection, and applicability. However, integration with microfluidics for PoC applications is still poorly understood, despite CRISPR-Cas being widely used. This study reviews recent developments in CRISPR-on-chip-based diagnostics and highlights its potential applications in infectious diseases, biosensors, and personalized medicine. Furthermore, challenges and future perspectives in achieving an ideal diagnostic solution are discussed.},
}
@article {pmid41527434,
year = {2026},
author = {Stewart, C and Liddle, TA and Tolla, E and Lewis, JE and Marshall, C and Evans, NP and Morgan, PJ and Ebling, FJP and Stevenson, TJ},
title = {Hypothalamic deiodinase type-3 establishes the period of circannual interval timing in mammals.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {41527434},
issn = {2050-084X},
support = {LT-RL-2019-06//Leverhulme Trust/ ; Institutional Strategic Support Fund/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; *Iodide Peroxidase/metabolism/genetics ; *Hypothalamus/enzymology/physiology ; Photoperiod ; Seasons ; Phodopus/physiology ; Male ; Cricetinae ; },
abstract = {Animals respond to environmental cues to time phenological events, but the intrinsic mechanism of circannual timing remains elusive. We used transcriptomic sequencing and frequent sampling of multiple hypothalamic nuclei in Djungarian hamsters to examine the neural and molecular architecture of circannual interval timing. Our study identified three distinct phases of transcript changes, with deiodinase type-3 (Dio3) expression activated during the early induction phase. Subsequent work demonstrated that targeted mutation of Dio3 using CRISPR-Cas resulted in a shorter period for circannual interval timing. Hamsters that are non-responsive to short photoperiods and fail to show any winter adaptations do not display changes in Dio3 expression and do not show any change in body mass or pelage. Our work demonstrates that changes in Dio3 induction are essential for setting the period of circannual interval timing.},
}
@article {pmid41526382,
year = {2026},
author = {Van Vu, T and Thi Nguyen, N and Kim, J and Hoai Nguyen, T and Kim, JY},
title = {Development of an ultra-efficient prime editing system in tomato.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {95},
pmid = {41526382},
issn = {2041-1723},
support = {RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; },
mesh = {*Solanum lycopersicum/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Genome, Plant ; },
abstract = {Prime editing (PE) enables precise genome modifications without donor DNA or double-strand breaks, but its application in dicot plants has faced challenges due to low efficiency, locus dependence, and poor heritability. Here, we develop an ultra-efficient prime editing (UtPE) system for dicots by integrating evolved PE6 variants (PE6c and PE6ec), an altered pegRNA (aepegRNA), an RNA chaperone, and a geminiviral replicon. UtPE significantly improves editing performance in tomatoes, with UtPEv1 excelling in simple edits (unstructured RTTs) and UtPEv3 effective for complex targets (structured RTTs or multiple nucleotide changes). Compared to a PE2max-based tool, UtPE increases desired editing efficiency by 3.39 to 8.89-fold, enables editing at previous inaccessible sites, achieves an average of 16.0% desired editing efficiency in calli, and produces high-frequency desired edits in up to 87.5% of T0 plants. Multiplexed editing at up to three loci and stable T1 inheritance are also achieved, resulting in traits such as jointless pedicels and glyphosate resistance, while minimizing off-target effects.},
}
@article {pmid41525963,
year = {2026},
author = {Habib, AH and Sain, ZM and Rafeeq, M and Karami, MM and Alsufyani, HA and Iqbal, J and Chaieb, K and Altayb, HN and Nadeem, MS and Al-Abbasi, FA and Kazmi, I},
title = {MicroRNA-CRISPR biosensors for cancer diagnostics.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {583},
number = {},
pages = {120837},
doi = {10.1016/j.cca.2026.120837},
pmid = {41525963},
issn = {1873-3492},
abstract = {Circulating microRNAs (miRNAs) are promising minimally invasive biomarkers for cancer and cardiovascular disorders. However, their low sequence length, low abundance, high sequence homology (including iso-miRs), and strong matrix and preanalytical effects in biofluids require highly sensitive and robust analytical technologies. CRISPR-Cas systems, particularly Cas12a, Cas12b, Cas13a, and Cas9, offer programmable nucleic acid recognition with high mismatch discrimination combined with collateral nuclease activity, enabling versatile signal amplification through fluorescence, electrochemical, electrochemiluminescent (ECL), photoelectrochemical (PEC), colorimetric, and lateral-flow readouts. This review critically evaluates the latest advances in CRISPR-based miRNA biosensors, emphasizing their analytical performance and translational potential in clinical diagnostics across plasma/serum, saliva, whole blood, and extracellular vesicle samples. The detection limits are typically within the femtomolar to attomolar range. The requirements for clinical translation are equally influenced by factors such as sample preparation, inhibitor tolerance, miRNA panel multiplexing, quantitative readout, and reagent stability. We compared CRISPR-based workflows with RT-qPCR and digital PCR and provided a roadmap for standardization and quality control, as well as the minimal analytical and clinical validation standards required for adopting CRISPR technology in clinical chemistry laboratories.},
}
@article {pmid41524770,
year = {2026},
author = {Basit, A and Zhu, J and Zheng, W},
title = {Assessing off-target effects in CRISPR/Cas9: challenges and strategies for precision DNA editing.},
journal = {Archives of microbiology},
volume = {208},
number = {2},
pages = {114},
pmid = {41524770},
issn = {1432-072X},
support = {YZ2024220//Work was partly supported by the 2024 Annual Special Funds for Municipal-School Cooperation Projects of Yangzhou City (YZ2024220) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)./ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The emergence of CRISPR/Cas9 technology has transformed the landscape of gene editing, allowing for precise alterations in DNA that hold great promise for research and potential therapies. However, a significant concern is the occurrence of off-target effects, which can lead to unintended genetic modifications with potentially harmful consequences. This paper explores the nature of off-target effects in CRISPR/Cas9, discussing how they arise and their implications for the reliability of gene editing. We identify the challenges faced in detecting and predicting these off-target interactions, including limitations in current detection techniques and the complexities of cellular biology. We present strategies aimed at minimizing off-target effects, such as careful design of guide RNAs, the use of computational tools for prediction, and improved delivery methods. Through a review of case studies, we highlight successful cases where off-target activity has been significantly reduced, offering insights into best practices for enhancing the accuracy of CRISPR/Cas9 applications. Moreover, we provide a comparative overview of Cas9, Cas12, and Cas13 systems, emphasizing their distinct target specificities, mechanisms of action, and off-target profiles. This comparison offers a broader understanding of how alternative CRISPR effectors may be leveraged to improve genome and transcriptome editing precision. This study underscores the importance of continued research to address the challenges of off-target effects, ultimately supporting the development of safer and more effective gene editing methods for clinical use.},
}
@article {pmid41524535,
year = {2026},
author = {Batra, SS and Cabrera, A and Spence, JP and Goell, J and Anand, SS and Hilton, IB and Song, YS},
title = {Predicting the effect of CRISPR-Cas9-based epigenome editing.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {41524535},
issn = {2050-084X},
support = {R35 GM134922/GM/NIGMS NIH HHS/United States ; R35 GM143532/GM/NIGMS NIH HHS/United States ; R35-GM143532/GM/NIGMS NIH HHS/United States ; R35-GM134922/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; HEK293 Cells ; *Epigenome ; Protein Processing, Post-Translational ; Machine Learning ; Histones/metabolism ; K562 Cells ; *Epigenesis, Genetic ; *Epigenomics/methods ; Epigenome Editing ; },
abstract = {Epigenetic regulation orchestrates mammalian transcription, but functional links between them remain elusive. To tackle this problem, we use epigenomic and transcriptomic data from 13 ENCODE cell types to train machine learning models to predict gene expression from histone post-translational modifications (PTMs), achieving transcriptome-wide correlations of ∼0.70-0.79 for most cell types. Our models recapitulate known associations between histone PTMs and expression patterns, including predicting that acetylation of histone subunit H3 lysine residue 27 (H3K27ac) near the transcription start site (TSS) significantly increases expression levels. To validate this prediction experimentally and investigate how natural vs. engineered deposition of H3K27ac might differentially affect expression, we apply the synthetic dCas9-p300 histone acetyltransferase system to 8 genes in the HEK293T cell line and to 5 genes in the K562 cell line. Further, to facilitate model building, we perform MNase-seq to map genome-wide nucleosome occupancy levels in HEK293T. We observe that our models perform well in accurately ranking relative fold-changes among genes in response to the dCas9-p300 system; however, their ability to rank fold-changes within individual genes is noticeably diminished compared to predicting expression across cell types from their native epigenetic signatures. Our findings highlight the need for more comprehensive genome-scale epigenome editing datasets, better understanding of the actual modifications made by epigenome editing tools, and improved causal models that transfer better from endogenous cellular measurements to perturbation experiments. Together, these improvements would facilitate the ability to understand and predictably control the dynamic human epigenome with consequences for human health.},
}
@article {pmid41524418,
year = {2026},
author = {Raftopoulou, O and Malmstrom, K and Pan, M and Barrangou, R},
title = {Enhanced editing of Bifidobacterium lactis using the endogenous Type I-G CRISPR-Cas system.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0183925},
doi = {10.1128/aem.01839-25},
pmid = {41524418},
issn = {1098-5336},
abstract = {Diverse Bifidobacterium animalis subsp. lactis strains are widely used as commercial probiotics. While proof-of-concept studies have shown that some strains can be edited using several CRISPR-Cas approaches, this species remains difficult to engineer, hindering functional genomic studies to establish their molecular mode of action and enhance their probiotic functionalities. Here, we show that >95% of available B. lactis genomes harbor a conserved Type I-G CRISPR-Cas system, which we leverage to develop and validate a broadly applicable genome editing framework. We redesigned backbone plasmids with different replicons and antibiotic resistance markers and evaluated performance across six commercial strains for transformation efficiency. A vector carrying the pBC1 origin coupled with a chloramphenicol resistance marker improved transformation in most strains. Using synthetic CRISPR arrays with self-targeting spacers in combination with homologous editing templates, we tested multiple spacers and evaluated short (600 bp) versus long (1,000 bp) homology arms. To demonstrate applicability, we generated knockouts in three glycoside hydrolases within the Balac 1593-1601 cluster, readily cured editing plasmids in non-selective medium, and performed iterative genome editing. Growth phenotyping across carbohydrates confirmed that the GH36 α-galactosidase Balac 1601 knockout abolished melibiose and raffinose utilization, and that deletions within Balac 1596 and Balac 1593 carbohydrate hydrolases produced non-canonical phenotypes, suggestive of a modulatory role associated with shift in carbon use and compensation by other pathways. These results establish a practical toolkit for editing diverse B. lactis strains, unravel the genomics underlying probiotic attributes, and provide a blueprint for genome engineering in other non-model probiotic bacteria.IMPORTANCEBifidobacterium animalis subsp. lactis strains are prominent probiotics widely formulated in foods and dietary supplements, yet remain difficult to engineer, limiting efforts to connect genes to probiotic traits and to build strains with enhanced functions. Here, we harness the native Type I-G CRISPR Cas system to enable genome editing across commercial B. lactis strains by optimizing a compact plasmid backbone, testing multiple spacers to achieve efficient editing, and selecting homology arms of the appropriate length for recombination. With this framework, we generate knockouts at multiple, functionally distinct loci, demonstrating target-agnostic applicability, and we cure the CRISPR-editing vectors efficiently, enabling sequential edits. This toolkit enables systematic genotype-to-phenotype mapping in B. lactis and provides a practical framework for strain improvement in organisms of industrial relevance.},
}
@article {pmid41523156,
year = {2025},
author = {Altpeter, F},
title = {Gene editing to enhance biotic stress tolerance in sugarcane.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1750169},
pmid = {41523156},
issn = {1664-462X},
}
@article {pmid41522810,
year = {2026},
author = {Heer, CD and Elia, JL and Menon, V and Johnson, SS and Arbelaez, SR and Friedman, S and Lopez-Giraldez, F and Sundaram, RK and Herzon, SB and Bindra, RS and Gueble, SE},
title = {Targeted CRISPR knockout screening identifies known and novel chemogenomic interactions between DNA damaging agents and DNA repair genes.},
journal = {NAR cancer},
volume = {8},
number = {1},
pages = {zcaf052},
pmid = {41522810},
issn = {2632-8674},
mesh = {Humans ; *DNA Repair/genetics/drug effects ; *DNA Damage/drug effects/genetics ; Cell Line, Tumor ; Gene Knockout Techniques ; CRISPR-Cas Systems ; *Glioma/genetics/drug therapy ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Genetic instability is a hallmark of cancer, often arising from mutations to DNA damage repair and response (DDR) genes. Classical genetic, biochemical, and structural approaches elucidated the foundational mechanisms of DDR pathways and provided a scientific understanding of their involvement in repair of lesions induced by broad classes of DNA-damaging agents (DDAs). However, given the chemical diversity of DDAs and resultant DNA lesions, along with the multitude of interconnected DDR factors, the chemogenomic landscape of DDA-DDR interactions remains incompletely mapped. To this end, we developed a DDR-targeted, CRISPR knockout screening approach and assessed relationships amongst 353 DNA repair genes and 15 DDAs in LN229 glioma cells. Within this dataset of 5295 DDR-related chemogenomic interactions, we identified many established interactions and discovered novel ones. For example, we observed a specific role of transcription-coupled nucleotide excision repair in the repair of adducts generated by monofunctional alkylating agents, a role for the Fanconi anemia pathway in addressing methyl lesions, overt differences in DSB repair following treatment with topoisomerase I versus II poisons, and repair dependencies associated with the imidazotetrazines temozolomide, mitozolomide, and KL-50. Future directions will continue to investigate the mechanisms of novel chemogenomic interactions that we have uncovered as well as work to identify chemogenomic interactions amenable to clinical translation.},
}
@article {pmid41522435,
year = {2025},
author = {Zalila-Kolsi, I},
title = {Engineered bacteria as living therapeutics: Next-generation precision tools for health, industry, environment, and agriculture.},
journal = {AIMS microbiology},
volume = {11},
number = {4},
pages = {946-962},
pmid = {41522435},
issn = {2471-1888},
abstract = {Synthetic biology has revolutionized precision medicine by enabling the development of engineered bacteria as living therapeutics, dynamic biological systems capable of sensing, responding to, and functioning within complex physiological environments. These microbial platforms offer unprecedented adaptability, allowing for real-time detection of disease signals and targeted therapeutic delivery. This review explores recent innovations in microbial engineering across medical, industrial, environmental, and agricultural domains. Key advances include CRISPR-Cas systems, synthetic gene circuits, and modular plasmid architectures that provide fine-tuned control over microbial behavior and therapeutic output. The integration of computational modeling and machine learning has further accelerated design, optimization, and scalability. Despite these breakthroughs, challenges persist in maintaining genetic stability, ensuring biosafety, and achieving reproducibility in clinical and industrial settings. Ethical and regulatory frameworks are evolving to address dual-use concerns, public perception, and global policy disparities. Looking forward, the convergence of synthetic biology with nanotechnology, materials science, and personalized medicine is paving the way for intelligent, responsive, and sustainable solutions to global health and environmental challenges. Engineered bacteria are poised to become transformative tools not only in disease treatment but also in diagnostics, biomanufacturing, pollution mitigation, and sustainable agriculture.},
}
@article {pmid41479230,
year = {2026},
author = {Zhang, J and Liu, Y and Cao, W and Ruan, R and Wang, M},
title = {Establishment of a Novel CRISPR/Cas9-Based Multiplex Editing System in the Citrus Postharvest Pathogen Penicillium digitatum.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {1},
pages = {1167-1174},
doi = {10.1021/acs.jafc.5c11144},
pmid = {41479230},
issn = {1520-5118},
mesh = {*Penicillium/genetics/pathogenicity/metabolism ; *Citrus/microbiology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Plant Diseases/microbiology ; Fungal Proteins/genetics/metabolism ; },
abstract = {Penicillium digitatum, the causal fungus of the citrus green mold, leads to substantial postharvest losses in the citrus industry. In this study, we engineered a versatile CRISPR/Cas9-mediated gene editing platform capable of generating multiple sgRNAs from a single polycistronic transcript. By incorporating endogenous tRNAs and the strong promoter PdMLE1 into the CRISPR/Cas9 system, the efficiency of single gene editing can reach up to 94.2%. By taking advantage of the efficient shearing and processing capabilities of tRNA, the platform enabled multiplex editing with efficiencies of 44.4% for two-gene and 33.3% for three-gene modifications, respectively. After two rounds of three-gene editing, we were able to successfully obtain hextuple-gene mutants. Finally, functional characterization revealed that the target polysaccharide-lyase-encoding genes play limited roles in pathogenicity in P. digitatum. Taken together, our results represent a powerful tool for genome engineering in P. digitatum, facilitating research into its pathogenesis.},
}
@article {pmid41447470,
year = {2026},
author = {Liu, T and Ye, B and Zhang, Y and Yan, X},
title = {Finely Tuned CRISPRi Module for Upgrading the Performance of Constitutive Promoters in the Bacillus subtilis Protein Expression System.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {1},
pages = {1046-1052},
doi = {10.1021/acs.jafc.5c03424},
pmid = {41447470},
issn = {1520-5118},
mesh = {*Bacillus subtilis/genetics/metabolism/growth &amp; development ; *Promoter Regions, Genetic ; *Bacterial Proteins/genetics/metabolism ; Xylose/metabolism ; Gene Expression Regulation, Bacterial ; CRISPR-Cas Systems ; Fermentation ; },
abstract = {Bacillus subtilis is a critical host for protein production, with many industrial strains relying on strong constitutive promoters. However, this kind of promoter typically imposes a heavy burden on the host from the early stage of fermentation, leading to reduced growth rate and biomass. To overcome the drawbacks of these promoters, we developed a xylose-inducible CRISPRi module to dynamically control the activity of these promoters. The strength of this module was finely tuned via promoter engineering and the xylose concentration. The addition of xylose inhibited the target promoter and favored cell growth at an early stage, while the consumption of xylose recovered the strength of the promoter and facilitated protein expression, resulting in better balance between cell growth and protein production. The yield of a target protein was increased by 38% using this module. Our work provides a simple and effective method to upgrade industrial strains driven by strong constitutive promoters.},
}
@article {pmid41397982,
year = {2025},
author = {Okuwa, T and Himeda, T and Kobayashi, K and Nomura, N and Utani, K and Koike, S and Nakamura, A and Higuchi, M},
title = {Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {534},
pmid = {41397982},
issn = {2041-1723},
support = {25K10386//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21K07045//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 24K10234//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP25fk0108716//Japan Agency for Medical Research and Development (AMED)/ ; S2023-4//Kanazawa Medical University/ ; K2024-3//Kanazawa Medical University/ ; },
mesh = {Humans ; *Integrins/metabolism/genetics ; *Glycosaminoglycans/metabolism ; HeLa Cells ; Virus Internalization ; CRISPR-Cas Systems ; *Receptors, Virus/metabolism ; Virus Attachment ; Host-Pathogen Interactions ; Gene Knockout Techniques ; },
abstract = {Saffold virus (SAFV), a member of the species Cardiovirus saffoldi within the Picornaviridae family, causes acute respiratory and gastrointestinal illnesses as well as hand, foot, and mouth disease. It is also suspected to be associated with neuronal disorders, such as encephalitis and meningitis, in severe cases. Despite its clinical significance, the virus-host interactions underlying SAFV pathogenicity remain largely unknown. Using a genome-wide CRISPR-Cas9 knockout screen, we identify the following receptors for SAFV infection: sulfated glycosaminoglycans (GAGs) and integrin αVβ8. Single knockouts of SLC35B2, an essential gene for sulfated GAG synthesis, or the integrin genes ITGAV or ITGB8 partially reduce SAFV-3 and SAFV-2 susceptibility in HeLa cells, and a double knockout confers complete resistance. Furthermore, we demonstrate that SAFV-3 virions bind directly to sulfated GAGs and integrin αVβ8. Based on these findings, we propose a model of SAFV infection in which sulfated GAGs and integrin αVβ8 act through dual and cooperative pathways to facilitate viral entry.},
}
@article {pmid41390353,
year = {2025},
author = {Howe, LJ and Aulchenko, YS and Davey Smith, G and Davies, NM and Esparza-Gordillo, J and Johnson, T and Liu, JZ and Richardson, TG and Sanseau, P and Scott, RA and Seaton, DD and Sharma, A and Cortes, A},
title = {Evaluating transportability of in vitro cellular models to in vivo human phenotypes using gene perturbation data.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {513},
pmid = {41390353},
issn = {2041-1723},
mesh = {Humans ; Phenotype ; Lysosomes/metabolism ; *Models, Biological ; Cholesterol, LDL/blood/metabolism ; CRISPR-Cas Systems ; Cholesterol/metabolism ; },
abstract = {Gene perturbation screens (e.g. CRISPR-Cas9) assess the impact of gene disruption on in-vitro cellular phenotypes (e.g., proliferation, anti-viral response). In-vitro experiments can be useful models for in-vivo (organismal) phenotypes (e.g., immune cell anti-viral response and infectious diseases). However, assessing whether an in-vitro cellular model effectively captures in-vivo biology is challenging. An in-vitro model is 'transportable' to an in-vivo phenotype if perturbations impacting the in-vitro phenotype also impact the in-vivo phenotype with mechanism-consistent directionality and effect sizes. We propose a framework; Gene Perturbation Analysis for Transportability (GPAT), to assess model transportability using gene perturbation effect estimates from perturbation screens (in-vitro) and loss-of-function burden tests (in-vivo). In hypothesis-driven analyses, GPAT provides evidence for model transportability of higher lysosomal cholesterol accumulation in-vitro to lower human plasma LDL-cholesterol (P = 0.0006), consistent with the known role of lysosomes in lipid biosynthesis. In contrast, there was limited evidence for other putative in-vitro models. In hypothesis-free analyses, we find evidence for transportability of cancer cell line proliferation to in-vivo human plasma cellular phenotypes (e.g. erythroleukemia proliferation and plasma lymphocyte percentage). Here we show that perturbation data can be used to evaluate transportability of in-vitro cellular models, informing assay prioritisation and supporting novel hypothesis generation.},
}
@article {pmid41387726,
year = {2025},
author = {Srinivasan, R and Sun, T and Sandles, A and Wu, D and Wang, L and Patel, H and Pabalate, R and Bader, M and Heidersbach, A and Ho, C and Xie, S and Ng, A and Haley, B},
title = {Chemically-inducible CRISPR/Cas9 circuits for ultra-high dynamic range gene perturbation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {504},
pmid = {41387726},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {CRISPR/Cas9 technologies provide unique capabilities for modeling disease and understanding gene-to-phenotype connections. In cultured cells, chemical-mediated control of Cas9 activity can limit off-target effects and enable mechanistic study of essential genes. However, widely-used Tet-On systems often show leaky Cas9 expression, leading to unintended edits, as well as weak activity upon induction. Leakiness can be problematic in the context of Cas9 nuclease activity, which may result in cumulative DNA damage and degradation of the target cell genome over time. To overcome these deficiencies, we have established transgenic platforms that minimize Cas9 functionality in the OFF-state along with maximized and uncompromised ON-state gene editing efficiency. By combining conditional destabilization and inhibition of Cas9, we have developed an all-in-one (one or multiple guide RNAs and Cas9) ultra-tight, Tet-inducible system with exceptional dynamic range (ON vs. OFF-state) across various cell lines and targets. As an alternative to Tet-mediated induction, we have created a Branaplam-regulated splice switch module for low-baseline and robust Cas9 activity control. Lastly, for circumstances where DNA damage needs to be avoided, we have constructed a dual-control, Tet-inducible CRISPRi module for tight and potent transcriptional silencing. This upgraded suite of inducible CRISPR systems has broad applications for numerous cell types and experimental conditions.},
}
@article {pmid41372159,
year = {2025},
author = {Pan, R and Ren, J and Chen, X and Flores, LF and Gonzalez, RVL and Adonnino, AA and Lofts, B and Waldo, J and Halmai, J and Devinsky, O and Fink, K and Liu, XS},
title = {Editing DNA methylation in vivo.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {527},
pmid = {41372159},
issn = {2041-1723},
support = {R01MH134519//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R01NS126185//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; },
mesh = {Animals ; *DNA Methylation/genetics ; *Gene Editing/methods ; Mice ; DNA Methyltransferase 3A ; Mice, Transgenic ; Promoter Regions, Genetic ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Proprotein Convertase 9/genetics/metabolism ; Liver/metabolism ; DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism ; CRISPR-Cas Systems ; Proto-Oncogene Proteins/genetics/metabolism ; Male ; DNA-Binding Proteins/genetics/metabolism ; Epigenesis, Genetic ; Neurons/metabolism ; },
abstract = {DNA methylation is a crucial epigenetic mechanism that regulates gene expression. Precise editing of DNA methylation has emerged as a promising tool for dissecting its biological function. However, challenges in delivery have limited most applications of DNA methylation editing to in vitro systems. Here, we develop two transgenic mouse lines harboring an inducible dCas9-DNMT3A or dCas9-TET1 editor to enable tissue-specific DNA methylation editing in vivo. We demonstrate that targeted methylation of the Psck9 promoter in the liver of dCas9-DNMT3A mice results in decreased Pcsk9 expression and a subsequent reduction in serum low-density lipoprotein cholesterol level. Targeted demethylation of the Mecp2 promoter in dCas9-TET1 mice reactivates Mecp2 expression from the inactive X chromosome and rescues neuronal nuclear size in Mecp2[+/-] mice. Genome-wide sequencing analyses reveal minimal transcriptional off-targets, demonstrating the specificity of the system. These results demonstrate the feasibility and versatility of methylation editing, to functionally interrogate DNA methylation in vivo.},
}
@article {pmid41365890,
year = {2025},
author = {Li, W and Liu, S and Fang, X and Zou, J and Jiang, Q and Min, X and Zhu, X and Cao, Y and Gao, X and Han, W and Azhar, M and Xing, X and Li, F and Zhang, Y and Liu, H and Cheng, L and Wang, C and Bao, J},
title = {Efficient high-precision transgene knock-in by Recombinases (Redα/β)-enhanced DNA integration-CRISPR-Cas9 (RED-CRISPR).},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {538},
pmid = {41365890},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; *Gene Knock-In Techniques/methods ; *Transgenes/genetics ; Mice ; Humans ; Recombinational DNA Repair/genetics ; *Gene Editing/methods ; *Recombinases/metabolism/genetics ; DNA/genetics/metabolism ; HEK293 Cells ; },
abstract = {CRISPR-Cas9 tools have revolutionized genetic engineering, yet the efficient precise integration of DNA cargos, particularly for large DNA payloads (>1 kilobase, kb), remains a technical bottleneck. Herein, we develop a Recombinases (Redα/β)-enhanced DNA integration-CRISPR-Cas9 approach, referred to as RED-CRISPR, which offers a versatile yet robust homology-directed repair (HDR) strategy enabling efficient and precise kb-scale DNA insertion across various cell types, including immortalized and primary cells of variable origins. RED-CRISPR significantly enhances HDR efficiencies by 2- to 5-fold change across diverse loci and further elevates HDR rates by 1.5- to 2.5-fold when synergizing with other HDR-enhancing strategies. We achieved up to 45% knock-in efficiency for CAR-T cell manufacturing, and attained 43% knock-in rate for generation of genetically modified mice using an 8-kb DNA cargo. Through a head-to-head comparison, RED-CRISPR profoundly mitigates off-target mutational burden and chromosomal translocations. We envision RED-CRISPR as a powerful genome-editing tool with broad biomedical and therapeutic applications.},
}
@article {pmid41521661,
year = {2026},
author = {Newman, A and Starrs, L and Burgio, G},
title = {Nuc domain electrostatics drive the trans cleavage activity of CRISPR-Cas12a.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41521661},
issn = {1362-4962},
support = {2018833//National Health and Medical Research Council/ ; 2027987//National Health and Medical Research Council/ ; //The Gordon and Gretel Bootes foundation/ ; //National Computing Infrastructure/ ; //Australian Government Research Training Program/ ; },
mesh = {Static Electricity ; *Endodeoxyribonucleases/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *Bacterial Proteins/chemistry/genetics/metabolism ; Catalytic Domain ; Gene Editing ; DNA Cleavage ; DNA/metabolism ; Protein Domains ; Kinetics ; Amino Acid Substitution ; Models, Molecular ; Mutation ; },
abstract = {The trans cleavage activity of Cas12a has been extensively used for the detection of biomolecules. Different Cas12a orthologues exhibit faster or slower trans cleavage kinetics, making some orthologues more suited for sensitive molecular detection. Ionic strength of reaction buffers and mutations that change the electrostatic environment near the RuvC active site have also been reported to strongly influence trans cleavage kinetics. Studying three commonly used Cas12a orthologues (FnCas12a, AsCas12a, and LbCas12a), we report that electrostatic interactions near the RuvC active site are critical for their trans cleavage activity. Alanine substitution of arginine and lysine residues in the Nuc domain can abolish trans cleavage while modestly reducing cis cleavage. Substitutions in the RuvC lid and substitutions to introduce positively charged residues in the Nuc could enhance both cis and trans cleavage. These Cas12a variants improved DNA detection and genome editing efficacy. Overall, this study provides a blueprint for rationally engineering the DNase activities of Cas12a.},
}
@article {pmid41521468,
year = {2026},
author = {Hsieh, JA and Wu, FH and Yang, DX and Wu, AE and Liu, CA and Chen, CH and Lin, SZ and Lin, YJ and Lin, CS},
title = {Protoplast-Based Functional Genomics and Genome Editing: Progress, Challenges and Applications.},
journal = {Plant, cell &amp; environment},
volume = {},
number = {},
pages = {},
doi = {10.1111/pce.70375},
pmid = {41521468},
issn = {1365-3040},
support = {//Academia Sinica/ ; //Buddhist Tzu Chi Medical Foundation/ ; NSTC 113-2313-B-001-006-//National Science and Technology Council, Taiwan/ ; NSTC 114-2628-B-002-008-//National Science and Technology Council, Taiwan/ ; NSTC 114-2314-B-303-006-//National Science and Technology Council, Taiwan/ ; },
abstract = {Protoplast-based systems provide a powerful and versatile platform for exploring how plants sense and respond to their environment. By enabling the direct delivery of proteins, DNA, and RNA into plant cells after cell wall removal, this approach facilitates precise molecular dissection of signaling, stress adaptation, and gene regulation across both model species and economically important crops. In this review, we analyzed 1050 published articles and categorizing them by delivery methods, research focus, plant species, and tissue types. We further highlight recent advances, including the application of single-cell transcriptomics, which provides unprecedented resolution for dissecting cellular responses and offers deeper insights into the mechanisms underlying stress resilience. Importantly, protoplast regeneration is gaining renewed attention not only as a model system for studying cellular reprogramming but also as a practical platform for crop improvement. Applications of protoplast regeneration include protoplast fusion, which integrates nuclear and organellar DNA/genomes from divergent parents to accelerate breeding and enhance tolerance to both biotic and abiotic stresses. Another important application is CRISPR/Cas ribonucleoprotein (RNP)-based editing targeting stress-resilience-related genes. In asexually propagated or highly heterozygous perennial crops with limited sexual reproduction, protoplast-based RNP delivery offers a viable and regulation-compliant strategy. This approach may help address public concerns over transgenic technologies while enabling the rapid development of stress-tolerant cultivars.},
}
@article {pmid41521358,
year = {2026},
author = {S Marques, B and Vitorino, C and V Ventura, F},
title = {CRISPR Applications in HIV Management - Prevention, Diagnosis, Monitoring and Treatment.},
journal = {Current HIV/AIDS reports},
volume = {23},
number = {1},
pages = {1},
pmid = {41521358},
issn = {1548-3576},
mesh = {Humans ; *HIV Infections/diagnosis/prevention &amp; control/therapy ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Despite global efforts to combat the human immunodeficiency virus (HIV) epidemic, acquired immunodeficiency syndrome (AIDS) still claims one life every minute, underscoring the persistent need for improved control strategies. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) technologies have emerged as promising tools that may transform HIV management. The objective of this review is to summarise recent advancements in CRISPR/Cas-based approaches for HIV prevention, diagnosis, monitoring, and treatment, and to evaluate their potential and current challenges. A systematic literature search was conducted to identify relevant CRISPR/Cas applications in HIV infection. In prevention, CRISPR/Cas strategies aim to hinder viral integration and enhance host immune response, although substantial development is required before clinical translation. In diagnosis, CRISPR/Cas methods show high specificity and sensitivity, yet their reliance on specialised equipment and expertise limits their accessibility. In HIV monitoring, CRISPR/Cas-based methods have not yet demonstrated superiority over the quantitative PCR. In treatment, two ongoing clinical trials - one targeting a viral co-receptor on hematopoietic stem cells (HSCs) and the other excising proviral DNA - illustrate the potential of CRISPR/Cas-mediated cures, despite challenges such as low editing efficiency and off-target effects. Overall, CRISPR/Cas technologies hold considerable promise for advancing HIV management, but issues of accessibility, affordability, and scalability must be addressed to ensure global impact.},
}
@article {pmid41431922,
year = {2026},
author = {Chen, K and Zhu, J and Fan, C and Zhou, A and Li, B and Ge, H and Ning, X},
title = {A Nanoimprinted Photothermal Chip for On-Demand Spatiotemporal Activation of CRISPR/Cas9 Gene Editing.},
journal = {Nano letters},
volume = {26},
number = {1},
pages = {532-542},
doi = {10.1021/acs.nanolett.5c05571},
pmid = {41431922},
issn = {1530-6992},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Mice ; Gold/chemistry ; Sheep ; Infrared Rays ; Humans ; },
abstract = {Precise control of gene editing in target cells is essential for CRISPR/Cas9 applications. Here, we present a nanoimprinted photothermal chip (NPC) engineered for on-demand delivery and activation of CRISPR/Cas9 complexes with high spatial and temporal precision. Fabricated by nanoimprint lithography and subsequent surface modification, NPC features a customized PEGylated plasmonic gold nanopillar array, which provides both optimal cellular adhesion and efficient photothermal conversion. Upon NIR irradiation, NPC generates spatially confined thermal microdomains that transiently permeabilizes cell membranes, thereby facilitating cytosolic delivery of CRISPR/Cas9 complexes and synchronously modulating genome-editing kinetics. In vitro studies demonstrate robust gene knockout in both mouse and sheep cell lines while preserving high cell viability and editing fidelity. Remarkably, NPC-mediated PD-1 gene disruption in cytotoxic T cells markedly enhance their antitumor activity. Overall, this work establishes NPC as a transformative platform for precise and controllable CRISPR/Cas9 gene editing with broad therapeutic potential.},
}
@article {pmid41416985,
year = {2026},
author = {Han, DH and Lee, SY and Kim, Y and Oh, J and Park, J and Park, YM and Kim, SG and Kim, TS and Park, JK},
title = {Ultrasensitive Detection of Multiple Foodborne Pathogens Using CRISPR-Cas12a on a Finger-Actuated Microfluidic Device Integrated with a Modular Pressurizing Pump.},
journal = {Analytical chemistry},
volume = {98},
number = {1},
pages = {531-542},
doi = {10.1021/acs.analchem.5c05295},
pmid = {41416985},
issn = {1520-6882},
mesh = {*Lab-On-A-Chip Devices ; *CRISPR-Cas Systems ; *Listeria monocytogenes/isolation &amp; purification/genetics ; Nucleic Acid Amplification Techniques ; Limit of Detection ; *Food Microbiology ; *Escherichia coli O157/isolation &amp; purification/genetics ; Salmonella/isolation &amp; purification/genetics ; *Microfluidic Analytical Techniques/instrumentation ; Milk/microbiology ; Foodborne Diseases/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Foodborne pathogens pose a serious threat to global health and the economy, causing gastrointestinal illnesses and potentially leading to fatalities. Here, we present a recombinase polymerase amplification (RPA)-CRISPR-Cas12a-based method for the detection of foodborne pathogens using target-specific CRISPR RNAs (crRNAs) on a reusable, reconfigurable finger-actuated microfluidic device. Unlike previous finger-actuated pushbutton-based microfluidic devices, the device incorporates a modular pressurizing pump (MoPP), a standalone, reconfigurable actuation module that not only enhances reusability and reduces cross-contamination risks but also provides a flexible interface that allows user-defined fluidic routing and multiplexed assay workflows. Using a MoPP-integrated finger-actuated microfluidic device, the RPA-CRISPR-Cas12a-based detection of three foodborne pathogens was validated with an optimized crRNA and RPA primer sequence. Genomic DNA (gDNA) extracted from pathogen-spiked milk samples further demonstrated real-world applicability, achieving a limit of detection (LOD) of 1.62, 1.84, and 1.01 CFU/mL for Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes, respectively. The developed microfluidic RPA-CRISPR-Cas12a-based detection platform is expected to be a reconfigurable, user-friendly, and highly sensitive point-of-care testing system for monitoring foodborne pathogens throughout the food supply chain.},
}
@article {pmid41362221,
year = {2026},
author = {Yao, X and Wang, L and Su, L and Rao, W and Luo, Z and Li, Y},
title = {Localized CRISPR/Cas13a powered DNA walker for sensitive and high-throughput detection of norovirus.},
journal = {Journal of materials chemistry. B},
volume = {14},
number = {2},
pages = {572-579},
doi = {10.1039/d5tb02131f},
pmid = {41362221},
issn = {2050-7518},
mesh = {*Norovirus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *DNA/chemistry/genetics ; Metal Nanoparticles/chemistry ; Gold/chemistry ; High-Throughput Screening Assays ; Limit of Detection ; RNA, Viral/analysis ; },
abstract = {The development of sensitive and high-throughput methods for detecting foodborne viruses is crucial for disease prevention and public health protection. In this study, we present a novel localized Cas13a-based DNA walker (LCas13a-DNA walker) for the ultrasensitive, stable, and rapid detection of norovirus (NoV). When the DNA walker was confined in AuNPs, the spatial confinement effect improved the local concentration of reaction substrates, accelerated the reaction speed, and enhanced the sensitivity of the DNA walker. Besides, an original design of uracil-rich hairpin (UH)-modified AuNPs as the walking track significantly improves the stability of the detection system. Meanwhile, employing CRISPR/Cas13a as the driving force streamlines viral RNA recognition and substantially reduces the reaction time down to 30 minutes by eliminating the reverse transcription step. Additionally, a biomimetic array, formed by photonic crystals (PCs), enabled high-throughput signal acquisition with a microplate reader, and concurrently amplified the fluorescence signal. The proposed assay realized ultra-sensitivity of NoV with a detection limit as low as 4.1 pM and a wide linear range from 10 pM to 5 nM. Due to the advantages of high sensitivity, high-throughput, stability, and rapid analysis, this proposed method provides a potential strategy for point-of-care detection of pathogenic viruses in food safety monitoring and disease diagnosis.},
}
@article {pmid41294335,
year = {2026},
author = {Paulin, OKA and Tsavou, A and Priest, EL and Griffiths, JS and Lortal, L and Kempf, A and Chow, EWL and Pang, LM and Wickramasinghe, DN and Lyon, CM and Hernday, AD and Wang, Y and Richardson, JP and Naglik, JR},
title = {The combinatorial action of hyphal growth and candidalysin is critical for promoting Candida albicans oropharyngeal infection.},
journal = {mBio},
volume = {17},
number = {1},
pages = {e0330425},
doi = {10.1128/mbio.03304-25},
pmid = {41294335},
issn = {2150-7511},
support = {214229_Z_18_Z/WT_/Wellcome Trust/United Kingdom ; R15AI185747//National Institute of Allergy and Infectious Diseases/ ; BBSRC: UKRI717/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Candida albicans/genetics/growth &amp; development/pathogenicity ; *Hyphae/growth &amp; development/genetics ; Animals ; *Fungal Proteins/genetics/metabolism ; Mice ; *Virulence Factors/genetics/metabolism ; Humans ; Gene Deletion ; Disease Models, Animal ; Aspartic Acid Endopeptidases/genetics/metabolism ; Epithelial Cells/microbiology ; Female ; *Candidiasis, Oral/microbiology ; Virulence ; Oropharynx/microbiology ; Candidiasis/microbiology ; CRISPR-Cas Systems ; },
abstract = {Candida albicans is one of the most common fungal pathogens, yet much remains unknown about how its virulence factors cooperate to promote pathogenicity. To investigate this, CRISPR-Cas9 technology was used to create a panel of 19 single, double, triple, and quadruple deletion mutant strains targeting four established virulence factors: ALS3 (adhesin/invasin), ECE1 (candidalysin toxin), HGC1 (hypha formation regulator), and SAP2 (protease). In vitro, the deletion of each gene had differing impacts across multiple characterization assays. The hgc1∆/∆ mutant was unable to form hyphae under inducing conditions, leading to downstream impairment of epithelial invasion. The als3∆/∆ mutant exhibited significantly reduced adhesion and invasion into epithelial cells, resulting in attenuated cellular damage. The ece1∆/∆ mutant displayed significantly reduced epithelial damage, cell signaling, and immune activation. The phenotype of the sap2∆/∆ mutant resembled that of wild type but was unable to degrade protein. In an immunocompromised murine model of oropharyngeal infection, hyphal growth and candidalysin production were the dominant drivers of elevated fungal burden, innate immune responses, and mortality. Following a 5-day infection with hgc1∆/∆ and ece1∆/∆ single gene deletion strains, mice had survival rates of 100% and 80%, respectively, compared to 15% in wild-type infected mice. Notably, 100% survival was also observed following challenge with all hgc1∆/∆ and ece1∆/∆ combination mutants. This study demonstrates that specific C. albicans virulence attributes act in combination to promote mucosal infection, with hyphal growth and candidalysin production being a critical driver of oropharyngeal infection.IMPORTANCECandida albicans has been classified by the WHO as a "critical priority" pathogen, highlighting the urgent need for a greater understanding of the mechanisms that enable it to cause disease. C. albicans possesses numerous virulence attributes, but how they synergize during infection is not well understood. Here, using reverse genetics, we dissect the individual and combinatorial roles of four C. albicans virulence factors (Als3p, candidalysin, hyphal growth, and Sap2p) in vitro and in an in vivo murine model of oropharyngeal candidiasis. Increasing the number of C. albicans gene deletions correlated with reduced oral fungal burden, with hyphal growth and candidalysin together being critical for infection, inflammation, and mortality during oropharyngeal infection. These findings demonstrate that virulence attributes act cooperatively as a collective network to promote pathogenicity, a finding also observed in plant fungal pathogens. Our approach has identified specific fungal virulence factors that can be targeted for new treatment strategies against C. albicans infections.},
}
@article {pmid41520043,
year = {2026},
author = {Senger, J and Keutgen, M and Roth, N and Seitl, I and Fischer, L},
title = {Toward food-grade production of the Bacteroides helcogenes protein-glutamine glutaminase with an optimized Bacillus subtilis strain.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {8},
pmid = {41520043},
issn = {1432-0614},
mesh = {*Bacillus subtilis/genetics/metabolism ; *Glutaminase/genetics/biosynthesis/metabolism ; *Bacteroides/enzymology/genetics ; *Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Bioreactors/microbiology ; },
abstract = {Protein-glutamine glutaminases (PGs; EC 3.5.1.44) have gained attention in the food industry due to their application in plant protein products. The recently discovered PG from Bacteroides helcogenes (PGB) has especially been shown to provide promising characteristics for improving the techno-functional properties of plant proteins. A prerequisite for food enzymes, such as the PG, is their production with an expression host that meets food safety and yield requirements. The antibiotic-free and secretory production of the PGB was targeted in this study using the undomesticated Bacillus subtilis 007. The CRISPR/Cas9-mediated approach enabled specific genomic PGB integrations, while simultaneously deleting unwanted B. subtilis traits. Firstly, the PGB expression cassette was integrated into the sigF gene, leading to an asporogenic strain and extracellular activity of 4.1 µkat/Lculture in bioreactor cultivations. However, excessive foaming hampered the production process tremendously. Consequently, a second PGB copy was integrated into the sfp locus, which is involved in the production of lipopeptides, such as surfactin. As a result, the PGB activity was increased to 5.4 µkat/Lculture, and foaming during cultivation was reduced significantly. The introduction of a third PGB copy for preventing cell motility did not increase production; however, the integration into the well-established amyE locus improved the PGB yield during reactor cultivations. A final extracellular activity of 9.5 µkat/Lculture was reached. The multiple genomic integrations of the PGB gene enabled the efficient PGB secretion in an optimized B. subtilis host without the need for antibiotics. KEY POINTS: • Site-specific PGB integration enabled by genome sequencing of B. subtilis 007. • Antibiotic-free and secretory PGB production with an optimized B. subtilis host. • Increased PGB production reaching 9.5 µkat/Lculture.},
}
@article {pmid41518500,
year = {2026},
author = {Álvarez, E and Franco-Zorrilla, JM},
title = {Targeted DNA Affinity Purification for Quantifying DNA-Binding Specificities of Transcription Factors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2985},
number = {},
pages = {89-104},
pmid = {41518500},
issn = {1940-6029},
mesh = {*Transcription Factors/metabolism/genetics ; Binding Sites ; *DNA, Plant/metabolism/genetics ; High-Throughput Nucleotide Sequencing/methods ; Protein Binding ; *Chromatography, Affinity/methods ; Gene Expression Regulation, Plant ; *DNA/metabolism ; Arabidopsis/genetics/metabolism ; },
abstract = {Plant growth relies on flexible gene regulation to adapt to environmental changes. This process is ultimately controlled by transcription factors (TFs), which bind to specific DNA motifs, known as TF-binding sites (TFBS), located in the gene regulatory regions to regulate their expression. These interactions play crucial roles in plant development and responses to environmental cues, as well as in plant evolution and domestication, making both cis- (i.e., TFBS) and trans-regulatory factors as potential molecular targets in plant breeding for traits such as yield, quality, and stress resilience. These biotechnological approaches require precise knowledge of the target gene sets and TFBS specifically recognized by TFs. Recent advances in high-throughput sequencing techniques have enabled precise identification of TF target genes, especially thanks to methodologies that combine the main features of both in vitro and in vivo approaches. However, small scale and targeted approaches are still needed to evaluate the relative contribution of specific nucleotide positions in TF recognition. In this chapter, we describe a modified version of DNA Affinity Purification sequencing (DAP-seq) that replaces genomic DNA with a PCR-generated library of TFBS variants. This approach, termed targeted-DAP, allows a flexible and quantitative analysis of TF-binding using next-generation sequencing. Additionally, expressing TFs in Escherichia coli provides an economical source of proteins, enabling scalable and cost-effective analysis of DNA-binding specificities. We showed the benefits of this technique to demonstrate the contribution of the genomic context around the TFBS for specific recognition of a bHLH TF. Development of targeted DAP-seq would be of interest for the evaluation of nucleotide variation-either allelic or generated by CRISPR/Cas-within TFBS in TF recognition with predictable consequences on plant phenotypes.},
}
@article {pmid41518477,
year = {2026},
author = {Ghazaei, C},
title = {The role of bacteriophages and CRISPR-Cas in combating multidrug-resistant bacteria.},
journal = {Natural products and bioprospecting},
volume = {16},
number = {1},
pages = {14},
pmid = {41518477},
issn = {2192-2195},
abstract = {The alarming increase of multidrug-resistant (MDR) bacteria presents a serious global health crisis, reducing the effectivenessof traditional antibiotics and requiring alternative therapeutic strategies. Among the most promising innovations are bacteriophages-viruses that specifically infect bacteria-and CRISPR-Cas systems, molecular tools enabling precise genome editing. These technologies individually offer targeted antibacterial activity with minimal disturbance to the host microbiota. When combined, they forma synergistic platform capable of overcoming many limitations of conventional antibiotics, including broad-spectrum activity, resistance development, and limited adaptability. This review examinesmechanisms of bacterial resistance, the biological foundation of bacteriophages and CRISPR-Cas systems, and their application in fighting MDR pathogens. However, significant challenges remain, including delivery barriers, off-target effects, regulatory uncertainty, and public acceptance of gene-editing tools. Antimicrobial resistance now tanks among the top threats to global health, with an estimated burden exceeding one million deaths annually, surpassing many other infectious diseases. The article concludes with a discussion of the clinical prospects of phage-CRISPR therapies and highlights key areas for future research. By merging the specificity of phages with the programmable strength of CRISPR, these biotechnological advances provide a powerful and approach to address the growing threat of antibiotic resistance.},
}
@article {pmid41518077,
year = {2026},
author = {Liu, Y and Wang, H and Chen, L and Wu, X and Xu, Z and Huang, Q and Zhang, H and Cao, X and Liang, X and Zhong, X and Luo, C},
title = {CRISPR-Cas9-Generated TXNDC15 c.560delA Homozygous Mouse Model Exhibits Meckel-Gruber Syndrome Phenotype.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {64},
number = {1},
pages = {e70040},
pmid = {41518077},
issn = {1526-968X},
support = {SZSM202311005//Sanming Project of Medicine in Shenzen Municipality/ ; 2021A1515012495//Natural Science Foundation of Guangdong Province/ ; JCYJ20210324135009024//Shenzhen Science and Technology Innovation Commission/ ; },
mesh = {Animals ; Mice ; *Polycystic Kidney Diseases/genetics/pathology ; CRISPR-Cas Systems ; *Ciliary Motility Disorders/genetics/pathology ; *Encephalocele/genetics/pathology ; Disease Models, Animal ; Phenotype ; Homozygote ; *Retinitis Pigmentosa/genetics/pathology ; Frameshift Mutation ; },
abstract = {To determine whether TXNDC15 variation causes Meckel-Gruber syndrome (MKS), we assessed the pathogenicity of the frameshift variant c.560delA. A CRISPR-Cas9 generated mouse model carrying the equivalent Txndc15 c.512delA mutation was analyzed at embryonic day 15.5. Homozygous Txndc15[mt/mt] embryos displayed the complete MKS phenotype-fetal lethality, exencephaly, omphalocele, post-axial polydactyly, and polycystic kidneys-together with markedly reduced TXNDC15 protein in brain, liver, and kidney. These findings confirm TXNDC15 as a bona fide MKS disease gene.},
}
@article {pmid41329281,
year = {2025},
author = {Kolesov, DE and Orlova, NA and Vorobiev, II},
title = {Generation of Long-Lived CHO Cells Suitable for Production of Afucosylated Antibodies and Fc-Fusion Proteins.},
journal = {Doklady biological sciences : proceedings of the Academy of Sciences of the USSR, Biological sciences sections},
volume = {525},
number = {1},
pages = {359-367},
pmid = {41329281},
issn = {1608-3105},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Immunoglobulin Fc Fragments/genetics/metabolism ; *Fucosyltransferases/genetics/metabolism ; CRISPR-Cas Systems ; *Recombinant Fusion Proteins/genetics/biosynthesis ; *Antibodies/metabolism ; Gene Knockout Techniques ; },
abstract = {Using genome editing, we created a homozygous α-(1,6)-fucosyltransferase (FUT8[-]/[-]) knockout in apoptosis-resistant CHO 4BGD cells, yielding the new 4BGD-F cell line. Combining CRISPR/Cas9 with paired gRNAs and non-specific puromycin selection yielded a cell population with an exceptionally high FUT8 knockout frequency, obviating the need for metabolic enrichment with lentil lectin (Lens culinaris agglutinin, LCA). Despite impaired clonogenicity of the knockout cells, we successfully isolated multiple clonal cell lines harboring extensive biallelic FUT8 deletions. Isolated clones with biallelic deletions retained key parental line characteristics: viability >90% in 17-day fed-batch cultures at high densities (>15 × 10[6] cells/mL), and rapid selectability using both dihydrofolate reductase and glutamine synthetase systems. Mass spectrometric analysis of the test protein GLP1-Fc secreted by 4BGD-F cells confirmed the absence of N-glycan fucosylation. The CHO 4BGD-F cell line provides a valuable platform for producing afucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity.},
}
@article {pmid41517680,
year = {2026},
author = {Rehman, SU and Abbas, GH},
title = {CRISPR/CAS9-based gene editing in cancer therapy: A systematic review and meta-analysis on current status and future directions.},
journal = {Medicine},
volume = {105},
number = {2},
pages = {e47114},
doi = {10.1097/MD.0000000000047114},
pmid = {41517680},
issn = {1536-5964},
mesh = {*Gene Editing/methods/trends ; *CRISPR-Cas Systems ; *Neoplasms/therapy/genetics ; Humans ; *Genetic Therapy/methods/trends ; },
abstract = {BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has recently been discovered for gene editing and cancer therapy and its applications are expanding. This review and meta-analysis aim to assess the present and future of CRISPR/Cas9 based gene editing in cancer treatment and the way forward.<br><br>METHODS: The search was conducted in PubMed from 2015 to 2025 and 89 relevant studies were identified. The study design, CRISPR/Cas9 targets, delivery methods, therapeutic efficacy and limitations were extracted from the studies.<br><br>RESULTS: We reviewed the efficacy, challenges, and potential for translation of CRISPR/Cas9 in oncogene and tumor suppressor gene targeting and immune modulation. Several preclinical researches showed that CRISPR/Cas9 mediated disruption of oncogenes or restoration of tumor suppressor genes led to significant tumor regression. The evaluation was also extended to off target effects and integration with immunotherapy.<br><br>CONCLUSION: From the findings of this work, it can be concluded that CRISPR/Cas9 is a promising tool, but there are several limitations including off target effects, delivery systems and ethical issues that need to be solved in order to improve the clinical significance.},
}
@article {pmid41516423,
year = {2026},
author = {Kongsomboonchoke, P and Ariyachet, C and Kaewsapsak, P and Sirichindakul, P and Tangkijvanich, P},
title = {Development of a Sensitive and Specific RPA-CRISPR/Cas12a Assay for Intrahepatic Quantification of HBV cccDNA.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516423},
issn = {1422-0067},
support = {//Second Century Fund (C2F)/ ; //Center of Excellence in Hepatitis and Liver Cancer/ ; },
mesh = {Humans ; *Hepatitis B virus/genetics ; *DNA, Circular/genetics/analysis ; *CRISPR-Cas Systems/genetics ; *DNA, Viral/genetics/analysis ; *Hepatitis B/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; *Liver/virology ; Sensitivity and Specificity ; Recombinases/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Hepatitis B virus (HBV) persists in infected hepatocytes through covalently closed circular DNA (cccDNA), a stable episomal form that serves as the transcriptional template for viral replication. Accurate and sensitive quantification of intrahepatic cccDNA is crucial for evaluating antiviral therapies, particularly those targeting a functional cure. Here, we report the development of a novel, cccDNA-specific detection system combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a-based fluorescence detection. We designed and validated CRISPR RNAs (crRNAs) targeting HBV cccDNA-specific regions conserved across genotypes A-D. Reaction conditions for both RPA and Cas12a detection were optimized to enhance sensitivity, specificity, and accuracy. The system reliably detected as few as 10 copies of cccDNA-containing plasmid per reaction and showed no cross-reactivity with non-cccDNA forms in serum or plasma, indicating assay specificity. When applied to liver tissue samples from 10 HBV-infected and 6 non-HBV patients, the RPA-CRISPR/Cas12a assay exhibited a high sensitivity (90%) and a strong correlation with qPCR results (R[2] = 0.9155), confirming its accuracy. In the conclusion, the RPA-CRISPR/Cas12a system provides a robust, cost-effective, and scalable platform for sensitive and specific quantification of intrahepatic HBV cccDNA. This method holds promises for research and high-throughput therapeutic screening applications targeting cccDNA clearance.},
}
@article {pmid41516375,
year = {2026},
author = {Firdaus, F and Yadav, V and Ramakrishnan, M and Wasi, A and Ganie, IB and Upadhyay, A and Shahzad, A and Ahmad, Z},
title = {2-Hydroxy-4-Methoxybenzaldehyde (2H4MB): Integrating Cell Culture, Metabolic Engineering, and Intelligent Genome Editing.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516375},
issn = {1422-0067},
support = {Y20240114//Young Foreign Talent Program/ ; },
mesh = {*Metabolic Engineering/methods ; *Gene Editing/methods ; *Benzaldehydes/metabolism/chemistry ; Cell Culture Techniques/methods ; CRISPR-Cas Systems ; Biosynthetic Pathways ; },
abstract = {2-Hydroxy-4-Methoxybenzaldehyde (2H4MB) is a valuable aromatic compound with applications in flavour, fragrance, and pharmaceuticals. Because of its endangered status and root-specific accumulation, its production in native plants is restricted. In order to increase 2H4MB yield, this study emphasises recent developments in metabolic engineering, synthetic biology, in vitro culture methods, and AI-assisted route prediction. This review discussed about how CRISPR-based genome editing can be used to modify important biosynthetic genes and regulatory components, as well as how predictive machine learning techniques can be used to improve production conditions. Inadequate genetic resources, poorly understood biosynthetic pathways, and a dearth of reliable transformation systems are among the present constraints. The work highlights the importance of using integrative plant biotechnology techniques to fully realise the industrial and medicinal potential of this underutilised chemical.},
}
@article {pmid41516361,
year = {2026},
author = {Deriglazova, IO and Shepelev, MV and Kruglova, NA and Georgiev, PG and Maksimenko, OG},
title = {The Chimeric Nuclease SpRYc Exhibits Highly Variable Performance Across Biological Systems.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516361},
issn = {1422-0067},
support = {075-15-2024-539//Ministry of Science and Education of the Russian Federation/ ; },
mesh = {Animals ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; HEK293 Cells ; Drosophila melanogaster/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {The CRISPR-Cas9 system has significantly advanced genome editing but remains constrained by its requirement for specific protospacer adjacent motifs (PAMs). To overcome this limitation, PAM-relaxed nucleases, including the novel near-PAMless chimeric SpRYc, have been developed. Here, we evaluated SpRYc editing activity across multiple experimental systems, including human HEK293 and CEM-R5 cells, as well as Drosophila melanogaster S2 cells and embryos. In HEK293 cells, SpRYc exhibited broad PAM compatibility, enabling editing at non-canonical PAMs, albeit with reduced and variable efficiency at canonical NGG sites compared to SpCas9. This context dependency was more pronounced in CEM-R5 T cells, where SpRYc activity at endogenous CXCR4 and B2M loci was largely restricted to NGG PAMs. In contrast, unlike SpCas9, SpRYc displayed negligible genome-editing activity in Drosophila embryos in vivo. Notably, the transcriptional activator dSpRYc-VPR showed robust activity in Drosophila S2 cells at both canonical and non-canonical PAMs. Reduced chromatin occupancy of dSpRYc-VPR suggests a balance between expanded PAM recognition and DNA-binding stability, providing a mechanistic explanation for context-dependent performance of SpRYc. Overall, our results highlight that expanded targeting flexibility comes at the cost of variable efficiency, underscoring the need for extensive locus- and context-specific validation of PAM-relaxed genome-editing tools.},
}
@article {pmid41516323,
year = {2025},
author = {Evseev, PV and Podoprigora, IV and Chaplin, AV and Khabadze, ZS and Malkov, AA and Kafarskaia, LI and Shagin, DA and Urban, YN and Borisova, OY and Efimov, BA},
title = {Bulleidia extructa PP_925: Genome Reduction, Minimalist Metabolism, and Evolutionary Insights into Firmicutes Diversification.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516323},
issn = {1422-0067},
mesh = {*Genome, Bacterial ; Phylogeny ; *Evolution, Molecular ; Humans ; *Firmicutes/genetics/metabolism/classification ; },
abstract = {Bulleidia extructa strain PP_925, isolated from the periodontal pocket of a patient with periodontitis, is a Gram-positive Bacillota with an unusually compact genome of 1.38 Mb. Phylogenomic analyses place PP_925 within Erysipelotrichales and show close relatedness of Bulleidia to Solobacterium and Lactimicrobium, as well as the existence of previously undescribed related clades. The metabolic repertoire of PP_925 is strongly reduced: it retains glycolysis, the phosphotransacetylase-acetate kinase pathway, and arginine catabolism but lacks the tricarboxylic acid cycle and most de novo biosynthetic pathways for amino acids, nucleotides, fatty acids, cofactors, and vitamins, implying reliance on salvage and cross-feeding. Phylogenetic inference indicates independent peptidoglycan losses in multiple mycoplasma Erysipelotrichia-related lineages, while PP_925 has retained an ancestral Gram-positive cell wall despite extensive genomic reduction. The genome preserves systems crucial for host interaction and adaptability, including a horizontally acquired tad locus encoding type IV pili, a comG competence system, and several adherence-associated virulence factors. Defense mechanisms are diverse and include a CRISPR-Cas II-A system, a type II restriction-modification module adjacent to Gao_Qat-like genes, and the Wadjet system in a genome without prophages; CRISPR spacers indicate repeated encounters with Bacillota phages. Comparative genomics of PP_925 and related strains reveals a small core genome with lineage-specific adhesion and defense modules, indicating recent shared ancestry combined with adaptive flexibility under substantial genome reduction.},
}
@article {pmid41516041,
year = {2025},
author = {Sahu, D and Ganguly, T and Mann, A and Gupta, Y and Nynatten, LRV and Fraser, DD},
title = {Emerging Technologies for Exploring the Cellular Mechanisms in Vascular Diseases.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516041},
issn = {1422-0067},
mesh = {Humans ; *Vascular Diseases/genetics/metabolism/pathology/diagnosis ; Animals ; Gene Editing/methods ; Artificial Intelligence ; CRISPR-Cas Systems ; Single-Cell Analysis/methods ; },
abstract = {Vascular diseases (VDs) and cardiovascular diseases (CVDs) are the leading causes of morbidity and mortality worldwide. Current diagnostic and therapeutic approaches are limited by insufficient resolution and a lack of mechanistic understanding at the cellular level. Traditional imaging and clinical assays do not fully capture the dynamic molecular and structural complexities underlying vascular pathology. Recent technological innovations, including single-cell and spatial transcriptomics, super-resolution and photoacoustic imaging, microfluidic organ-on-chip platforms, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based gene editing, and artificial intelligence (AI), have created new opportunities for investigating the cellular and molecular basis of VDs. These techniques enable high-resolution mapping of cellular heterogeneity and functional alterations, facilitating the integration of large-scale data for biomarker discovery, disease modeling, and therapeutic development. This review focuses on evaluating the translational readiness, limitations, and potential clinical applications of these emerging technologies. Understanding the cellular and molecular mechanisms of VDs is essential for developing targeted therapies and precise diagnostics. Integrating single-cell and multiomics approaches highlights disease-driving cell types and gene programs. Optogenetics and organ-on-chip platforms allow for controlled manipulation and physiologically relevant modeling, while AI enhances data integration, risk prediction, and clinical interpretability. Future efforts should prioritize multi-center, large-scale validation studies, harmonization of assay protocols, and integration with clinical datasets and human samples. Multi-omics approaches and computational modeling hold promise for unraveling disease complexity, while advances in regulatory science and digital simulation (such as digital twins) may further accelerate personalized medicine in vascular disease research and treatment.},
}
@article {pmid41513250,
year = {2026},
author = {Zhu, Y and Luo, S and Cui, X and Wu, J and Cheng, W and Wang, X and Zhang, Q and Tan, G and Yang, H and Zheng, Y and Peng, W},
title = {Converting an Untransformable Vibrio parahaemolyticus Isolate into a Fast Genetic Engineering Platform.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00771},
pmid = {41513250},
issn = {2161-5063},
abstract = {The pathogenic bacterium Vibrio parahaemolyticus represents a substantial economic and public health concern; however, elucidating its virulence mechanisms has been significantly impeded by its inherent resistant to genetic manipulation, primarily attributed to sophisticated immune defense systems including restriction-modification (R-M) modules, CRISPR-Cas systems, standalone DNases, and DdmDE systems. Paradoxically, while genetic modification is essential for overcoming these barriers, the very barriers themselves obstruct DNA introduction. Our investigation focused on the V. parahaemolyticus X1 strain, where initial plasmid transformation attempts proved unsuccessful. However, low-efficiency conjugation allowed knockout of defense genes, thereby silencing the host's defense mechanisms. Our findings revealed a standalone DNase, Vpn, as the predominant obstacle to foreign DNA entry in the X1 strain, while a DdmDE system executes elimination of invaded plasmids. Leveraging these insights, we created the V. parahaemolyticus X2 strain via sequential depletion of the Vpn nuclease and the DdmDE system. Capitalizing on the bacterium's exceptional growth rate, characterized by a generation time of approximately 10.5 min, we established a highly efficient molecular cloning platform capable of creating a new plasmid construct within a single day. This work not only presents a strategic framework for genetic manipulation of previously recalcitrant bacterial species but also underscores the potential of fast-growing marine bacteria as promising candidates for next-generation biotechnological applications.},
}
@article {pmid41512012,
year = {2026},
author = {Chaudhari, VR and Lin, MT and Hines, KM and Hanson, MR},
title = {Rewinding evolution in planta: A Rubisco-null platform validates high-performance ancestral enzymes.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {3},
pages = {e2523360123},
doi = {10.1073/pnas.2523360123},
pmid = {41512012},
issn = {1091-6490},
support = {DE-SC0020142//DOE Office of Basic Energy Sciences, Chemical Sciences, Geosciences, and Biosciences Division/ ; MCB-2131582//NSF (NSF)/ ; },
mesh = {*Ribulose-Bisphosphate Carboxylase/genetics/metabolism ; *Nicotiana/genetics/enzymology ; Plants, Genetically Modified/genetics ; Photosynthesis/genetics ; CRISPR-Cas Systems ; Chloroplasts/genetics ; Evolution, Molecular ; Kinetics ; Gene Knockout Techniques ; },
abstract = {Improving the photosynthetic enzyme Rubisco is a key target for enhancing C3 crop productivity, but progress has been hampered by the difficulty of evaluating engineered variants in planta without interference from the native enzyme. Here, we report the creation of a Rubisco-null Nicotiana tabacum platform by using CRISPR-Cas9 to knock out all 11 nuclear-encoded small subunit (rbcS) genes. Knockout was achieved in a line expressing cyanobacterial Rubisco from the plastid genome, allowing the recovery of viable plants. We then developed a chloroplast expression system for coexpressing both large and small subunits from the plastid genome. We expressed two resurrected ancestral Rubiscos from the Solanaceae family. The resulting transgenic plants were phenotypically normal and accumulated Rubisco to wild-type levels. Importantly, kinetic analyses of the purified ancestral enzymes revealed they possessed a 16 to 20% higher catalytic efficiency (kcat,air/Kc,air) under ambient conditions, driven by a significantly faster turnover rate (kcat,air). We have demonstrated that our system allows robust in vivo assessment of novel Rubiscos and that ancestral reconstruction is a powerful strategy for identifying superior enzymes to improve photosynthesis in C3 crops.},
}
@article {pmid41511319,
year = {2025},
author = {Xu, J and Zhang, J and Yang, D},
title = {Editorial: CRISPR-Based Genome Editing in Translational Research-2nd Edition.},
journal = {Cells},
volume = {15},
number = {1},
pages = {},
pmid = {41511319},
issn = {2073-4409},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Translational Research, Biomedical ; Humans ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Genome editing technologies represented by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) have transformed biomedical research and therapeutic development [...].},
}
@article {pmid41510784,
year = {2026},
author = {Hejlesen, R and Bakkeren, C and Damsgaard, C and Laursen, LS and Kjær-Sørensen, K and Corti, P and Malte, H and Oxvig, C and Fago, A},
title = {Myoglobin Affects Tissue-Specific Transcriptome, Heart Regeneration and Whole Animal Metabolic Rates.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {1},
pages = {e71424},
doi = {10.1096/fj.202503482RR},
pmid = {41510784},
issn = {1530-6860},
support = {NNF19OC0057938//Novo Nordisk Fonden (NNF)/ ; 3103-0011B//Danmarks Frie Forskningsfond (DFF)/ ; DMMTF24081545//Company of Biologists (Co_Biologists)/ ; //Graduate School Natural Science Aarhus University/ ; },
mesh = {Animals ; Zebrafish/genetics/metabolism ; *Myoglobin/genetics/metabolism ; *Regeneration/physiology ; *Transcriptome ; *Heart/physiology ; Muscle, Skeletal/metabolism ; Myocytes, Cardiac/metabolism ; *Myocardium/metabolism ; *Zebrafish Proteins/genetics/metabolism ; },
abstract = {Myoglobin (Mb) is a small haem-containing protein traditionally associated with oxygen carrier functions in cardiac and skeletal muscle. However, studies using Mb knockout mice have yielded conflicting results regarding its functional roles in vivo. Here, we used a CRISPR-Cas-generated zebrafish Mb knockout model to investigate the consequences of Mb loss across skeletal muscle types, transcriptomics profiles, and whole-animal metabolic rates under both resting and maximal exercise conditions. Mb deficiency did not alter skeletal muscle fiber composition or overall mitochondrial respiratory capacity but induced multiple tissue-specific transcriptomic changes, including downregulation of gene sets involved in respiration and differentiation pathways in the heart, while upregulating those associated with respiration and glycogen metabolism in the skeletal muscle. During cardiac regeneration following ventricle amputation in wild-type zebrafish, Mb expression was transiently suppressed, consistent with a role in maintaining the cardiomyocytes in a differentiated state. Physiologically, Mb knockout zebrafish displayed a reduced standard metabolic rate at rest, enhanced hypoxia tolerance (i.e., a lower critical oxygen tension), and increased maximal swimming speed, while maintaining unchanged maximal metabolic rate and aerobic scope relative to wild-type counterparts. Collectively, these findings show that loss of Mb in zebrafish elicits coordinated tissue-specific transcriptional changes, potentially facilitates cardiac regeneration, lowers standard metabolic rate, and enhances maximal swimming speed and hypoxia tolerance, thereby providing new insights into the multifaceted in vivo functions of Mb.},
}
@article {pmid41386334,
year = {2026},
author = {Svane, N and Kurosawa, T and Schmid, B and Saaby, L and Kristensen, M and Tabata, H and Kubo, Y and Terasaki, T and Brodin, B and Deguchi, Y},
title = {Elucidating the roles of TM7SF3 and LHFPL6 in the putative H[+]/OC antiporter function in the human brain capillary endothelial cell line, hCMEC/D3.},
journal = {European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences},
volume = {217},
number = {},
pages = {107409},
doi = {10.1016/j.ejps.2025.107409},
pmid = {41386334},
issn = {1879-0720},
mesh = {Humans ; *Endothelial Cells/metabolism ; Blood-Brain Barrier/metabolism ; *Brain/metabolism/blood supply ; Cell Line ; *Antiporters/metabolism/genetics ; Biological Transport ; *Membrane Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: The putative proton/organic cation (H[+]/OC) antiporter has been shown to mediate transport of CNS drug compounds like oxycodone and pyrilamine across the blood-brain barrier (BBB). This transporter has a broad substrate profile and is able to transport substrates against their concentration gradient, making it an interesting target for brain drug delivery. However, the molecular identity of this transporter remains unknown. Recent studies have indicated that the two proteins TM7SF3 and LHFPL6 might be components of this transporter. The present study aimed to investigate the roles of TM7SF3 and LHFPL6 in the H[+]/OC antiporter function to advance understanding of its molecular identity and potential in CNS drug delivery.<br><br>METHODS: CRISPR-Cas9 gene-editing was used to generate three hCMEC/D3 knockout (KO) cell lines: TM7SF3 KO (TM-KO), LHFPL6 KO (LH-KO), and a double KO of TM7SF3 and LHFPL6 (TMLH-KO). The uptake of pyrilamine analogue (EDMPG) and [[3]H]-pyrilamine was assessed in wild type (WT) and KO lines. Quantitative Realtime Polymerase Chain Reaction (qRT-PCR) confirmed successful gene knockouts. Passive diffusion properties and the expression and functionality of known BBB transporters, including LAT1 (SLC7A5), GLUT1 (SLC2A1), and MCT1 (SLC16A1), were also examined.<br><br>RESULTS: The EDMPG uptake was significantly reduced in TM-, LH-, and TMLH-KO cells, suggesting that TM7SF3 and LHFPL6 contribute to the H[+]/OC antiporter function. However, [[3]H]-pyrilamine uptake remained unchanged across all KOs, indicating a TM7SF3- and LHFPL6-independent transport mechanism. This was further supported by the persistent inhibition of [[3]H]-pyrilamine uptake in the presence of known H[+]/OC antiporter substrates. While passive diffusion and GLUT1- and MCT1-mediated transport were unaffected, LAT1-mediated uptake of [[3]H]L-leucine and gabapentin (Neurontin) was significantly reduced in LH- and TMLH-KO cells, correlating with decreased LAT1 mRNA expression in these cells.<br><br>CONCLUSIONS: This study suggests that the H+/OC antiporter operates via two distinct mechanisms: a high-capacity, TM7SF3- and LHFPL6-independent pathway and a low-capacity, TM7SF3- and LHFPL6-dependent pathway. These findings underscore the complexity of the H[+]/OC antiporter molecular composition and highlight the need for further research to fully elucidate its identity.},
}
@article {pmid41366211,
year = {2025},
author = {Nuccio, SP and Cadoni, E and Nikoloudaki, R and Galli, S and Ler, AJ and Sanchez-Cabanillas, C and Maher, TE and Fan, E and Guneri, D and Flint, G and Zhu, M and Liu, LS and Fullenkamp, CR and Waller, Z and Magnani, L and Schneekloth, JS and Di Antonio, M},
title = {Chemically modified CRISPR-Cas9 enables targeting of individual G-quadruplex and i-motif structures, revealing ligand-dependent transcriptional perturbation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {385},
pmid = {41366211},
issn = {2041-1723},
support = {Lister Prize 2022//Lister Institute of Preventive Medicine/ ; BB/R011605/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; },
mesh = {*G-Quadruplexes ; *CRISPR-Cas Systems/genetics ; Humans ; Ligands ; *Transcription, Genetic ; Promoter Regions, Genetic ; DNA/chemistry/genetics/metabolism ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Nucleotide Motifs ; CRISPR-Associated Protein 9/metabolism/genetics ; Gene Editing/methods ; },
abstract = {The development of selective ligands to target DNA G-quadruplexes (G4s) and i-motifs (iMs) has revealed their relevance in transcriptional regulation. However, most of these ligands are unable to target individual G4s or iMs in the genome, limiting their scope. Herein, we describe an Approach to Target Exact Nucleic Acid alternative structures (ATENA) that relies on the chemical conjugation of established G4 and iM ligands to a catalytically inactive Cas9 protein (dCas9), enabling their individual targeting in living cells. ATENA demonstrates that the selective targeting of the G4 present in the oncogene c-MYC leads to the suppression of transcripts regulated exclusively by one of its promoters (P1). Conversely, targeting the c-MYC iMs on the opposite strand leads to the selective increase of P1-driven transcripts. ATENA reveals that G4-mediated transcriptional responses are highly ligand-specific, with different ligands eliciting markedly different effects at the same G4 site. We further demonstrate that the basal expression levels of the gene targeted can be used to predict the transcriptional impact associated with G4-stabilization. Our study provides a platform for investigating G4- and iM-biology with high precision, unveiling the therapeutic relevance of individual DNA structures with selectivity.},
}
@article {pmid41353207,
year = {2025},
author = {Sun, H and Teng, Q and Liu, W and Guo, R and Li, M and Xiong, W and Huang, Q and Yu, Q and Luo, N and Li, Y and Song, J and Gong, S and Shi, X and Yi, C and Liu, K},
title = {CRISPR-free RNA base editing mediated PTC-readthrough restores hearing in mice with Otof nonsense mutation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {413},
pmid = {41353207},
issn = {2041-1723},
support = {no. 81770997//National Natural Science Foundation of China (National Science Foundation of China)/ ; no. 82460223//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Codon, Nonsense/genetics ; Mice ; *Deafness/genetics/therapy ; *Genetic Therapy/methods ; Disease Models, Animal ; *Membrane Proteins/genetics/metabolism ; *RNA Editing/genetics ; *Hearing/genetics ; Humans ; Cochlea/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Male ; Female ; Mice, Inbred C57BL ; },
abstract = {The gene therapy achieved by AAV-mediated otoferlin-overexpression is an effective therapeutic strategy for congenital deafness. However, achieving its physiological and endogenous patterns of expression remains challenging. Here, we generate the homologous mutation Otof c.1315 C > T (p.R439*), equivalent to OTOF c.1273 C > T (p.R425*) found in humans with profound deafness, to create a nonsense mutation-induced deaf mouse model. We then deliver the 'RESTART v3' system, which is a CRISPR-free RNA base editor for nonsense mutation suppression, into the cochlea of the mice. We achieve physiological otoferlin expression, and the edited premature termination codon is reverse-mutated to the original amino acid. We observe significant hearing restoration and enhancement of the behavioral auditory startle reflex. Thus, our study presents a successful RNA editing strategy to significantly restore hereditary deafness in mice carrying the specific Otof nonsense mutation, which holds great promise for future clinical translation.},
}
@article {pmid41339636,
year = {2025},
author = {Agnarelli, A and Buckley-Benbow, L and Ozgencil, M and Lad, M and Ampah, KK and Kalinka, A and Belan, O and Maslen, S and Skehel, MJ and Walter, D and Day, M and Bellelli, R},
title = {The genetic and biochemical basis of human leading strand synthesis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {412},
pmid = {41339636},
issn = {2041-1723},
mesh = {Humans ; *DNA Replication/genetics ; Genomic Instability ; *DNA Polymerase II/metabolism/genetics/chemistry ; Proliferating Cell Nuclear Antigen/metabolism ; DNA/biosynthesis/genetics ; Iron/metabolism ; Iron-Sulfur Proteins/metabolism ; CRISPR-Cas Systems ; },
abstract = {The maintenance of genome stability requires efficient leading strand synthesis by DNA Polymerase Epsilon (Polε). By performing CRISPR genetic screens in cells lacking the POLE4 subunit of Polε we define a genetic map of the factors required to support Polε function in the absence of its accessory subunits. A set of genes involved in iron metabolism emerge as required to sustain Iron Sulphur Cluster (ISC)-dependent Polε activity. We then dissect a synthetic lethal interaction between POLE3-POLE4 and the CHTF18-RFC2/5 complex. By combining cell biology, structural modelling and biochemistry, we define the existence of two tiers of regulation of Polε processivity: leading strand-specific loading of PCNA by CHTF18-RFC2/5 and "gripping" of newly synthesised dsDNA by POLE3-POLE4. The combined loss of these functions is incompatible with leading strand synthesis and viability. In summary, we describe the biochemical basis of human leading strand synthesis and the consequence of its dysfunction in genome stability.},
}
@article {pmid41025871,
year = {2026},
author = {Frangedakis, E and Yelina, NE and Eeda, SK and Romani, F and Fragkidis, A and Haseloff, J and Hibberd, JM},
title = {A tRNA-gRNA multiplexing system for CRISPR genome editing in Marchantia polymorpha.},
journal = {Journal of experimental botany},
volume = {77},
number = {2},
pages = {330-344},
doi = {10.1093/jxb/eraf433},
pmid = {41025871},
issn = {1460-2431},
support = {//BBSRC/ ; BB/L014130/1//EPSRC OpenPlant Synthetic Biology Research Centre/ ; BB/F011458/1//EPSRC OpenPlant Synthetic Biology Research Centre/ ; BBP0031171//EPSRC OpenPlant Synthetic Biology Research Centre/ ; },
mesh = {*Marchantia/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *RNA, Transfer/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Plant ; },
abstract = {The liverwort Marchantia polymorpha is a widely used model organism for studying land-plant biology, and it has also proven to be a promising testbed for bioengineering. CRISPR/Cas9 technology has become a transformative tool for precise genome modifications in M. polymorpha; however, a robust method for the simultaneous expression of multiple gRNAs, which is crucial for enhancing the versatility of CRISPR/Cas9-based genome editing, has yet to be fully developed. In this study, we introduce an adaptation from the OpenPlant kit CRISPR/Cas9 tools that facilitates expression of multiple gRNAs from a single transcript through incorporation of tRNA sequences. The ability to deliver multiple gRNAs simultaneously significantly improves the capacity and scalability of genome editing in M. polymorpha. Additionally, by combining this vector system with a simplified and optimized protocol for thallus transformation, we further streamline the generation of CRISPR/Cas9 mutants in M. polymorpha. The resulting gene-editing system offers a multipurpose, time-saving, and straightforward tool for advancing functional genomics in M. polymorpha, enabling more comprehensive genetic modifications and genome engineering.},
}
@article {pmid41510594,
year = {2026},
author = {Wei, J and Duan, Y and Xue, C and Zheng, L and Wei, Q and Wu, Z and Xin, H and Zeng, T and Deng, H and Fan, S and Xiong, W and Zeng, Z and Li, M and Zhou, M},
title = {Targeted demethylation of the BRD7 promoter based on CRISPR/dCas9 system inhibits the malignant progression of nasopharyngeal carcinoma.},
journal = {Clinical and translational medicine},
volume = {16},
number = {1},
pages = {e70583},
pmid = {41510594},
issn = {2001-1326},
support = {82473262//the National Natural Science Foundation of China/ ; 82403005//the National Natural Science Foundation of China/ ; 2023SK2008//the Human Provincial key Research and Development Program/ ; 2024ZZTS0160//the Free Exploration Program of Central South University/ ; 20256508//the scientific research plan project of Hunan Provincial Health Commission/ ; 111-2-12//the program of Introducing Talents of Discipline to Universities/ ; },
mesh = {Humans ; *Nasopharyngeal Carcinoma/genetics/pathology ; Promoter Regions, Genetic/genetics ; Mice ; Animals ; *Nasopharyngeal Neoplasms/genetics/pathology ; *Chromosomal Proteins, Non-Histone/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Disease Progression ; Cell Line, Tumor ; DNA Methylation/genetics ; Cell Proliferation/genetics ; Female ; Bromodomain Containing Proteins ; },
abstract = {BACKGROUND: BRD7 has been confirmed to be lowly expressed in nasopharyngeal carcinoma (NPC) tissues and exerts tumour suppressive roles. However, the molecular mechanism of the downregulation of BRD7 expression and whether the strategy of activating BRD7 expression plays anti-tumour effects still needs to be clarified.<br><br>METHODS: Methylation-specific polymerase chain reaction (PCR) was used to identify the methylation levels of BRD7 promoter. In vitro experiments were used to evaluate the effects of BRD7-targeted demethylation system on the malignant progression of NPC cells. Chromatin immunoprecipitation (ChIP)-qPCR experiment was employed to examine the regulatory mechanisms underlying the demethylation system. Xenograft tumour models were used to assess impact of this demethylation system on tumour growth in vivo and the anti-tumour effects of the lentivirus-mediated demethylation system in NPC.<br><br>RESULTS: There was hypermethylation modification in BRD7 promoter, which was negatively correlated with BRD7 expression. Next, we constructed a LentiCRISPRv2/dCas9-TET1CD-sgRNAs system targeting specific methylation sites of BRD7 promoter based on five sgRNAs, and confirmed that all five sgRNA-guided CRISPR/dCas9 systems could activate BRD7 and inhibit cell proliferation to varying degrees, among which sgRNA2&amp;sgRNA5 were the most significant. Further, we constructed NPC cell lines stably transfected with LentiCRISPRv2/dCas9-TET1CD-sgRNA2&amp;5, and confirmed that both sgRNA2&amp;sgRNA5 could promote the transcriptional activation by reducing its methylation, and inhibit the cell proliferation, migration, invasion and tumour growth in vivo of NPC, and the combination of them has a more significant demethylation, transcriptional activation and anti-tumour effect. In addition, BRD7 had hypermethylation modification in its promoter and decreased expression in NPC tissues, and both of them were negatively correlated, making it a potential diagnostic marker for NPC diagnosis.<br><br>CONCLUSIONS: The hypermethylation modification of BRD7 is an important mechanism leading to the inactivation of BRD7, and targeting demethylation of BRD7 inhibits the malignant progression of NPC, which might be a promising targeted therapeutic approach for treating NPC.},
}
@article {pmid41510162,
year = {2026},
author = {Pan, J and Li, B and Wang, Y and Han, Y and Liu, G and Sun, SK},
title = {Physical stimuli-responsive CRISPR-Cas9 systems for spatiotemporally precise control of genome engineering.},
journal = {Theranostics},
volume = {16},
number = {6},
pages = {2984-3010},
pmid = {41510162},
issn = {1838-7640},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Animals ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 (CRISPR-Cas9) endonuclease system has revolutionized biology research by enabling precise, efficient, and versatile genome editing. However, achieving spatiotemporally controlled gene editing within specific organs, tissues, or cells remains a major challenge, as unregulated CRISPR-Cas9 activity can lead to severe off-target effects, hindering its clinical translation. To enhance the on-target precision and reduce the unwanted consequences of aberrant or premature CRISPR-Cas9 activation, various strategies have been developed to regulate its function at translational or post-translational stages using diverse external physicochemical stimuli. While chemical molecule-inducible CRISPR-Cas9 systems have demonstrated significant progress, most of them still suffer from inherent deficiencies, such as unsatisfactory spatiotemporal precision, irreversibility, pharmacokinetic dependence, internal disturbance, and safety concerns related to chemical inducers. By contrast, externally applied physical stimuli provide distinct advantages for triggering CRISPR-Cas9 activity, offering superior spatiotemporal precision, reversibility, and biocompatibility. These features significantly enhance the controllability, target specificity, and practical applicability of CRISPR-Cas9 systems across diverse biological settings. This review systematically explores recent advances in physical stimuli-responsive CRISPR-Cas9 platforms, detailing their design strategies, activation mechanisms, and proof-of-concept applications. Furthermore, we provide a comparative analysis of different stimulation strategies, highlighting their respective characteristics, current limitations, and future prospects. A discussion on the persistent bench-to-bedside gap is also included, aiming to guide future development toward clinically viable solutions.},
}
@article {pmid41483499,
year = {2026},
author = {Jin, G and Yang, C and Deng, Q and Wu, L and Chen, W and Chen, Z},
title = {Establishing a chimeric tRNA-sgRNA scaffold and computational basis for enhanced CRISPR interference.},
journal = {Biochemical and biophysical research communications},
volume = {798},
number = {},
pages = {153222},
doi = {10.1016/j.bbrc.2025.153222},
pmid = {41483499},
issn = {1090-2104},
mesh = {*RNA, Transfer/genetics/chemistry ; *CRISPR-Cas Systems/genetics ; Escherichia coli/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Humans ; Molecular Docking Simulation ; Gene Editing/methods ; Molecular Dynamics Simulation ; Nucleic Acid Conformation ; },
abstract = {The CRISPR/Cas9 system revolutionizes genome engineering, yet optimizing the stability and expression levels of single-guide RNA (sgRNA) is crucial for achieving more effective gene regulation. Transfer RNAs (tRNA), known for their inherent stability, present a valuable solution. In this study, we developed a chimeric tRNA-sgRNA (tgRNA) by integrating sgRNA into the anticodon stem of a Sephadex aptamer-human HBV ε tRNA (SeptRNA) scaffold, resulting in the formation of SeptgRNA. When applied to target the E. coli ampC and ompA genes, SeptgRNA exhibited significantly increased accumulation compared to conventional sgRNAs. To overcome potential steric hindrance from the tRNA scaffold, we utilized CRISPR interference (CRISPRi) by co-expressing SeptgRNA with deactivated Cas9 (dCas9), which effectively suppressed DNA transcription. This approach demonstrated superior gene expression suppression compared to traditional sgRNA-based CRISPRi. Molecular docking and molecular dynamics simulations revealed that the SeptRNA scaffold stabilizes the sgRNA stem-loop architecture and enhances the stability of the dCas9-tgRNA-DNA ternary complex. Our findings provide proof-of-concept for the use of chimeric tgRNAs in gene knockdown, highlighting their potential for increased expression levels and improved stability. This study advances the CRISPR/Cas9 toolkit and underscores the versatility of tRNA scaffolds in genetic engineering applications.},
}
@article {pmid41421070,
year = {2026},
author = {Salodkar, D and Dongarwar, S and Nair, A and Ashtaputre, P and Reddy, S and Somkuwar, S and Begde, D},
title = {Single-Step CRISPR/Cas13a Assay for detection of small RNAs in Saliva : a proof-of-concept study.},
journal = {Cancer genetics},
volume = {300-301},
number = {},
pages = {67-71},
doi = {10.1016/j.cancergen.2025.12.003},
pmid = {41421070},
issn = {2210-7762},
mesh = {Humans ; *Saliva/chemistry/metabolism ; *CRISPR-Cas Systems/genetics ; *Biomarkers, Tumor/genetics ; Proof of Concept Study ; *MicroRNAs/genetics/analysis ; Sensitivity and Specificity ; *Mouth Neoplasms/genetics/diagnosis ; Leptotrichia/genetics ; Real-Time Polymerase Chain Reaction ; },
abstract = {OBJECTIVE: We describe a proof-of-concept study of a rapid, single-step CRISPR/Cas13a assay using Leptotrichia wadei (LwCas13a) for the detection of small RNA (miRNA) biomarkers in saliva, and compare its performance to real-time PCR (RT-PCR).<br><br>METHODS: The single-step Cas13a assay was evaluated against RT-PCR for its detection efficiency, sensitivity, specificity, and its ability to function in a complex biological matrix. A proof-of-concept test was conducted on patient saliva samples to detect a known oral cancer biomarker, hsa-miR-21-3p RESULTS: The Cas13a assay successfully detected candidate miRNA at picomolar concentrations in both in vitro and saliva samples, demonstrating sensitivity and specificity comparable to RT-PCR. Notably, the assay provided discernible detection of the cancer biomarker directly in patient saliva without the need for RNA extraction or reverse transcription steps.<br><br>CONCLUSION: The proposed single-step CRISPR/Cas13a assay may be developed into a promising platform for developing quick and affordable point-of-care diagnostics for cancer and other diseases, circumventing the need for expensive and time-consuming sample preparation steps.},
}
@article {pmid41508907,
year = {2026},
author = {Liu, CF and Leon, S and Herrig, I and Wessely, O and Tang, WHW},
title = {Generation of Mice Harboring Bicc1 Conditional Null Alleles.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {64},
number = {1},
pages = {e70038},
pmid = {41508907},
issn = {1526-968X},
support = {MF-2203-02413//G. Harold and Leila Y. Mathers Charitable Foundation/ ; R01DK080745/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Mice ; Alleles ; Mice, Knockout ; CRISPR-Cas Systems ; *Polycystic Kidney Diseases/genetics ; *RNA-Binding Proteins/genetics ; Gene Editing ; Exons ; Female ; },
abstract = {Bicaudal C1 (Bicc1) encodes an RNA-binding protein critical for many organ development and epithelial tissue homeostasis. Bicc1 null mutations have been shown to lead to the development of polycystic kidney disease (PKD) and death at an early prenatal stage. To elucidate the tissue-specific functions of Bicc1, we engineered two independent conditional knockout (cKO) mouse lines targeting distinct exonic regions of the gene. The first line was generated using a traditional embryonic stem (ES) cell-based approach, wherein loxP sites were inserted flanking exon 4 (E4), enabling Cre-mediated excision of a functionally essential coding region. The second line was created using CRISPR/Cas9 genome editing, introducing loxP sites around both exon 4 and exon 5 (E4-5) in a double-step zygote injection strategy. Both alleles were validated by PCR genotyping, sequencing, and functional recombination was confirmed via a tissue-specific Cre driver. These independent cKO models provide a robust platform for dissecting the role of Bicc1 in specific tissues and developmental stages, and offer new avenues for studying the mechanistic basis of PKD and other Bicc1-related pathologies.},
}
@article {pmid41508607,
year = {2026},
author = {Wang, F and Lu, S and Zhu, C and Yang, L},
title = {PCdb: A comprehensive plant genome editing database integrating sgRNA efficiency, off-target predictions, and epigenomic landscapes.},
journal = {Plant communications},
volume = {},
number = {},
pages = {101708},
doi = {10.1016/j.xplc.2026.101708},
pmid = {41508607},
issn = {2590-3462},
abstract = {CRISPR/Cas-based genome editing has revolutionized plant biotechnology, enabling precise genomic modifications for crop improvement and functional genomics. The success of these applications hinges on designing single guide RNAs (sgRNAs) that maximize on-target efficiency while minimizing off-target effects. Current resources for sgRNA design and performance evaluation in plants are fragmented and lack integration with genomic and epigenomic context, which influences both editing efficacy and specificity. Here, we present PCdb (Plant CRISPR Database; https://gmo.sjtu.edu.cn/pcdb), a comprehensive plant-focused database by integrating experimentally validated sgRNAs, their annotated genomic contexts, genome-wide off-target predictions, and multi-layered epigenomic annotations. PCdb encompasses 6,172 manually curated editing records from 2,132 publications, covering 4,320 unique sgRNAs and 6,117,424 predicted off-target sites across nine major plant species. Uniquely, PCdb contextualizes potential editing outcomes-both on-target and off-target-within the chromatin landscape by incorporating DNA methylation profiles, chromatin accessibility data, and histone modification patterns. The database features an intuitive web interface supporting flexible queries, interactive visualization tools, and comprehensive analytical modules for both sgRNA efficiency assessment and off-target analysis. A case study reanalysis of Oryza sativa yield-related genes demonstrates PCdb's capability to provide a comprehensive performance profile, evaluating both on-target characteristics and off-target risks within their native epigenomic context. Through systematic analysis of the database, we reveal critical sequence and chromatin features influencing editing outcomes, providing novel insights for improved gene editing efficacy and specificity.},
}
@article {pmid41506049,
year = {2026},
author = {Yaşar, S and Gehrke, F and Capdeville, N and Puchta, H},
title = {Recent progress in plant genome engineering: from large insertions to chromosome number changes.},
journal = {Current opinion in biotechnology},
volume = {97},
number = {},
pages = {103426},
doi = {10.1016/j.copbio.2025.103426},
pmid = {41506049},
issn = {1879-0429},
abstract = {The adaptation of the CRISPR/Cas system as a biotechnological tool has enabled a wide spectrum of targeted genome modifications. Whereas earlier approaches focused on small sequence changes, recent years have seen a shift toward larger-scale alterations. Advances in homology-directed gene targeting now enable efficient, scar-free kilobase insertions, while combining nuclease-deficient Cas effectors with recombinases or transposases allows the integration of much larger sequences. Prime editing further expands this scope, enabling inversions, replacements, and deletions spanning hundreds of kilobases to several megabases. More recently, genome engineering has reached a new stage with chromosome fission and fusion, demonstrating the feasibility of controlled karyotype restructuring. Together, these advances open new opportunities for crop improvement, from establishing reproductive barriers and mimicking evolutionary processes to trait stacking on Plant Artificial Chromosomes.},
}
@article {pmid41506042,
year = {2026},
author = {Jiang, H and Yang, J and Qian, C and Li, A and Liu, Y and Zhang, F and Deng, Y and Duan, J and Lv, X},
title = {Logic-gated CRISPR-Cas12a assay with engineered signal amplification for sensitive multiplexed detection of HCC miRNAs.},
journal = {Biosensors &amp; bioelectronics},
volume = {297},
number = {},
pages = {118376},
doi = {10.1016/j.bios.2026.118376},
pmid = {41506042},
issn = {1873-4235},
abstract = {Rapid, sensitive, and accurate multi-target analysis is of great significance in biomedical detection. Traditional methods, however, often function as a mere "one-pot" collection of individual assays, ignoring the intrinsic relationships among biomarkers. To address this, we established a novel biosensing platform that integrates circle-to-circle AND logic gate circuit with an engineered CRISPR-Cas system for the early screening of hepatocellular carcinoma. In this design, the logic gate processes multiple miRNA inputs (e.g., miRNA 122 and miRNA 223), and only upon co-recognition, generates a unified DNA output. This output then directly activates a CRISPR-Cas12a system, which has been enhanced by a multi-legged crRNA assembled on a DNA tetrahedra and a cube-based framework probe to enhance the trans-cleavage activity and reaction kinetics. This integration converts complex multi-target recognition into a single, amplified detection signal, minimizing systemic interference. Under optimal conditions, the method achieved detection limits as low as 78.88 fM for miRNA 122 and 65.26 fM for miRNA 223, with serum recovery rates of 89.66 %-108.08 %. Clinical validation using 36 samples showed that excellent correlation with RT-qPCR (all R[2] > 0.98) and areas under the ROC curves of 0.8514 and 0.9244, effectively distinguishing liver cancer patients from healthy individuals. Combining high sensitivity, specificity, and clinical applicability, this strategy provides a universal platform for logic-operated multiple biomarkers analysis. Looking forward, integration with microfluidic systems could enable automated, high-throughput testing, further enhancing its utility in point-of-care diagnostics. This approach holds great promise not only for early hepatocellular carcinoma screening but also, with adaptation of the input logic, for the detection of a broad spectrum of cancers and other diseases.},
}
@article {pmid41505099,
year = {2026},
author = {Xu, B and Li, S and Li, Y and Zhao, S and Li, X and Han, J and Wu, D and Li, S and Chen, L and Xie, S and Han, X and Zhao, C},
title = {Enhanced SfaTnpB enables single-base-specific, one-pot nucleic acid detection for high-sensitivity diagnostics.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41505099},
issn = {1362-4962},
support = {2023YFF1001000//National Key Research &amp; Developmental Program of China/ ; SH23YCKY01//Basic Research Project of Yazhouwan National Laboratory/ ; 32202634//Natural Science Foundation of China/ ; //Yazhouwan National Laboratory/ ; },
mesh = {Humans ; Polymorphism, Single Nucleotide ; CRISPR-Cas Systems ; *Transposases/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Papillomavirus Infections/diagnosis/virology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR/Cas12-based nucleic acid detection has revolutionized molecular diagnostics but shows limited single-nucleotide specificity, limited high-fidelity subtype discrimination, and limited compatibility with one-pot assays, restricting its broader clinical application. Here, we report a transposon-associated transposase B (TnpB) ortholog, SfaTnpB, with high trans-cleavage activity, robust single-base mismatch discrimination, and broad temperature tolerance. By stepwise engineering of its guide RNA (ωRNA), we developed an enhanced SfaTnpB (enSfaTnpB) system with markedly improved trans-cleavage efficiency. In combination with a TAM-independent split-activator strategy, this system enables precise detection of single-nucleotide polymorphisms. We further developed TOPIC (TnpB-based One-Pot nucleIC acid detection), a one-pot detection platform coupling enSfaTnpB with recombinase-aided amplification (RAA) or loop-mediated isothermal amplification that enables ultrasensitive detection of human papillomavirus (HPV) subtypes 16 and 18 (∼4 copies/μl) and African swine fever virus DNA (∼3 copies/μl). Finally, RAA-TOPIC accurately detected and genotyped 14 high-risk HPV subtypes with high-fidelity subtype discrimination, showing complete concordance with quantitative real-time PCR-based clinical diagnostics. These findings establish TOPIC as a compact, programmable, and scalable molecular detection tool with broad potential for precision diagnostics and point-of-care testing, particularly in resource-limited settings.},
}
@article {pmid41505097,
year = {2026},
author = {Wang, L and Xiang, X and Yin, G and Shu, H and Wu, Y and Chen, H and Na, R and Gijzen, M and Hou, Y and Dong, S},
title = {Effector gene silencing coordinated by histone methylation and small RNAs enhances host adaptation in a plant pathogen.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41505097},
issn = {1362-4962},
support = {32400164//National Natural Science Foundation of China/ ; 32488302//National Natural Science Foundation of China/ ; 25ZR1401177//Natural Science Foundation of Shanghai/ ; //Shanghai Jiao Tong University/ ; },
mesh = {*Histones/metabolism/genetics ; *Gene Silencing ; *Phytophthora/genetics/pathogenicity ; Plant Diseases/microbiology/genetics/parasitology ; Methylation ; *Host-Pathogen Interactions/genetics ; Epigenesis, Genetic ; CRISPR-Cas Systems ; Virulence/genetics ; *RNA, Small Untranslated/genetics/metabolism ; },
abstract = {Pathogen adaptability driven by epigenetic processes remains poorly understood and poses a significant challenge to sustainable disease management. Histone 3 lysine 27 trimethylation (H3K27me3) and small RNA (sRNA)-mediated silencing of avirulence (Avr) genes are two major strategies that pathogens employ to evade recognition by host resistance (R) proteins. Here, we demonstrate that these two epigenetic mechanisms operate in a coordinated manner to silence Avr genes in the oomycete Phytophthora sojae. CRISPR/Cas9-mediated editing of PsSu(z)12, a core component of the Polycomb repressive complex 2 (PRC2), abolished H3K27me3 deposition at Avr1b and Avr3a, leading to transcriptional reactivation and loss of avirulence. Complementation with PsSu(z)12 restored H3K27me3 and silencing at Avr1b, but not at Avr3a. This prompted sRNA profiling at both loci, revealing differential co-enrichment of sRNA and H3K27me3. Integrated analysis of H3K27me3-enriched chromatin immunoprecipitation, RNA and sRNA sequencing data uncovered a strong locus-specific co-silencing pattern, with 11 out of 12 H3K27me3-regulated arginine-X-leucine-arginine effectors also targeted by sRNAs. Notably, epigenetic variation among field isolates indicated regulatory heterogeneity and plasticity in effector control. Together, our findings establish PsSu(z)12 as a central hub coordinating H3K27me3 and sRNA-mediated effector gene silencing, revealing a dual-layered epigenetic mechanism that enables immune evasion and promotes pathogen adaptation.},
}
@article {pmid41505088,
year = {2026},
author = {Lee-Yow, YC and Valbuena, RC and Richter, CS and Chang, HY and Engreitz, JM},
title = {Junction-targeting designs limit the application of CRISPR-Cas13d in circular RNA perturbation studies.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
doi = {10.1093/nar/gkaf1447},
pmid = {41505088},
issn = {1362-4962},
support = {5T32GM007790/GF/NIH HHS/United States ; DGE-1656518//GRFP/ ; //Stanford Interdisciplinary Graduate Fellowship/ ; R35HG011324/HG/NHGRI NIH HHS/United States ; NNF21SA0072102//Novo Nordisk Foundation/ ; //Lucile Packard Children's Hospital at Stanford University/ ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {RNA, Circular/genetics ; Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA/genetics/metabolism ; K562 Cells ; Gene Knockdown Techniques/methods ; Exons ; },
abstract = {Circular RNAs (circRNAs) are RNA molecules formed through the backsplicing of linear exons. Several thousand have been identified, yet relatively few are functionally characterized due to challenges in distinguishing effects of circular from linear RNA targets. Recently, CRISPR-Cas13 systems have been utilized to directly target unique junctions formed through backsplicing, potentially allowing for selective degradation of circular isoforms. Applying this approach in pooled screens has indeed identified circRNAs proposed to affect viability in several cancer cell lines. However, the design limitations of applying Cas13d to study circRNAs are not fully characterized. Here, we assessed the limitations of Cas13d-mediated circRNA knockdowns by performing essentiality screens on 900 highly expressed circRNAs in K562, an ENCODE tier 1 cell line. We observed consistent off-target knockdown of linear isoforms by certain circRNA-targeting single-guide RNAs (sgRNAs). Re-analysis of existing Cas13d screens in other cell types revealed similar off-target effects. Using machine learning models that predict Cas13d sgRNA efficacy, we further found that most circRNA-targeting sgRNAs are unlikely to induce strong knockdown. After accounting for these design constraints, 0 of 346 circRNAs testable in our screens had detectable effects on proliferation. Our findings highlight key limitations of junction-targeting strategies, with implications for future circRNA perturbation studies.},
}
@article {pmid41505087,
year = {2026},
author = {Di Pede, AC and Bagheri, N and Belforte, E and Palone, A and Rossetti, M and Porchetta, A},
title = {Triplex DNA clamp regulates Cas12a activation for ssDNA and RNA sensing.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41505087},
issn = {1362-4962},
support = {MFAG 2022-ID. 27151//AIRC/ ; //National Recovery and Resilience Plan/ ; //Italian Ministry of University and Research/ ; D53D23009090001//European Union/ ; 2022FPYZ2N//European Union/ ; PNRR M4C2-Investimento 1.4- CN00000041//Italian Ministry of Ministry of University and Research/ ; //Fondazione Umberto Veronesi/ ; },
mesh = {*DNA, Single-Stranded/metabolism/genetics/chemistry ; *CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *RNA/metabolism/genetics/chemistry ; *DNA/chemistry/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {We present a molecular strategy that enables the programmable activation of the CRISPR-Cas12a system in response to triplex DNA formation triggered by single-stranded DNA (ssDNA) or RNA inputs. Our triplex-controlled Cas12a assay leverages the high specificity of clamp-like triplex structures to control a toehold-based strand displacement reaction within a rationally designed DNA hairpin (PAM-Switch). Upon displacement and protospacer adjacent motif (PAM) complementation, the Cas12a ribonucleoprotein (RNP) is activated, initiating trans-cleavage and producing a concentration-dependent fluorescent signal. By decoupling target recognition (via triplex formation) from direct hybridization with the Cas12a-crRNA complex, the assay eliminates the need for target-specific crRNAs. This design also allows multiple detection of distinct nucleic acid (NA) targets using a single Cas12a reaction mix. Through the use of triplex-based clamps, the proposed platform achieves enhanced specificity for single-nucleotide variants and supports the detection of both ssDNA and RNA targets across a broad range of lengths (10-20 nucleotides), addressing key limitations in current Cas12a-based diagnostics and opening new avenues for NA sensing.},
}
@article {pmid41504765,
year = {2026},
author = {Osei, EK and O'Mahony, AK and O'Hea, R and Moriarty, J and O'Doherty, &#xc1; and Wilson, M and Garcia Manzanilla, E and Mahony, J and Kenny, JG},
title = {Genomics of Irish swine-derived Streptococcus suis: population structure, prophages and anti-viral defence mechanisms.},
journal = {Microbial genomics},
volume = {12},
number = {1},
pages = {},
doi = {10.1099/mgen.0.001590},
pmid = {41504765},
issn = {2057-5858},
mesh = {Animals ; *Streptococcus suis/genetics/virology/classification/isolation &amp; purification ; Swine/microbiology ; *Prophages/genetics ; Ireland ; Phylogeny ; *Streptococcal Infections/microbiology/veterinary ; Whole Genome Sequencing ; Genomics ; *Swine Diseases/microbiology ; Multilocus Sequence Typing ; Genome, Bacterial ; Genetic Variation ; Serogroup ; },
abstract = {Streptococcus suis is a major pig pathogen with zoonotic potential, posing an occupational risk to farmers and meat handlers. We characterized 110 S. suis strains from diseased pigs in Ireland (2005-2022) using whole-genome sequencing to investigate population structure and phage-host dynamics. We identified 15 distinct serotypes, with serotypes 9 and 2 being the most dominant. In silico multi-locus sequence typing revealed high diversity within the collection, identifying several sequence types (STs), including 26 novel STs. Investigation of strain-level genomic clustering using PopPUNK against global S. suis genomes showed that the Irish isolates were phylogenetically dispersed across the broader global S. suis population rather than clustering in a single clonal group. The majority of Irish isolates fall within the ten established pathogenic lineages, including the highly virulent zoonotic lineage 1. A locally persistent clonal lineage was identified among Irish isolates, showing minimal genetic variation over a decade.Prophage analysis revealed novel viral taxa that were interspersed among known streptococcal phages, rather than clustering distinctly. Restriction-modification systems were the predominant anti-viral defence systems identified across genomes. CRISPR-Cas systems were present in limited strains but showed substantial targeting bias toward full-length prophages, indicating ongoing phage pressure. CRISPR spacers matched non-S. suis streptococcal phages, and phylogenomic analysis revealed that Vansinderenvirus phages clustered with S. suis rather than other Streptococcus thermophilus phages, suggesting evolutionary connections between phage lineages infecting different streptococci.This study presents the first comprehensive genomic characterization of S. suis in Ireland, revealing a diverse population with significant implications for animal and human health.},
}
@article {pmid41503690,
year = {2026},
author = {Ma, N and Zhang, M and Ghonaim, AH and Zhou, P and Wang, C and Zhou, J and Guo, G and Lebbink, RJ and Bosch, BJ and Zhu, H and Li, W and He, Q},
title = {The essential role of heparan sulfate in the entry of PDCoV and other porcine coronaviruses.},
journal = {Virulence},
volume = {17},
number = {1},
pages = {2614154},
doi = {10.1080/21505594.2026.2614154},
pmid = {41503690},
issn = {2150-5608},
mesh = {Animals ; *Heparitin Sulfate/metabolism ; Swine ; *Deltacoronavirus/physiology ; *Virus Internalization/drug effects ; Swine Diseases/virology ; Humans ; *Porcine epidemic diarrhea virus/physiology ; *Coronavirus Infections/virology/veterinary ; Cell Line ; CRISPR-Cas Systems ; Receptors, Virus/metabolism ; Transmissible gastroenteritis virus/physiology ; Alphacoronavirus ; },
abstract = {Porcine enteric coronaviruses, including porcine deltacoronavirus (PDCoV), porcine epidemic diarrhea virus (PEDV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and transmissible gastroenteritis coronavirus (TGEV), can cause acute diarrhea, vomiting, dehydration, and high mortality in suckling piglets. Recent studies revealing human PDCoV infections and the potential of SADS-CoV to penetrate human cell lines have heightened apprehensions about the zoonotic transmission risks of these viruses. While heparan sulfate (HS) serves as a receptor in PDCoV binding, the key host genes involved in HS biogenesis and the specific molecular mechanisms underlying this process have not been fully examined. Enzymes involved in HS biosynthesis, including SLC35B2, EXT1, and NDST1, were identified as critical host factors via the use of CRISPR-Cas9 knockout cells. Moreover, inhibition assays using heparin sodium, a competitive HS mimic, demonstrated dose-dependent reductions in PDCoV infection in vitro. Additionally, mitoxantrone, an HS-binding drug, reduced PDCoV infection. Furthermore, HS was confirmed to facilitate the entry of other porcine enteric coronaviruses (SeCoVs), including PEDV, SADS-CoV, and TGEV, underscoring the conserved role of HS in CoV pathogenesis. These insights contribute to the understanding of porcine coronavirus-host interactions and support the development of innovative antiviral interventions.},
}
@article {pmid41453683,
year = {2026},
author = {Akhter, R and Kitab, B and Kayesh, MEH and Shimizu, R and Onuma, H and Yamamoto, N and Ogawa, S and Sugiyama, M and Tanaka, Y and Sato, Y and Kohara, M and Tsukiyama-Kohara, K},
title = {Optimization of lipid nanoparticles loaded with ribonucleoprotein-oligonucleotide complexes for in vivo delivery of a CRISPR/Cas9 system targeting hepatitis B virus.},
journal = {Virus research},
volume = {363},
number = {},
pages = {199682},
doi = {10.1016/j.virusres.2025.199682},
pmid = {41453683},
issn = {1872-7492},
mesh = {*Hepatitis B virus/genetics/drug effects/physiology ; *CRISPR-Cas Systems ; Animals ; *Nanoparticles/chemistry/administration &amp; dosage ; Mice ; Humans ; *Ribonucleoproteins/administration &amp; dosage/genetics/chemistry ; Virus Replication/drug effects ; *Lipids/chemistry ; Genetic Therapy/methods ; *Hepatitis B, Chronic/therapy/virology ; RNA, Guide, CRISPR-Cas Systems/genetics/administration &amp; dosage ; CRISPR-Associated Protein 9 ; Liposomes ; },
abstract = {Patients with chronic hepatitis B virus (HBV) infection may benefit from clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based gene therapy. We previously identified a guide RNA (WJ11) that suppressed HBV replication in vitro and in vivo; however, we were unable to achieve delivery at clinically feasible doses in vivo using an adeno-associated virus (AAV) vector. Lipid nanoparticle (LNP)-based WJ11/Cas9 ribonucleoprotein-oligonucleotide complex delivery suppressed HBV replication by 2-3-fold more than did AAV-based delivery. In the present study, we investigated the HBV replication-suppressive effects of LNP/WJ11/Cas9 complexes after intravenous administration to persistently HBV genotype C-infected humanized chimeric mice. CL4H6 (ionizable lipid) LNPs were selected as the first candidate for WJ11/Cas9 delivery based on their reported high encapsulation efficiency; however, no significant anti-HBV effect was noted in serum or hepatic tissue. The ionizable lipid candidate CL4F11_ε-3 improved absolute serum HBV values to a certain degree but had no significant effect on hepatic HBV DNA or covalently closed circular (ccc)DNA levels. CL4F11_ζ-2 LNP/WJ11/Cas9, a new complex prepared through structural optimization of the ionizable lipid and heat treatment of WJ11, showed suppressive effect for serum viral load along with a reduction of hepatic HBV DNA, HBV cccDNA, HBsAg, and HBcrAg levels when compared with controls. Therefore, LNP-based delivery of this CRISPR/Cas9 formula holds promise for the treatment of chronic HBV infection.},
}
@article {pmid41423170,
year = {2026},
author = {Sheri, V and Verma, PK and Lekkala, S and Janga, MR},
title = {Application of digital PCR and CRISPR/Cas13a-based fluorescent assay for accurate and on-site detection of cotton leafroll dwarf virus.},
journal = {Journal of virological methods},
volume = {341},
number = {},
pages = {115332},
doi = {10.1016/j.jviromet.2025.115332},
pmid = {41423170},
issn = {1879-0984},
mesh = {*Plant Diseases/virology ; *Gossypium/virology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Plant Leaves/virology ; Viral Load ; *Closteroviridae/isolation &amp; purification/genetics ; RNA, Viral/genetics ; Texas ; *Polymerase Chain Reaction/methods ; },
abstract = {Cotton leafroll dwarf virus (CLRDV) is an emerging viral pathogen posing a significant threat to cotton production in the United States. Early and accurate detection is critical for effective disease surveillance and management. Although traditional reverse transcription PCR (RT-PCR) is commonly employed for CLRDV diagnosis, it suffers from limitations in sensitivity, quantification accuracy, and involves labor-intensive workflows. In this study, we evaluated two advanced molecular diagnostic approaches for detecting CLRDV, digital PCR (dPCR) and CRISPR/Cas13a-based fluorescent assay. Symptomatic cotton leaf samples from Lubbock and Brownfield, Texas, were screened and confirmed positive by RT-PCR. Digital PCR analysis enabled absolute quantification of viral load, revealing significantly higher titers in Brownfield (F2) samples and offered improved sensitivity over RT-PCR, particularly in samples with low viral loads. However, dPCR is resource-intensive and requires specialized instrumentation. To address the need for rapid, field-deployable diagnostics, we developed a CRISPR/Cas13a-based assay targeting the conserved ORF3, ORF2, and ORF3a regions of the CLRDV genome. Adapted from the SHERLOCK platform, this fluorescence-based assay uses collateral cleavage activity of Cas13a to enable highly specific visual detection. While the assay successfully enabled direct detection from crude leaf extracts without RNA purification, the sensitivity analysis was conducted using purified, in vitro transcribed RNA. Fluorescence signals were reliably observed with as few as 50 RNA copies, defining the assay's practical limit of detection. While dPCR is optimal for quantitative laboratory analysis, the CRISPR/Cas13a-based assay offers a rapid, sensitive, and cost-effective tool for field-level detection. Together, these complementary tools enhance CLRDV surveillance and management in cotton.},
}
@article {pmid40879055,
year = {2026},
author = {Wang, M and Liao, Q and Bai, S and Liu, X and Peng, Y and Liu, P and Lu, H and Zhu, JK and Zeng, C},
title = {Co-Delivery of Multiple RNAs via Lipid Nanoparticles Enables Precise Gene Editing of CAR-T Cells.},
journal = {Advanced healthcare materials},
volume = {15},
number = {1},
pages = {e01475},
doi = {10.1002/adhm.202501475},
pmid = {40879055},
issn = {2192-2659},
support = {KJZD20240903102703005//Shenzhen Science and Technology Program/ ; 2024B1111130001//Guangdong S&amp;T Program/ ; 82402147//National Natural Science Foundation of China/ ; NCTIB2023XB01002//"Open Competition to Select the Best Candidates" Technology Breakthrough Project for Cell Therapy of NCTIB/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Nanoparticles/chemistry ; Animals ; Mice ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Lipids/chemistry ; *Receptors, Chimeric Antigen/genetics/metabolism ; *T-Lymphocytes/metabolism ; *Immunotherapy, Adoptive/methods ; Programmed Cell Death 1 Receptor/genetics/metabolism ; *RNA ; Gene Transfer Techniques ; Liposomes ; },
abstract = {Over the past decade, CAR-T cell therapy has achieved remarkable success in treating hematological malignancies. However, traditional CAR-T cell engineering employs viral vectors, which has several limitations. Additionally, the immunosuppressive tumor microenvironment, particularly mediated by the PD-1/PD-L1 pathway, significantly restricts CAR-T cell efficacy. CRISPR/Cas9-mediated PD-1 knockout can enhance CAR-T cell anti-tumor activity, but traditional electroporation (EP) method often damages T cells. Herein, a novel lipid nanoparticles (LNPs)-mediated delivery technology are introduced to engineer CAR-T cells. The LNPs platform enables the simultaneous expression of CAR cassette and CRISPR/Cas9 gene editor in T cells via co-delivery of multiplex RNAs (CD19 CAR mRNA+Cas9 mRNA+sgRNA targeting PD-1). Importantly, LNPs exhibit higher transfection efficiency and superior cell viability compared to traditional electroporation method. The engineered CAR-T cells with PD-1 knockout, which express anti-CD19 CAR, can specifically kill CD19+ Nalm-6 tumor cells in vitro and display enhanced anti-tumor activity in vivo. Furthermore, LNPs-mediated co-delivery of Cas9 mRNA and sgRNAs targeting PD-1, TRAC, and B2M enables triple-knockout of T cells with high editing efficiencies (76% for PD-1, 86% for TRAC, and 80% for B2M), highlighting the ability for multiplex gene editing. This LNP-mediated delivery strategy has great potentials for the development of safer and more efficacious CAR-T cells.},
}
@article {pmid41503845,
year = {2026},
author = {Li, MF and Zubair, A and Wdidi, S and He, S},
title = {Disrupting Viral Persistence: CRISPR/Cas9-Based Strategies for Hepatitis B and C Treatment, and Challenges.},
journal = {Journal of cellular and molecular medicine},
volume = {30},
number = {1},
pages = {e70986},
pmid = {41503845},
issn = {1582-4934},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Hepatitis B virus/genetics ; *Hepatitis B/therapy/virology/genetics ; Gene Editing/methods ; *Hepatitis C/therapy/virology/genetics ; *Hepacivirus/genetics ; Animals ; Antiviral Agents/therapeutic use/pharmacology ; Genome, Viral ; DNA, Circular/genetics ; DNA, Viral/genetics ; Genetic Therapy ; Virus Replication/genetics ; },
abstract = {Hepatitis B and C viruses (HBV and HCV) remain among the leading causes of liver disease worldwide. Current antiviral drugs, such as nucleotide analogues (NAs), can reduce the replication of new HBV and HCV infections but cannot completely eliminate chronic infections. This is primarily because a stable form of viral DNA, known as covalently closed circular DNA (cccDNA), persists in liver cells and continues to sustain the infection. In recent years, the CRISPR/Cas9 gene-editing system has emerged as a powerful tool for precisely cutting and inactivating specific DNA sequences. Due to its efficiency and ease of use, researchers have applied CRISPR/Cas9 in numerous studies to directly target and disrupt the HBV genome, demonstrating promising antiviral effects in both cell cultures and animal models. Targeting multiple sites within the HBV genome has been shown to further enhance its effectiveness, paving the way for potential combination therapies aimed at disabling both cccDNA and HBV and HCV DNA integrated into the host genome. Despite its potential, CRISPR/Cas9 still faces significant challenges before clinical application, most notably the risk of off-target effects-unintended cleavage of non-target DNA sequences-and the difficulty of delivering the system efficiently into liver cells in vivo. Future progress will depend on improving the tool's precision, efficiency, flexibility and delivery methods. In this review, we explore recent advances in designing guide RNAs (gRNAs) for targeting HBV and HCV, as well as the delivery systems used to transport CRISPR/Cas9 into cells. We also discuss the remaining challenges and potential strategies for advancing CRISPR/Cas9 from the laboratory toward a viable clinical cure for HBV and HCV.},
}
@article {pmid41502163,
year = {2026},
author = {Manojkumar, C and Limbola, M and Paul, S and Thangadurai, K and Rajendran, KV and Roy, A and Mandal, B and Jeena, K and Bedekar, MK},
title = {CRISPR-Cas12a-based lateral flow detection of white spot syndrome virus: a dual-target approach for detection of early and latent infection.},
journal = {Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc},
volume = {},
number = {},
pages = {10406387251399147},
doi = {10.1177/10406387251399147},
pmid = {41502163},
issn = {1943-4936},
abstract = {White spot syndrome virus (WSSV; family Nimaviridae; taxon species White spot syndrome virus) is a major viral pathogen that poses a significant threat to the global shrimp industry, with early detection being the most effective strategy for disease control. We developed a CRISPR-Cas12a-based dual-target detection assay for WSSV, specifically targeting the VP28 gene (gene product is a major envelope protein) and WSSV366 (a latency-associated gene), optimized using Indian WSSV isolates. Our CRISPR RNAs for both targets had high efficiency, and we evaluated the assay using fluorescence-based and lateral flow strip (LFS) endpoint detection. In fluorescence assays, the Cr-WSSV assay (without recombinase polymerase amplification, RPA) detected WSSV at 3 × 10[5] copies/μL; RPA integration significantly enhanced sensitivity, allowing detection at as low as 20 and 200 copies for VP28 and WSSV366, respectively, with 100% specificity. We developed a CRISPR-based LFS assay with optimized FAM-biotin reporter concentrations of 100 nM and 250 nM, yielding robust and reproducible results for improved field applicability. Performance evaluation confirmed lack of cross-reactivity to other WOAH-listed shrimp pathogens, while maintaining detection limits of 20 and 200 copies of VP28 and WSSV366. Clinical validation further demonstrated that the RPA-Cr-WSSV-LFS assay successfully detected WSSV366 even in VP28-negative samples, underscoring the importance of detecting WSSV366 in latent infections. Our rapid, cost-effective, and highly sensitive CRISPR-Cas-based assay enhances WSSV surveillance and biosecurity in shrimp aquaculture by incorporating structural and latency-associated gene markers, making it a promising alternative to conventional molecular testing.},
}
@article {pmid41501880,
year = {2026},
author = {Zhu, M and Wu, Y and Ou, H and Liu, X and Wang, Y and Liu, X and Zou, C and Yang, G and Du, M and Yu, D and Zheng, D and He, L and Zhang, K and Zhang, W and Wang, S and Qin, H and Hao, Q and He, Y and Lin, W and Zhang, Y and Gu, J and Li, M and Qin, W and Cao, Z},
title = {Targeting TRIM25 as a therapeutic strategy to enhance ferroptosis in glioblastoma cells.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {20},
pmid = {41501880},
issn = {1477-3155},
support = {01-SWKJYCJJ17//the National Defense Biotechnology Fund for Outstanding Young Talents/ ; 2025GTKP008//the National Key Laboratory of Oncology/ ; },
mesh = {*Ferroptosis/drug effects ; Humans ; *Glioblastoma/metabolism/pathology/drug therapy ; Animals ; Cell Line, Tumor ; *Tripartite Motif Proteins/metabolism/genetics ; *Ubiquitin-Protein Ligases/metabolism/genetics ; Mice ; *Brain Neoplasms/metabolism/pathology ; CRISPR-Cas Systems ; Gene Editing ; Mitochondria/metabolism ; Nanoparticles/chemistry ; Mice, Nude ; Ubiquitination ; },
abstract = {BACKGROUND: Glioblastoma (GBM) is the most common malignant brain tumor with a dismal prognosis (< 7% 5-year survival) under current first-line treatment. While inducing programmed cell death (PCD) is a promising antitumor strategy, its effectiveness in GBM remains controversial. Ferroptosis emerged as the most enriched PCD process and was highly correlated with GBM malignant progression.<br><br>METHODS: We performed a CRISPR-Cas9 loss-of-function screen to identify critical ferroptosis contributors. Mechanistic studies involved assessing mitochondrial function and morphology. Protein interaction and degradation pathways were investigated using immunoprecipitation and ubiquitination assays. We developed a blood-brain-barrier-penetrating genome editing delivery system, Angiopep-2-modified nanoparticles with disulfide bonds (ANPSS), loaded with Cas9/sgRNA complexes.<br><br>RESULTS: Voltage-dependent anion channel 2 (VDAC2) was identified as a critical contributor to ferroptosis. VDAC2 overexpression induced mitochondrial dysfunction and characteristic ferroptotic mitochondrial morphology. The E3 ubiquitin ligase TRIM25 was identified as a key suppressor of VDAC2, directly interacting with it and inducing its K48-linked polyubiquitination and subsequent proteasomal degradation. In vivo, the ANPSS(sgTRIM25) system effectively targeted GBM cells, significantly promoted ferroptosis, and inhibited GBM progression.<br><br>CONCLUSIONS: Our findings demonstrate that TRIM25 is a critical negative regulator of VDAC2-dependent ferroptosis in GBM. Targeting TRIM25 using the ANPSS(sgTRIM25) genome editing system effectively overcomes ferroptosis resistance and suppresses tumor growth, representing a viable therapeutic approach for GBM.},
}
@article {pmid41501459,
year = {2026},
author = {Dmytrenko, O and Yuan, B and Crosby, KT and Krebel, M and Chen, X and Nowak, JS and Chramiec-Głąbik, A and Filani, B and Gribling-Burrer, AS and van der Toorn, W and von Kleist, M and Achmedov, T and Smyth, RP and Glatt, S and Bravo, JPK and Heinz, DW and Jackson, RN and Beisel, CL},
title = {RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41501459},
issn = {1476-4687},
abstract = {In all domains of life, tRNAs mediate the transfer of genetic information from mRNAs to proteins. As their depletion suppresses translation and, consequently, viral replication, tRNAs represent long-standing and increasingly recognized targets of innate immunity[1-5]. Here we report Cas12a3 effector nucleases from type V CRISPR-Cas adaptive immune systems in bacteria that preferentially cleave tRNAs after recognition of target RNA. Cas12a3 orthologues belong to one of two previously unreported nuclease clades that exhibit RNA-mediated cleavage of non-target RNA, and are distinct from all other known type V systems. Through cell-based and biochemical assays and direct RNA sequencing, we demonstrate that recognition of a complementary target RNA by the CRISPR RNA triggers Cas12a3 to cleave the conserved 5'-CCA-3' tail of diverse tRNAs to drive growth arrest and anti-phage defence. Cryogenic electron microscopy structures further revealed a distinct tRNA-loading domain that positions the tRNA tail in the RuvC active site of the nuclease. By designing synthetic reporters that mimic the tRNA acceptor stem and tail, we expanded the capacity of current CRISPR-based diagnostics for multiplexed RNA detection. Overall, these findings reveal widespread tRNA inactivation as a previously unrecognized CRISPR-based immune strategy that broadens the application space of the existing CRISPR toolbox.},
}
@article {pmid41500141,
year = {2026},
author = {Zheng, S and Tie, H and Chai, S and Wang, M and Wang, S and Zeng, YY and Wu, G and Zhang, TG},
title = {Molecular mechanisms and biotechnological advances in herbicide resistance: Insights into the development of herbicide-tolerant crops.},
journal = {Journal of plant physiology},
volume = {317},
number = {},
pages = {154690},
doi = {10.1016/j.jplph.2026.154690},
pmid = {41500141},
issn = {1618-1328},
abstract = {Herbicides play a pivotal role in modern agriculture by controlling weed populations and safeguarding crop yields. However, the long-term and extensive use of herbicides has accelerated the evolution of herbicide-resistant weeds, thereby diminishing their efficacy and posing a serious threat to global food security. Recent advances in molecular biology and plant biotechnology have greatly expanded our understanding of herbicide resistance mechanisms and enabled the development of crops with enhanced herbicide resistance. Herbicide resistance genes function primarily by encoding detoxifying enzymes, modifying herbicide target sites, or activating specific metabolic pathways that mitigate herbicidal toxicity. Emerging genetic tools, including transgenic approaches and CRISPR/Cas-mediated genome editing, have further facilitated the precise introduction of resistance traits into major crops. It is noteworthy that this review offers novel insights into the latest CRISPR/Cas applications, including base editing and prime editing for developing novel, non-transgenic herbicide-resistant crops. Furthermore, it provides a systematic overview of advanced strategies for engineering multi-gene stacking traits to combat complex or evolving weed resistance. This review integrates recent progress in elucidating the molecular targets of herbicides and the underlying resistance mechanisms, and highlights the potential of modern biotechnological strategies for engineering herbicide-resistant crops to promote sustainable and environmentally responsible weed management.},
}
@article {pmid41499404,
year = {2026},
author = {Wan, X and Cohen, SM and Yu, Y and Hoan Le, H and Park, H and Groaz, A and Moreno, R and Tan, M and Schneider, J and Gronquist, MR and Shinya, R and Schroeder, FC and Sternberg, PW},
title = {O-acyltransferase genes involved in the production of volatile sex pheromones in Caenorhabditis elegans.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {2},
pages = {e2524778123},
doi = {10.1073/pnas.2524778123},
pmid = {41499404},
issn = {1091-6490},
support = {R24OD023041//HHS | NIH (NIH)/ ; DGE 1745301//NSF | NSF Graduate Research Fellowship Program (GRFP)/ ; 00000//Tianqiao and Chrissy Chen Institute for Neuroscience/ ; 00000//Chuck Lorre Research Scholars Program/ ; },
mesh = {Animals ; *Sex Attractants/biosynthesis/metabolism/genetics ; *Caenorhabditis elegans/genetics/metabolism/enzymology ; Male ; *Acyltransferases/genetics/metabolism ; Female ; *Caenorhabditis elegans Proteins/genetics/metabolism ; Gas Chromatography-Mass Spectrometry ; CRISPR-Cas Systems ; Sexual Behavior, Animal ; },
abstract = {Gene family expansions are critical for functional diversification, yet the contributions of paralogs to metabolic pathways are often unclear. In Caenorhabditis, the expanded O-acyltransferase (OAC) family-enzymes that transfer acyl groups to hydroxylated substrates-remains poorly characterized despite having been implicated in lipid metabolism. Using CRISPR-Cas9 mutagenesis, behavioral assays, gas chromatographic-mass spectral (GC-MS) analyses, and metabolomics, we systematically analyzed 59 OAC-family protein-coding genes to define their roles in regulating signaling molecules. We found that four adjacent paralogs (oac-13, oac-16, oac-25, and oac-28) on chromosome I are required for synthesizing volatile sex pheromones-airborne signals critical for male mate-searching. Specifically, oac-13 and oac-16 are necessary for producing both major pheromone components, while the identical tandem paralogs oac-25 and oac-28 regulate the production of the later-eluting component in gas chromatography. Disruption of these genes reduced production of key pheromone components and impaired male attraction. Metabolomics revealed that oac-16 and other OACs also modulate the synthesis and secretion of nonvolatile ascaroside pheromones, indicating dual roles in chemical signaling. This work uncovers functional specialization within an expanded gene family, illustrating how redundancy and divergence enable adaptive evolution of communication systems.},
}
@article {pmid41498982,
year = {2026},
author = {Mangal, H and Mathur, S and Kumar, S and Chaurasia, A and Ranjan, R},
title = {Role of CRISPR in bioremediation of heavy metal(loid): a breakthrough in environmental biotechnology.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {1},
pages = {34},
pmid = {41498982},
issn = {1573-0972},
mesh = {*Metals, Heavy/metabolism ; *Biodegradation, Environmental ; *CRISPR-Cas Systems ; *Biotechnology/methods ; Gene Editing/methods ; Bacteria/genetics/metabolism ; Biosensing Techniques ; Plants/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {A number of technological advancements have made bioremediation an emerging and innovative technology, including its economic viability, increased competence, and natural environment friendliness. The efficiency, scalability, and specificity of conventional physical, chemical, and biological remediation techniques are still limited, despite their partial success. Recent developments in CRISPR-based genome engineering have made it possible to precisely manipulate metal transporters, detoxification enzymes, and stress-response pathways in microorganisms and plants, opening up new possibilities to improve bioremediation. This review offers a thorough and integrated examination of enzyme engineering, biosensing systems, microbial bioremediation, and CRISPR-enabled phytoremediation. This work is novel because it presents a unified roadmap for next-generation bioremediation technologies by integrating CRISPR editing with multi-omics, synthetic biology, and emerging CRISPR-based biosensors. We also go over ecological risks, current difficulties, legal issues, and potential field deployment scenarios in the future. These revelations collectively demonstrate the revolutionary potential of CRISPR in creating highly effective, sustainable, and scalable remedies for heavy metal pollution.},
}
@article {pmid41498549,
year = {2026},
author = {Gopalaswamy, R and Subbian, S},
title = {The power of resistance: mechanisms of antimicrobial resistance in Mycobacterium tuberculosis and its impact on tuberculosis management.},
journal = {Clinical microbiology reviews},
volume = {},
number = {},
pages = {e0019425},
doi = {10.1128/cmr.00194-25},
pmid = {41498549},
issn = {1098-6618},
abstract = {SUMMARYThe global resurgence of drug-resistant tuberculosis (DR-TB) presents a formidable challenge to public health, driven by a complex interplay of mycobacterial evolution, dynamics and outcomes of host-pathogen interactions and systemic gaps in diagnosis and treatment strategies. This comprehensive review delineates the multifactorial basis of antimicrobial resistance (AMR) in Mycobacterium tuberculosis (Mtb), integrating molecular, immunological, and pharmacological perspectives to inform next-generation strategies for effective TB control. We reconceptualize TB as a dynamic clinical spectrum-ranging from asymptomatic infection to overt disease-shaped by granuloma biology and bacterial adaptation. This spectrum underpins both diagnostic ambiguity and therapeutic failure, particularly in the context of phenotypic drug tolerance/resistance to current anti-TB drugs. We discuss Mtb's intrinsic and extrinsic resistance mechanisms, including the lipid-rich cell envelope, efflux systems, and enzymatic drug modification, which are compounded by acquired mutations that disrupt drug activation, alter targets, and confer cross-resistance. These adaptations are further potentiated by granuloma-induced pharmacokinetic heterogeneity and host-induced metabolic quiescence. We highlight the emerging role of therapeutic drug monitoring and pharmacokinetic/pharmacodynamic modeling in optimizing individualized therapy, particularly for novel regimens incorporating bedaquiline, pretomanid, and linezolid. Moreover, we underscore the diagnostic limitations in detecting heteroresistance and early-stage disease, advocating for expanded deployment of advanced and targeted molecular diagnostic modalities. Finally, we propose a paradigm shift toward integrated, precision-based TB management, leveraging host-directed therapies, biofilm-disrupting agents, and real-time pharmacokinetics-guided dosing to preempt resistance emergence and improve clinical outcomes. This review provides a translational framework for addressing the biological and operational complexities of DR-TB in the era of AMR.},
}
@article {pmid41475346,
year = {2026},
author = {Behera, AK and Kim, JJ and Kordale, S and Pekovic, F and Damodaran, AP and Kumari, B and Vidak, S and Dickson, E and Xiao, MS and Duncan, G and Andresson, T and Misteli, T and Valkov, E and Gonatopoulos-Pournatzis, T},
title = {RNA-coupled CRISPR screens reveal ZNF207 as a regulator of LMNA aberrant splicing in progeria.},
journal = {Molecular cell},
volume = {86},
number = {1},
pages = {41-59.e15},
doi = {10.1016/j.molcel.2025.12.003},
pmid = {41475346},
issn = {1097-4164},
mesh = {Humans ; *Progeria/genetics/metabolism/pathology ; *Lamin Type A/genetics/metabolism ; *Alternative Splicing ; *CRISPR-Cas Systems ; Ribonucleoprotein, U1 Small Nuclear/metabolism/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; RNA Precursors/genetics/metabolism ; Zinc Fingers ; },
abstract = {Despite progress in understanding pre-mRNA splicing, the regulatory mechanisms controlling most alternative splicing events remain unclear. We developed CRASP-seq (CRISPR-based identification of regulators of alternative splicing with phenotypic sequencing), a method that integrates pooled CRISPR-based genetic perturbations with deep sequencing of splicing reporters, to quantitatively assess the impact of all human genes on alternative splicing from a single RNA sample. CRASP-seq identified both known and untested regulators, enriched for proteins involved in RNA splicing and metabolism. As a proof-of-concept, CRASP-seq analysis of the LMNA cryptic splicing event linked to progeria uncovered ZNF207, primarily known for mitotic spindle assembly, as a regulator of progerin splicing. ZNF207 depletion enhances canonical LMNA splicing and decreases progerin protein levels in patient-derived cells. We further show that ZNF207's zinc-finger domain broadly impacts alternative splicing through direct interactions with U1 small nuclear ribonucleoprotein (snRNP) components. These findings position ZNF207 as a U1 snRNP auxiliary factor and demonstrate the power of CRASP-seq to uncover key regulators and domains of alternative splicing.},
}
@article {pmid41421338,
year = {2026},
author = {Du, R and Flynn, MJ and Mahe, K and Honsa, M and Gu, B and Li, D and McGeary, SE and Gradinaru, V and Jungmann, R and Elowitz, MB},
title = {miRNA modules for precise, tunable control of gene expression.},
journal = {Molecular cell},
volume = {86},
number = {1},
pages = {194-212.e7},
doi = {10.1016/j.molcel.2025.11.028},
pmid = {41421338},
issn = {1097-4164},
mesh = {Animals ; *MicroRNAs/genetics/metabolism ; Mice ; Humans ; Dependovirus/genetics ; Transgenes ; *Gene Expression Regulation ; Gene Editing/methods ; Neurons/metabolism ; CRISPR-Cas Systems ; Gene Dosage ; HEK293 Cells ; },
abstract = {Accurate control of transgene expression is important for research and therapy but is challenging to achieve in most settings. MicroRNA (miRNA)-based regulatory circuits can be incorporated within transgenes for improved control. However, the design principles, performance limits, and applications of these circuits in research and biotechnology have not been systematically determined. Here, combining modeling and experiments, we introduce miRNA-based circuit modules, termed "dosage invariant miRNA-mediated expression regulators" (DIMMERs), that establish precise, tunable control of transgene expression across diverse cell types to facilitate imaging, editing, and gene therapy. The circuits use multivalent miRNA regulatory interactions to achieve nearly uniform, tunable protein expression over two orders of magnitude variation in gene dosage. They function across diverse cell types and can be multiplexed for the independent regulation of multiple genes. DIMMERs reduce off-target CRISPR base editing, improve single-molecule imaging, and allow live tracking of adeno-associated virus (AAV)-delivered transgene expression in mouse cortical neurons. DIMMERs thus enable accurate regulation for research and biotechnology applications.},
}
@article {pmid41369550,
year = {2026},
author = {Smith, DJ},
title = {Complementary human gene interaction maps from radiation hybrids and CRISPRi.},
journal = {Physiological genomics},
volume = {58},
number = {1},
pages = {42-57},
doi = {10.1152/physiolgenomics.00075.2025},
pmid = {41369550},
issn = {1531-2267},
support = {C25CR8562//University of California Cancer Research Coordinating Committee/ ; //Norton Simon Research Foundation/ ; },
mesh = {Humans ; Genome-Wide Association Study ; *Gene Regulatory Networks/genetics ; *CRISPR-Cas Systems/genetics ; *Radiation Hybrid Mapping/methods ; *Protein Interaction Maps/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Alleles ; },
abstract = {The only comprehensive human genetic interaction map was constructed using increased gene copy numbers in radiation hybrid (RH) cells. Recently, a second map restricted to essential genes was created using CRISPR interference (CRISPRi)-induced loss-of-function alleles. Here, the two maps are compared to understand their similarities and differences. Both maps showed significant overlap with protein-protein interaction databases and identified a shared set of interacting genes, although the specific gene pairs differed between approaches. Notably, the RH map exhibited strong overlap with genome-wide association study (GWAS) networks, whereas the CRISPRi map did not. These findings demonstrate how gain- and loss-of-function alleles reveal distinct yet complementary genetic interaction landscapes.NEW &amp; NOTEWORTHY This study compared two mammalian genetic interaction networks for cell growth: the radiation hybrid (RH) network used extra gene copies and the CRISPRi network used partial gene suppression. Both networks overlapped with protein-protein interaction data and identified common interacting genes, yet specific gene pair interactions differed dramatically. Only the RH network predicted genome-wide association study (GWAS) networks. As the first comparison of large-scale mammalian genetic interaction networks, this work reveals how gain- and loss-of-function variants capture diverse biological perspectives.},
}
@article {pmid41355773,
year = {2026},
author = {Tian, S and Yao, L and Gong, F and Li, Y and Zhao, Y and Yang, Y},
title = {Rapid and sensitive detection of circulating tumor DNA via a CRISPR/Cas12a-based catalytic hairpin assembly.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {1},
pages = {115-123},
doi = {10.1039/d5ay01624j},
pmid = {41355773},
issn = {1759-9679},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Circulating Tumor DNA/blood/genetics ; Limit of Detection ; *Endodeoxyribonucleases/metabolism/genetics ; Biomarkers, Tumor/blood/genetics ; Biosensing Techniques/methods ; Neoplasms/blood/diagnosis/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Cancer is one of the major diseases that endanger the human health. Circulating tumor DNA (ctDNA) is an ideal biomarker for the real-time monitoring of cancer. In the present work, a rapid and sensitive assay coupled with CRISPR/Cas12a and CHA (Cas12a-CHA) was constructed for the detection of ctDNA. We designed and prepared a trigger, which was the substrate of Cas12a. On the addition of ctDNA, crRNA-guided ctDNA activated the trans-endonuclease activity of Cas12a. After being activated, Cas12a exhibited a high trans-cleavage activity on the trigger, which resulted in a decrease in fluorescence. Owing to this design, the Cas12a-CHA assay enabled the sensitive detection of ctDNA with a linear range of 10 fM to 50 pM. Furthermore, a limit-of-detection of 5.8 fM was achieved within 40 min. Besides, the proposed assay had an excellent base mismatch recognition ability and worked well in human serum samples. Conclusively, this detection platform holds significant potential for application in early cancer diagnosis.},
}
@article {pmid41345100,
year = {2025},
author = {Bai, M and Zhang, J and Lin, W and Zhou, Y and Jiang, M and Wu, H and Peng, C and Lin, J and He, F and Kuang, H and Guan, Y},
title = {A flanking-nicks prime editor (FLICK-PE) system to boost prime editing in dicots.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {337},
pmid = {41345100},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; *Glycine max/genetics/drug effects ; *Nicotiana/genetics ; Plants, Genetically Modified/genetics ; Glyphosate ; Glycine/analogs &amp; derivatives/pharmacology ; CRISPR-Cas Systems/genetics ; Herbicide Resistance/genetics ; 3-Phosphoshikimate 1-Carboxyvinyltransferase/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Plant ; },
abstract = {Prime editing (PE) enables precise genome modifications to mammalian cells and monocot staple crops, but remains relatively challenging in dicot plants. Here, we develop a Flanking-Nicks Prime Editor (FLICK-PE) system that boosts editing efficiency in soybean and tobacco. We show that optimization for PE by adding a nicking sgRNA could dramatically enhance intended-editing efficiency in soybean. Inspired by this observation, we design a FLICK-PE strategy to confer a pair of nicks flanking the target site. In soybean, FLICK-PE achieves on average a 15.7-fold increase in intended-editing efficiency compared to PE2, and a 2.2-fold increase compared to PE3. Using FLICK-PE, we efficiently engineer glyphosate resistance in soybean by introducing TAP-IVS mutations in EPSPS1a, achieving three amino-acid substitutions and an intended editing efficiency of 21.1%. This approach yields stable edited soybean varieties with vigorous glyphosate tolerance and minimal growth penalties in a field trial. FLICK-PE also demonstrates efficacy in tobacco, underscoring its broad applicability and versatility for rapid, precision breeding in agriculturally vital crops.},
}
@article {pmid41344285,
year = {2026},
author = {Dasanayaka, BP and Pathirana, SL and Jayawardana, A and Handunnetti, SM and Fernando, N and Galhena, BP and Weerasena, SJ and Nitsche, A and Iddamaldeniya, SS and Dietzsch, AK},
title = {A roadmap in detecting frequently reported bovine babesiosis: From blood smear to CRISPR.},
journal = {Veterinary parasitology},
volume = {342},
number = {},
pages = {110662},
doi = {10.1016/j.vetpar.2025.110662},
pmid = {41344285},
issn = {1873-2550},
mesh = {Animals ; Cattle ; *Babesiosis/diagnosis/parasitology/blood ; *Cattle Diseases/diagnosis/parasitology/blood ; *CRISPR-Cas Systems ; *Babesia/isolation &amp; purification/genetics ; Babesia bovis/isolation &amp; purification/genetics ; Real-Time Polymerase Chain Reaction/veterinary ; Sensitivity and Specificity ; },
abstract = {Current diagnosis of Babesia bovis and B. bigemina relies on direct microscopy, nucleic acid detection, and serology. Light-microscopic analysis of Giemsa-stained smears still serves as the primary diagnostic modality at the point of care. However, carrier cattle, particularly those harbouring B. bovis, often carry parasites at levels far below the detection threshold, and such levels are sensitive only to DNA-based detection approaches. Early probe-hybridization techniques have been largely replaced by conventional Polymerase Chain Reaction (PCR), nested formats, and real-time quantitative PCR (qPCR)-which enable species-specific discrimination within closed-tube systems, thereby minimizing contamination risk. Species‑level identification is essential for clinical management, surveillance, and experimental studies. Duplex TaqMan qPCRs simultaneously distinguish B. bovis from B. bigemina, surpassing the analytical sensitivity offered by nested PCR (nPCR) methodologies. Reverse-line-blot (RLB) hybridisation broadens the diagnostic scope by concurrently detecting co-infections of B. bovis and B. bigemina and mixed haemoparasitic species in one workflow. However, the analytical sensitivity of RLB remains inferior to that of qPCR in detecting low-density and carrier-state infections of B. bigemina. RLB remains useful for retrospective genotyping when amplification is impractical or fails. Field-ready isothermal approaches have expanded the scope of molecular diagnostics beyond laboratory settings, facilitating field-level application and rapid on-site detection. Coupling Loop-mediated isothermal amplification (LAMP) with a lateral-flow dipstick (LFD) (LAMP-LFD) enables pen-side direct visual detection. Antibody tests are essential tools for herd-level surveillance. A recent chimeric Enzyme-Linked Immunosorbent Assay (ELISA) that combines three immunodominant B. bovis antigens broaden strain coverage and boosts diagnostic reliability. Recombinase polymerase amplification coupled to CRISPR-Cas12a cleavage has achieved single-target detection of B. bigemina from tick salivary-gland DNA, paving the way for innovative pen-side platforms, once cost and technical hurdles are overcome. Importantly, vector-based detection using appropriate tissues bearing an optimum level of ticks is species-dependent. B. bigemina sporozoites concentrate in nymph/adult salivary glands, whereas B. bovis sporozoites are produced mainly in larval salivary glands, so monitoring programs should stratify sampling accordingly.},
}
@article {pmid41338106,
year = {2026},
author = {Paenkaew, S and Euppayo, T and Tungtrakanpoung, R and Teapunvong, W and Nganvongpanit, K and Buddhachat, K},
title = {Rapid and specific detection of Babesia vogeli using RPA/CRISPR-Cas12a: A feasible field-friendly diagnostic for canine babesiosis.},
journal = {Veterinary parasitology},
volume = {342},
number = {},
pages = {110660},
doi = {10.1016/j.vetpar.2025.110660},
pmid = {41338106},
issn = {1873-2550},
mesh = {Animals ; Dogs ; *Babesiosis/diagnosis/parasitology ; *Dog Diseases/diagnosis/parasitology ; *Babesia/isolation &amp; purification/genetics ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Recombinases/metabolism ; },
abstract = {Babesia vogeli is a protozoan parasite causing canine babesiosis, a tick-borne disease prevalent in tropical and subtropical regions. Its microscopic identification is challenging due to morphological similarity with other Babesia spp., and serological assays often yield inaccurate results. To address this issue, we developed a rapid, equipment-minimal diagnostic method combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a (RPA/CRISPR-cas12a) for B. vogeli-specific detection. The RPA assay enables DNA amplification for both B. vogeli and Hepatozoon canis, while CRISPR/Cas12a using gRNA_Bab ensures specificity for B. vogeli, even in co-infections and other pathogens. This approach detects as few as 10[5] copies within two hours for both readout platforms such as fluorescence and lateral flow dipstick (LFD). Forty canine blood samples were detected by RPA/CRISPR-cas12a to examine its performance. Results showed high concordance with qPCR-high resolution melting (HRM) (Cohen's kappa: 0.93 for fluorescence, 0.81 for LFD), outperforming conventional PCR. The clinical sensitivity and specificity of RPA/CRISPR-cas12a were 100 % and 96.8 %, respectively and the concordance with qPCR-HRM was 97.5 %. RPA/CRISPR-cas12a for Babesia spp. detection provided a simple, rapid, and accurate method, demonstrating promise for point-of-care diagnosis of canine babesiosis in resource-limited settings. This method showed high potential as a practical diagnostic tool in veterinary clinics, with accelerated surveillance to control outbreaks of Babesia-associated canine babesiosis.},
}
@article {pmid41145673,
year = {2026},
author = {Benz, T and Larghero, P and Meyer, C and Hanewald, T and Brüggmann, D and Hentrich, AE and Louwen, F and Marschalek, R},
title = {CRISPR/Cas9-mediated t(4;11) translocation in human hematopoietic stem/precursor cells demonstrates plasticity to differentiate into either the myeloid or lymphoid lineage.},
journal = {Leukemia},
volume = {40},
number = {1},
pages = {72-86},
pmid = {41145673},
issn = {1476-5551},
support = {2022.070.1//Wilhelm Sander-Stiftung (Wilhelm Sander Foundation)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Translocation, Genetic ; *Hematopoietic Stem Cells/metabolism/cytology ; *Chromosomes, Human, Pair 11/genetics ; *Cell Differentiation/genetics ; *Chromosomes, Human, Pair 4/genetics ; Myeloid-Lymphoid Leukemia Protein/genetics ; Histone-Lysine N-Methyltransferase/genetics ; *Myeloid Cells/cytology/metabolism ; *Lymphocytes/cytology/metabolism ; Cell Lineage/genetics ; Oncogene Proteins, Fusion/genetics ; DNA-Binding Proteins/genetics ; Transcriptional Elongation Factors ; },
abstract = {The chromosomal translocation t(4;11)(q21;q23) is frequently diagnosed in KMT2A-r Acute Leukemia patients. Although we understand much about the function of both wildtype KMT2A and AFF1 multiprotein complexes, little is known about the molecular actions the two fusion proteins KMT2A::AFF1 and AFF1::KMT2A during the very early steps of disease onset and progression. Most published data have been generated in t(4;11) cell lines or transplanted mouse models, where exactly this process remains a black box. Here, we present the results of our efforts to establish a t(4;11) chromosomal translocation in human hematopoietic stem/precursor cells by CRISPR/Cas9. These genetically modified cells can be expanded over 5-6 months in vitro and their potential to differentiate was examined with IL-7 supplementation. The benefit of this model system is that (1) both reciprocal fusion proteins are concomitantly present, and (2) a molecular surveillance is possible at any timepoint through analysis of RNA, DNA or protein. Thus, the CRISPR/Cas9 technique allowed us to create a bona fide model system to study the very early steps of leukemia onset at the molecular level. In conclusion, this approach is the fastest way to investigate and characterize KMT2A-r fusions in primary human cells.},
}
@article {pmid40759332,
year = {2025},
author = {Yang, J and Huang, C and Feng, Y and He, J and Liu, Y and Zhang, P and Liu, C},
title = {Adaptor protein complex 1 gamma 1 subunit is an important host factor involved in both Zika virus and dengue virus infections.},
journal = {Virologica Sinica},
volume = {40},
number = {6},
pages = {874-883},
doi = {10.1016/j.virs.2025.07.012},
pmid = {40759332},
issn = {1995-820X},
mesh = {Humans ; *Dengue Virus/physiology/genetics ; *Zika Virus/physiology/genetics ; Virus Replication ; *Zika Virus Infection/virology/metabolism ; *Dengue/virology/metabolism ; Cell Line ; Virus Internalization ; *Host-Pathogen Interactions ; Viral Envelope Proteins/metabolism ; CRISPR-Cas Systems ; Animals ; RNA Interference ; },
abstract = {Mosquito-borne flaviviruses, such as Zika virus (ZIKV) and dengue virus (DENV), cause diverse severe clinical manifestations including fever, rash, hepatitis, arthralgia, and congenital anomalies. Here, we identified a host factor, the adaptor protein complex 1 gamma 1 subunit (AP1G1), which plays an important role in both ZIKV and dengue virus 2 (DENV2) infections. We explored the role of AP1G1 in ZIKV and DENV2 infections using CRISPR/Cas9 gene editing technology and RNA interference (RNAi) techniques. Knockout or silencing of AP1G1 decreases the replication of ZIKV and DENV2 in multiple human cell lines. Intriguingly, depletion of AP1G1 results in a significant reduction in ZIKV at an early stage, but decreases DENV2 replication levels during the late stage, suggesting that AP1G1 plays distinct roles in the infection by ZIKV and DENV2. Furthermore, we determined that AP1G1 mediates ZIKV-endosomal membrane fusion through inhibitor experiments and fluorescence labeling assays. Mechanistically, we found that AP1G1 exerts its pro-viral effect through binding to the ZIKV envelope glycoprotein (E protein). This interaction promotes the fusion of viral and endosomal membranes, during which the ZIKV genomic RNAs are released from the endosome into the cytoplasm, a process that facilitates viral replication. However, for DENV2 infection, AP1G1 primarily affects its viral RNA replication stage, rather than the fusion of virus-endosomal membrane. Taken together, our work demonstrates that AP1G1 plays a pro-viral role in both ZIKV and DENV2 infections via distinct mechanisms, highlighting its potential as a therapeutic target for antiviral strategies.},
}
@article {pmid41497669,
year = {2025},
author = {Kumbara, A and Tognon, M and Carone, G and Fontanesi, A and Bombieri, N and Giugno, R and Pinello, L},
title = {CRISPR-HAWK: Haplotype- and Variant-aware guide design toolkit for CRISPR-Cas.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41497669},
issn = {2692-8205},
abstract = {MOTIVATION: Current CRISPR guide RNA design tools rely on reference genomes, overlooking how genetic variation impacts editing outcomes. As genome editing advances toward clinical applications, incorporating population diversity becomes essential for ensuring therapeutic efficacy across diverse populations.<br><br>RESULTS: We present CRISPR-HAWK, a framework integrating individual- and population-scale variants and haplotypes into gRNA design. Analyzing therapeutic targets across 79,648 genomes reveals that genetic variants substantially alter guide performance. For the clinically approved sickle cell disease therapeutic guide targeting BCL11A, we identify haplotypes that completely abolish predicted cutting activity. Across seven therapeutic loci, 82.5% of guides contain variants modifying on-target activity. Variants also create novel protospacer adjacent motif sites generating individual-specific guides invisible to reference-based design. These findings demonstrate that variant-aware selection is critical for equitable genome editing.<br><br>AVAILABILITY: CRISPR-HAWK is available at https://github.com/pinellolab/CRISPR-HAWK and https://github.com/InfOmics/CRISPR-HAWK.},
}
@article {pmid41497644,
year = {2025},
author = {Stringer, AM and Wade, JT},
title = {Identification of PAM Requirements for the Vibrio cholerae type I-E CRISPR-Cas System.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41497644},
issn = {2692-8205},
abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems that use RNA-guided protein complexes to target invading nucleic acid. A surveillance complex consisting of protein and a CRISPR-RNA (crRNA) binds target nucleic acid via base-pairing interactions, typically leading to processing of the target nucleic acid by a nuclease. CRISPR-Cas systems are classified based on their mechanism of action, with type I systems being the most prevalent in nature. Type I CRISPR-Cas systems target DNA, and require extensive complementarity between the crRNA and the target DNA. Moreover, type I systems require the presence of a "Protospacer Adjacent Motif" (PAM) sequence in the target DNA immediately adjacent to the expected region of base-pairing with the crRNA. Classical biotypes of the bacterial pathogen Vibrio cholerae have active type I-E CRISPR-Cas systems. While the optimal PAM sequence for this CRISPR-Cas system is known to be AAY, the activity of other sequences as possible PAMs has not been determined. Here, we quantify the effectiveness of all possible trinucleotide sequences in the PAM position for the V. cholerae type I-E CRISPR-Cas system. Our data indicate a hierarchy of PAM efficacy, with 15 of the 64 trinucleotide sequences functioning as a PAM.},
}
@article {pmid41496934,
year = {2026},
author = {Khizar, M and Aminpoor, H and Zaib, M and Ali, Q and Karimi, H},
title = {Nanoparticle-enhanced CRISPR delivery: paving the path for in vivo tumor gene editing.},
journal = {Annals of medicine and surgery (2012)},
volume = {88},
number = {1},
pages = {1054-1055},
pmid = {41496934},
issn = {2049-0801},
abstract = {Nanoparticle-based delivery systems are redefining how CRISPR/Cas technology can be used in cancer treatment. By encapsulating CRISPR components within lipid, polymeric, or inorganic nanoparticles, researchers have improved their stability, circulation time, and tumor-targeting precision. The NTLA-2001 trial demonstrated the first successful use of lipid nanoparticles for in vivo CRISPR delivery in humans, paving the way for potential applications in oncology. Preclinical studies have shown promising results, with efficient gene knockout and tumor suppression across multiple models. Despite these advances, barriers remain, including limited delivery to solid tumors, potential off-target effects, and inconsistent nanoparticle formulations. Global research efforts spanning the United States, China, Europe, and India are now focused on refining delivery platforms and standardizing protocols. This letter highlights current progress, ongoing challenges, and the need for transparent, globally coordinated development. Nanoparticle-enhanced CRISPR delivery has the potential to bring genetic precision therapy from the laboratory to the clinic, offering a new avenue for durable and accessible cancer care.},
}
@article {pmid41496894,
year = {2026},
author = {Kantor, B and Duke, L and Bhide, PG},
title = {CRISPR-Cas editing technologies for viral-mediated gene therapies of human diseases: Mechanisms, progress, and challenges.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {1},
pages = {102786},
pmid = {41496894},
issn = {2162-2531},
abstract = {The gene therapy landscape has evolved substantially in recent years, beginning with the approval of the first adeno-associated virus-based gene therapy, Luxterna, in 2017. Since then, the US FDA has approved nearly 30 new viral gene therapy programs, with notable examples including Zolgensma, Spinraza, Hemgenix, Zynteglo, Lyfgenia, Kymriah, Skysona, and Tecelra. Remarkably, all these products rely on delivery via adeno-associated vectors (AAVs) and lentiviral vectors (LVs). Improvements in viral-mediated gene transfer efficiency and clinical-scale manufacturing, together with immense commercial interest, have greatly propelled the clinical adoption of gene therapy products. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) and its related Cas proteins (CRISPR-Cas) have made significant advances in gene therapy, offering next-generation approaches for curative gene editing to treat genetic diseases and disorders. In this review, we examine the range of these therapeutics and their viral carriers, focusing primarily on LVs and AAVs. We provide a snapshot of the current status of the field and highlight some of the current challenges in the clinical application of gene therapy, with particular emphasis on viral CRISPR-Cas-based technologies and their future potential.},
}
@article {pmid41496629,
year = {2026},
author = {Li, J and Wang, L and Yang, S and Zhou, X and Gou, Q and Cai, J and Yang, H and Wang, Q and Li, S},
title = {Chicken Shank Color Determined by Inhibition of Dermal Melanin (ID) is Mediated by a Structural Variation Regulating CDKN2A Expression.},
journal = {Pigment cell &amp; melanoma research},
volume = {39},
number = {1},
pages = {e70072},
doi = {10.1111/pcmr.70072},
pmid = {41496629},
issn = {1755-148X},
support = {2022hszd006//Major Project of Hubei Hongshan Laboratory/ ; 31772585//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Chickens/genetics ; *Melanins/metabolism ; *Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism ; *Skin Pigmentation/genetics ; Melanocytes/metabolism ; *Gene Expression Regulation ; Polymorphism, Single Nucleotide/genetics ; Female ; Male ; CRISPR-Cas Systems/genetics ; Genome-Wide Association Study ; *Skin/metabolism ; },
abstract = {Shank color in chickens is a classic quantitative trait governed by four genetic loci. Among these, the Inhibition of dermal melanin (ID) locus, which suppresses dermal melanogenesis in the shank, is the sole sex-linked mutation and its molecular mechanisms remain elusive. To identify the causal mutation, we established a resource population segregating for shank colors. A genome-wide association study utilizing FarmCPU software identified a top-associated SNP on the Z chromosome. Linkage mapping subsequently narrowed the candidate region, within which we discovered a candidate structural variant associated with the yellow shank phenotype. This variant is characterized by a 143 bp deletion coupled with a 2 bp insertion. CDKN2A was the only gene within the same topologically associating domain to exhibit differential expression. Functional validation via CRISPR/Cas9-edited cells demonstrated that this mutation regulates CDKN2A transcription and is responsible for the ID shank color in chickens. We propose that the resulting absence of melanocytes is likely due to apoptosis. This work resolves the molecular basis of the ID locus, thereby completing the genetic puzzle of chicken shank color. This discovery enables the development of molecular markers for auto-sexing of day-old chicks, a tool with significant potential for the poultry industry.},
}
@article {pmid41495909,
year = {2026},
author = {Derollez, E and Roson-Calero, N and Rouzé, P and Dedieu-Berne, A and Ballesté-Delpierre, C and Fraikin, N and Iorga, BI and Huang, TD and Bigot, S and Vila, J and Bogaerts, P and Lesterlin, C},
title = {Specific killing and resensitization of pathogenic Escherichia coli strains carrying blaCTX-M-15 β-lactamase using targeted-antibacterial-plasmids (TAPs).},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41495909},
issn = {1362-4962},
support = {//Joint Programming Initiative on Antimicrobial Resistance/ ; JPIAMR2021-194//JPIAMR/ ; FRM-EQU202103012587//Foundation for Medical Research/ ; ANR-20-PAMR-0010//French Priority Research Program/ ; //Agence Nationale de la Recherches/ ; },
mesh = {*beta-Lactamases/genetics/metabolism ; *Escherichia coli/genetics/drug effects/enzymology ; *Plasmids/genetics ; CRISPR-Cas Systems ; Humans ; Anti-Bacterial Agents/pharmacology ; Conjugation, Genetic ; Drug Resistance, Multiple, Bacterial/genetics ; Escherichia coli Infections/microbiology ; },
abstract = {Targeted-Antibacterial-Plasmids (TAPs) offer a precise approach to combat multidrug-resistant bacteria by selectively removing resistant strains while preserving commensals. Here, we assess TAPs that deliver CRISPR/Cas systems via conjugation to kill or resensitize extended-spectrum β-lactamase (ESBL)-producing Escherichia coli carrying the blaCTX-M-15 gene. We systematically tested multiple variables in the TAP approach, including two commensal E. coli donors, three distinct helper plasmids encoding the transfer machineries, and six recipient strains harbouring the resistance gene either chromosomally or on a plasmid. Cas9-based TAPs induced double-stranded breaks in chromosomal blaCTX-M-15 genes, resulting in immediate bacterial death. When the target gene was plasmid-borne, Cas9 cleavage triggered plasmid loss and partial toxin-antitoxin-mediated killing. In contrast, dCas9-based TAPs inhibited blaCTX-M-15 expression without affecting cell viability, thereby restoring third-generation cephalosporin susceptibility. In mixed-culture experiments, TAPs specifically eliminated only blaCTX-M-15-carrying E. coli while sparing other non-targeted bacterial species. Conjugation assays in human faeces demonstrated substantial suppression of cefotaxime-resistant (CtxR) E. coli by both Cas9- and dCas9-based TAPs, underscoring their efficacy in complex microbial environments. These findings highlight TAPs' decolonization promise, paving the way for future microbiome-editing interventions against multidrug-resistant carriage or infection.},
}
@article {pmid41495894,
year = {2026},
author = {Ishihara, K and Matsumoto, S and Gerle, C and Gopalasingam, CC and Shigematsu, H and Shirai, T and Numata, T},
title = {Sequential structural rearrangements at the PAM-distal site of a type I-F3 CRISPR-Cas effector enabling RNA-guided DNA transposition.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41495894},
issn = {1362-4962},
support = {20H02916//Japan Society for the Promotion of Science/ ; 24H00505//Japan Society for the Promotion of Science/ ; //Japan Foundation for Applied Enzymology/ ; //Naito Foundation/ ; //Institute for Fermentation, Osaka/ ; //Noda Institute for Scientific Research/ ; 23KJ1734//JSPS/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *DNA Transposable Elements/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; *Bacterial Proteins/chemistry/genetics/metabolism ; Vibrio parahaemolyticus/genetics ; DNA/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; Models, Molecular ; },
abstract = {Some prokaryotes carry CRISPR-associated transposons (CASTs), Tn7-like elements that incorporate genes encoding CRISPR-Cas effectors. CAST insertion is directed by CRISPR-Cas effectors through RNA-guided DNA binding and interactions with transposition-associated proteins. Although efficient sequence-specific DNA integration requires both precise target DNA recognition and coordinated interactions between effectors and transposition-associated proteins, the underlying mechanism remains elusive. Here, we determined three cryo-EM structures of target DNA-bound type I-F3 TniQ-Cascade from Vibrio parahaemolyticus, revealing how Cas8/5 recognizes the protospacer adjacent motif (PAM) and identifying a key residue responsible for the cytidine preference at position -2 of the PAM. We revealed mismatch tolerance at the PAM-proximal site. Structural analyses showed that correct base pairing at the PAM-distal site correlates with conformational changes in the Cas8/5 helical bundle and TniQ, bending the DNA to guide its downstream region toward the transposition machinery. Together, these dynamic rearrangements at the PAM-distal region provide insights into the licensing mechanism of type I-F3 CAST transposition and highlight its potential for genome engineering applications.},
}
@article {pmid41495892,
year = {2026},
author = {Zhang, Z and Wang, J and Guo, T and Yu, X and Wang, F and Zhang, H and Liu, Y and Li, W and Cheng, Y and Peng, Y and Yan, G and Cui, J and Ma, L},
title = {Argonaute-mediated RNA editing selectively repairs point mutations.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41495892},
issn = {1362-4962},
support = {2022YFA0911800//National Key Research and Development Program of China/ ; 2024BCA001//Technological Innovation Plan in Hubei Province/ ; 2024040701010046//Natural Science Foundation of Wuhan City/ ; 2025CSA051//Science and Technology Program Project of Hubei Province/ ; 32300517//National Natural Science Foundation of China/ ; 32400513//National Natural Science Foundation of China/ ; NYWSWZX2025-2027-10//Hubei Provincial Major Special Project for the Development of the Agricultural Microbiology Industry/ ; 2021CSA066//Hubei Province Central Government Guided Special Fund for Local Science and Technology Development/ ; },
mesh = {*Argonaute Proteins/metabolism/genetics ; Humans ; *RNA Editing ; *Point Mutation ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; Ribonucleoproteins/metabolism/genetics ; Adenosine Deaminase/genetics/metabolism ; },
abstract = {RNA editing enzymatically modifies RNA molecules post-transcriptionally, enabling precise sequence alterations. Advantages include reversibility and temporal control without genomic DNA changes, allowing dynamic regulation of gene expression while preserving original genetic information. In this study, we characterized McAgo derived from Monosporascus cannonballus, which functions as a programmable nuclease guided by 14-30 nt gRNAs, demonstrating robust RNA cleavage activity at physiological temperature. Furthermore, we delivered McAgo RNP (ribonucleoprotein) complexes into mammalian cells, achieving >90% RNA knockdown efficiency with minimal innate immune responses. A catalytically inactive mutant (dMcAgo) using a gRNA as short as 20 nt, conjugated to the hADAR2 deaminase domain (hADAR2dd E488Q), achieved up to 90% RNA editing efficiency in vitro. This study establishes, for the first time, the effective targeting of endogenous RNA by a heterologous Argonaute in mammalian cells, alongside its demonstrated utility for RNA editing-thereby expanding the functional repertoire of Argonaute proteins.},
}
@article {pmid41494892,
year = {2026},
author = {Wang, MR and Mu, W and Zhen, A and Kitchen, SG},
title = {CRISPR/Cas strategies to enhance CAR T-cell function and persistence via metabolic reprogramming.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.12.001},
pmid = {41494892},
issn = {1879-3096},
abstract = {While chimeric antigen receptor (CAR) T-cell therapy has become a standard of care in various blood cancers, its full curative potential for other diseases has yet to be maximized. One key limiting factor is progressive T-cell exhaustion and differentiation over time, leading to the loss of the CAR-expressing cells. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) gene manipulation to enhance CAR T-cell therapy has revolutionized the field in recent years. In this review, we will examine the application of CRISPR/Cas aimed at improving CAR T-cell function and persistence to combat the issues of exhaustion and dysfunction, with a focus on metabolic reprogramming. Understanding current preclinical CRISPR/Cas strategies for modulating CAR T-cell metabolism is critical in advancing CAR-T therapies to clinical applications.},
}
@article {pmid41494604,
year = {2026},
author = {Zeng, Y and Zhao, G and Wu, S and Hu, B and Forn-Cuní, G and Knol, R and El Ghalbzouri, A and Snaar-Jagalska, E and Kros, A},
title = {CD44-targeted lipid nanoparticles for enhanced CRISPR/Cas9 delivery in cancer gene editing.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {391},
number = {},
pages = {114598},
doi = {10.1016/j.jconrel.2025.114598},
pmid = {41494604},
issn = {1873-4995},
abstract = {Skin cancer is the third most common malignancy, with melanoma being the most challenging due to its resistance to current therapies. Gene editing technologies like CRISPR/Cas9 offer a promising strategy for targeting cancer-specific genes, but the efficient delivery of these tools to tumor sites remains a significant challenge. Lipid nanoparticles (LNPs) have emerged as the leading platform for gene editing tools due to their ability to protect and transport large payloads. To enhance the precision of gene editing in melanoma, we developed CD44-specific peptide-modified LNPs for targeted delivery of CRISPR/Cas9 mRNA and guide RNA against polo-like kinase 1 (sgPLK1). Our approach led to enhanced targeting and gene editing efficacy by specifically delivering CRISPR/Cas9 and sgPLK1 to melanoma tumor cells, resulting in significant inhibition of tumor growth in both in vitro and in vivo skin melanoma models. Moreover, this platform showed the capacity to reach metastatic melanoma in the brain and resulting in substantial suppression of tumor growth in brain metastasis models. We envision that this peptide-modification strategy could be further employed to improve the targeting capabilities and therapeutic outcomes of LNPs for CRISPR/Cas9-based gene editing, paving the way for more precise and effective cancer treatments.},
}
@article {pmid41461227,
year = {2026},
author = {Su, S and Zhang, X and Wang, X and Qiu, C and Xu, Z and Piñero, JC and Peng, X and Li, F and Zuo, Y and Chen, M},
title = {CRISPR/Cas9-based evidence that overexpression of Gm-mGST1 mediates abamectin resistance in the oriental fruit moth, Grapholita molesta.},
journal = {Insect biochemistry and molecular biology},
volume = {187},
number = {},
pages = {104472},
doi = {10.1016/j.ibmb.2025.104472},
pmid = {41461227},
issn = {1879-0240},
mesh = {Animals ; *Ivermectin/analogs &amp; derivatives/pharmacology ; *Moths/genetics/drug effects/metabolism ; *Insecticide Resistance/genetics ; CRISPR-Cas Systems ; *Insecticides/pharmacology ; *Glutathione Transferase/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Chloride Channels/genetics/metabolism ; Larva/genetics/growth &amp; development/drug effects ; },
abstract = {Abamectin-based insecticides are widely used in integrated pest management and are particularly effective against fruit borers such as the oriental fruit moth, Grapholita molesta. However, rapid resistance evolution threatens their long-term efficacy. This study elucidates the role of the glutathione S-transferase gene Gm-mGST1 in abamectin resistance in G. molesta. A laboratory-selected resistant strain (AB-R) exhibited an 85.5-fold increase in resistance compared with a susceptible strain (AB-S). Sequencing of glutamate-gated chloride channel (GmGluCl) gene revealed no target-site mutations, implicating a metabolic resistance mechanism. In AB-R, GST enzymatic activity was significantly elevated. GST synergist diethyl maleate (DEM) increased the toxicity of abamectin more strongly in the abamectin-resistant G. molesta strain than in the susceptible strain, indicating that GSTs contribute to abamectin resistance. Gm-mGST1 showed strong and stage-specific overexpression under abamectin exposure. Functional analysis using CRISPR/Cas9 knockout of Gm-mGST1 in the AB-R strain reduced resistance 16.3-fold, providing the definitive evidence that a GST gene directly mediates abamectin resistance in G. molesta. The catalytic activity of recombinant Gm-mGST1 was verified in vitro using CDNB as the substrate. Additionally, abamectin exhibited a certain degree of inhibitory effect on the activity of Gm-mGST1. HPLC analysis further revealed that the peak area of abamectin significantly decreased in the presence of recombinant Gm-mGST1, while ectopic expression in Drosophila melanogaster increased abamectin tolerance by 1.97-fold. There is a significant positive correlation between the abamectin resistance levels and the expression levels of Gm-mGST1 in field populations of G. molesta. These findings identify Gm-mGST1 as a critical gene involved in abamectin resistance and establish it as a potential molecular marker for monitoring resistance in field populations. More broadly, this study sets a precedent for integrating CRISPR/Cas9 gene editing into insecticide resistance research, bridging the gap between correlative evidence and functional validation, and providing a framework for developing GST-targeted resistance management strategies in orchard pests. This study provides evidence using CRISPR/Cas9 to confirm the contribution of GST to abamectin resistance in insects.},
}
@article {pmid41423037,
year = {2026},
author = {Ma, L and Wu, B and Li, S and Zhang, X and Zhao, X and Zhang, J and Zhang, M and Zhang, M and Ma, L and Guo, C and Zhang, T},
title = {CRISPR/Cas9-mediated LmSerpin5 knockout causes midgut dysplasia and leads to embryonic lethality in Locusta migratoria.},
journal = {Journal of insect physiology},
volume = {168},
number = {},
pages = {104925},
doi = {10.1016/j.jinsphys.2025.104925},
pmid = {41423037},
issn = {1879-1611},
mesh = {Animals ; *Locusta migratoria/genetics/growth &amp; development/embryology/immunology ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; Nymph/growth &amp; development/genetics ; Immunity, Innate/genetics ; Gene Knockout Techniques ; },
abstract = {Serpins play a crucial role in in various physiological processes of insects. Previous studies have suggested that Serpins regulated processes like egg diapause, melanization, and antimicrobial peptide synthesis in Locusta migratoria, but their overall functional characterization remains insufficient. In this study, the functions of LmSerpin5 in regulating developmental processes and innate immunity were investigated via CRISPR/Cas9-mediated knockout. Homozygous LmSerpin5 mutation caused complete embryonic lethality. By contrast, chimeric mutants showed elevated mortality during embryonic-to-first-instar nymph transition, though chitinous tissue development remained unaffected. Additionally, adult mutants exhibited no external malformations but displayed pathological changes in immune organs, including fat body cells with enlarged lipid droplets and nuclei, and midgut absorptive cells lacking brush borders. Furthermore, pro-nymphal midguts exhibited reduced microvilli density, structural defects, and inflammatory intestinal folds. Molecular analysis confirmed upregulation of Toll pathway downstream genes (LmMyd88, LmPelle and LmTube) in mutant tissues, with midgut-specific activation of LmTube and LmPelle linking structural damage to immune dysregulation. These results demonstrated LmSerpin5 maintains homeostasis through dual mechanisms: ensuring embryonic survival and suppressing excessive Toll activation.},
}
@article {pmid41353342,
year = {2025},
author = {He, L and Yao, Y and You, Y and Wei, X and Ma, Y and Yuan, W and Lang, Z and Zhu, JK},
title = {Versatile molecular tools enabling customizable DNA methylation editing in Arabidopsis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {251},
pmid = {41353342},
issn = {2041-1723},
support = {32188102//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32100458//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Arabidopsis/genetics ; *DNA Methylation/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Genome, Plant ; },
abstract = {Tools to edit DNA methylation in a targeted manner are vital for establishing causal relationships between DNA methylation and its function, as well as for plant breeding and gene therapy. Here, by constructing dCas9 fusions to a panel of effectors and cofactors, we develop a range of highly effective tools for editing DNA methylation in Arabidopsis, including five tools for DNA methylation and six tools for DNA demethylation. Our tools show a diversity of performance features in terms of specificity and efficiency, offering either the capacity to edit DNA methylation in a target-specific manner or the ability to edit DNA methylation genome-wide due to potent off-target effect. Importantly, DNA methylation edited by these tools is inherited in the absence of transgene. These versatile tools pave the way for diverse applications of DNA methylation editing in not only research but also epigenetic breeding of crops.},
}
@article {pmid41213129,
year = {2026},
author = {Ajdanian, L and Villot, S and Karikari, B and Torkamaneh, D},
title = {Technological advances in trait development: from conventional breeding and untargeted mutagenesis to precision genome editing.},
journal = {Genome},
volume = {69},
number = {},
pages = {1-13},
doi = {10.1139/gen-2025-0020},
pmid = {41213129},
issn = {1480-3321},
mesh = {*Gene Editing/methods ; *Plant Breeding/methods ; *Crops, Agricultural/genetics ; *Mutagenesis ; Plants, Genetically Modified/genetics ; *Genome, Plant ; CRISPR-Cas Systems ; },
abstract = {Plant biotechnology has revolutionized modern agriculture by enabling precise and efficient crop improvement strategies. This review explores the evolution of selective breeding, mutation breeding, and precision breeding, highlighting their applications in Canada's agricultural sector. Conventional selective breeding has been instrumental in developing high-yielding and disease-resistant cultivars, while mutation breeding, through physical and chemical mutagenesis, has introduced valuable genetic diversity. The advent of transgenic breeding allowed for the direct insertion of foreign genes, leading to the development of crops with herbicide tolerance, pest resistance, and improved nutritional content. However, concerns over regulatory restrictions and public acceptance have driven the rapid adoption of genome editing tools, which enable precise modifications without introducing foreign DNA. Canada has played a key role in applying these biotechnological innovations, successfully developing genetically modified canola, CRISPR-edited wheat, stress-resistant soybean, and barley and oat cultivars improved for stress resistance and yield. While each breeding approach presents distinct advantages and limitations, integrating conventional and molecular techniques is essential for maximizing genetic potential, ensuring agriculture, and effectively food security challenges.},
}
@article {pmid41047629,
year = {2026},
author = {Zhang, C and Chen, Z and Cao, J and Zhang, Z and Li, WK and Zhang, X and Chen, J and Liu, J and Yuan, Z and Gao, F and Shi, Z and Zhao, XM and Chen, J and Zhan, C and Cheng, TL},
title = {RNA inosine sensor-guided TadA mutational scanning for toxicity minimization of adenine base editors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {1},
pages = {380-396},
doi = {10.1016/j.ymthe.2025.10.011},
pmid = {41047629},
issn = {1525-0024},
mesh = {*Inosine/genetics/metabolism ; Humans ; *RNA Editing ; Animals ; *Adenine/metabolism ; *Mutation ; *Gene Editing/methods ; Mice ; HEK293 Cells ; CRISPR-Cas Systems ; Biosensing Techniques/methods ; },
abstract = {The TadA component of adenine base editors (ABEs) induces widespread RNA off-target edits and raises safety concerns for their applications. However, the extent of RNA editing-related toxicity remains elusive, and high-throughput engineering of ABEs focusing on RNA editing activities remains challenging. Here, we demonstrate that RNA off-target editing of classical ABEs leads to substantial toxicity in vitro and in vivo. We then design a rapid, cost-effective, and sensitive fluorescent RNA inosine sensor to accelerate RNA off-target editing evaluation and high-throughput screening in mammalian cells. Deep mutation scanning with the RNA sensor identifies various TadA8e mutants displaying minimized RNA editing activity, with the representative H52L/D53R mutant compatible with both SpCas9 and the compact IscB nickase. We show that the engineered ABEs could efficiently target clinically relevant sites in vitro and in vivo with enhanced precision, thereby providing promising tools for applications in which RNA editing-related toxicity should be carefully evaluated and minimized.},
}
@article {pmid41017153,
year = {2026},
author = {Ye, C and Ma, Y and Shrestha, R and Cai, J and Liu, Y and Peng, L and Yu, J and Cai, H},
title = {Extracellular vesicle-mediated delivery of CRISPR machinery silences androgen receptor in castration-resistant prostate cancer cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {1},
pages = {281-299},
doi = {10.1016/j.ymthe.2025.09.045},
pmid = {41017153},
issn = {1525-0024},
mesh = {Humans ; Male ; *Receptors, Androgen/genetics ; *Extracellular Vesicles/metabolism/genetics ; *Prostatic Neoplasms, Castration-Resistant/genetics/therapy/pathology/metabolism ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Silencing ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Proliferation ; CRISPR-Associated Protein 9 ; Ribonucleoproteins/genetics ; },
abstract = {CRISPR-mediated gene editing is a promising technology for treatment of diseases by silencing a driver gene at the genomic DNA level. However, delivery of CRISPR machinery remains challenging for potential therapeutic application. Here, we developed a platform using extracellular vesicles (EVs) as a vehicle to deliver Cas9/single-guide RNA (sgRNA) ribonucleoprotein (RNP) complex to silence androgen receptor (AR) gene in prostate cancer (PCa) cells. A genetic modification conferred the N-myristoylation to the Cas9 protein, which enhanced the encapsulation of Cas9/sgRNA RNP into EVs and silenced both ectopic and endogenous AR gene. Interestingly, gene editing efficiency varied across PCa cell lines, associated with different chromatin accessibility at the target site. Functional analyses demonstrated that Cas9/sgRNA RNP (targeting the N-terminal domain of the AR gene) did not change gene-edited AR mRNA levels, but significantly inhibited expression levels of AR downstream genes, thereby attenuating PCa cell proliferation. Importantly, EV-mediated delivery of the Cas9/sgRNA RNP introduced indels into the AR gene and inhibited proliferation of enzalutamide-resistant PCa cells. This study highlights a therapeutic strategy for treatment of castration-resistant PCa using a programmable EV-mediated delivery platform.},
}
@article {pmid40994008,
year = {2026},
author = {Marco, E and Sousa, P and Janoudi, T and de Dreuzy, E and Heath, JM and Viswanathan, R and Zuris, JA and Gotta, GM and Giannoukos, G and Hansen, S and Wood, DK and Walters, MC and Tisdale, JF and Wilson, CJ and Chang, KH},
title = {Nonclinical evaluation of renizgamglogene autogedtemcel for SCD and TDT.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {1},
pages = {249-265},
doi = {10.1016/j.ymthe.2025.09.031},
pmid = {40994008},
issn = {1525-0024},
mesh = {*Anemia, Sickle Cell/genetics/therapy ; Humans ; *Gene Editing/methods ; Animals ; Mice ; *beta-Thalassemia/genetics/therapy ; Fetal Hemoglobin/genetics/metabolism ; CRISPR-Cas Systems ; Promoter Regions, Genetic ; Repressor Proteins ; Erythroid Cells/metabolism ; Carrier Proteins/genetics/metabolism ; },
abstract = {Sickle cell disease and transfusion-dependent β-thalassemia can be treated by fetal hemoglobin upregulation. Disruption of the distal BCL11A binding site at the HBG1/2 promoters to induce fetal hemoglobin using either SpCas9 or AsCas12a mimics multiple hereditary persistence of fetal hemoglobin mutations. AsCas12a showed higher editing efficiency, higher specificity, and increased fetal hemoglobin induction potential compared with SpCas9. AsCas12a-edited healthy donor CD34[+] cells exhibited long-term, multi-lineage, and polyclonal engraftment in immunocompromised mice. High-level fetal hemoglobin induction was observed in erythroid progeny derived in vivo from edited healthy donor CD34[+] cells and sickle cell disease or transfusion-dependent β-thalassemia donor CD34[+] cells in vitro. In erythroid cells from patients with sickle cell disease, gene editing reduced sickling and improved rheological behaviors under deoxygenated conditions. In erythroid cells from patients with β-thalassemia, gene editing ameliorated ineffective erythropoiesis and significantly increased hemoglobin content per cell. A comprehensive off-target editing evaluation in edited CD34[+] cells showed AsCas12a to be highly specific, with no off-target editing detected. In summary, editing CD34[+] cells at the HBG1/2 promoter distal BCL11A binding site using AsCas12a phenocopied hereditary persistence of fetal hemoglobin mutations, demonstrating its potential as a gene editing approach for the treatment of β-hemoglobinopathies.},
}
@article {pmid40914806,
year = {2026},
author = {Liu, H and Singh, S and Mullen, TJ and Bullock, C and Keegan, S and Patterson, T and Thakur, S and Lundberg, A and Shenker, S and Couto, R and Yadav, C and Dastagir, S and Li, L and Bainter, W and Liberzon, E and Malloy, CR and Lazzarotto, CR and Ohsumi, TK and Chilakala, S and Chen, HM and Kshirsagar, R and Hohmann, AF and Arlauckas, SP and Lazorchak, A and Scull, C and Morgan, RA},
title = {A precision gene-engineered B cell medicine producing sustained levels of active factor IX for hemophilia B therapy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {1},
pages = {266-280},
doi = {10.1016/j.ymthe.2025.09.001},
pmid = {40914806},
issn = {1525-0024},
mesh = {*Hemophilia B/therapy/genetics ; *Factor IX/genetics/metabolism ; Animals ; *Genetic Therapy/methods ; Mice ; Humans ; *B-Lymphocytes/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Dependovirus/genetics ; Receptors, CCR5/genetics ; Genetic Vectors/genetics ; Genetic Engineering ; Transgenes ; Disease Models, Animal ; },
abstract = {Hemophilia B gene therapy treatments have not addressed the need for predictable, durable, active, and redosable factor IX (FIX). Unlike conventional gene therapy, engineered B cell medicines (BCMs) are durable, redosable, and titratable and thus have the potential to address significant unmet needs in the hemophilia B treatment paradigm. BE-101 is an autologous BCM comprising expanded and differentiated B lymphocyte lineage cells genetically engineered ex vivo to secrete factor IX (FIX)-Padua. CRISPR-Cas9-mediated gene editing at the C-C chemokine receptor type 5 (CCR5) locus was used to facilitate transgene insertion of an adeno-associated virus 6-encoded DNA template via homology-directed repair. Transgene insertion did not alter B cell biology, viability, or differentiation into plasma cells. Appreciable levels of BE-101-derived FIX-Padua were detected within 1 day after IV administration in mice, and steady state was reached within 2 weeks and persisted for over 184 days. Redosing produced an increase in FIX-Padua production close to linear dose proportionality. Comprehensive genotoxicity analysis found no off-target issues of concern. No safety signals were observed in animal tolerability and Good Laboratory Practice toxicology studies. In conclusion, BE-101 produces sustained levels of active FIX-Padua with the ability to engraft without host preconditioning and with the potential for redosing and titratability.},
}
@article {pmid40911442,
year = {2026},
author = {Tang, J and Yang, S and Li, S and Yue, X and Jin, T and Yang, X and Zhang, K and Yang, Q and Liu, T and Zhao, S and Gai, J and Li, Y},
title = {Editing a gibberellin receptor gene improves yield and nitrogen fixation in soybean.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {1},
pages = {75-95},
doi = {10.1111/jipb.70026},
pmid = {40911442},
issn = {1744-7909},
support = {32372192//National Natural Science Foundation of China/ ; JBGS-2021-014//Core Technology Development for Breeding Program of Jiangsu Province/ ; BM2024005//Jiangsu Key Laboratory of Soybean Biotechnology and Intelligent Breeding/ ; },
mesh = {*Glycine max/genetics/metabolism/growth &amp; development ; *Nitrogen Fixation/genetics ; *Gene Editing ; *Gibberellins/metabolism ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; Seeds ; },
abstract = {Soybean is an important source of oil, protein, and feed. However, its yield is far below that of major cereal crops. The green revolution increased the yield of cereal crops partially through high-density planting of lodging-resistant semi-dwarf varieties, but required more nitrogen fertilizers, posing an environmental threat. Genes that can improve nitrogen use efficiency need to be integrated into semi-dwarf varieties to avoid the overuse of fertilizers without the loss of dwarfism. Unlike cereal crops, soybean can assimilate atmospheric nitrogen through symbiotic bacteria. Here, we created new alleles of GmGID1-2 (Glycine max GIBBERELLIN INSENSITIVE DWARF 1-2) using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) editing, which improved soybean architecture, yield, seed oil content, and nitrogen fixation, by regulation of important pathways and known genes related to branching, lipid metabolism, and nodule symbiosis. GmGID1-2 knockout reduced plant height, and increased stem diameter and strength, number of branches, nodes on the primary stem, pods, and seeds per plant, leading to an increase in seed weight per plant and yield in soybean. The nodule number, nodule weight, nitrogenase activity, and nitrogen content were also improved in GmGID1-2 knockout soybean lines, which is novel compared with the semi-dwarf genes in cereal crops. No loss-of-function allele for GmGID1-2 was identified in soybean germplasm and the edited GmGID1-2s are superior to the natural alleles, suggesting the GmGID1-2 knockout mutants generated in this study are valuable genetic resources to further improve soybean yield and seed oil content in future breeding programs. This study illustrates the pleiotropic functions of the GID1 knockout alleles with positive effects on plant architecture, yield, and nitrogen fixation in soybean, which provides a promising strategy toward sustainable agriculture.},
}
@article {pmid40898613,
year = {2026},
author = {Yuan, L and Xiong, Y and Zhang, Y and Gu, S and Lei, Y},
title = {Epigenome editing based treatment: Progresses and challenges.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {1},
pages = {46-67},
doi = {10.1016/j.ymthe.2025.08.047},
pmid = {40898613},
issn = {1525-0024},
mesh = {Humans ; *Gene Editing/methods ; DNA Methylation ; *Epigenome ; *Epigenesis, Genetic ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Animals ; *Epigenomics/methods ; Gene Expression Regulation ; Epigenome Editing ; },
abstract = {Epigenome editing is emerging as a transformative approach in clinical treatment, enabling precise modifications to gene expression without altering the underlying DNA sequence. The ongoing transition of epigenome editing techniques from foundational research to clinical applications highlights several key strategies. These include targeted DNA methylation/demethylation, histone modification, and transcriptional regulation. These approaches offer the potential for durable and reversible gene expression modulation, paving the way for precisely tailored therapies for genetic and complex diseases. Here, we review pioneering research, technological advancements, granted patents, and clinical trials that have been reported during the past decade. By synthesizing current research and development efforts, this review aims to provide insights into the promising landscape of epigenome editing and its potential to promote therapeutic interventions.},
}
@article {pmid41492065,
year = {2026},
author = {Wang, Z and Wang, Y and Gao, H and Dai, J and Tang, N and Wang, Y and Ji, Q},
title = {Phage-associated Cas12p nucleases require binding to bacterial thioredoxin for activation and cleavage of target DNA.},
journal = {Nature microbiology},
volume = {11},
number = {1},
pages = {81-93},
pmid = {41492065},
issn = {2058-5276},
mesh = {*Thioredoxins/metabolism/genetics ; *CRISPR-Cas Systems ; *Bacteriophages/genetics/enzymology/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; Protein Binding ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; DNA Cleavage ; DNA/metabolism ; Escherichia coli/genetics/virology/metabolism ; },
abstract = {The evolutionary competition within phage-host systems led to the emergence of CRISPR-Cas defence mechanisms in bacteria and anti-CRISPR elements in bacteriophages. Although anti-CRISPR elements are well characterized, the role of bacterial factors that influence CRISPR-Cas efficacy has been comparatively overlooked. Type V CRISPR-Cas12 systems display striking functional and mechanistic diversity for nucleic acid targeting. Here we use a bioinformatic approach to identify Cas12p, a phage-associated nuclease that forms complexes with the bacterial thioredoxin protein TrxA to enable target DNA degradation. This represents an unexpected phage-bacteria interaction, in which the bacteriophage co-opts a bacterial factor to augment its own genome degradation machinery, potentially against competing phages. Biochemical characterization, cryo-EM-based structural analysis of the Cas12p-TrxA-sgRNA-dsDNA complex at 2.67 Å and bacterial defence assays reveal that TrxA directly binds and activates Cas12p, enabling its nuclease activity and subsequent CRISPR immunity. These findings expand our understanding of the multilayered intricacies of phage-bacteria molecular interactions.},
}
@article {pmid41489362,
year = {2026},
author = {Shin, J and Barrangou, R},
title = {Occurrence and applications of CRISPR-Cas systems in bifidobacteria.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0170325},
doi = {10.1128/aem.01703-25},
pmid = {41489362},
issn = {1098-5336},
abstract = {Bifidobacterium is a key member of the human gut microbiota, and many strains are widely used as probiotics due to their health-promoting properties. Despite growing interest, genetic studies in Bifidobacterium have been relatively limited, primarily due to the lack of available genome editing tools. Recent advances in genomics and CRISPR-Cas systems provide opportunities for targeted genome modification in this genus. In this review, we provide an overview of the occurrence, diversity, and distribution of CRISPR-Cas systems across Bifidobacterium species and examine the editing tools developed and implemented to date. We also highlight practical challenges such as strain variability and low transformation efficiency and introduce future avenues of research such as large-payload insertion and in situ editing. Expanding the genetic toolbox for Bifidobacterium will broaden our understanding of this important genus and enable the development of next-generation probiotics.},
}
@article {pmid41488985,
year = {2026},
author = {Kim, K and Lee, J and Lee, N and Cho, BK},
title = {CRISPR-Based Approaches to Engineer Nonmodel Bacteria for Bioproduction and Biotherapeutics.},
journal = {Biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.biochem.5c00613},
pmid = {41488985},
issn = {1520-4995},
abstract = {Microbial diversity encompasses vast genetic and functional capacities, with immense potential for biotechnological applications. Yet, most biotechnological advances have been confined to a narrow set of model organisms, leaving the broader repertoire of nonmodel microbes largely untapped due to species-specific barriers that hinder genetic manipulation. Over the past decade, the advent of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems has transformed microbial engineering by enabling precise, programmable, and scalable control of genomes and gene expression. Importantly, the relative independence of many CRISPR effectors from host cofactors has facilitated their use in microbes previously challenging to engineer, thus expanding opportunities to exploit their unique metabolic and biosynthetic traits. In this review, we summarize the major CRISPR-Cas toolkits and highlight recent innovations, with particular emphasis on translational applications in nonmodel organisms such as C1-gas-fixing acetogens, antibiotic-producing Streptomyces, and gut commensal Bacteroides. We emphasize three areas of emerging impact: engineering microbial cell factories for sustainable biomanufacturing, accelerating natural product discovery, and development of next-generation live biotherapeutics. Finally, we discuss current limitations and future opportunities, underscoring how the integration of genome editing, synthetic biology, and systems-level approaches is reshaping the landscape of microbial biotechnology.},
}
@article {pmid41488303,
year = {2025},
author = {Tsolakidou, PJ},
title = {CRISPR-Cas systems against carbapenem resistance: from proof-of-concept to clinical translation.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1725247},
pmid = {41488303},
issn = {1664-302X},
abstract = {Carbapenem-resistant Enterobacterales (CRE) pose a major global threat, driven by plasmid-borne carbapenemase genes such as bla KPC, bla NDM and bla OXA-48. CRISPR-Cas systems offer programmable strategies to selectively eliminate these resistance determinants. This mini-review summarizes recent advances in Cas9-based plasmid curing, RNA-targeting approaches such as Cas13a and Cas13d, and DNA-targeting Cas3-enhanced bacteriophage therapeutics that have entered early clinical evaluation. Particular attention is given to conjugative CRISPR-Cas9 plasmid systems, which enable targeted plasmid eradication without laboratory transformation and broaden the delivery toolbox beyond phage vectors. We further discuss major translational challenges, including delivery efficiency, phage host-range constraints, ecological risks of horizontal CRISPR dissemination, and off-target effects. Finally, we highlight emerging delivery platforms-outer membrane vesicles, lipid and polymeric nanoparticles, conjugative plasmids with containment circuits, and engineered live biotherapeutics-that may complement or overcome current limitations. Collectively, these developments illustrate the potential of CRISPR-based antimicrobials to augment traditional therapies through precise gene-level suppression of carbapenem resistance.},
}
@article {pmid41487293,
year = {2025},
author = {},
title = {Erratum: Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing [Corrigendum].},
journal = {Biologics : targets &amp; therapy},
volume = {19},
number = {},
pages = {745-746},
doi = {10.2147/BTT.S585961},
pmid = {41487293},
issn = {1177-5475},
abstract = {[This corrects the article DOI: 10.2147/BTT.S326422.].},
}
@article {pmid41486484,
year = {2025},
author = {Boogari, M and Mohebbi, M and Hadidi, N},
title = {Genetically Engineered Probiotics: Design, Therapeutics, and Clinical Translation.},
journal = {Iranian biomedical journal},
volume = {29},
number = {6},
pages = {374-383},
doi = {10.61882/ibj.5197},
pmid = {41486484},
issn = {2008-823X},
abstract = {Genetically engineered probiotics aim to address transient colonization and the intra- and inter-subject variability that limit conventional probiotics. These strains utilizes CRISPR/Cas editing, programmable gene circuits, and biosensors in chassis such as E. coli Nissle 1917 and L. lactis. This narrative review summarizes the current engineering toolkits and standards (e.g., SEVA), chassis selection criteria, biocontainment strategies, and translational requirements under CMC/GMP frameworks and discusses regulatory considerations for clinical translation. Representative examples include IL-10-secreting L. lactis and phenylalanine-metabolizing strains for PKU (SYNB1618/SYNB1934), which illustrate pharmacodynamic target engagement and short-term preclinical safety. We outline clinical advancements in predefined pharmacodynamics, durability of function, monitoring shedding and HGT, and genomic-microbiome-informed patient stratification. Systems modeling approaches (GEM/ABM) are discussed as tools to guide rational design. GEPs offer programmable "sense-and-respond" therapeutics, with successful clinical adoption depending on durable efficacy, long-term safety, and clearly defined regulatory pathways.},
}
@article {pmid41484149,
year = {2026},
author = {Kremer, N and Mueller, F and Nguyen, H and Schulz, L and Popp, T and Artes, E and Wolters, J and Renner, M and Vetter, I and Maffini, S and Robles, MS and Musacchio, A and Bange, T},
title = {CUL4A-DDB1-DCAF10 is an N-recognin for N-terminally acetylated Src kinases.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {132},
pmid = {41484149},
issn = {2041-1723},
support = {5041 140321//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Cullin Proteins/metabolism/genetics ; Humans ; Acetylation ; *src-Family Kinases/metabolism/genetics ; HEK293 Cells ; *DNA-Binding Proteins/metabolism/genetics ; Proteolysis ; Protein Processing, Post-Translational ; Ubiquitination ; Ubiquitin-Protein Ligases/metabolism ; CRISPR-Cas Systems ; },
abstract = {Co-translational N-terminal modifications such as methionine excision, acetylation, and myristoylation govern protein stability, localization, and folding. Disruption can expose N-terminal degrons that trigger ubiquitin-mediated degradation, safeguarding the proteome. N-terminal acetylation usually protects proteins from degradation, but can also promote it through the Ac/N-degron pathway. Src-family kinases (SFKs), signaling enzymes implicated in tumorigenesis, require N-terminal myristoylation for function. Using peptide pull-downs, mass spectrometry, and AlphaFold 3 predictions, we identify DCAF10 as the E3 ligase substrate receptor for alternatively N-terminally acetylated SFKs. Combining siRNA-mediated knockdown and CRISPR/Cas9-mediated knockout of endogenous Lyn with inducible Lyn-GFP variants confirms that DCAF10 regulates SFK levels by recognizing an N-terminal acetylated glycine residue. In vitro, a CUL4A-DDB1-DCAF10 complex ubiquitinates N-terminally acetylated SFKs. Thus, we define a novel N-degron pathway that monitors replacement of myristoylation by acetylation and activates degradation of SFKs upon acetylation. This mechanism may extend to other N-terminally myristoylated proteins beyond SFKs.},
}
@article {pmid41481841,
year = {2026},
author = {Jin, YM and Li, XD and Zhu, JK and Shao, CY and Huang, BB and Huang, HL and Wang, XW and Jiang, HB and Chen, W},
title = {Programmable adenine base editing in cyanobacteria using an engineered TadA-Cas9 fusion.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {1},
pages = {e70655},
doi = {10.1111/tpj.70655},
pmid = {41481841},
issn = {1365-313X},
support = {32470092//National Natural Science Foundation of China/ ; 32170108//National Natural Science Foundation of China/ ; 2024QL060//Ningbo Youth Leading Talent Project/ ; //Ningbo University Startup Funding/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Adenine/metabolism ; *Synechocystis/genetics ; *Cyanobacteria/genetics ; Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Anabaena/genetics ; },
abstract = {Cyanobacteria are photosynthetic prokaryotes with great potential in green biomanufacturing and basic research. Despite decades of pioneering achievements, the application of advanced genome editing tools, particularly CRISPR-based systems, has remained limited in cyanobacteria. In this study, we developed pCyABE, a new adenine base editor for efficient and precise A·T to G·C editing in cyanobacteria. This system utilizes a TadA-Cas9 nickase fusion and functions without double-strand breaks or donor templates. We demonstrated its high editing efficiency in Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, highlighting its broad usability. pCyABE supports multiplex editing and enables start codon disruption for gene functional studies. Furthermore, this tool exhibits low off-target activity and can be effectively removed via sucrose counterselection. In conclusion, pCyABE provides a versatile and efficient genome editing platform that significantly expands the genetic toolbox for cyanobacterial research and biotechnology applications.},
}
@article {pmid41481737,
year = {2026},
author = {Qi, S and Wei, L and Ding, Z and Zhong, F and Yang, S and Wu, L and Yang, X and Kang, B and Dan, M and Gan, J and Li, C and Su, X},
title = {Targeting transthyretin by one Cas9 variant with superfidelity and broad compatibility.},
journal = {Science advances},
volume = {12},
number = {1},
pages = {eadu6505},
pmid = {41481737},
issn = {2375-2548},
mesh = {*Prealbumin/genetics/metabolism ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; Amyloid Neuropathies, Familial/genetics/therapy ; HEK293 Cells ; },
abstract = {Amyloid transthyretin (ATTR) amyloidosis is a fatal disease caused by the accumulation of misfolded transthyretin proteins. Although knocking down the TTR gene by CRISPR-Cas9 represents a promising strategy for treating ATTR amyloidosis, its efficiency and safety remain to be further investigated. Here, we report a systematic investigation of SpCas9-based TTR editing. Besides the target site, wild-type SpCas9 and the reported variants induced extensive off-target edits. To improve the fidelity, we performed structural analysis and designed a series of SpCas9 variants. Studies demonstrated that SpCas9-Mut5 is an ultrahigh-fidelity variant, which induces extremely low levels of off-target edits and translocations without substantial impairment of on-target editing activity. SpCas9-Mut5 is compatible with the adenine base editor (ABE) system, markedly reducing off-target edits and narrowing the editing window. In conclusion, our study suggests that SpCas9-Mut5 is an excellent candidate for TTR gene editing. Besides ATTR amyloidosis, SpCas9-Mut5 and its derivative ABE could be widely used in the treatment of other diseases.},
}
@article {pmid41481232,
year = {2026},
author = {Li, Y and Zhang, W and Wei, Z and Li, H and Liu, X and Zheng, T and Aziz, T and Xing, C and Meng, A and Wu, X},
title = {Stage- and tissue-specific gene editing using 4-OHT-inducible Cas9 in whole organism.},
journal = {The Journal of cell biology},
volume = {225},
number = {4},
pages = {},
pmid = {41481232},
issn = {1540-8140},
support = {#3258820001//National Natural Science Foundation of China/ ; #2023YFA1800300//National Key Research and Development Program of China/ ; 2018YFC1003304//National Key Research and Development Program of China/ ; #202302AO370011//Southwest United Graduate School/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Zebrafish/genetics/embryology ; *Tamoxifen/analogs &amp; derivatives/pharmacology ; *CRISPR-Cas Systems/genetics ; Mice ; Female ; *CRISPR-Associated Protein 9/genetics/metabolism ; Germ Cells/metabolism ; Organ Specificity ; Humans ; Receptors, Estrogen/genetics/metabolism ; Animals, Genetically Modified ; },
abstract = {Vertebrate genes function in specific tissues and stages, so their functional studies require conditional knockout or editing. In zebrafish, spatiotemporally inducible genome editing, particularly during early embryogenesis, remains challenging. Here, we establish inducible Cas9-based editing in defined cell types and stages. The nCas9ERT2 fusion protein, consisting of Cas9 and an estrogen receptor flanked by two nuclear localization signals, is usually located in the cytoplasm and efficiently translocated into nuclei upon 4-hydroxytamoxifen (4-OHT) treatment in cultured cells or embryos. As a proof of concept, we demonstrate that genes in primordial germ cells in embryos and germ cells in adult ovaries from a transgenic line with stable expression of nCas9ERT2 and gRNAs can be mutated by 4-OHT induction. The system also works in early mouse embryos. Thus, this inducible nCas9ERT2 approach enables temporospatial gene editing at the organismal level, expanding the tissue- and stage-specific gene-editing toolkit.},
}
@article {pmid41479566,
year = {2025},
author = {Antipenko, ID and Venedyukhina, SA and Sorokina, NP and Kucherenko, IV and Smirnova, TS and Rogov, GN and Shkurnikov, MY},
title = {Whole-Genome Sequencing Uncovers Metabolic and Immune System Variations in Propionibacterium freudenreichii Isolates.},
journal = {Acta naturae},
volume = {17},
number = {4},
pages = {72-82},
pmid = {41479566},
issn = {2075-8251},
abstract = {Propionibacterium freudenreichii plays a crucial role in the production of Swiss-type cheeses; however, genomic variability among strains, which affects their technological traits, remains insufficiently explored. In this study, whole-genome sequencing and comparative analysis were performed on five industrial P. freudenreichii strains. Despite their overall high genomic similarity, the strains proved different in gas production and substrate metabolism. Phylogenetic analysis revealed a close relationship between strain FNCPS 828 and P. freudenreichii subsp. shermanii (z-score = 0.99948), with the latter being unable to reduce nitrates but being able to metabolize lactose. The narG gene encoding the nitrate reductase alpha subunit was detected in only one of the five analyzed strains - FNCPS 828 - and in 39% of previously described P. freudenreichii genomes, suggesting its potential as a marker of nitrate-reducing capability. Analysis of 112 genomes showed that the I-G CRISPR-Cas system was present in more than 90% of the strains, whereas the type I-E system was found in approximately 25%. All the five study strains harbored the type I-G system; strain FNCPS 3 additionally contained a complete type I-E system with the highest number of CRISPR spacers, some of which matched previously published bacteriophage sequences. The most prevalent anti-phage defense systems included RM I, RM IV, AbiE, PD-T4-6, HEC-06, and ietAS. These findings highlight the genetic diversity of P. freudenreichii strains, which is of great importance in their industrial applications. The identification of narG as a potential marker of nitrate-reducing activity, along with detailed mapping of CRISPR- Cas systems, boosts opportunities for the rational selection and engineering of starter cultures with tailored metabolic properties and increased resistance to bacteriophages.},
}
@article {pmid41479062,
year = {2026},
author = {Lee, NJ and Matsuoka, RL},
title = {CRISPR/Cas9-Based Mutagenesis Strategies for Efficient Biallelic Gene Inactivation and Consistent Phenotypic Detection in F0 Zebrafish.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2989},
number = {},
pages = {319-332},
pmid = {41479062},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems/genetics ; Phenotype ; *Mutagenesis ; Alleles ; *Gene Silencing ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The zebrafish is a valuable animal model for investigating the genetic basis of vertebrate evolution, development, behavior, and regeneration. However, the existence of numerous gene paralogs in the zebrafish genome represents a major challenge, complicating functional genomic research using reverse-genetics approaches. To facilitate reverse genetics-based phenotypic screens, we recently presented simple methods that enable efficient induction of biallelic gene disruptions in F0 zebrafish, providing a rapid avenue for screening potential gene functions through consistent phenotypic detection. Here, we describe detailed protocols for these CRISPR/Cas9-based mutagenesis strategies to achieve highly effective biallelic gene inactivation in F0 zebrafish. The high consistency of these strategies, combined with a streamlined workflow, offers a robust phenotypic screening platform for a quick and reliable functional assessment of genes of interest, both individually and in a scalable manner. These strategies enhance the efficacy of successful F0 zebrafish phenotypic screening, thereby accelerating functional genetic studies using this powerful model organism.},
}
@article {pmid41389602,
year = {2026},
author = {Kumari, P and Gupta, V and Chhikara, A and Dalal, J},
title = {Revolutionizing forensic DNA analysis: The potential of CRISPR-Cas9 technology in genetic investigations.},
journal = {Journal of forensic and legal medicine},
volume = {117},
number = {},
pages = {103047},
doi = {10.1016/j.jflm.2025.103047},
pmid = {41389602},
issn = {1878-7487},
mesh = {Humans ; *CRISPR-Cas Systems ; *DNA Fingerprinting/methods ; *Forensic Genetics/methods ; *Gene Editing ; },
abstract = {The newest achievements in the field of molecular biology and gene-editing technologies have transformed the paradigm of forensic DNA analysis. However, there are still great difficulties in interpreting degraded, low-template, mixed genetic samples. The review critically evaluates the transformative potential of Clustered Regularly Interspaced Short Palindromic Repeats and an associated protein 9 (CRISPR-Cas9) as an accurate, effective, and cost-efficient system of genome-editing in the field of forensic science. Based on the evidence of the current literature, the paper critically analyzes the mechanisms of CRISPR-Cas9 activity, its RNA-guided specificity, dual-strand cleavage, and high-fidelity targeting, and compares its functionality with other standard methods like the STR and SNP profiling. The review also discusses more complex CRISPR-based diagnostic systems, such as SHERLOCK, DETECTR, and HOLMES that allow the analysis of DNA rapidly, without amplification, and in a portable format. Among major discoveries, there is the ability of CRISPR to increase the accuracy of DNA profiling, resolve mixture, recapitulate damaged genetic material, and reduce the possibility of contamination. In addition to genetic analysis, it has applications in forensic epigenetics, prediction of phenotypes, microbial forensics and environmental trace analysis. The review also covers the ethical, legal and governance implications of implementing CRISPR-based evidence in the judicial process especially in as far as data privacy; admissibility and equity of access are concerned. In general, CRISPR-Cas9 is a paradigm shift in forensic genomics, the one that has the potential to transform personal identification, reconstruction of the crime scene, and the interpretation of molecular evidence. Future efforts should focus on method validation, standardization, and ethical governance to ensure the responsible and sustainable implementation of this technology in forensic practice.},
}
@article {pmid41372414,
year = {2026},
author = {Habtewold, T and Lwetoijera, DW and Hoermann, A and Mashauri, R and Matwewe, F and Mwanga, R and Kweyamba, P and Maganga, G and Magani, BP and Mtama, R and Mahonje, MA and Tambwe, MM and Tarimo, F and Chennuri, PR and Cai, JA and Del Corsano, G and Capriotti, P and Sasse, P and Moore, J and Hudson, D and Manjurano, A and Tarimo, B and Vlachou, D and Moore, S and Windbichler, N and Christophides, GK},
title = {Gene-drive-capable mosquitoes suppress patient-derived malaria in Tanzania.},
journal = {Nature},
volume = {649},
number = {8096},
pages = {442-448},
pmid = {41372414},
issn = {1476-4687},
mesh = {Tanzania/epidemiology ; Animals ; *Plasmodium falciparum/isolation &amp; purification/genetics/drug effects/growth &amp; development ; *Anopheles/genetics/parasitology ; Humans ; *Malaria, Falciparum/prevention &amp; control/parasitology/transmission ; *Gene Drive Technology/methods ; *Mosquito Vectors/genetics/parasitology ; Female ; Animals, Genetically Modified/genetics ; Male ; Child ; CRISPR-Cas Systems/genetics ; },
abstract = {Gene drive technology presents a transformative approach to combatting malaria by introducing genetic modifications into wild mosquito populations to reduce their vectorial capacity. Although effective modifications have been developed, these efforts have been confined to laboratories in the global north. We previously demonstrated that modifying Anopheles gambiae to express two exogenous antimicrobial peptides inhibits the sporogonic development of laboratory-cultured Plasmodium falciparum, with models predicting substantial contributions to malaria elimination in Africa when integrated with gene drive[1-3]. However, the effectiveness of this modification against genetically diverse, naturally circulating parasite isolates remained unknown. To address this critical gap, we adapted our technology for an African context by establishing infrastructural and research capacity in Tanzania, enabling the engineering of local A. gambiae under containment. Here we report the generation of a transgenic strain equipped with non-autonomous gene drive capabilities that robustly inhibits genetically diverse P. falciparum isolates obtained from naturally infected children. These genetic modifications were efficiently inherited by progeny when supplemented with Cas9 endonuclease provided by another locally engineered strain. Our work brings gene drive technology a critical step closer to application, providing a locally tailored and powerful tool for malaria eradication through the targeted dissemination of beneficial genetic traits in wild mosquito populations.},
}
@article {pmid41313207,
year = {2026},
author = {Wang, Y and Qin, Z and Wang, Q and Yang, Y and Gu, C and Yu, F and Wu, Y and Zhang, Lx},
title = {An RPA-CRISPR/Cas12a-based rapid and sensitive nucleic acid method for detection of Toxoplasma gondii in tissue and blood samples.},
journal = {Microbiology spectrum},
volume = {14},
number = {1},
pages = {e0155025},
pmid = {41313207},
issn = {2165-0497},
support = {231111111500//Key Research and Development Special Project of Henan Province of China/ ; 2022YFD1800200, 2023YFD1801200//National Basic Research Program of China/ ; },
mesh = {*Toxoplasma/genetics/isolation &amp; purification ; Animals ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; DNA, Protozoan/genetics ; *Toxoplasmosis/diagnosis/parasitology/blood ; Limit of Detection ; *Toxoplasmosis, Animal/diagnosis/parasitology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Toxoplasma gondii is a zoonotic pathogen that can infect humans and a wide range of warm-blooded animals, posing a significant threat to human health and the livestock industry. The development of a time-saving, highly sensitive, and specific method for the detection of T. gondii in tissue and blood samples is crucial to the monitoring, prevention, and control of toxoplasmosis. In this study, we evaluated the efficiency of a previously described method, termed REPORT, that integrates recombinase polymerase amplification with CRISPR/Cas12a for the detection of T. gondii nucleic acids. We evaluated the limit of detection (LOD) and specificity of the extended REPORT method using prepared target DNA in addition to tissue and blood samples. Furthermore, we validated the accuracy of T. gondii detection in clinical samples using the REPORT-based method in comparison with nested PCR based on the B1 gene. Sensitivity tests showed that the LOD of the REPORT-based fluorescence method and the lateral flow strip method were 3.7 copies /μL for target DNA, 3.1 tachyzoites/g for tissue samples, and five tachyzoites/mL for blood samples. Specificity tests suggested that the REPORT method had good specificity and did not cross-react with several common parasites. The method performed well for clinical DNA samples, demonstrating its ability for use in on-site detection.IMPORTANCEToxoplasma gondii can infect over 200 species of warm-blooded animals, including humans, posing not only a significant threat to public health systems but also causing substantial economic losses to the global livestock industry. Current diagnostic methods are slow, equipment-dependent, and impractical for field use. This study addresses these limitations by developing REPORT, a rapid, ultrasensitive nucleic acid test combining recombinase polymerase amplification and CRISPR/Cas12a. The REPORT detects T. gondii in tissue and blood samples within 1 h at low cost, requiring only a portable heater. Its visual results (fluorescence or test strips) enable on-site use without specialized training, achieving 100% accuracy versus nested PCR. With a sensitivity of 3.1 parasites per gram of tissue and five parasites per milliliter of blood, this method revolutionizes toxoplasmosis screening in resource-limited clinics, farms, and food safety inspections, empowering timely interventions to curb transmission and improve public health outcomes.},
}
@article {pmid41225007,
year = {2026},
author = {Noh, H and Hashem, Z and Boms, E and Najafov, A},
title = {SIGLEC12 mediates plasma membrane rupture during necroptotic cell death.},
journal = {Nature},
volume = {649},
number = {8096},
pages = {460-466},
pmid = {41225007},
issn = {1476-4687},
mesh = {*Necroptosis/genetics ; Humans ; Animals ; *Cell Membrane/metabolism/pathology ; Mice ; Protein Kinases/metabolism ; Phosphorylation ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Necroptosis is a form of lytic cell death that is overactivated during infections and in inflammatory pathologies[1]. NINJ1 was recently found to be a mediator of plasma membrane rupture (PMR) during pyroptosis, toxin-induced necrosis, apoptosis, and ferroptosis[2,3], but the mediator of PMR during necroptotic cell death remained unknown. Here, using a CRISPR-Cas9-based genome-wide knockout approach, we identify SIGLEC12 as a key mediator of necroptosis downstream of MLKL at the PMR step. Cells with knockdown or knockout of SIGLEC12 are defective in necroptosis-induced PMR and demonstrate ballooning morphology. During necroptosis, SIGLEC12 undergoes dephosphorylation, interacts with MLKL, forms cytosolic puncta and assembles into fibrils. Notably, SIGLEC12 is cleaved by TMPRSS4 during necroptosis to produce a 20-kDa fragment highly homologous to NINJ1, and this cleavage event is required and sufficient to induce PMR during necroptosis. A SIGLEC12 variant associated with cancer (Ser458Phe) and a variant found in the general human population (Arg528Trp) attenuate SIGLEC12 cleavage by TMPRSS4. Knockout of Siglec12 in mouse cells does not affect PMR, suggesting a species-specific role. Our identification of SIGLEC12 as a mediator of PMR expands our understanding of how programmed necrosis is executed and offers new approaches for targeting this proinflammatory form of cell death in human diseases.},
}
@article {pmid40673390,
year = {2026},
author = {Guerra, F and De Rouck, S and Verhulst, EC},
title = {SYNCAS-mediated CRISPR-Cas9 genome editing in the Jewel wasp, Nasonia vitripennis.},
journal = {Insect molecular biology},
volume = {35},
number = {1},
pages = {48-55},
doi = {10.1111/imb.70002},
pmid = {40673390},
issn = {1365-2583},
support = {OCENW.M.22.140//Dutch Research Council (NWO)/ ; G035420N//Research Foundation Flanders (FWO)/ ; 101123162/ERC_/European Research Council/International ; },
mesh = {Animals ; *Wasps/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Female ; Gene Knockout Techniques ; Insect Proteins/genetics ; },
abstract = {Genetic engineering is a formidable approach to studying biology. The development of CRISPR-Cas9 has allowed the genetic engineering of insect species from several orders, and in some species, this tool is used routinely for genetic research. However, insect gene editing often relies on the delivery of CRISPR-Cas9 components via embryo injection. This technique has a limitation: some species lay their eggs inside hard substrates or living hosts, making embryo collection impossible or labour intensive. Recently, a variety of techniques that exploit maternal injection of nucleases have been developed to circumvent embryo injection. Yet, despite this variety of maternal delivery techniques, some insects remain refractory to gene editing. One of these is the parasitoid wasp, Nasonia vitripennis, an important hymenopteran model species. In this study, a recently developed method termed SYNCAS was used to perform knock-out (KO) of the cinnabar gene in this wasp, obtaining KO efficiencies up to 10 times higher than reported for other maternal injection approaches. We found up to 2.73% of all offspring to display a KO phenotype, and we obtained up to 68 KO offspring per 100 injected mothers. The optimal timing of injection and provision of hosts for egg laying was determined. With this protocol, routine applications of CRISPR-Cas9 become feasible in this species, allowing reverse genetics studies of genes with unknown associated phenotypes and paving the way for more advanced editing techniques.},
}
@article {pmid39901548,
year = {2025},
author = {Yang, F and Wang, H and Fan, S and Qiu, H and Li, X and Shi, G and Li, Z and Luan, X and Wu, H},
title = {Advances in Synthetic Lethality in Potential Oncology Therapeutic Approaches.},
journal = {Current topics in medicinal chemistry},
volume = {25},
number = {15},
pages = {1868-1881},
pmid = {39901548},
issn = {1873-4294},
support = {ZR2022MB140//Natural Science Foundation of Shandong Province/ ; ZYQR201810168//National funding of postdoctoral innovative talents support program of Henan Province/ ; },
mesh = {Humans ; *Neoplasms/genetics/drug therapy/pathology ; *Antineoplastic Agents/pharmacology/chemistry/therapeutic use ; *Synthetic Lethal Mutations/drug effects ; Molecular Targeted Therapy ; CRISPR-Cas Systems ; },
abstract = {Synthetic lethality represents a novel paradigm in molecular targeted cancer therapy. In synthetic lethality, perturbation of one gene alone does not hinder cell viability, yet simultaneous perturbation of both genes results in a loss of cellular viability. The presence of gene mutations in cancer cells, as opposed to normal cells, provides an opportunity for targeted therapies that mimic the effects of the second genetic mutation, enabling selective eradication of cancer cells. Recent advances in high-throughput screening technologies, such as CRISPR-Cas9 and RNA interference, have significantly enhanced the identification of synthetic lethal interactions, expanding the potential targets for therapeutic intervention. Challenges in exploiting synthetic lethality for cancer treatment include the complexities of tumor biology, limited comprehension of synthetic lethal interactions, drug resistance, and impediments in screening and clinical translation. Emerging strategies, such as combination therapies and novel drug designs, are being developed to overcome these obstacles. By virtue of its selective lethality towards cancer cells bearing specific genetic alterations, targeting synthetic lethal genes holds the promise to provide wider therapeutic windows compared to traditional cytotoxic chemotherapy. This review describes the current state of synthetic lethality applications in cancer treatment, encompassing both biological and methodological perspectives. It highlights the latest advancements in synthetic lethality with emerging interventional strategies. Furthermore, it explores future directions for research and clinical implementation, aiming to refine and expand the therapeutic potential of synthetic lethality in oncology.},
}
@article {pmid41478990,
year = {2026},
author = {Hu, O and Provvido, A and Zhu, Y},
title = {Generation of IL17RB Knockout Cell Lines Using CRISPR/Cas9-Based Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2983},
number = {},
pages = {361-370},
pmid = {41478990},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Gene Knockout Techniques/methods ; *Receptors, Interleukin-17/genetics ; Cell Line ; HEK293 Cells ; },
abstract = {CRISPR/Cas9-based genome editing is an inexpensive and efficient tool for genetic modification. Here, we present a methodological approach for establishing interleukin-17 receptor B (IL17RB) knockout cell lines using CRISPR/Cas9-mediated genomic deletion. The IL17RB gene encodes for a cytokine receptor that specifically binds to IL17B and IL17E and is overexpressed in various cancers. The method involves CRISPR design, CRISPR cloning, delivery of the CRISPR clone into cells, and verification of IL17RB gene deletion by deletion screening primer design, genomic DNA extraction, and polymerase chain reaction (PCR). A similar approach can be used for generating mammalian cell lines with gene knockout for other genes of interest.},
}
@article {pmid41478989,
year = {2026},
author = {Huo, Z and Tu, J and Shoemaker, R and Lee, DF and Zhao, R},
title = {Engineering Mutation Clones in Mammalian Cells with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2983},
number = {},
pages = {343-359},
pmid = {41478989},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; Cell Line ; *Tumor Suppressor Protein p53/genetics ; *Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Human Embryonic Stem Cells/metabolism/cytology ; Exons ; Gene Knock-In Techniques/methods ; *Genetic Engineering/methods ; },
abstract = {CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat, as a powerful genome engineering system, has been widely accepted and employed in gene editing of a vast range of cell types. Compared to zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), CRISPR shows a less complicated process and higher efficiency. With the development of different CRISPR systems, it can be used not only to knock out a gene but also to make precise modifications, activate or repress target genes with epigenetic modifications, and even for genome wide screening. Here we will describe the procedure of generating a stable cell line with a knock-in mutation created by CRISPR. Specifically, this protocol demonstrated how to apply CRISPR to create the point mutation of R249 to S249 on TP53 exon 7 in human embryonic stem cells (hESC) H9 line, which includes three major steps: (1) design CRISPR system targeting TP53 genomic region, (2) deliver the system to H9 hESC and clone selection, and (3) examination and selection of positive clones.},
}
@article {pmid41478695,
year = {2026},
author = {Zhang, H and Han, M and Pang, Z and Li, W and Li, X and Sun, B},
title = {The lldD lactate dehydrogenase is a determinant of lactic acid tolerance in Pichia kudriavzevii by pyruvate metabolism pathway.},
journal = {Food microbiology},
volume = {136},
number = {},
pages = {104989},
doi = {10.1016/j.fm.2025.104989},
pmid = {41478695},
issn = {1095-9998},
mesh = {*Pichia/genetics/metabolism/enzymology/drug effects ; *Lactic Acid/metabolism/pharmacology ; Fermentation ; *L-Lactate Dehydrogenase/genetics/metabolism ; Metabolic Networks and Pathways ; *Pyruvic Acid/metabolism ; *Fungal Proteins/genetics/metabolism ; Ethanol/metabolism ; CRISPR-Cas Systems ; Glucose/metabolism ; Gene Knockout Techniques ; },
abstract = {Lactic acid stress is common in traditional fermented foods. Pichia kudriavzevii owes its industrial prevalence to its superior acid tolerance, making deciphering its response mechanism imperative for sustainable fermentation processes. This work explored the remarkable acid tolerance of P. kudriavzevii, which can tolerate 80 g/L lactic acid. The key lactic acid tolerance gene lldD of P. kudriavzevii was determined through transcriptomic analysis. Importantly, this work knocked out the key gene lldD related to lactic acid tolerance for the first time by using the CRISPR-Cas9 technology. Meanwhile, the lldD knockout strain (pk-ΔlldD) was successfully constructed. There were significant differences between pk-ΔlldD and P. kudriavzevii in terms of tolerance to lactic acid, metabolism of lactic acid, utilization of glucose and ethanol production. In addition, the deletion of the gene lldD has a significant impact on the ABC transporter and metabolites of the amino acid metabolic pathway in P. kudriavzevii. In conclusion, this work provides a theoretical basis for engineering high acid-tolerant industrial yeast strains through targeted genetic modification. It helps enhance the stability of fermentation processes under lactic acid stress and ultimately lays a foundation for promoting efficient and low-loss production in the fermentation industry.},
}
@article {pmid41477891,
year = {2026},
author = {Luo, G and Song, J and Fu, Y and Jiang, Y and Gao, Y and Zhong, Z and Li, L and Wei, Y and Jia, HR and Guo, L and Fu, T and Wu, Q and Tan, W},
title = {SPARK-seq: A high-throughput platform for aptamer discovery and kinetic profiling.},
journal = {Science (New York, N.Y.)},
volume = {391},
number = {6780},
pages = {eadv6127},
doi = {10.1126/science.adv6127},
pmid = {41477891},
issn = {1095-9203},
mesh = {*Aptamers, Nucleotide/chemistry/genetics ; Kinetics ; Humans ; *High-Throughput Nucleotide Sequencing/methods ; *Single-Cell Analysis/methods ; *Membrane Proteins/metabolism/genetics/chemistry ; RNA, Messenger/genetics ; CRISPR-Cas Systems ; },
abstract = {Cell surface proteins are key disease biomarkers and therapeutic targets, yet high-throughput methods for aptamer discovery targeting these proteins in situ remain limited. We introduce single-cell perturbation-driven aptamer recognition and kinetics sequencing (SPARK-seq), a high-throughput platform integrating single-cell messenger RNA and aptamer sequencing with CRISPR-based surface protein perturbation. In a single experiment, SPARK-seq simultaneously mapped 5535 distinct aptamers to eight surface proteins, capturing interactions across more than two orders of magnitude in protein abundance and spanning diverse biophysical classes. The method discriminated closely related paralogous proteins with no detectable cross-reactivity and provided kinetic information that enabled the prioritization of aptamers with slow dissociation rates. Leveraging this kinetic diversity, we engineered variants with improved off-rate properties. SPARK-seq establishes a platform for high-efficiency discovery and rational variant design of aptamers and functional nucleic acids, unlocking possibilities in diagnostics and therapeutics.},
}
@article {pmid41477825,
year = {2026},
author = {Song, LCT and Alker, ATP and Oromí-Bosch, A and Swartz, SE and Martinson, JNV and Arora, J and Wang, AM and Rovinsky, R and Smith, SJ and Pierce, EC and Deutschbauer, AM and Doudna, JA and Cress, BF and Rubin, BE},
title = {Identification of proteins influencing CRISPR-associated transposases for enhanced genome editing.},
journal = {Science advances},
volume = {12},
number = {1},
pages = {eaea1429},
pmid = {41477825},
issn = {2375-2548},
mesh = {*Gene Editing/methods ; *Transposases/genetics/metabolism ; Escherichia coli/genetics ; Vibrio cholerae/genetics ; *CRISPR-Cas Systems ; *Genome, Bacterial ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {CRISPR-associated transposases (CASTs) hold tremendous potential for microbial genome editing because of their ability to integrate large DNA cargos in a programmable, site-specific manner. However, their widespread application has been hindered by poorly understood host factor requirements for transposition. To address this gap, we conducted the first genome-wide screen for host factors affecting Vibrio cholerae CAST (VchCAST) activity using an Escherichia coli RB-TnSeq library and identified 15 genes affecting VchCAST transposition. Of these, seven factors were validated to improve VchCAST activity, and two were inhibitory. Guided by the identification of homologous recombination effectors, RecD and RecA, we tested the λ-Red recombineering system in our VchCAST editing vectors and increased editing efficiency by 55.2-fold in E. coli, 5.6-fold in Pseudomonas putida, and 10.8-fold in Klebsiella michiganensis while maintaining high target specificity and similar insertion arrangements. This study improves the understanding of factors affecting VchCAST activity and enhances its efficiency as a bacterial genome editor.},
}
@article {pmid41406662,
year = {2026},
author = {Schalper, KT and Yang, R and Guan, X and Zhang, J and Schreiber, D and Moon, J and Liu, C},
title = {Programmable CRISPR-mediated gold nanoparticle adhesion for visual colorimetric detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {295},
number = {},
pages = {118319},
pmid = {41406662},
issn = {1873-4235},
support = {U01 CA269147/CA/NCI NIH HHS/United States ; },
mesh = {*Colorimetry/methods ; *Gold/chemistry ; *Biosensing Techniques/methods ; Humans ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; *Papillomavirus Infections/diagnosis/virology ; *DNA, Viral/isolation &amp; purification/genetics/analysis ; *Papillomaviridae/isolation &amp; purification/genetics ; Female ; Limit of Detection ; DNA, Single-Stranded/chemistry ; },
abstract = {While the aggregation behavior of gold nanoparticles (AuNPs) has been extensively studied in biosensing, catalysis, and nanomedicine, their potential for programmable surface adhesion via tunable surface chemistry remains largely untapped. Here, a programmable CRISPR-mediated hydrophobic adhesion phenomenon using streptavidin-coated AuNPs functionalized with Cy5-ssDNA-biotin probes is introduced. Hydrophobic Cy5 moieties on the AuNP surface induce localized aggregation and strong adhesion to hydrophobic surfaces. This unique behavior was leveraged by coupling CRISPR-Cas12a-mediated ssDNA cleavage with Cy5-labeled ssDNA-coated AuNPs to develop a simple, visual-readout colorimetric assay for nucleic acid detection. When combined with recombinase polymerase amplification, the method achieved ultrasensitive detection of human papillomavirus (HPV) DNA down to 10 aM, without the need for complex instrumentation. The platform's clinical utility was validated by detecting HPV DNA in cervical swab samples, highlighting its promise for low-cost, sensitive, and accessible point-of-care diagnostics in resource-limited settings. Unlike conventional aggregation-dispersion systems, this platform introduces a fundamentally distinct signal transduction mechanism based on surface adhesion, defining a new modality within CRISPR-based colorimetric diagnostics and offering a simple, low-cost solution for point-of-care testing.},
}
@article {pmid41402283,
year = {2025},
author = {Sivanandan, S and Leitmann, B and Lubeck, E and Sultan, MM and Stanitsas, P and Ranu, N and Ewer, A and Mancuso, JE and Phillips, ZF and Kim, A and Bisognano, JW and Cesarek, J and Ruggiu, F and Feldman, D and Koller, D and Sharon, E and Kaykas, A and Salick, MR and Chu, C},
title = {A pooled Cell Painting CRISPR screening platform enables de novo inference of gene function by self-supervised deep learning.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {77},
pmid = {41402283},
issn = {2041-1723},
mesh = {*Deep Learning ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Regulatory Networks ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Pooled CRISPR screening enables large-scale interrogation of gene functions but typically measures simple phenotypes such as fitness. High-content methods like Perturb-seq extend dimensionality to transcriptomics but are costly and limited in scope. Optical pooled screening (OPS) combines pooled CRISPR screening with imaging to yield scalable, information-rich readouts, yet existing implementations remain pathway-specific. Here we describe an OPS-compatible Cell Painting platform that enables hypothesis-free reverse genetic screening through multiplexed morphological profiling. We validate this technique using a well-defined morphological gene set, compare classical image analysis to self-supervised learning methods using a mechanism-of-action library, and perform discovery screening with a druggable genome library. By combining rich morphological data with deep learning, gene networks emerge without the need for target-specific biomarkers, leading to unbiased discovery of gene functions.},
}
@article {pmid41381248,
year = {2026},
author = {Adamopoulos, PG and Athanasopoulou, K and Scorilas, A},
title = {A versatile type VI CRISPR-based approach for targeted m[6]A demethylation in mRNAs.},
journal = {Genome research},
volume = {36},
number = {1},
pages = {169-182},
pmid = {41381248},
issn = {1549-5469},
mesh = {Humans ; *RNA, Messenger/genetics/metabolism ; *Adenosine/analogs &amp; derivatives/metabolism ; HeLa Cells ; AlkB Homolog 5, RNA Demethylase/genetics/metabolism ; Demethylation ; *CRISPR-Cas Systems ; *RNA Processing, Post-Transcriptional ; },
abstract = {Epitranscriptomics, a rapidly evolving field mainly driven by massive parallel sequencing technologies, explores post-transcriptional RNA modifications. N [6]-methyladenosine (m[6]A) has emerged as the most prominent and dynamically regulated modification in human mRNAs, being implicated in the regulation of diverse biological processes, including spermatogenesis, heat shock response, ultraviolet-induced DNA damage response and maternal mRNA clearance. Despite the recognized significance of m[6]A in mRNA regulation, limited studies have focused on the targeted and efficient manipulation of this modification in mRNAs. Here, we present Dem6A-Vec, an "all-in-one" plasmid vector designed for site-specific m[6]A demethylation in human mRNAs. Dem6A-Vec integrates the expression of a catalytically inactive RfxCas13d fused to the m[6]A demethylase ALKBH5 and a U6-driven customizable guide RNA in a single construct, simplifying experimental workflows and enhancing targeting efficiency. Using nanopore direct RNA sequencing, we identify high-confident m[6]A sites in HeLa cells, which serve as targets for Dem6A-Vec. We validate the targeted demethylation of m[6]A sites in the EEF2 and RRAGA genes using the established SELECT-qPCR method, confirming the impacts on mRNA stability and highlighting the tool's precision and versatility. The presented approach is implemented in multiple mRNA sites with diverse methylation stoichiometries, underscoring its adaptability to various transcriptomic contexts. This study provides a robust and scalable method for investigating the functional roles of m[6]A modifications, offering a transformative platform for advancing epitranscriptomic research and potential therapeutic applications.},
}
@article {pmid41380236,
year = {2026},
author = {Yin, Z and Yin, H and Zhou, Y and Liu, H},
title = {CRISPR/Cas13a-mediated photoelectrochemical-colorimetric dual-mode biosensor for RNA N-acetyltransferase 10 detection based on Bi2O2S/Ag2S and laccase-like nanozyme.},
journal = {Biosensors &amp; bioelectronics},
volume = {295},
number = {},
pages = {118298},
doi = {10.1016/j.bios.2025.118298},
pmid = {41380236},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Laccase/chemistry ; Electrochemical Techniques/methods ; Colorimetry/methods ; *Acetyltransferases/isolation &amp; purification/analysis/chemistry ; Humans ; CRISPR-Cas Systems ; Limit of Detection ; Bismuth/chemistry ; },
abstract = {N-Acetyltransferase 10 (NAT10) is a crucial protein that catalyzes RNA acetylation modification and plays a significant role in biological activities. Accurate detection of NAT10 is of great importance in clinical testing and drug development. To achieve this goal, a novel biosensing platform was developed for NAT10 detection, relying on an RNA acetylation-inhibited CRISPR/Cas13a system, a Bi2O2S/Ag2S type-II heterojunction, and a laccase-mimetic nanocatalyst, using a photoelectric-colorimetric dual-mode strategy. Based on the catalysis effect of laccase-mimetic nanocatalyst, its substrates (hydroquinone and 2,4-dichlorophenol) were oxidized to form p-benzoquinone and 2,4-dichloroquinone, respectively. This oxidation process not only impaired the electron-donating ability of hydroquinone but also induced the coupling of 2,4-dichloroquinone with 4-antipyrine to generate a red product-enabling both photoelectrochemical and colorimetric detection of the NAT10 protein. The biosensor exhibited wide linear ranges of 0.1-1000 μg/L (photoelectrochemical mode) and 1-1000 μg/L (colorimetric mode), and the low detection limit of 0.056 and 0.348 μg/L for the two modes, respectively. Additionally, this biosensor was used to investigate the inhibitory effects of plasticizers and phosphorus-containing flame retardants on NAT10. Furthermore, molecular docking simulations were employed to explore the underlying inhibition mechanism. The developed biosensor not only provided a novel detection technique for NAT10, but also offers an alternative method for NAT10 inhibitor screening and a new tool for evaluating the ecotoxicological effects of organic pollutants.},
}
@article {pmid41330302,
year = {2026},
author = {Xu, W and Lin, Y and Huang, Z and Li, Y and Lu, Y and Liu, M and Cui, S and Zhang, T and Shi, N and Sheng, Y and Hu, J},
title = {Split proximity circuit initiated CRISPR-Cas12a system profiling exosomal surface proteins for early cancer detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {295},
number = {},
pages = {118280},
doi = {10.1016/j.bios.2025.118280},
pmid = {41330302},
issn = {1873-4235},
mesh = {Humans ; *Exosomes/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Breast Neoplasms/diagnosis/blood/genetics ; *Early Detection of Cancer/methods ; Female ; Epithelial Cell Adhesion Molecule/genetics/isolation &amp; purification ; *Biomarkers, Tumor/genetics/blood ; Limit of Detection ; Mucin-1/genetics ; Aptamers, Nucleotide/chemistry ; },
abstract = {Early diagnosis of breast cancer is critical for improving prognosis, but traditional methods have limitations. Herein, we propose an SPC-CRISPR system for the sensitive and specific detection of multiple breast cancer exosomal proteins without prior exosome isolation. This system couples CRISPR system with an enzyme-free amplification method to achieve dual-signal amplification. SPC-CRISPR is based on a split proximity circuit (SPC) that triggers catalytic hairpin assembly (CHA), converting protein signals on the surface of exosomes into nucleic acid signals, and the CRISPR-Cas12a system enabling further signal amplification and output. The system targets phosphatidylserine (PS), MUC1, and EpCAM on exosomes: Tim4-modified magnetic beads capture PS-expressing exosomes, and dual-aptamers recognize MUC1 and EpCAM, enabling SPC assembly and subsequent amplification. In buffer and cell-derived exosomes, the SPC-CRISPR system showed a detection limit of 10 particles/μL (R[2] = 0.990). Clinical tests utilizing merely 1 μL of serum samples successfully distinguished breast cancer patients from healthy donors (AUC = 0.9778, accuracy = 91.23 %), detected stage 0 breast cancer patients against healthy controls (accuracy = 92.59 %), and differentiated metastatic from non-metastatic cases (p < 0.001). The combination of high sensitivity, minimal sample requirements, and an exosome isolation-free workflow positions the SPC-CRISPR system as a promising tool for the clinical early detection and classification of breast cancer, with broader applicability to other cancers by swapping the corresponding aptamers.},
}
@article {pmid41318543,
year = {2025},
author = {Pernaci, C and Johnson, A and Gillette, S and Warden, AS and McCormick, C and Weiser-Novak, S and Ramirez, G and Broersma, EH and Mishra, P and Sivakumar, A and Cherqui, S and Coufal, NG},
title = {Microgliopathy as a primary mediator of neuronal death in models of Friedreich's Ataxia.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {81},
pmid = {41318543},
issn = {2041-1723},
support = {TRAN1-13983//California Institute for Regenerative Medicine (CIRM)/ ; EDUC2-08388//California Institute for Regenerative Medicine (CIRM)/ ; R01NS135162//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {*Friedreich Ataxia/pathology/genetics/metabolism ; Animals ; *Microglia/pathology/metabolism ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Mice ; Frataxin ; Disease Models, Animal ; Iron-Binding Proteins/genetics/metabolism ; Cell Death ; Mitochondria/metabolism/pathology ; *Neurons/pathology/metabolism ; Purkinje Cells/pathology/metabolism ; Trinucleotide Repeat Expansion ; Iron/metabolism ; Male ; Female ; White Matter/pathology ; CRISPR-Cas Systems ; },
abstract = {Friedreich's ataxia (FRDA) is an incurable neurodegenerative disorder caused by a GAA repeat expansion in the frataxin (FXN) gene, leading to a severe reduction of the mitochondrial FXN protein, crucial for iron metabolism. While microglial inflammation is observed in FRDA, it remains unclear whether immune dysfunction is a primary disease mediator or a secondary reactionary phenotype. Utilizing patient-derived induced pluripotent stem cells (iPSCs), we report an intrinsic microglial phenotype of stark mitochondrial defects, iron overload, lipid peroxidation, and lysosomal abnormalities. These factors drive a pro-inflammatory state that contributes to neuronal death in co-culture systems. In a murine xenograft model, transplanted human FRDA microglia accumulate in white matter and the Purkinje cell layer, resulting in Purkinje neuron loss in otherwise healthy brains. Notably, CRISPR/Cas9-mediated correction of the GAA repeat reverses microglial defects and mitigates neurodegeneration. Here, we suggest that microglial dysfunction serve as a disease driver and a promising therapeutic target in FRDA.},
}
@article {pmid41277417,
year = {2026},
author = {Choi, MY and Li, C and Choi, JH and Choi, JW},
title = {Intracellular biosensors by functional nanomaterial-integrated CRISPR technologies for real-time molecular sensing.},
journal = {Chemical communications (Cambridge, England)},
volume = {62},
number = {1},
pages = {58-70},
doi = {10.1039/d5cc05016b},
pmid = {41277417},
issn = {1364-548X},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Nanostructures/chemistry ; Gene Editing ; Nanotechnology ; },
abstract = {CRISPR technology, originally developed as a genome-editing tool, has recently emerged as a powerful platform for intracellular biosensing. By harnessing the programmability and target specificity of CRISPR-associated proteins, such as Cas9, Cas12, and Cas13, researchers have engineered biosensors capable of detecting a wide array of intracellular signals, including nucleic acids, non-coding RNAs, and small-molecule metabolites. This review discusses the recent advancements in CRISPR-powered biosensors for real-time, dynamic monitoring of cellular processes and molecular events. Particular focus is given to the integration of nanotechnology, which plays a crucial role in enhancing the delivery efficiency, signal amplification, and sensor stability. Nanomaterials such as gold nanoparticles, quantum dots, DNA nanostructures, and upconversion nanoparticles have been strategically employed to improve the intracellular transport of CRISPR components, facilitate signal readouts, and enable multimodal sensing in complex cellular environments. Additionally, we delve into how CRISPR-nanotechnology hybrids can be adapted for multiplex analysis and single-cell resolution. This review also addresses the current challenges in intracellular biosensing, including precise delivery, biocompatibility, and long-term monitoring, and outlines future directions for the application of these systems in precision medicine, synthetic biology, and advanced therapeutic monitoring. Through the convergence of gene-editing systems and nanotechnology, CRISPR-based intracellular biosensors are anticipated to revolutionize next-generation diagnostic and therapeutic strategies.},
}
@article {pmid41261774,
year = {2026},
author = {Sun, W and Zhu, Y and Zhang, X and Meng, X and Xia, K and Zhang, X and Han, X and Pan, W and Guo, J and Li, J and Zhang, H},
title = {A mortise-tenon joint system facilitates precise targeted DNA insertion and replacement in rice.},
journal = {Molecular plant},
volume = {19},
number = {1},
pages = {36-47},
doi = {10.1016/j.molp.2025.11.006},
pmid = {41261774},
issn = {1752-9867},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *Mutagenesis, Insertional/methods ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; DNA, Plant/genetics ; },
abstract = {Precise and scarless DNA insertion and replacement represent two of the major challenges in plant genome editing. Numerous tools have been developed, including homology-directed repair-dependent tools and prime-editing-mediated systems. However, none has fully resolved these challenges. In this study, we develop the "mortise-tenon joint system" (MT), a novel strategy that enables precise and efficient targeted DNA insertion and replacement. By leveraging the APOBEC-Cas9-uracil DNA glycosylase/(apurinic or apyrimidinic site) lyase within our previously reported APOBEC-Cas9 fusion-induced deletion system, which performs single-strand cleavage on the non-target strand and double-strand cleavage on the target strand, we generated a unique "mortise" structure, consisting of double-strand breaks with single or double non-complementary 5' overhangs. We further designed "tenons," double-stranded DNA donors containing 5' sticky ends precisely matching the 5' overhangs of the mortises. The end-capture interaction between mortises and tenons facilitates precise targeted insertion and replacement, achieving frequencies of 16.30%-59.47% across seven tested targets using 21-85 bp donor inserts in rice. If long DNA donors with sticky ends complementary to the mortise structure can be generated, the MT system may enable highly precise targeted insertion and replacement of large DNA fragments.},
}
@article {pmid41148632,
year = {2026},
author = {Jin, Z and Yang, Y and Chen, C and Zhang, Z and Ren, Q and Cui, Z and Huang, CZ and Zuo, H},
title = {Gene silencing regulated by aggregates of Corn aptamer at 3' UTR of mRNA.},
journal = {Nanoscale horizons},
volume = {11},
number = {1},
pages = {232-242},
doi = {10.1039/d5nh00510h},
pmid = {41148632},
issn = {2055-6764},
mesh = {*RNA, Messenger/genetics/chemistry/metabolism ; *3' Untranslated Regions/genetics ; *Gene Silencing ; *Aptamers, Nucleotide/chemistry/genetics ; Humans ; CRISPR-Cas Systems ; *Zea mays/genetics/chemistry ; },
abstract = {Gene therapy, as a cutting-edge approach for disease intervention, relies heavily on advancements in gene silencing techniques. For instance, CRISPR-Cas9 has emerged as a leading gene-editing tool due to its ability to introduce precise cuts at specific genomic loci, enabling targeted gene insertion, deletion, or modification. In this study, we developed a simple and effective gene silencing strategy by introducing a nucleic acid self-assembly module into the 3' untranslated region (UTR) of mRNA. This module demonstrated significant gene silencing efficacy in eukaryotic cells through the formation of RNA aggregates. To systematically investigate its regulatory mechanism on translation efficiency through the formation of higher-order RNA structures, we quantitatively analyzed both mRNA and protein expression levels. Furthermore, our modular 3' UTR sequences can be integrated with classical 5' UTR elements (e.g., TOP sequences) to construct a multidimensional post-transcriptional regulatory network. This technology expands the diversity of existing UTR element libraries and offers a reservoir of programmable regulatory elements for applications in synthetic biology. It enables the construction of orthogonal combinations of multidimensional elements, tailored to specific gene expression regulation needs.},
}
@article {pmid41121528,
year = {2026},
author = {Horsley, N and Sythoff, AVB and Delgado, M and Liu, S and Cabernard, C},
title = {CrisprBuildr: an open-source application for CRISPR-mediated genome engineering in Drosophila melanogaster.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {1},
pages = {},
pmid = {41121528},
issn = {2160-1836},
support = {R35GM148160/NH/NIH HHS/United States ; },
mesh = {Animals ; *Drosophila melanogaster/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Software ; *Genome, Insect ; *Genetic Engineering/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR/Cas9 is a powerful tool for targeted genome editing experiments. Using CRISPR/Cas9, genes can be deleted or modified by inserting specific DNA sequences, encoding for fluorescent proteins, small peptide tags, or other modifications. Such experiments are essential for detailed gene and protein characterization. However, designing and cloning the corresponding constructs can be repetitive, time-consuming, and laborious. To assist users in CRISPR/Cas9-based genome engineering, we developed CrisprBuildr, an open-source, web-based application for designing modifications to their target genes. CrisprBuildr guides users through creating guide RNAs and repair template vectors to generate cloning maps. The application is designed for the Drosophila melanogaster genome but can serve as a template for other available genomes. We also created new tagging vectors using EGFP and mCherry combined with the small peptide SspB-Q73R for use in iLID-based optogenetic experiments.},
}
@article {pmid41477711,
year = {2025},
author = {Rozov, SM and Deineko, EV},
title = {[Recombinase-Based Engineering of Plant Genomes in the Era of Genome Editing].},
journal = {Molekuliarnaia biologiia},
volume = {59},
number = {6},
pages = {873-890},
doi = {10.7868/S3034555325060016},
pmid = {41477711},
issn = {0026-8984},
mesh = {*Gene Editing/methods ; *Genome, Plant ; *CRISPR-Cas Systems ; *DNA Nucleotidyltransferases/genetics/metabolism ; *Recombinases/genetics/metabolism ; *Plants/genetics ; DNA Transposable Elements ; },
abstract = {The rapidly evolving CRISPR/Cas-based genome editing technologies, which have dominated nearly all areas of molecular biology over the past decade, still face several unresolved challenges. One of the major limitations of current genome editing tools is the low efficiency of targeted long-sequence insertions. This issue is particularly critical in plant systems, where genome editing efficiency is hindered by specific cellular characteristics. Site-specific recombinases (SSRs), which have long been employed in genetic engineering to mediate various genomic rearrangements-including deletions, duplications, insertions, and inversions-are limited in their application by the requirement for preexisting recombination recognition sites in the genome. However, CRISPR/Cas and recombinase tools complement each other, and their combined use offers a powerful strategy to overcome key limitations of genome editing. The discovery of CRISPR-associated transposons such as CAST and OMEGA, which naturally utilize their own recombinases, marks a significant advance in genome engineering, providing an elegant example of the natural convergence between CRISPR and recombinase technologies.},
}
@article {pmid41475352,
year = {2025},
author = {Martinho, C and Hoshino, M and Raphalen, M and Bukhanets, V and Kerur, A and Bogaert, KA and Luthringer, R and Coelho, SM},
title = {Efficient CRISPR-Cas genome editing in brown algae.},
journal = {Cell reports methods},
volume = {},
number = {},
pages = {101273},
doi = {10.1016/j.crmeth.2025.101273},
pmid = {41475352},
issn = {2667-2375},
abstract = {Brown algae represent the third most complex lineage to have independently evolved multicellularity, distinct from plants and animals. Yet, functional studies of their development and evolution have been limited by the absence of efficient genome editing tools. Here, we present a robust, high-efficiency, and transgene-free CRISPR-based genome editing platform applicable across four ecologically and biotechnologically important brown algal species. Using Ectocarpus as a model, we optimized a polyethylene glycol (PEG)-mediated ribonucleoprotein (RNP) delivery system that achieves reproducible editing across multiple loci without cloning or specialized equipment. As proof of concept, we recreated the hallmark imm mutant phenotype by precisely editing the IMMEDIATEUPRIGHT (IMM) locus. APT/2-fluoroadenine (2-FA) selection further enhanced specificity with minimal false positives. The method was easily transferable to other species, including kelps. This platform now enables functional genomics in brown algae, providing powerful tools for investigating development, life cycle regulation, and the independent evolution of complex multicellularity.},
}
@article {pmid41475348,
year = {2025},
author = {Gao, X and Zhu, K and Zhang, W and Wang, L and Wang, L and Hua, L and Niu, T and Qin, B and Yu, X and Zhu, H and Cui, S},
title = {RNA anti-CRISPRs deplete Cas proteins to inhibit the CRISPR-Cas system.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2025.12.005},
pmid = {41475348},
issn = {1097-4164},
abstract = {RNA-based anti-CRISPRs (Racrs) interfere with the type I-F CRISPR-Cas system by mimicking the repeats found in CRISPR arrays. Here, we determined the cryo-electron microscopy (cryo-EM) structures of the type I-F crRNA-guided surveillance complex (Csy complex) from Pectobacterium atrosepticum and three RacrIF1-induced aberrant subcomplexes. Additionally, we observed that Cas7f proteins could bind to non-specific nucleic acids, forming right-handed superhelical filaments composed of different Cas7 copies. Mechanistically, RacrIF1 lacks the specific S-conformation observed in the corresponding position of the 5' handle in canonical CRISPR complexes, and it instead adopts a periodic "5 + 1" pattern. This conformation creates severe steric hindrance for Cas5f-Cas8f heterodimer and undermines their binding. Furthermore, Cas7f nonspecifically binds nucleic acids and can form infinite superhelical filaments along Racrs molecules. This oligomerization sequesters Cas6f and Cas7f from binding, therefore blocking the formation of functional CRISPR-Cas effector complexes and ultimately blocking antiviral immunity. Our study provides a structural basis underlying Racrs-mediated CRISPRs inhibition.},
}
@article {pmid41475278,
year = {2025},
author = {Kolanchi, P and Saminathan, N and Selvaraj, D and Krishnamoorthy, A and Palanivelu, K and Aruchalam, A},
title = {Bioprocess and genetic advances enhancing Beauveria bassiana biocontrol efficacy.},
journal = {Microbial pathogenesis},
volume = {211},
number = {},
pages = {108272},
doi = {10.1016/j.micpath.2025.108272},
pmid = {41475278},
issn = {1096-1208},
abstract = {Beauveria bassiana is a widely exploited entomopathogenic fungus that has emerged as a central component of ecologically sustainable pest management. Recent years have witnessed rapid progress across its biological understanding, technological development, and application potential. This review synthesizes contemporary advances spanning infection biology, secondary metabolite biosynthesis, strain development, bioprocess engineering, formulation science, and genetic improvement. At the molecular level, multi-omics studies have elucidated the coordinated regulation of surface adhesion, cuticular penetration, host immune modulation, dimorphic transitions, and toxin production, revealing gene networks that govern virulence, stress tolerance, and ecological adaptation. These insights have informed improved strategies for strain isolation and high-throughput phenotypic screening, enabling the selection of isolates with enhanced pathogenicity, environmental robustness, and endophytic competence. Parallel advances in solid-state and submerged fermentation, supported by agro-industrial substrates and data-driven optimization, have strengthened large-scale production of infective propagules with consistent quality. Such gains are further reinforced by modern formulation approaches, including oil-based dispersions, encapsulation systems, nanoemulsions, and seed-coating technologies, which collectively improve spore stability, persistence, and delivery under heterogeneous field conditions. More recently, CRISPR/Cas-based genome editing and pathway engineering have opened new avenues for precision enhancement of virulence traits, metabolic output, and abiotic stress resilience. Despite these achievements, the broader adoption of B. bassiana remains constrained by variable field performance, slower speed of action relative to chemical insecticides, strain-dependent efficacy, and regulatory and quality-control challenges. By integrating fundamental biology with technological innovation and practical limitations, this review provides a coherent framework for advancing B. bassiana from laboratory optimization to reliable field implementation, underscoring its promise as a next-generation, environmentally aligned biocontrol agent in modern agriculture.},
}
@article {pmid41474879,
year = {2025},
author = {Cheng, M and Wang, Y and Lin, W and Ye, J and Wu, M and Xiang, B and Liu, L and Sun, B},
title = {A Universal Light-Activated CRISPR-RNA Based on Split Direct Repeat for One-Pot Cas12a Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c05722},
pmid = {41474879},
issn = {1520-6882},
abstract = {Spatiotemporal regulation of CRISPR-Cas systems holds significant promise for precision gene editing and molecular diagnostics. While photochemical strategies for CRISPR activity control have advanced, a universal regulatory approach remains elusive. Here, we report a modular light-activated CRISPR-RNA design through splitting conventional crRNA within the direct repeat (DR) into two functional domains: a conserved 5' split direct repeat (5' SDR) and a variable 3' split direct repeat (SDR) + spacer (3' SDR-Spacer) module. Double-stranded extensions were introduced at the cleavage site to preserve functional integrity. Through screening of light-sensitive caging group modification sites in the universal 5' SDR, a novel light-activated CRISPR-RNA system was developed. This system only requires spacer redesign of the 3' SDR-Spacer for new targets, while the caged 5' SDR is universal. Thereupon, we established a universal light-activated CRISPR-RNA assisted one-pot RAA-Cas12a detection system (UniLight-CRISPR). When applied to Mycoplasma pneumoniae detection using qPCR-validated clinical samples, UniLight-CRISPR demonstrated 95.45% sensitivity and 100% specificity, matching the performance of conventional two-step Cas12a assays. This universal photo regulation strategy not only addresses current limitations in CRISPR diagnostics but also provides a blueprint for adapting other Cas enzymes. We anticipate broad applications of our universal light-activated CRISPR-RNA system, extending from molecular diagnostics to gene-editing research.},
}
@article {pmid41048051,
year = {2026},
author = {Huang, LT and Gao, RJ and Zhang, D and Nian, C and Martzke, W and Shapiro, AMJ and Kin, T and Tahamtani, Y and Lynn, FC},
title = {Truncated CD19 as a selection marker for the isolation of stem cell-derived β-cells.},
journal = {Disease models &amp; mechanisms},
volume = {19},
number = {1},
pages = {},
doi = {10.1242/dmm.052376},
pmid = {41048051},
issn = {1754-8411},
support = {5-SRA-2020-1059-S-B, 3-COE-2022-1103-M-B//Juvenile Diabetes Research Foundation United States of America/ ; ASD-173663//Institute of Nutrition, Metabolism and Diabetes/ ; 5238 BIOM//Michael Smith Health Research BC/ ; IGAP//BC Children's Hospital/ ; 5-SRA-2020-1059-S-B//Breakthrough T1D Canada/ ; 3-COE-2022-1103-M-B//Breakthrough T1D Canada/ ; ASD-173663/CAPMC/CIHR/Canada ; //BC Children's Hospital/ ; 6563//National Science and Engineering Research Council of Canada/ ; },
mesh = {Humans ; *Insulin-Secreting Cells/cytology/metabolism ; *Antigens, CD19/metabolism ; Biomarkers/metabolism ; *Cell Separation/methods ; Human Embryonic Stem Cells/cytology/metabolism ; CRISPR-Cas Systems/genetics ; Insulin/metabolism ; C-Peptide/metabolism ; Cell Differentiation ; },
abstract = {Stem cell-derived β-cells (SCβ-cell) are a renewable and scalable alternative to cadaveric islets as a cell-replacement therapy for type 1 diabetes (T1D). However, heterogeneity within SCβ-cell cultures remains problematic for graft safety and function. Magnetic selection of SCβ-cells expressing a unique cell-surface marker may help deplete undesirable cell types and facilitate functional maturation. Here, we explored the transmembrane glycoprotein CD19 as a potential cell-surface marker for the enrichment of insulin-expressing SCβ-cells. Using CRISPR/Cas9 technology, we created a knock-in add-on of CD19-mScarlet downstream of insulin (INS) coding sequence exon 2 in human embryonic stem cells (hESCs). We developed and optimized a magnetic-activated cell sorting protocol for CD19-mScarlet-expressing cells, forming enriched SCβ-cell clusters with improved glucose-stimulated C-peptide secretion. This strategy holds promise to facilitate large-scale production of functional SCβ-cells for disease modeling and cell-replacement therapy.},
}
@article {pmid41316351,
year = {2025},
author = {Qin, Z and Wang, Y and Sun, M and Wang, Q and Duan, J and Gu, C and Zhang, X and Yu, F and Wu, Y and Xu, H and Li, J and Zhang, L},
title = {Development of a field-deployable RPA-CRISPR/Cas12a assay for the detection of Cyclospora cayetanensis in human feces.},
journal = {Parasites &amp; vectors},
volume = {19},
number = {1},
pages = {4},
pmid = {41316351},
issn = {1756-3305},
support = {232102110088//Henan Province Scientific and Technological Project/ ; 2023YFD1801200//National Key Research and Development Program of China/ ; 231111111500//Key Research and Development Project of Henan Province/ ; },
mesh = {*Cyclospora/isolation &amp; purification/genetics ; Humans ; *Feces/parasitology ; *Cyclosporiasis/diagnosis/parasitology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases/genetics/metabolism ; Biosensing Techniques/methods ; *Molecular Diagnostic Techniques/methods ; },
abstract = {BACKGROUND: Cyclospora is an emerging intestinal pathogenic protozoan transmitted through foodborne and waterborne routes. At least 19 countries in the world have recorded outbreaks of cyclosporiasis, mainly associated with the consumption of contaminated fresh agricultural products. The lack of a sensitive immediate test is one of the major obstacles to the rapid diagnosis of cyclosporiasis. The target interference mechanisms of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have been adapted into versatile and efficient genome manipulation and disease-curing technologies, while also being promising for point-of-care testing (POCT) applications. It can serve as an excellent rapid and specific detection tool.<br><br>METHODS: The recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system were combined to develop a detection method for C. cayetanensis (termed RECCT-Cay) via visual observation of fluorescent readings under blue light and field diagnosis using lateral flow strip (LFS) biosensors.<br><br>RESULTS: The detection limit of the established RECCT-Cay was 7 copies/&#x3bc;L. Under simulated clinical conditions, the detection limit was 30 oocysts per gram of stool. At the same time, the established detection platform can distinguish C. cayetanensis from the closely related Eimeria spp. The results of our constructed assay were compared with nested PCR, and the detection results of 30 clinical stool samples were consistent, with three samples positive for C. cayetanensis. Based on the RECCT-Cay detection principle, a portable suitcase-sized device has been designed, which can conduct rapid on-site detection of clinical samples.<br><br>CONCLUSIONS: The RECCT-Cay platform features rapid speed, high sensitivity, and the capability for field detection, making it a promising tool for use in remote areas.},
}
@article {pmid41474623,
year = {2025},
author = {Banh, DV and Goldberg, GW and Marraffini, LA},
title = {Phage induction of Staphylococcus aureus pathogenicity islands promotes the CRISPR-Cas adaptive immune response.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116776},
doi = {10.1016/j.celrep.2025.116776},
pmid = {41474623},
issn = {2211-1247},
abstract = {Staphylococcus aureus pathogenicity islands (SaPIs) are mobile genetic elements carrying virulence genes that spread upon infection by helper phages that induce their transfer. Staphylococci also carry type II and III CRISPR-Cas systems that mount an adaptive immune response against phages through the acquisition of spacer sequences from viral genomes, directing Cas nucleases to their targets. Whether and how SaPIs and CRISPR interact with each other during helper phage infection is not known. Here we report that, as a result of the packaging of incomplete helper phage genomes into SaPI particles, defective viral DNA delivered into new hosts stimulates spacer acquisition in both CRISPR types. Once immunized, staphylococci target the helper phage and prevent SaPI mobilization. Our work reveals an unexpected synergy between CRISPR-Cas systems and SaPIs that enhances antiphage immunity and could favor the retention of beneficial elements within the population.},
}
@article {pmid41474367,
year = {2026},
author = {Rasool, HMH and Gong, X and Jin, Y and Liu, M and Yanyan, W and Qu, M and Anxiang, Z and Lingling, X and Li, Z and Zhou, J and Chen, Q},
title = {Development of Visual and Fluorescence Detection Method of Brucella by RPA-CRISPR/Cas12a Assay.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {1},
pages = {e71397},
doi = {10.1096/fj.202503610R},
pmid = {41474367},
issn = {1530-6860},
support = {2022YFC2304001//MOST | National Key Research and Development Program of China (NKPs)/ ; 2024BEG-02029//The Key Research and Development Program of Ningxia Hui Autonomous Region/ ; 2024312885//Comprehensive Control Technology For Brucellosis and Hydatic Disease in Xinjiang/ ; CAAS-ZDRW202410//The Agricultural Science and Technology Innovation Program (ASTIP)/ ; },
mesh = {*Brucella/genetics/isolation &amp; purification ; Animals ; *Brucellosis/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; Sensitivity and Specificity ; Cattle ; Fluorescence ; Limit of Detection ; Humans ; },
abstract = {Brucella is a significant pathogen in the livestock industry, causing Brucellosis, a zoonotic disease that leads to considerable health and economic losses in both humans and animals. Current diagnostic methods for Brucella, including culture, serological assays, and PCR/qPCR, are valuable tools but have inherent limitations. These include the requirement for BSL-3 laboratories, trained personnel, complex procedures, expensive equipment, issues with sensitivity and specificity, and the time-consuming nature of assays, making them unsuitable for large-scale epidemiological screening. Therefore, there is a critical need to develop a rapid, portable, and cost-effective diagnostic method with high specificity and sensitivity. In this study, we established a rapid, portable, reliable, and inexpensive detection method for Brucella genus identification based on RPA-CRISPR/Cas12a technology. Specific RPA primers and crRNA sequences were designed targeting the bcsp31 gene of Brucella. Subsequently, both a fluorescence assay and a lateral flow strip (LFS) assay were developed after optimizing the conditions using the RPA-CRISPR/Cas12a system. The limit of detection (LoD) was 1 copy/μL for RPA-CRISPR/Cas12a-F and 10 copies/μL for RPA-CRISPR/Cas12a-LFS and the entire assay was completed in less than 30 min. This method demonstrated excellent specificity in distinguishing Brucella from other closely related pathogens. Moreover, the RPA-CRISPR/Cas12a assay showed high concordance with classical quantitative real-time PCR when testing diverse clinical samples (blood, serum, milk, semen, vaginal secretions). Together, these findings make this method a promising tool for Brucella detection, with potential applications in both field surveillance and clinical diagnostics.},
}
@article {pmid41472258,
year = {2025},
author = {Gladue, DP and O'Mahony, A},
title = {CRISPR Treatments for AI-Designed Synthetic Viruses: Rapid Programmable Countermeasures for Emerging and Engineered Viruses.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472258},
issn = {1999-4915},
mesh = {Humans ; *CRISPR-Cas Systems ; Genome, Viral ; *Artificial Intelligence ; *Synthetic Biology/methods ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Viruses/genetics ; Animals ; Genetic Engineering/methods ; },
abstract = {The convergence of artificial intelligence and synthetic biology is innovating and accelerating the design of novel viral genomes, expanding both therapeutic opportunities and dual-use risk. This review articulates a countermeasure strategy for emerging and engineered viruses leveraging the programmable CRISPR modality. Building on mounting in vitro and in vivo evidence that Cas9 degrades DNA viruses (e.g., Orthopoxviruses, HSV-1, ASFV), while Cas13 targets RNA viral genomes (e.g., Influenza A, Dengue, RSV), both leading to reduced viremia, diminished disease burden, and alleviated symptoms. Here, we outline a rapid-response pipeline to position CRISPR-based countermeasures in translational and pandemic-response frameworks, linking real-time sequencing to AI-assisted gRNA selection and multiplexed cassette design to achieve viral targeting efficacy. To minimize resistance and off-target risk, we emphasize multi-gRNA cocktails, continuous genomic surveillance, and adaptive gRNA rotation. We also propose governance mechanisms, such as pre-cleared gRNA repositories, transparent design logs, standardized off-target/safety screening, and alignment with evolving nucleic-acid-synthesis screening frameworks to enable emergency deployment while preserving security. Furthermore, compressing the time from sequence to treatment and complementary to vaccines and small-molecule antivirals, CRISPR represents a technologically agile and strategically essential capability to combat both natural outbreaks and AI-enabled biothreats. Collectively, programmable CRISPR antivirals represent an auditable, rapidly adaptable foundation for next-generation biodefense preparedness.},
}
@article {pmid41472250,
year = {2025},
author = {Tan, C and Xing, S and Xie, X and Chen, X and Liu, X and Wang, W and Liu, L and Zhou, X and Wu, J and Li, C},
title = {Development and Application of a Rapid Field Detection Technology for DENV-2 Based on the HUDSON Nucleic Acid Extraction-Free/RT-RAA/CRISPR-Cas12a System.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472250},
issn = {1999-4915},
support = {2024YFC2607800//Chunxiao-Li/ ; },
mesh = {*Dengue Virus/genetics/isolation &amp; purification/classification ; *Dengue/diagnosis/virology ; *CRISPR-Cas Systems ; Humans ; Sensitivity and Specificity ; Animals ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Dengue fever has become a major global public health challenge due to its rapidly in-creasing incidence. Rapid on-site detection of dengue virus (DENV) is critical for early diagnosis, timely patient isolation, and outbreak control. In this study, dengue virus serotype 2 (DENV-2), the predominant strain circulating in tropical and subtropical regions, was selected as the target pathogen. We established a one-tube rapid detection assay that integrates the HUDSON nucleic acid extraction-free protocol, reverse transcription recombinase-aided amplification (RT-RAA), and CRISPR/Cas12a-mediated trans cleavage activity. The method achieved a detection limit of 1 × 10[2] copies/μL for simulated infected samples and exhibited no cross-reactivity with other DENV serotypes (DENV-1, DENV-3, DENV-4) or with other arboviruses, including Zika, Japanese encephalitis, yellow fever, and chikungunya viruses. The assay demonstrated high sensitivity and specificity across various sample types, including mosquitoes, rodents, blood, and cultured cells, with results consistent with quantitative PCR (qPCR). Requiring only basic equipment such as a water bath, the system enables on-site detection of DENV-2 within 1 h. This simple, cost-effective, and reliable assay provides a practical tool for field-based DENV-2 surveillance and supports effective public health responses in resource-limited settings.},
}
@article {pmid41472204,
year = {2025},
author = {Dong, J and He, X and Bao, S and Wei, Z},
title = {Diagnostic Methods for Bovine Coronavirus: A Review of Recent Advancements and Challenges.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472204},
issn = {1999-4915},
support = {KJZC-2024-15//Gansu Provincial Department of Agriculture and Rural Affairs Science and Technology Support Project/ ; },
mesh = {Animals ; Cattle ; *Coronavirus, Bovine/genetics/isolation &amp; purification ; *Cattle Diseases/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; *Coronavirus Infections/diagnosis/veterinary/virology ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; CRISPR-Cas Systems ; },
abstract = {Bovine coronavirus(BCoV) is a significant pathogen causing substantial economic losses in the cattle industry through increased calf mortality, reduced growth performance, and decreased milk yield. Rapid and accurate diagnostic methods are therefore essential for controlling BCoV transmission. Current diagnostic methods comprise two primary categories: conventional techniques and cutting-edge innovations. Conventional approaches, including molecular methods like RT-PCR/qRT-PCR and immunological assays such as ELISA and neutralization tests, remain the main diagnostic methods. However, they are limited by laboratory dependency as well as the necessary balance between speed and sensitivity. These limitations have promoted the development of innovative methods, including isothermal amplification, CRISPR/Cas systems, droplet digital PCR, and integrated platforms. This review comprehensively analyzes the advantages, limitations, and applications of current diagnostic methods, highlighting integrated platforms such as RPA-CRISPR-LFA and microfluidics-based LFA. These innovations bridge critical performance gaps by enhancing sensitivity and specificity while enabling field application, demonstrating significant potential as next-generation point-of-care diagnostics for managing this economically critical pathogen.},
}
@article {pmid41472049,
year = {2025},
author = {Slukin, PV and Fursov, MV and Volkov, DV and Sizova, AA and Detushev, KV and Dyatlov, IA and Fursova, NK},
title = {Diversity of CRISPR-Cas Systems Identified in Urological Escherichia coli Strains.},
journal = {Microorganisms},
volume = {13},
number = {12},
pages = {},
pmid = {41472049},
issn = {2076-2607},
support = {075-15-2025-525 of 30.05.2025.//Ministry of Science and Higher Education of the Russian Federation/ ; },
abstract = {Type I-E and I-F CRISPR-Cas systems were identified in 237 E. coli strains isolated from patients with urinary tract infections (UTIs) between 2004 and 2019. The strains were classified into nine distinct groups (I-IX) based on the presence or absence of cas genes and repeat regions (RRs). Within the type I-E systems, two sequence variants were identified, distinguished by polymorphisms in the casB, cas3, cas7, cas5, and cas6 genes. The direct repeats (DRs) also differed, with I-E-associated RRs ranging from 26 to 32 bp and I-F-associated RRs consistently being 28 bp. We identified 762 unique spacers (29-35 bp in length) across the strain collection, while the number of spacers per strain varied from 1 to 47, and potential DNA targets were determined for 65 spacers, targeting 38 bacteriophage genomes, 19 plasmids, and 8 cas genes of the I-F type CRISPR-Cas system. Multilocus sequence typing (MLST) revealed 68 sequence types and 24 clonal complexes (CCs), with the most prevalent being ST131, CC10, CC69, CC405, CC14, CC38, CC73, and CC648. Significant correlations were observed between specific phylogroups/CCs, the type of CRISPR-Cas system present, and distinct profiles of virulence and antibiotic resistance genes.},
}
@article {pmid41472024,
year = {2025},
author = {Yi, M and Hu, Y and Fan, B and Pan, Y and Pan, B and Wang, J and Liu, Z},
title = {Advances in Novel Detection Technologies for Occult Hepatitis B Virus Infection: Building an Ultra-Sensitive Barrier for Transfusion Safety.},
journal = {Microorganisms},
volume = {13},
number = {12},
pages = {},
pmid = {41472024},
issn = {2076-2607},
abstract = {Occult hepatitis B virus infection (OBI), characterized by extremely low viral loads and the persistent intrahepatic presence of cccDNA, poses a profound challenge to global public health security. With a prevalence ranging from 0.06% to over 15% in different donor populations, OBI maintains a risk of transmission and can progress to hepatocellular carcinoma. Its prevention and control have long been limited by the sensitivity constraints of conventional detection methods, highlighting the urgent need for more sensitive diagnostic innovations. Emerging technologies offer distinct breakthroughs: ddPCR facilitates absolute quantification; CRISPR-Cas systems coupled with isothermal amplification enable rapid, point-of-care testing; third-generation sequencing resolves viral integration and mutations; and nanomaterials enhance the signal detection. This review synthesises advancements in OBI diagnostic technologies and provides a comparative overview of their strengths, limitations, and transfusion safety implications, as well as their potential applications in blood transfusion. Recommendations are also proposed to inform the advancement of OBI risk control in blood transfusion and to guide the development of novel diagnostic technologies, particularly relevant to regions with high HBV endemicity, such as China.},
}
@article {pmid41251053,
year = {2026},
author = {Shrestha, M and Kim, Y and Park, S and Jiang, HL and Lee, JY and Kim, D and Jeong, JH},
title = {eVLP-Mediated Cas9 Delivery for Preventing IBMIR in Islet Transplantation.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {1},
pages = {e08128},
doi = {10.1002/smll.202508128},
pmid = {41251053},
issn = {1613-6829},
support = {//National Research Foundation of Korea/ ; RS-2025-02303064//Korean government/ ; RS-2024-02507183//Ministry of Health and Welfare/ ; 22A0205L1//Ministry of Health and Welfare/ ; 23A0205L1//Ministry of Health and Welfare/ ; 21A0202L1//Ministry of Health and Welfare/ ; 21A0202L1//Ministry of Science and ICT, South Korea/ ; },
mesh = {Animals ; *Islets of Langerhans Transplantation/adverse effects/methods ; Thromboplastin/metabolism/genetics ; Plasminogen Activator Inhibitor 1/metabolism/genetics ; Rats ; Diabetes Mellitus, Experimental/therapy ; Male ; *Inflammation/prevention &amp; control ; *CRISPR-Associated Protein 9/metabolism ; Mice ; Islets of Langerhans ; *CRISPR-Cas Systems/genetics ; },
abstract = {Islet transplantation is a promising strategy for effective β-cell replacement in patients with type 1 diabetes. However, its success is hindered significantly by instant blood-mediated inflammatory reaction (IBMIR), which leads to rapid graft loss. IBMIR is triggered when the transplanted islets come in contact with blood, activating the coagulation cascade, complement pathways, and innate immune responses. Tissue factor (TF), abundantly expressed on the islet surface, initiates the coagulation cascade, leading to thrombin formation, platelet activation, and neutrophil infiltration. Plasminogen activator inhibitor-1 (PAI-1) plays a critical role in IBMIR by inhibiting fibrinolysis and causing ischemic injury in the graft. TF and PAI-1 contribute significantly to IBMIR, thus making them critical targets for genetic interventions to prevent IBMIR. In this study, an engineered virus-like particle (eVLP)-mediated Cas9 nuclease is employed to knock out TF and PAI-1 genes in rat islets. TF and PAI-1 expression are effectively downregulated without inducing any off-target effects or without compromising the viability and functionality of the islets. Streptozotocin-induced diabetic mice transplanted with TF- and PAI-1-knockout islets exhibited improved glycemic control and a significant reduction in the plasma levels of thrombin-antithrombin (TAT) complex and complement component 3a (C3a), indicating the successful inhibition of IBMIR post-transplantation.},
}
@article {pmid40899441,
year = {2026},
author = {Patel, UA and Shi, MY and Kazan, JM and Nixon, KCJ and Ran, X and Nair, SN and Huang, O and Song, L and Aparnathi, MK and He, MY and Bakhtiari, M and Krishnan, R and Hessenow, RK and Philip, V and Ketela, T and Jendrossek, V and Hakem, R and He, HH and Kridel, R and Lok, BH},
title = {CRISPR Screen Identifies HDAC3 as a Novel Radiosensitizing Target in Small Cell Lung Cancer.},
journal = {Molecular cancer therapeutics},
volume = {25},
number = {1},
pages = {183-195},
pmid = {40899441},
issn = {1538-8514},
support = {U01 CA253383/CA/NCI NIH HHS/United States ; UL1 TR000457/TR/NCATS NIH HHS/United States ; //Terry Fox Research Institute (TFRI)/ ; //Canada Foundation for Innovation (CFI)/ ; //Cancer Research Society (CRS)/ ; //Canadian Institutes of Health Research (CIHR)/ ; U01CA253383//National Cancer Institute (NCI)/ ; UL1TR00457//Clinical and Translational Science Center, Weill Cornell Medicine (CTSC)/ ; GRK 2762/1//Deutsche Forschungsgemeinschaft (DFG)/ ; },
mesh = {Humans ; Animals ; *Small Cell Lung Carcinoma/genetics/pathology/radiotherapy/drug therapy ; *Histone Deacetylases/genetics/metabolism ; Mice ; *Lung Neoplasms/genetics/pathology/radiotherapy ; *Radiation-Sensitizing Agents/pharmacology ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Radiation Tolerance/genetics/drug effects ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Acrylamides ; Phenylenediamines ; },
abstract = {Small cell lung cancer (SCLC) is an aggressive malignancy, with most patients presenting with prognostically poor extensive-stage disease. Limited progress in standard care stresses the urgent need for novel therapies. Radiotherapy offers some survival benefit for selected patients with SCLC but could be enhanced with radiosensitizers. In this study, we identify HDAC3 as a novel radiosensitizing target in SCLC using a CRISPR knockout screen and demonstrate its efficacy and mechanism. SBC5 cells were transduced with a custom EpiDrug single-guide RNA library and treated with ionizing radiation (IR) to identify radiosensitizing genes. HDAC3 emerged as a candidate and was validated through genetic knockdown and pharmacologic inhibition (RGFP966) in multiple SCLC cell lines. Both approaches enhanced radiosensitivity, as shown by cell viability (dose modification factor10 = 1.14-1.69) and clonogenic assays (dose modification factor10 = 1.16-1.41). We assessed changes in chromatin accessibility by assay for transposase-accessible chromatin using sequencing and IR-induced DNA damage and repair using γH2AX foci detection, double-strand break (DSB) repair assays, and immunoblotting of repair proteins. HDAC3-deficient cells exhibited increased chromatin accessibility, greater IR-induced DSBs, and impaired repair capacity, resulting in persistent DNA damage. This repair defect sensitized cells to PARP inhibitors, for which combining RGFP966 with olaparib or talazoparib produced additive to synergistic effects. In SCLC xenograft models, HDAC3 knockdown or RGFP966, combined with IR, achieved significant tumor growth inhibition. Collectively, we identified HDAC3 as a novel radiosensitizing target in SCLC. Its functional loss increased the generation and persistence of IR-induced DNA DSBs, effectively sensitizing SCLC cell lines and xenografts to IR, providing a potential radiosensitization strategy to treat SCLC.},
}
@article {pmid41471222,
year = {2025},
author = {Nass, NM and Zaher, KA},
title = {From Methylomes to CRISPR Epigenetic Editing: New Paths in Antibiotic Resistance.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {12},
pages = {},
doi = {10.3390/pathogens14121267},
pmid = {41471222},
issn = {2076-0817},
mesh = {*Gene Editing/methods ; *Epigenesis, Genetic ; *Bacteria/genetics/drug effects ; *CRISPR-Cas Systems ; Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Humans ; DNA Methylation ; *Epigenome ; *Drug Resistance, Microbial/genetics ; Gene Expression Regulation, Bacterial ; Epigenome Editing ; },
abstract = {Antibiotic resistance (AR) has long been interpreted through the lens of genetic mutations and horizontal gene transfer. Yet, mounting evidence suggests that epigenetic regulation, including DNA and RNA methylation, histone-like proteins, and small non-coding RNAs, plays a similarly critical role in bacterial adaptability. These reversible modifications reshape gene expression without altering the DNA sequence, enabling transient resistance, phenotypic heterogeneity, and biofilm persistence under antimicrobial stress. Advances in single-molecule sequencing and methylome mapping have uncovered diverse DNA methyltransferase systems that coordinate virulence, efflux, and stress responses. Such epigenetic circuits allow pathogens to survive antibiotic exposure, then revert to susceptibility once pressure subsides, complicating clinical treatment. Parallel advances in CRISPR-based technologies now enable direct manipulation of these regulatory layers. CRISPR interference (CRISPRi) and catalytically inactive dCas9-fused methyltransferases can silence or reactivate genes in a programmable, non-mutational manner, offering a new route to reverse resistance or sensitize pathogens. Integrating methylomic data with transcriptomic and proteomic profiles further reveals how epigenetic plasticity sustains antimicrobial tolerance across environments. This review traces the continuum from natural bacterial methylomes to engineered CRISPR-mediated epigenetic editing, outlining how this emerging interface could redefine antibiotic stewardship. Understanding and targeting these reversible, heritable mechanisms opens the door to precision antimicrobial strategies that restore the effectiveness of existing drugs while curbing the evolution of resistance.},
}
@article {pmid41471176,
year = {2025},
author = {Bhowmik, S and Rivu, S and Bari, ML and Ahmed, S},
title = {Genome Mining of Cronobacter sakazakii in Bangladesh Reveals the Occurrence of High-Risk ST83 and Rare ST789 Lineages.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {12},
pages = {},
doi = {10.3390/pathogens14121220},
pmid = {41471176},
issn = {2076-0817},
support = {BIO-34//University Grant Commission, Bangladesh/ ; },
mesh = {Bangladesh/epidemiology ; Humans ; *Cronobacter sakazakii/genetics/isolation &amp; purification/classification/pathogenicity ; *Genome, Bacterial ; *Enterobacteriaceae Infections/microbiology/epidemiology ; Virulence Factors/genetics ; Infant ; Food Microbiology ; Plasmids/genetics ; Infant, Newborn ; Whole Genome Sequencing ; Phylogeny ; Infant Formula/microbiology ; },
abstract = {Cronobacter sakazakii is a foodborne pathogen of major concern due to its link with severe neonatal infections through powdered infant formula (PIF). However, its genomic epidemiology in Bangladesh remains uncharacterized. We report the first whole-genome analysis of three isolates from PIF. Two isolates (S41_PIFM and S44_RUTF) belonged to ST83, a lineage repeatedly associated with neonatal meningitis, septicemia, and persistence in PIF production environments, while the third (S43_TF) represented ST789, a recently described and rare lineage of unknown pathogenic potential. Pan-genome and comparative analyses identified 39 virulence determinants, 19 antimicrobial-resistance genes, and diverse mobile genetic elements. ST83 isolates harbored plasmid replicons IncFII(pCTU2) and pESA2, while the ST789 isolate carried insertion sequence ISKpn34, indicating horizontal gene transfer potential. All strains encoded I-E CRISPR-Cas systems. The detection of globally recognized high-risk ST83 clones alongside the novel ST789 lineage highlights emerging public health risks. This study provides the first genomic insights into C. sakazakii in Bangladesh and underscores the urgent need for genomic surveillance and strengthened food safety monitoring to protect infant health in low- and middle-income countries.},
}
@article {pmid41468306,
year = {2026},
author = {Wang, Y and Li, Y and Li, J and Li, M and Qiu, X},
title = {Silencing of optogenetic and chemogenetic transgenes in human iPSCs involves promoter methylation and methylation-independent mechanisms.},
journal = {Epigenetics},
volume = {21},
number = {1},
pages = {2606983},
doi = {10.1080/15592294.2025.2606983},
pmid = {41468306},
issn = {1559-2308},
mesh = {Humans ; *Promoter Regions, Genetic ; *Induced Pluripotent Stem Cells/metabolism/cytology ; HEK293 Cells ; *DNA Methylation ; Optogenetics ; *Transgenes ; *Gene Silencing ; CRISPR-Cas Systems ; DNA Transposable Elements ; Gene Editing ; Chemogenetics ; },
abstract = {The transplantation of neural progenitor cells derived from induced pluripotent stem cells (iPSCs) has therapeutic potential for the treatment of neurological diseases. However, the functional integration of transplanted iPSC-derived neurons into host neural networks remains controversial. Optogenetic and chemogenetic tools offer the means to assess such integration. However, constructing modifiable iPSC-derived neurons requires efficient gene editing. Here, we used CRISPR/Cas9 (targeting the AAVS1 safe harbor) and PiggyBac transposon systems to insert optogenetic and chemogenetic receptors (ChR2/hM4Di) into human iPSCs. While both systems successfully integrated genes into the genomes of HEK293T cells and iPSCs, receptor expression was detected only in HEK293T cells. Bisulfite sequencing revealed extensive methylation of the TRE3G BI promoter (95.3-98.2%) in iPSCs, in contrast to low methylation (5.9%) in HEK293T cells. For PiggyBac, the methylation of CMV/EF1α promoters in iPSCs exhibited integration site-dependent variability (0-95.2%). Notably, even hypomethylated clones failed to show gene expression, suggesting that additional regulatory mechanisms, such as histone modifications or chromatin remodeling, may contribute to transcriptional silencing. Differentiation into neural stem cells does not reverse methylation nor restore protein expression. Our findings demonstrate that the CRISPR/Cas9 and PiggyBac systems enable the integration of optochemical receptor genes into iPSCs. However, promoter methylation or other epigenetic and non-epigenetic gene-silencing mechanisms could pose barriers to efficient protein expression from the integrated transgene in iPSCs.},
}
@article {pmid41467478,
year = {2026},
author = {Niazian, M and de Ronne, M and Beauchamp, CJ and Belzile, F and Torkamaneh, D},
title = {CRISPR-induced knockouts reveal a dual role for the soybean NFR5α gene in symbiotic nitrogen fixation and root hair development.},
journal = {The plant genome},
volume = {19},
number = {1},
pages = {e70143},
pmid = {41467478},
issn = {1940-3372},
support = {6548//Genome Canada/ ; 337003//Fonds de recherche du Qu&#xe9;bec - Nature et technologies (FRQNT)/ ; },
mesh = {*Glycine max/genetics/growth &amp; development/microbiology ; *Nitrogen Fixation/genetics ; *Plant Roots/growth &amp; development/genetics ; *Symbiosis/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Plant Root Nodulation/genetics ; },
abstract = {Nitrogen fixation in soybeans, facilitated by symbiotic interactions with rhizobia, is a cornerstone of sustainable agriculture, reducing reliance on synthetic fertilizers. However, the efficiency of symbiotic nitrogen fixation (SNF) varies due to natural genetic variation in SNF-related genes. Our study underscores the pivotal role of the GmNFR5α gene not only in nodulation but also in root hair development, which is crucial for effective nutrient uptake and plant yield. Through detailed genetic analyses and clustered regularly interspaced short palindromic repeats (CRISPR)-based manipulations, we identified and characterized multiple knockout mutants, notably GmNFR5α-KO and combined GmNFR5α+GmROP6-KO, which exhibited significant reductions in root hair density and nodulation. These phenotypic changes correspond with the downregulation of key root hair development genes such as TTG, RHD1, RHD2, and KJK, establishing a clear link between GmNFR5α function and root hair formation. The potential of leveraging these genetic insights to improve nitrogen fixation in legumes and introduce SNF capabilities into cereal crops could revolutionize crop fertilization strategies, offering a sustainable solution to global agricultural challenges.},
}
@article {pmid41270510,
year = {2026},
author = {Li, J and Li, X and Li, X and Xu, W and Yuan, L and Shi, H and Lei, Z and Li, N and Wei, Y and Hua, J},
title = {CRISPR/Cas9-generated CD46-knockout spermatogonial stem cells reveal mechanisms of BVDV-induced reproductive dysfunction in male livestock.},
journal = {Veterinary microbiology},
volume = {312},
number = {},
pages = {110807},
doi = {10.1016/j.vetmic.2025.110807},
pmid = {41270510},
issn = {1873-2542},
mesh = {Animals ; Male ; Goats ; Cattle ; CRISPR-Cas Systems ; *Diarrhea Viruses, Bovine Viral/physiology/pathogenicity ; *Bovine Virus Diarrhea-Mucosal Disease/virology ; *Adult Germline Stem Cells/virology ; Reproduction ; Gene Knockout Techniques ; *Spermatogonia/virology ; },
abstract = {Bovine viral diarrhea virus (BVDV) is a major viral pathogen that affects ruminants, resulting in significant economic losses due to issues such as immunosuppression, reproductive disorders, and growth retardation. Bulls infected with this virus may become infertile within a few months and can transmit the virus to susceptible cattle during mating. However, the mechanism of BVDV impairing the reproductive function of male livestock is not clear, as there is no suitable cell model. This study used spermatogonial stem cells(SSCs) from cattle and goats as research materials to explore the mechanism by which BVDV affects the reproductive function of male livestock. The results of this study indicate that both cytopathic (cp) and noncytopathic (ncp) BVDV can replicate in SSCs and that SSCs are capable of producing infectious BVDV. Giemsa staining showed significant changes in the morphology of SSCs after BVDV infection. Western blot and mRNA analysis showed that proliferation-related genes (PCNA, CCND1, CDK2) and SSC functional genes (Lin28A, OCT4, SOX2) were down regulated after infection. In addition, BVDV infection can induce ferroptosis in SSCs. Furthermore, CRISPR-Cas9 mediated editing of CD46 in goat SSCs resulted in a decrease in BVDV infection rate and alleviated the negative impact of the virus on cell survival and proliferation. This study provides new insights into the mechanism of reduced reproductive function in male livestock infected with BVDV, and lays the foundation for developing targeted disease resistant breeding strategies.},
}
@article {pmid41218271,
year = {2026},
author = {Fang, T and Peng, C and Ding, R and Fan, Y and Jia, J and Chen, J and Zhang, X and Wang, D and Li, J},
title = {Biomimetic nanoplatform-mediated CRISPR/Cas9 delivery for dual-pathway metabolic blockade in head and neck squamous cell carcinoma.},
journal = {Biomaterials},
volume = {328},
number = {},
pages = {123837},
doi = {10.1016/j.biomaterials.2025.123837},
pmid = {41218271},
issn = {1878-5905},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Squamous Cell Carcinoma of Head and Neck/metabolism/drug therapy/pathology ; Cell Line, Tumor ; *Nanoparticles/chemistry ; *Head and Neck Neoplasms/metabolism/drug therapy/pathology ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; *Biomimetic Materials/chemistry ; Glycolysis/drug effects ; Mice ; Mice, Nude ; },
abstract = {Head and neck squamous cell carcinoma (HNSCC) continues to exhibit a poor prognosis, largely due to late diagnosis and the development of cisplatin resistance. Tumor proliferation in HNSCC is closely associated with upregulation of key glycolytic enzymes. However, monotherapeutic targeting of glycolysis paradoxically enhances compensatory glutaminolysis via glutamate overproduction. To overcome this metabolic adaptation, we developed biomimetic nanoparticles P-T-p@CM, fabricated from the pH/reduction dual-responsive copolymer poly(lactic acid)-polyhistidine-polyethylenimine (PLA-pHis-ss-PEI). This nanoplatform enables coordinated co-delivery of telaglenastat (a glutaminase 1 (GLS1) inhibitor) and a CRISPR-Cas9 plasmid encoding sgRNA targeting HIF-1α. This system utilizes homologous cancer cell membrane coating for precise tumor homing, with stimuli-responsive release enabling simultaneous dual metabolic blockade: CRISPR-mediated HIF-1α knockout attenuates glycolysis while telaglenastat suppresses glutamine-to-glutamate conversion. Metabolic analyses confirmed significant reduction in Glycolysis proton efflux rate (GlycoPER), the oxygen consumption rate (OCR) and ATP generation, as well as the related metabolites including the lactate production and glutamate. This dual-starvation strategy depleted energy reserves and biosynthetic precursors, inducing severe metabolic disruption. Notably, in vivo studies showed a 90 % tumor inhibition rate (TIR) after 15 days of treatment, through enhanced apoptosis, reduced proliferation, and tumor glucose/glutamate depletion. Collectively, P-T-p@CM establishes a paradigm-shifting approach to disrupt metabolic compensation in the treatment of HNSCC.},
}
@article {pmid41213207,
year = {2026},
author = {Chen, J and Wang, Z and Du, Y and Liu, B and Chen, Y and Chen, G and Cui, W},
title = {Injectable CRISPRa-microspheres for targeted A20 activation rescue age-related osteogenic impairment via senescence mitigation.},
journal = {Biomaterials},
volume = {328},
number = {},
pages = {123830},
doi = {10.1016/j.biomaterials.2025.123830},
pmid = {41213207},
issn = {1878-5905},
mesh = {Animals ; *Osteogenesis/genetics ; *Cellular Senescence/genetics ; Mice ; *Microspheres ; *Tumor Necrosis Factor alpha-Induced Protein 3/metabolism/genetics ; Mesenchymal Stem Cells/cytology/metabolism ; *Aging ; *CRISPR-Cas Systems/genetics ; Mice, Inbred C57BL ; Gene Editing/methods ; Bone Regeneration ; Male ; Nanoparticles/chemistry ; Injections ; Humans ; },
abstract = {Age-related osteogenic failure in bone defect repair remains a significant clinical challenge, primarily due to persistent chronic inflammation-induced stem cell senescence. To address this, we engineered injectable CRISPRa-based gene-editing microspheres (GEMs), utilizing microfluidic-synthesized lipid nanoparticles (cLNPs) to co-deliver dCas9-VP64/sgRNA. This platform allows for precise spatiotemporal activation of tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) within bone marrow stromal cells (BMSCs), effectively reprogramming the senescence-osteogenesis axis. Our study identifies A20 as a key regulator of the senescence-associated secretory phenotype (SASP) and osteogenic impairment in aged BMSCs. In vitro, GEMs reduced senescence markers (p16 and p21) by over 30 %, while increasing osteogenic gene expression (RUNX2 and ALP) by 4 ∼ 5-fold, and suppressed inflammatory cytokines IL-6 and TNF-α by more than 30 %. In vivo, in aged mice with critical-sized bone defects, GEMs achieved a significant bone regeneration and promoted vascularization 3.1 times faster (CD31[+] staining) compared to controls. This GEM system offers a promising, clinically viable strategy for recalibrating age-related skeletal disorders, demonstrated by the precise targeting of host stem cells in situ and achieving approximately 80 % defect healing in aged bone defect models.},
}
@article {pmid41187596,
year = {2026},
author = {Doherty-Boyd, WS and Tsimbouri, PM and Jayawarna, V and Walker, M and Taqi, AF and Mahon, N and Meek, D and Young, P and Miller, A and West, A and Salmeron-Sanchez, M and Dalby, MJ and Donnelly, H},
title = {Synthetic peptide hydrogels as a model of the bone marrow niche demonstrate efficacy of a combined CRISPR-CAR T-cell therapy for acute myeloid leukaemia.},
journal = {Biomaterials},
volume = {328},
number = {},
pages = {123803},
doi = {10.1016/j.biomaterials.2025.123803},
pmid = {41187596},
issn = {1878-5905},
mesh = {Humans ; *Leukemia, Myeloid, Acute/therapy/pathology ; *Hydrogels/chemistry ; *Peptides/chemistry ; *CRISPR-Cas Systems ; *Bone Marrow/pathology ; Mesenchymal Stem Cells/cytology ; Gene Editing ; *Receptors, Chimeric Antigen ; T-Lymphocytes ; *Immunotherapy, Adoptive/methods ; },
abstract = {Leukaemias, driven by mutations in haematopoietic stem cells (HSCs), rely on interactions with the bone marrow (BM) niche and other cell populations such as mesenchymal stromal cells (MSCs) for growth and survival. While chimeric antigen receptor (CAR) T-cell therapy shows promise for other haematological malignancies, its application to acute myeloid leukaemia (AML) is hindered by tumour heterogeneity and off-target toxicity. Combining CRISPR-Cas9 gene editing with CAR T-cell therapy has potential for selectively targeting AML cells while sparing healthy tissue. However, validating the efficacy of these treatments prior to clinical trial is hampered by the differences between humans and animal models typically used for pre-clinical testing. Furthermore, traditional in vitro models fail to replicate the complexity of the BM niche and often overestimate treatments' efficacy. Here, we present a bioengineered human-cell containing endosteal BM niche model combining a fibronectin-presenting polymeric surface and a synthetic peptide hydrogel (PeptiGel) that mimics native BM tissue's mechanical properties. This platform supports niche phenotypes in MSCs and HSCs and enables the evaluation of combined CRISPR-CAR T-cell therapy, demonstrating potential as a preclinical human model for testing novel therapies.},
}
@article {pmid41465590,
year = {2025},
author = {Nguyen, PD and Nakanishi, K and Nguyen, HP and Nguyen, HV and Kitao, M and Yoshimoto, M and Kamei, K},
title = {Characterisation of the Novel Cutibacterium acnes Phage KIT09 and First Report of CRISPR-Cas-Independent Bacteriophage Resistance in Phylotype IA1.},
journal = {International journal of molecular sciences},
volume = {26},
number = {24},
pages = {},
pmid = {41465590},
issn = {1422-0067},
support = {JPJSCCB20230005//Japan Society for the Promotion of Science/ ; },
mesh = {*Bacteriophages/genetics/isolation &amp; purification/physiology ; *Propionibacterium acnes/virology/genetics ; *CRISPR-Cas Systems ; Humans ; Mutation ; Genome, Viral ; *Propionibacteriaceae/virology ; },
abstract = {Despite being a commensal bacterium, Cutibacterium acnes has been widely considered a major opportunistic pathogen due to its capacity for biofilm production and inflammatory induction, causing device-related, post-implant infections, and skin inflammatory diseases. In this study, we isolated and characterised the novel bacteriophage Cutibacterium acnes phage KIT09 as a potential antimicrobial candidate for the treatment of Cutibacterium acnes-related infections such as acne vulgaris and postsurgical infections. Subsequently, phage-resistant bacterial mutants were generated through phage KIT09 exposure and characterised. Wastewater samples were collected for the isolation of C. acnes phages, followed by their characterisation using C. acnes National Institute of Technology and Evaluation (NITE) Biological Resources Center (NBRC) 107605 (phylotype IA1). Resistant mutants were isolated after prolonged exposure of the newly isolated phage to host bacteria and then characterised. A novel C. acnes phage, designated KIT09, was isolated, demonstrating prolonged bacteriolysis lasting up to 96 h at a multiplicity of infection of 10, and exhibiting high thermal and pH stability. Following sustained selective pressure by phage KIT09, three phage-resistant bacterial isolates were obtained, forming smaller colonies than the wild-type strain, but maintaining a high phage adsorption capacity (>90% after 20 min). Whole-genome sequencing revealed 12 nucleotide mutations across five genes, including six non-synonymous substitutions. Three genes encoding a two-component histidine kinase, DNA processing protein A (DprA), and a ThuA-containing domain protein were mutated in all resistant isolates. Characterisation of the novel phage KIT09 demonstrated its robust lytic activity and environmental stability against C. acnes phylotype IA1. Isolated resistant mutants retained high phage adsorption, accompanied by recurrent mutations in genes encoding a two-component histidine kinase, DprA, and a ThuA-domain protein, suggesting the presence of alternative, CRISPR-Cas-independent resistance mechanisms in C. acnes.},
}
@article {pmid41465565,
year = {2025},
author = {Mikhaylova, E and Khusnutdinov, E and Terekhov, M and Pozdeev, D and Gusev, O},
title = {Pig Genome Editing for Agriculture: Achievements and Challenges.},
journal = {International journal of molecular sciences},
volume = {26},
number = {24},
pages = {},
pmid = {41465565},
issn = {1422-0067},
support = {075-15-2025-014 (075-15-2024-666)//The Ministry of Science and Higher Education of Russian Federation/ ; },
mesh = {*Gene Editing/methods ; Animals ; Swine/genetics ; *Agriculture/methods ; CRISPR-Cas Systems ; Breeding/methods ; *Genome ; },
abstract = {The remaining problems in pig farming may no longer be solved with traditional methods. The search for genetic variants associated with desired characteristics and involvement of animals with superior genetics in breeding programs is rarely effective for polygenic traits and pleiotropic genes. The lack of diversity in the germplasm also limits the use of breeding, but some beneficial mutations that did not occur naturally can be introduced manually via genome editing methods. Mutations discovered in other species, such as cattle, can be reproduced in pigs. Traits that were previously pursued for centuries might be achieved by genome editing in a few years. Enormous progress has been made in producing pigs resistant to viruses and in increasing meat productivity and quality. But there are still pressing problems such as lameness and damaging behaviors that probably cannot be solved without genome editing techniques. Their wider application is complicated by the requirement for large amounts of biomaterial, surgical manipulations and cell culture, as well as by the shift towards biomedical research. This review concentrates on the main achievements and challenges in pig agricultural genetics that can be addressed by genome editing.},
}
@article {pmid41465342,
year = {2025},
author = {Sattarov, R and Kuznetsov, A and Klimko, V and Ignatyeva, E and Ivanov, R and Karabelsky, A and Fizikova, A},
title = {The Template-Jumping Editing Approach in F9-Associated Hemophilia B Gene Therapy.},
journal = {International journal of molecular sciences},
volume = {26},
number = {24},
pages = {},
pmid = {41465342},
issn = {1422-0067},
support = {Agreement No. 18-03 on 10 September 2024//"Sirius" Federal Territory "Scientific and technological development of the "Sirius" Federal Territory"/ ; },
mesh = {*Hemophilia B/therapy/genetics ; *Genetic Therapy/methods ; *Gene Editing/methods ; *Factor IX/genetics ; Humans ; CRISPR-Cas Systems ; Mutation ; Animals ; Genetic Vectors/genetics ; },
abstract = {Hemophilia B is a hereditary bleeding disorder caused by mutations localized throughout the F9 gene. Existing gene therapy products containing AAV vectors have significant limitations. Replacement therapy with coagulation factor FIX infusions is not an optimal way of treatment, as patients still have periodic bleeding and require frequent transfusions. Moreover, approximately 5% of adult patients with hemophilia B develop inhibitory antibodies to recombinant forms of FIX. Therefore, it is important to develop universal CRISPR/Cas gene therapy approaches for F9 editing using non-viral delivery systems to enable gene reversion to a functional sequence at an early stage of disease development and establishment of the patients' immune system. In this study, a unique approach of F9 prime-editing was tested for the first time. This method is estimated to edit 7.3% of pathogenic F9 mutation types. Specifically, it targets the gene region encoding amino acids 374 V to 408 Q, which accounts for approximately 9.35% of patients with hemophilia B. An advantage of this gene therapy approach is the absence of the need to change Primer Binding Site (PBS) or Reverse Transcriptase Template (RTT) sequences until going from preclinical to clinical trials, as well as the introduction of gain of function mutations in order to compensate for the low prime-editing frequencies and enhance the effect of treatment in vivo.},
}
@article {pmid41463573,
year = {2025},
author = {Li, Y and Gong, K and Wang, X and Sun, Z and Ding, F},
title = {Heat Shock Transcription Factors as Central Integrators of Plant Stress Responses: From Thermotolerance to Multi-Stress Resilience.},
journal = {Biology},
volume = {14},
number = {12},
pages = {},
pmid = {41463573},
issn = {2079-7737},
support = {ZR2025MS427//Shandong Provincial Natural Science Foundation/ ; 3247180862//National Natural Science Foundation of China/ ; },
abstract = {Heat shock transcription factors (HSFs) have long been recognized for their essential role in mediating thermotolerance via the activation of heat shock proteins (HSPs). Recent studies, however, have significantly broadened this view, revealing that HSFs function as versatile transcriptional regulators orchestrating plant adaptation to a wide range of abiotic and biotic stresses. This review synthesizes current knowledge of HSF structure, activation, and canonical roles in the heat shock response, while emphasizing emerging insights into their diverse functions beyond heat stress. Evidence from both model and crop species demonstrates that many HSFs confer tolerance to a broad range of stresses, including drought, cold, salinity, oxidative stress, and pathogen attack, through intricate crosstalk with hormonal (e.g., ABA, SA, JA) and redox signaling pathways, as well as MAPK-mediated phosphorylation. We also discuss biotechnological strategies such as CRISPR/Cas-mediated genome editing, stress-inducible promoter engineering, and synthetic transcriptional circuits that offer promising avenues for fine-tuning HSF expression and enhancing multi-stress resilience in crops. A deeper understanding of HSF multifunctionality not only advances our comprehension of plant stress biology but also provides a foundation for engineering resilient crops in the context of global climate change.},
}
@article {pmid41461563,
year = {2026},
author = {Riley, SE and Noskova Fairley, M and Xia, S and Cunningham, R and Cholewa-Waclaw, J and Feng, Y and Hansen, CG},
title = {In vivo screen reveals specific roles of Hippo pathway components in development and regeneration.},
journal = {Life science alliance},
volume = {9},
number = {3},
pages = {},
pmid = {41461563},
issn = {2575-1077},
mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Regeneration/genetics/physiology ; *Zebrafish Proteins/metabolism/genetics ; Signal Transduction/genetics ; Hippo Signaling Pathway ; *Protein Serine-Threonine Kinases/metabolism/genetics ; YAP-Signaling Proteins ; CRISPR-Cas Systems/genetics ; Macrophages/metabolism ; Larva ; Animal Fins/physiology ; Gene Expression Regulation, Developmental ; Transcription Factors/metabolism/genetics ; Transcriptional Coactivator with PDZ-Binding Motif Proteins ; },
abstract = {The Hippo signalling pathway is a major regulator of regeneration and development. However, the comparative importance and functional roles of individual Hippo pathway components in vivo are greatly unknown, particularly within the vertebrate lineage. To gain direct and comparable insights, we took advantage of the zebrafish larva model system. We generated individual and combined CRISPR/Cas9 F0 knockouts of a range of core Hippo pathway genes, including upstream regulators, the co-transcriptional regulators Yap1/Taz, and Yap1/Taz target genes. We analysed and compared the resulting developmental and regenerative phenotypes. Our findings highlight that paralogues of core components have distinct, but in some instances overlapping, functions. Intriguingly, we find that Yap1 and Taz have differential roles during development and regeneration. In addition, we characterise and compare two tail fin regenerative paradigms: after both severe and mild injury. These injury paradigms are drastically different and elicit diverse resolution processes. We confirm critical roles of the immune system in the regenerative process. Macrophage recruitment is reduced during severe tail fin regeneration after Yap1 and Taz loss, appearing earlier in yap1 than wwtr1 Crispants and correlating with defective regenerative function. This defective macrophage involvement might therefore be one of the mediators of the deficient regeneration in these two Crispants. Overall, our analysis emphasises distinct requirements and responses of the Hippo pathway during development and across different regenerative paradigms.},
}
@article {pmid41461407,
year = {2026},
author = {Shahid, M},
title = {Molecular engineering and in-silico biotechnological innovations for microbial degradation of persistent pesticides.},
journal = {Pesticide biochemistry and physiology},
volume = {217},
number = {},
pages = {106833},
doi = {10.1016/j.pestbp.2025.106833},
pmid = {41461407},
issn = {1095-9939},
mesh = {*Pesticides/metabolism ; Biodegradation, Environmental ; *Biotechnology ; Metabolic Engineering ; Soil Microbiology ; Bacteria/metabolism/genetics ; Computer Simulation ; },
abstract = {The persistence of recalcitrant pesticides in agricultural soils poses a serious threat to environmental and public health. Conventional remediation methods often have limited efficiency and, sustainability. Whereas, microbial degradation provides an eco-friendly and attractive alternative. This review highlights advances in molecular and biotechnological tools driving microbial pesticide degradation. It also emphasizes key genes, enzymatic pathways, and resilient microbes driving recalcitrant pesticide degradation. This review discusses the integration of next-generation sequencing, multi-omics platforms, CRISPR-Cas editing, synthetic biology, and AI-driven metabolic engineering in advancing microbial pesticide degradation. It also highlights progress in rhizosphere microbiome research, bioinformatics pipelines, and field-scale validation. The transition from lab to field highlights precision bioremediations' potential for sustainable pesticide management.},
}
@article {pmid41459216,
year = {2025},
author = {Sun, Y and Zhao, Q and Li, W and Kwok, LY and Zhang, H},
title = {Genomic diversity and functional adaptation of Limosilactobacillus reuteri isolated from diverse ecological niches.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1732127},
pmid = {41459216},
issn = {1664-302X},
abstract = {Limosilactobacillus reuteri is a widely utilized probiotic, however, the genomic diversity and evolutionary mechanisms underlying its adaptation to various hosts and environments remain incompletely understood. This study employed comparative genomics to analyze 176 L. reuteri genomes from animal (rodents, mammals, ruminants, and birds), human intestinal, and food sources (dairy products, fermented foods; 89 newly sequenced and 92 retrieved, 5 excluded by ANI < 95%). We assessed genomic features, average nucleotide identity, pan/core genomes, carbohydrate-active enzymes, bacteriocin production, CRISPR-Cas systems, and antibiotic resistance genes. The pan-genome consisted of 16,814 genes, while the core genome contained 553 genes. Core-gene phylogeny revealed seven clades, rodents isolates were positioned closer to the root. The clustering trend of fermented foods isolates in the phylogenetic tree may indicate that these strains have undergone convergent evolution or adaptive evolution in a specific environment. CAZymes varied across sources, and the predicted bacteriocin clusters were enriched in animal-derived, particularly in rodent isolates. CAZy functional composition in L. reuteri is shaped by the ecological niche and host environment, reflecting a pattern of host-driven evolutionary adaptation. CRISPR-Cas systems were present in 23.3% of genomes, predominantly in rodents isolates, indicating strong anti-phage capabilities. The heterogeneity of CRISPR-Cas systems among sources suggests that subpopulations of L. reuteri have been subjected to different evolutionary pressures. The predominance of Type II systems agrees with their widespread occurrence in lactobacilli. The presence of multiple probiotic function-related genes across all separation sources confirms the robust probiotic potential of L. reuteri. Antibiotic resistance genes, including tet, ermB, and vatE, were most prevalent among animal-derived isolates, with the highest numbers occurring in mammals and the lowest in rodents. Therefore, strain-specific safety assessments are necessary prior to clinical or food applications. The findings underscore the significance of host-specific adaptations in shaping the genetic and functional profiles of L. reuteri, offering valuable implications for its application in food-derived, human-derived, animal-derived and therapeutics.},
}
@article {pmid41458398,
year = {2025},
author = {Kang, J and Koo, J and Oh, H and Bae, E},
title = {Structural characterization of anti-CRISPR protein AcrIE9.},
journal = {Structural dynamics (Melville, N.Y.)},
volume = {12},
number = {6},
pages = {064701},
pmid = {41458398},
issn = {2329-7778},
abstract = {The arms race between bacteria and bacteriophages has driven the evolution of both CRISPR-Cas systems and anti-CRISPR (Acr) proteins. AcrIE9, a type I-E Acr protein identified in Pseudomonas aeruginosa, inhibits Cascade-mediated DNA binding by interacting with the Cas7e subunit. However, its structural basis and precise inhibitory mechanism have remained unclear. Here, we report the crystal structure of AcrIE9 at 1.73 Å resolution, along with additional structural and biochemical analyses. AcrIE9 exists as both monomer and dimer in solution, while the crystal structure reveals a homodimeric assembly. Each protomer adopts a unique α/β architecture, and structural similarity searches indicate that AcrIE9 represents a previously uncharacterized protein fold. In vitro binding assays using individually purified type I-E Cas subunits from P. aeruginosa did not detect direct interaction with AcrIE9, including with Cas7e. These findings suggest that AcrIE9 may recognize a composite interface formed only within the intact Cascade complex, consistent with the AlphaFold3 prediction of multivalent interactions with Cas7e subunits. Taken together, this study provides the structural characterization of AcrIE9 and supports an inhibitory mechanism involving a multi-subunit binding surface on Cascade.},
}
@article {pmid41458320,
year = {2025},
author = {Ghiotto, G and Francescato, L and Biancalani, MA and Treu, L and Campanaro, S},
title = {Hydrogen excess drives metabolic reprogramming and viral dynamics in syngas-converting microbiomes.},
journal = {Environmental science and ecotechnology},
volume = {28},
number = {},
pages = {100637},
pmid = {41458320},
issn = {2666-4984},
abstract = {Microbial communities drive essential bioprocesses, including the conversion of synthesis gas into biomethane, a sustainable energy source that supports circular carbon economies. In anaerobic environments, specialized consortia of bacteria and archaea facilitate syngas methanation through syntrophic interactions, where hydrogenotrophic methanogens play a central role in reducing carbon dioxide and monoxide with hydrogen. However, imbalances in gas ratios, particularly excess hydrogen, can disrupt these interactions and impair overall efficiency. Yet, the molecular mechanisms underlying microbial responses to such imbalances remain poorly understood. Here we show that hydrogen excess triggers profound metabolic and viral remodeling in a thermophilic anaerobic microbiome, leading to reduced methane yields and ecological instability. This reprogramming involves transcriptional downregulation of methanogenesis genes in the dominant archaeon Methanothermobacter thermautotrophicus, coupled with upregulation of CRISPR-Cas and restriction-modification systems that correlate with diminished activity of an associated phage, indicating activated host defenses against viral threats. Concurrently, bacterial species such as those from Tepidanaerobacteraceae enhance carbon fixation via the Wood-Ljungdahl pathway, serving as electron sinks to mitigate redox imbalance. These adaptive responses highlight the microbiome's resilience mechanisms under stress, revealing viruses as both stressors and selective forces in syntrophic systems. Such insights advance our understanding of microbiome dynamics in bioconversion processes and guide the engineering of more stable microbial consortia for optimized syngas-to-methane conversion amid variable feedstocks.},
}
@article {pmid41457595,
year = {2025},
author = {Chang, Y and Fan, S and Hao, T and Dai, J and He, W},
title = {[Characterization of cbm2813 encoding the cytochrome P450 enzyme in the biosynthetic gene cluster of carbomycin].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {11},
pages = {4125-4137},
doi = {10.13345/j.cjb.250364},
pmid = {41457595},
issn = {1872-2075},
mesh = {*Multigene Family/genetics ; *Streptomyces/genetics/metabolism/enzymology ; *Cytochrome P-450 Enzyme System/genetics/metabolism ; Escherichia coli/genetics/metabolism ; Macrolides/metabolism ; *Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Anti-Bacterial Agents/biosynthesis ; },
abstract = {Carbomycin, a 16-membered macrolide antibiotic produced in Streptomyces thermotolerans, comprises two components, carbomycin A (CA) and carbomycin B (CB). CB is converted into CA through epoxidation of the C12-C13 double bond. The gene cbm2813, located in the biosynthetic gene cluster of carbomycin, encodes a cytochrome P450 enzyme considered to catalyze this epoxidation. In this study, the functional and enzymatic properties of the cytochrome P450 enzyme Cbm2813 in the carbomycin biosynthesis gene cluster were characterized by in vivo gene inactivation and in vitro enzymatic reactions. We employed the CRISPR-Cas9 system to delete cbm2813 and obtained the mutant Δcbm2813. The fermentation products of the mutant contained CB but not CA. Complementation of Δcbm2813 restored CA production. Cbm2813 was successfully expressed in Escherichia coli and then purified. In vitro enzyme assays confirmed that Cbm2813 specifically recognized CB but not structurally similar 16-membered macrolide antibiotics, such as josamycin, midecamycin, and isovalerylspiramycin I. Cbm2813 exhibited the maximal activity at pH 5.5 and 36 ℃, with the catalytic efficiency kcat/Km of 4.39×10[3] L/(mol·s). Molecular docking suggested that the C9 carbonyl group of CB coordinated with the heme iron in the active site of the enzyme, ensuring strict substrate specificity. This study expands the toolbox of characterized P450 enzymes and advances the understanding of carbomycin biosynthesis.},
}
@article {pmid41457550,
year = {2025},
author = {Sun, Y and Li, W and Wang, P and Pei, J and Yang, L and Qiu, L and Liu, H},
title = {[CRISPRi-mediated regulation of nitrate metabolism genes in Chlamydomonas reinhardtii enhances lipid accumulation].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {12},
pages = {4794-4809},
doi = {10.13345/j.cjb.250363},
pmid = {41457550},
issn = {1872-2075},
mesh = {*Chlamydomonas reinhardtii/genetics/metabolism ; *Lipid Metabolism/genetics ; *Nitrates/metabolism ; Nitrate Reductase/genetics/metabolism ; Nitrogen/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nitrite Reductases/genetics ; },
abstract = {The global energy crisis and environmental pollution are becoming increasingly serious. The development of sustainable and clean renewable energy has become a key direction of scientific research. Microalgae are ideal raw materials for biodiesel production due to their efficient photosynthetic ability, fast growth rate, and rich lipid content. Chlamydomonas reinhardtii, as a model organism of unicellular eukaryotic green algae, has the advantages of a clear genetic background and convenient operation, which makes it an ideal target for the study of lipid metabolism in microalgae. Nitrogen stress can induce lipid accumulation in microalgae, while its molecular mechanism has not been fully elucidated. In this study, we used a CRISPR interference (CRISPRi) system to regulate key genes of nitrogen metabolism in a targeted manner and thus simulated the nitrogen stress environment to investigate its effect on lipid accumulation in C. reinhardtii, aiming to provide a new technological strategy for the efficient production of microalgal lipids. The CRISPRi system was constructed to inhibit the expression of the nitrate reductase gene (CrNIT1) and the nitrite reductase gene (CrNII1) in C. reinhardtii FACHB-2220. We evaluated the effects of nitrogen metabolism inhibition on lipid accumulation by measuring the cell growth, lipid content, and expression levels of key genes. The algal strain ΔNIT1-4 with inhibited CrNIT1 expression showed the CrNIT1 expression 10.27% that of the wild type (WT, and the strain ΔNII1-4 with inhibited CrNII1 expression showed the CrNII1 expression16.02% that of WT, indicating that the CRISPRi system effectively inhibited the transcription of the target genes. Under the condition of nitrogen abundance, the cell density of ΔNIT1-4 and ΔNII1-4 was only 33.7% and 40.2%, respectively, of that of WT. The total lipid content of ΔNIT1-4 and ΔNII1-4 was 34.41% and 33.45% of the dry weight, respectively, which was significantly higher than that of WT. In this study, we successfully simulated the nitrogen stress effect by suppressing the key genes of nitrogen metabolism through the CRISPRi system and significantly improved the lipid accumulation efficiency of C. reinhardtii. This study elucidates the regulatory relationship between nitrogen metabolism and lipid synthesis, providing a theoretical basis and technical support for the industrial application of microalgae in bioenergy production.},
}
@article {pmid41457547,
year = {2025},
author = {Song, S and Yao, D and Cai, Z and Yue, X and Qiao, C and Xue, C},
title = {[Construction of a CRISPR-Cas6-mediated lycopene synthase assembly regulation method].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {12},
pages = {4745-4758},
doi = {10.13345/j.cjb.250396},
pmid = {41457547},
issn = {1872-2075},
mesh = {Lycopene ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Intramolecular Lyases/genetics/metabolism ; Plasmids/genetics ; Carotenoids/metabolism ; },
abstract = {A CRISPR-Cas6-mediated lycopene synthase assembly regulation strategy was developed to optimize the metabolic pathway of lycopene biosynthesis in Escherichia coli and enhance production efficiency. Leveraging the orthologous properties of EcCas6e and Csy4 within the Cas6 protein family, along with RNA scaffolding, we constructed a protein-RNA complex for enzyme assembly. Sixteen plasmids (LYC-1 to LYC-16) were designed, and the assembly strategy was systematically optimized by varying the gene arrangement, linker length, and RNA scaffold expression. The performance of RNA scaffold-based enzyme assembly was compared with conventional protein linker-based approaches. Lycopene production was quantified via high-performance liquid chromatography (HPLC) to evaluate system performance. The recombinant strain LYC-3-4, which co-localized CrtB and CrtI via EcCas6e-Csy4 protein-RNA complexes, achieved the highest lycopene yield (4.02 mg/L), 58% higher than the control strain LYC-3-5 (2.55 mg/L) with mismatched RNA hybridization regions, and 41% higher than strain LYC-6 (2.86 mg/L), in which the enzymes were expressed separately. This result indicates that protein-RNA-mediated spatial co-localization significantly enhanced the substrate channeling effect, whereas other assembly configurations either failed to improve or even reduced lycopene production. In summary, we exploited the protein assembly capability of CRISPR-Cas6 proteins in combination with RNA scaffolds to achieve efficient enzyme co-localization within the lycopene biosynthetic pathway. This approach offers a convenient, flexible, and scalable tool for enzyme assembly regulation in metabolic engineering, with potential applications in microbial production of lycopene and other valuable metabolites.},
}
@article {pmid41457318,
year = {2025},
author = {Brandt, D and Dörrich, AK and Persicke, M and Kemmler, A and Leonhard, T and Haak, M and Nölting, S and Ruwe, M and Schmid, N and Thormann, KM and Kalinowski, J},
title = {A pentose, as a cytosine nucleobase modification in Shewanella phage Thanatos genomic DNA, mediates enhanced resistance toward host restriction systems.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0133325},
doi = {10.1128/aem.01333-25},
pmid = {41457318},
issn = {1098-5336},
abstract = {UNLABELLED: Co-evolution of bacterial defense systems and phage counter-defense mechanisms has resulted in an intricate biological interplay between bacteriophages and their prey. In order to evade nuclease-based mechanisms that target DNA, various bacteriophages modify their nucleobases, which impedes or even inhibits the recognition and restriction by endonucleases. We found that Shewanella phage Thanatos DNA is insensitive to multiple restriction enzymes and also to Cas I-Fv and Cas9 cleavage. Furthermore, with nanopore sequencing, the phage DNA showed severely impaired basecalling. In addition to an adenine methylation, the data indicated an additional, much more substantial nucleobase modification. Using liquid chromatography-mass spectrometry (LC-MS), we identified an unknown configuration of a deoxypentose attached to cytosine as an undiscovered modification of phage DNA, which is present in Thanatos genomic DNA and likely mediates resistance to restriction endonucleases, as well as reducing Cas nuclease activity significantly. To elucidate the underlying enzyme functions, we identified structural homologs of Thanatos proteins among known glycosyltransferase folds and experimentally proved a UDP-xylose pyrophosphorylase function of the phage protein TH1_063 by in vitro. Inactivation of TH1_060 leads to an almost complete inhibition of phage propagation, indicating an important role of the cytosine modification in phage survival and/or proliferation.<br><br>IMPORTANCE: Several phages extensively decorate their DNA building blocks, providing an effective protection against various host and phage-produced restriction systems. These modifications allow the phages to distinguish between their own DNA and that of the host, significantly increasing the establishment of the phage chromosome upon entry into the host and subsequent phage proliferation. Several different modifications have been previously identified and characterized. Here, we describe a hitherto unknown cytosine modification, consisting of a deoxypentose-putatively xylose-that provides protection against various bacterial restriction systems, including DNA-targeting CRISPR/Cas systems. Our findings expand the range of DNA modifications that phages use for protection.},
}
@article {pmid41403193,
year = {2025},
author = {Su, S and Zuo, Y and Ma, B and Zhao, Z and Wang, X and Zhang, X and Ignatus, AD and Piñero, JC and Peng, X and Li, F and Chen, M},
title = {Functional Validation of GmGSTs2 in the Resistance to Abamectin in the Oriental Fruit Moth, Grapholita molesta (Lepidoptera: Tortricidae).},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {52},
pages = {33033-33045},
doi = {10.1021/acs.jafc.5c12427},
pmid = {41403193},
issn = {1520-5118},
mesh = {Animals ; *Ivermectin/analogs &amp; derivatives/pharmacology ; *Moths/genetics/drug effects/enzymology/growth &amp; development ; *Insecticides/pharmacology ; *Glutathione Transferase/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Insecticide Resistance ; CRISPR-Cas Systems ; Larva/drug effects/genetics/growth &amp; development/enzymology ; },
abstract = {Abamectin has been used for decades as an insecticide and acaricide in arthropod pest management. However, there is no direct evidence from CRISPR/Cas9 studies confirming the involvement of GSTs in insect resistance to abamectin. The oriental fruit moth, Grapholita molesta, is a destructive pest of fruit trees worldwide. The role of GSTs in the oriental fruit moth remains unclear. In this study, an abamectin-resistant strain (AB-R) was derived from a susceptible laboratory strain (AB-S) of G. molesta. Synergist bioassays showed that the GST inhibitor diethyl maleate (DEM) significantly increased abamectin toxicity in AB-R. Biochemical assays indicated that glutathione S-transferase (GST) activity in AB-R was 1.63-fold higher than in AB-S. Among 25 GST genes examined, GmGSTs2 showed the largest expression difference between AB-R and AB-S and was expressed across developmental stages and body parts. Recombinant GmGSTs2 significantly reduced the effective quantity of abamectin in vitro. CRISPR/Cas9 knockout of GmGSTs2 in both genetic backgrounds increased susceptibility to abamectin and significantly affected the development and survival of G. molesta. The transgenic Drosophila melanogaster strain expressing GmGSTs2 showed an LC50 of 74.12 mg L[-1] (34.59-126.63) versus 25.48 mg L[-1] (12.28-39.82) in W[1118] controls, indicating a 2.91-fold difference. Together, synergism assays, enzyme activity measurements, in vitro metabolism, CRISPR knockout in both resistant and susceptible backgrounds, and a heterologous in vivo assay identify GmGSTs2 as a key metabolic driver of abamectin resistance in G. molesta, providing a practical target for resistance management.},
}
@article {pmid41456387,
year = {2025},
author = {Zhang, J and Liu, WJ and Ma, F and Zhang, CY},
title = {Self-priming amplification-integrated orthogonal CRISPR-Cas system for multiplexed profiling of piRNAs in clinical samples.},
journal = {Biosensors &amp; bioelectronics},
volume = {297},
number = {},
pages = {118334},
doi = {10.1016/j.bios.2025.118334},
pmid = {41456387},
issn = {1873-4235},
abstract = {CRISPR/Cas-based biosensors hold great diagnostic potential, but they are often limited in clinical practice by insufficient sensitivity and the lack of multiplexed analysis capability. To address these issues, we develop a self-priming amplification-integrated orthogonal CRISPR-Cas (SPA-OCRISPR) system for multiplexed detection of piRNAs. This assay employs an innovative four-way junction probe that seamlessly integrates target recognition with a primer-free self-priming amplification, significantly simplifying the workflow and enhancing the ligation efficiency. High-fidelity SplitR ligase-mediated transduction guarantees the excellent specificity, efficiently eliminating the need for reverse transcription. The orthogonal trans-cleavage activities of Cas12a and Cas13a are harnessed to generate distinct fluorescent signals with minimal cross-interference for multiplexed analysis. This strategy can achieve attomolar-level sensitivity and good specificity. Moreover, it can successfully quantify breast cancer-associated piRNAs (piR-651 and piR-36026) in clinical tissues, and accurately discriminate cancerous samples from healthy ones. Importantly, this strategy exhibits good generality and it can be extended to detect colorectal cancer-related piRNAs (piR-823 and piR-54265) through simply modifying the recognition sequences of split probes, underscoring its broad potential in multiplexed profiling and clinical cancer diagnostics.},
}
@article {pmid41455873,
year = {2025},
author = {Bibi, R and George, M and Sarkar, K},
title = {RNA-guided STAT3 modification fine tunes the epigenetic and epitranscriptomic regulation of CD4 + T helper cell differentiation during non-small cell lung cancer (NSCLC).},
journal = {Medical oncology (Northwood, London, England)},
volume = {43},
number = {2},
pages = {102},
pmid = {41455873},
issn = {1559-131X},
support = {11019/07/2018-Sch//Ministry of Tribal Affairs, Govt. of India/ ; EMDR/SG/15/2023-5901//Indian Council of Medical Research/ ; },
mesh = {Humans ; *STAT3 Transcription Factor/genetics/metabolism ; *Carcinoma, Non-Small-Cell Lung/genetics/immunology/pathology ; *Lung Neoplasms/genetics/immunology/pathology ; *Epigenesis, Genetic ; Cell Differentiation/genetics ; Tumor Microenvironment/immunology ; *CD4-Positive T-Lymphocytes/immunology ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Transcriptome ; },
abstract = {The accurate control of immune responses in the tumor microenvironment is crucial for augmenting anti-cancer immunity. This work examined the function of STAT3 in modulating epigenetic and epitranscriptomic pathways during the differentiation of CD4 + T helper cells in non-small cell lung cancer (NSCLC). Employing CRISPR/Cas9 genome editing, STAT3 was specifically eliminated in CD4[+]T cells derived from NSCLC patients. Functional investigations demonstrated that the reduction of STAT3 markedly enhanced the production of T helper 1 (TH1) cytokines, notably IFN-γ, while concurrently diminishing immunosuppressive signaling. Epigenetic analysis revealed significant modifications in DNA and RNA methylation patterns, along with heightened R-loop formation-alterations linked to augmented transcriptional activity of anti-tumor immune genes. Moreover, STAT3-deficient CD4[+]T cells demonstrated an enhanced ability to activate cytotoxic T lymphocytes, facilitating the targeted eradication of tumor cells. All of these effects together made the NSCLC microenvironment's immune system better at fighting cancer. Our results identify STAT3 as a crucial regulator of the genetic and epigenetic frameworks that influence T cell functionality in lung cancer. By combining RNA-guided genome editing with immune functional tests, we show that blocking STAT3 in a specific way could bring back strong anti-tumor immunity. This research underscores the therapeutic potential of STAT3-targeted therapies, presenting an innovative approach to alter T cell destiny and improve immune-mediated tumor eradication in non-small cell lung cancer (NSCLC). These methods could lead to the next generation of immunotherapies that improve clinical outcomes by fine-tuning both epigenetic and epitranscriptomic circuits.},
}
@article {pmid41454921,
year = {2025},
author = {Sanjay, G and Seetharam, RN and Singdevsachan, SK and Sathya, M},
title = {Microbial Systems Enhancing CAR-Based Therapies: A Synthetic Biology Paradigm for Next-Generation Cancer Immunotherapy.},
journal = {Current microbiology},
volume = {83},
number = {2},
pages = {106},
pmid = {41454921},
issn = {1432-0991},
support = {Hoynoza Technologies Pvt. Ltd//Hoynoza Technologies Pvt. Ltd/ ; },
mesh = {Animals ; Humans ; *Immunotherapy/methods ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology ; *Receptors, Chimeric Antigen/genetics/immunology ; *Synthetic Biology/methods ; },
abstract = {Chimeric antigen receptor (CAR)-based immunotherapies face significant translational challenges in solid tumor applications, particularly regarding manufacturing scalability, tumor targeting specificity, and antigen heterogeneity. This systematic review evaluates microbial systems as innovative platforms to address these limitations through synthetic biology-driven approaches, with a focus on bridging preclinical advances to clinical implementation. Analysis of 389 peer-reviewed studies (2015-2025) reveals that engineered probiotic strains (e.g., Escherichia coli Nissle 1917) achieve selective tumor colonization while functioning as programmable factories for:1. Synthetic antigen production and single-chain variable fragment (scFv) expression,2. Costimulatory domain delivery enabling antigen-agnostic CAR-T activation,3. Tumor microenvironment modulation via immunostimulatory chemokines. Microbial platforms demonstrate superior manufacturing economics (70-90% cost reduction vs. conventional methods) and enhance CAR-T functionality through epigenetic reprogramming by microbial metabolites (e.g., short-chain fatty acids). CRISPR/Cas-engineered genetic circuits further enable precise spatiotemporal control of therapeutic payloads.Microbial systems represent transformative platforms for scalable, programmable CAR immunotherapy with significant potential for solid tumor targeting. Key barriers to clinical translation include biocontainment challenges, incomplete mechanistic understanding of tumor homing specificity, and safety validation requirements. Strategic integration of synthetic biology with microbial chassis offers a viable pathway toward accessible next-generation cancer therapies.},
}
@article {pmid41454508,
year = {2025},
author = {Li, J and Wang, X and Wang, X and Qu, H and Gao, L and Zhao, Z and Luo, P and Zheng, Y},
title = {A Rapid and Sensitive CRISPR-Cas12a for the Detection of Legionella pneumophila.},
journal = {Polish journal of microbiology},
volume = {74},
number = {4},
pages = {484-493},
pmid = {41454508},
issn = {2544-4646},
mesh = {*Legionella pneumophila/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Sensitivity and Specificity ; Legionnaires' Disease/microbiology/diagnosis ; Water Microbiology ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Legionella pneumophila is a common environmental bacterium that can cause severe respiratory disease. In this study, a reliable, rapid, and convenient detection method for L. pneumophila was established using a combination of recombinase polymerase amplification (RPA) and CRISPR/Cas12a technology. First, we designed three pairs of RPA primers and two types of crRNA based on the L. pneumophila-specific mip gene. Subsequently, we optimized the primers and amplification time for the RPA reaction, the crRNA for the CRISPR/Cas12a reaction, as well as the concentration of the fluorescent probe. We successfully constructed an RPA-CRISPR/Cas12a fluorescence detection system and a portable RPA-CRISPR/Cas12a LFB. The detection systems achieved a sensitivity of 5 copies/μl and high specificity. One hundred sixty environmental water samples tested by RPA-CRISPR/Cas12a LFB showed no significant difference compared to the qPCR method, providing a reliable tool for future on-site detection.},
}
@article {pmid41453247,
year = {2025},
author = {Park, SE and Jeong, JH and Kim, YG and Park, HH},
title = {Structural analysis of predicted anti-CRISPR, ACZ01644.},
journal = {Biochemical and biophysical research communications},
volume = {797},
number = {},
pages = {153199},
doi = {10.1016/j.bbrc.2025.153199},
pmid = {41453247},
issn = {1090-2104},
abstract = {The CRISPR-Cas system provides adaptive immunity in bacteria and archaea against invading genetic elements, while anti-CRISPR (Acr) proteins have evolved in phages to counteract this defense. Here, we report the first structural and biochemical characterization of ACZ01644, a protein previously predicted to be an Acr. The crystal structure of ACZ01644 reveals a unique cone-shaped architecture composed of five α-helices and five β-strands forming a compact core, which represents a fold distinct from any known Acr family. Biochemical analyses demonstrated that ACZ01644 assembles as a trimer in solution, suggesting a potential functional relevance of this oligomeric state. However, in vitro assays revealed that ACZ01644 does not inhibit Cas9-mediated DNA cleavage, indicating that its inhibitory activity, if present, may involve other CRISPR subtypes or yet unidentified cofactors. Our findings reveal an unprecedented structural scaffold among putative Acr proteins and provide a foundation for future studies to elucidate its biological role in CRISPR-Cas regulation.},
}
@article {pmid41452514,
year = {2025},
author = {Lal, SK and Khatoon, G and Kumar, A and Kumar, K and Kumar, R and Pan, X and Kumar, S and Bhadana, VP and Pandey, A and Kumar, M and Soren, KR and Panditi, V},
title = {Genome Editing Enhanced Abiotic Stress ToleranceIn Cereal Crops.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {13},
pmid = {41452514},
issn = {1438-7948},
mesh = {*Gene Editing/methods ; *Edible Grain/genetics/growth &amp; development ; *Stress, Physiological/genetics ; CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; *Genome, Plant ; Plants, Genetically Modified/genetics ; },
abstract = {Cereals are crucial sources of food for human and animal populations worldwide. Their grain and fodder primarily serve as sources of energy and nutrition. Cereal production is hampered because of the prevalent abiotic stress worldwide. Abiotic stresses such as drought, salinity, extreme temperatures, and heavy metal toxicity significantly reduce global cereal crop production. Previously, traditional breeding and transgenic technology have been promising and potent approaches used to mitigate unfavourable abiotic stresses, enhancing crop production to some extent. The recent advent of more potent genome-editing technologies, particularly Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), has revolutionized the pace of crop improvement programs. Genome-editing technology using engineered nucleases offers significant opportunities for crop improvement. Genome editing tools include Meganucleases, Zinc Finger Nucleases (ZFN), Transcription activator-like effector nucleases (TALENs), and CRISPR/CRISPR-associated protein (Cas). Among all genome-editing tools, CRISPR/Cas9 has been widely used to improve crop cultivars due to its specificity, simplicity, robustness, and flexibility. Recent progress in genome-editing technology have improved various plant traits in cereals. Among these traits, cereal genotypes have shown substantial advances in the last decade, particularly in enhanced tolerance to abiotic stress, enabled by genome-editing tools. This review summarizes the recently developed cereal cultivars for abiotic stress tolerance that employ different genome-editing technologies, including the most recent additions, prime editing and base editing. These improved cereal cultivars perform better and maintain higher yields under adverse abiotic stresses.},
}
@article {pmid41430049,
year = {2025},
author = {Dong, Q and Chen, P and Guo, Z and Wei, H and Zeng, Y and Zhang, J and Men, Y and Liu, W and Sun, Y and Yang, J},
title = {Computational design of allulose-responsive biosensor toolbox for auto-inducible protein expression and CRISPRi mediated dynamic metabolic regulation.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11562},
pmid = {41430049},
issn = {2041-1723},
support = {32271545//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Biosensing Techniques/methods ; *Transcription Factors/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; Escherichia coli/metabolism/genetics ; Synthetic Biology/methods ; Glucose/metabolism ; },
abstract = {Biosensors based on transcription factors (TFs) have shown extensive applications in synthetic biology. Due to the complex multi-domain structure of effector-TF-DNA, computational design of TFs remains a challenge. Here, we present the successful structure-guided computational design of the access tunnel, ligand binding, allosteric transition process for an allulose-responsive PsiR. It enables a 20-fold increase in sensitivity, reducing the EC50 of PsiR-allulose biosensors (PABs) from 16 mM to 0.8 mM, and delivers a PAB box possessing the detection range from 10 μM to 100 mM. We further validate its broader applicability in enhancing sensitivity of LacI-IPTG biosensor. Based on the developed PABs, we present the inducer-free allulose-mediated auto-inducible protein expression system, and demonstrate an allulose-triggered CRISPR interference circuit for dynamic metabolic regulation. It facilitates a 68% increase in allulose titer and achieves a high yield of 0.43 g/g glucose. This work provides the versatile TF toolbox for developing allulose-triggered regulation circuits in biotechnology application.},
}
@article {pmid41389205,
year = {2025},
author = {Zobel, M and Damaggio, G and Mignogna, ML and Besusso, D and Scalzo, D and Cossu, A and Trovesi, C and Crosti, M and Cortina, F and Campus, I and Formenti, G and Mazzara, S and Gregoretti, F and Antonelli, L and Oliva, G and Zuccato, C and Colonna, V and Conforti, P and Cereda, M and Rossi, RL and Maestri, S and Scolz, A and Iennaco, R and Cattaneo, E},
title = {A human CAGinSTEM platform for decoding HTT repeats' somatic instability links CAG interruption to HD pathology in neurons.},
journal = {Cell reports},
volume = {44},
number = {12},
pages = {116685},
pmid = {41389205},
issn = {2211-1247},
mesh = {Humans ; *Huntington Disease/genetics/pathology ; *Huntingtin Protein/genetics/metabolism ; *Neurons/metabolism/pathology ; *Genomic Instability ; *Trinucleotide Repeats/genetics ; Trinucleotide Repeat Expansion ; CRISPR-Cas Systems/genetics ; },
abstract = {Somatic CAG instability in the mutant Huntingtin (HTT) gene is increasingly recognized as a key hallmark of Huntington's disease (HD). Using our novel human CAGinSTEM platform, we manipulated cis genetic elements influencing instability in human HD neurons, monitoring repeat length. Quality-controlled CRISPR-engineered stem cells with increasing CAG lengths and clinical haplotypes were analyzed using third-generation sequencing. Our findings link interruptions in the CAG repeat, especially the loss or duplication of the penultimate CAA of canonical alleles, to significant instability modulation. Notably, four internal CAA interruptions completely abolish CAG instability, reversing HD phenotypes such as altered striatal fate acquisition and nuclear disorganization. This platform highlights the role of cis modifiers, emphasizing the direct influence of HTT DNA repeat composition on CAG instability and providing a robust framework for modeling HTT repeat instability in vitro.},
}
@article {pmid41298993,
year = {2025},
author = {Heu, CC and Benowitz, KM and Matzkin, LM and Allan, CW and LeRoy, DM and Li, X and Tabashnik, BE and Carrière, Y and Fabrick, JA},
title = {Editing the kinesin-12 gene affects responses to Bt toxin Cry1Ac in Helicoverpa zea.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {45378},
pmid = {41298993},
issn = {2045-2322},
support = {2020-33522-32268//USDA, National Institute of Food and Agriculture, Biotechnology Risk Assessment Research Grants Program/ ; 2020-67013-31924//USDA, National Institute of Food and Agriculture, Agriculture and Food Research Initiative/ ; 2020-22620-023-000D//USDA, Agricultural Research Service/ ; },
mesh = {Animals ; Bacillus thuringiensis Toxins ; *Endotoxins/pharmacology ; *Gene Editing ; *Hemolysin Proteins/pharmacology ; *Kinesins/genetics ; *Moths/genetics/drug effects ; *Bacterial Proteins/pharmacology ; *Insecticide Resistance/genetics ; CRISPR-Cas Systems ; Bacillus thuringiensis/genetics ; *Insect Proteins/genetics ; },
abstract = {Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) are used globally to manage key insect pests. However, the evolution of resistance to Bt proteins in at least 11 pest species has reduced the effectiveness of Bt crops. Resistance to crystalline (Cry) Bt proteins including Cry1Ac produced by Bt cotton is a major problem in Helicoverpa zea (also known as bollworm and corn earworm), one of the most economically damaging pests in the United States. A previous genome-wide association study identified a nonsense point mutation in a kinesin-12 gene that was associated with resistance to Cry1Ac in a lab-selected strain of H. zea. Here, we used CRISPR/Cas9 gene editing to knock out the kinesin-12 gene in a Cry1Ac-susceptible laboratory strain, which caused a 4.0-fold increase in resistance to Cry1Ac. Conversely, gene editing that repaired the natural kinesin-12 nonsense mutation in a lab-selected resistant strain increased susceptibility to Cry1Ac by 3.8-fold. These complementary results provide compelling evidence that kinesin-12 plays a role in the mode of action of Cry1Ac against H. zea.},
}
@article {pmid41298482,
year = {2025},
author = {Aguado-Alvaro, LP and Garitano, N and Esser-Skala, W and Sayers, J and Del Valle, C and Alameda, D and Mendieta-Esteban, J and Calleja-Cervantes, ME and Goñi-Salaverri, A and Zazpe, J and de Vito, AR and Marchese, F and Alignani, D and Cudini, J and Gross, T and Rábago, G and Narayan, N and Martinez, L and Martinez, S and Huntly, B and Riley, P and Gonzalez, A and Taylor-King, JP and Fortelny, N and Pelacho, B and Lara-Astiaso, D},
title = {Identification of epigenetic regulators of fibrotic transformation in cardiac fibroblasts through bulk and single-cell CRISPR screens.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11660},
pmid = {41298482},
issn = {2041-1723},
mesh = {Humans ; Fibrosis/genetics ; *Epigenesis, Genetic ; Single-Cell Analysis/methods ; Myofibroblasts/metabolism/pathology ; *Myocardium/pathology/metabolism/cytology ; *Fibroblasts/metabolism/pathology ; Animals ; Chromatin/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cells, Cultured ; Mice ; Transcription Factors/metabolism/genetics ; },
abstract = {Cardiac fibrosis is mediated by the persistent activity of myofibroblasts, which differentiates from resident cardiac fibroblasts in response to tissue damage and stress signals. The signaling pathways and transcription factors regulating fibrotic transformation have been thoroughly studied. In contrast, the roles of chromastin factors in myofibroblast differentiation and their contribution to pathogenic cardiac fibrosis remain poorly understood. Here, we combined bulk and single-cell CRISPR screens to characterize the roles of chromatin factors in the fibrotic transformation of primary cardiac fibroblasts. We uncover strong regulators of fibrotic states including Srcap and Kat5 chromatin remodelers. We confirm that these factors are required for functional processes underlying fibrosis including collagen synthesis and cell contractility. Using chromatin profiling in perturbed cardiac fibroblasts, we demonstrate that pro-fibrotic chromatin complexes facilitate the activity of well-characterized pro-fibrotic transcription factors. Finally, we show that KAT5 inhibition alleviates fibrotic responses in patient-derived human fibroblasts.},
}
@article {pmid41276526,
year = {2025},
author = {Murray, R and Chowdhury, MR and Botticello-Romero, NR and Desai, K and Chilakapati, SR and Chong, B and Xia, Y and Messana, A and Sobon, H and Rocha, J and Musenge, F and Camblin, A and Ciaramella, G and Sitkovsky, MV and Maldini, CR and Hatfield, SM},
title = {Multiplex gene-editing strategy to engineer allogeneic EGFR-targeting CAR T-cells with improved efficacy against solid tumors.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11593},
pmid = {41276526},
issn = {2041-1723},
mesh = {Animals ; Humans ; ErbB Receptors/metabolism/genetics/immunology ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; Mice ; *Gene Editing/methods ; Tumor Microenvironment/immunology ; *Immunotherapy, Adoptive/methods ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; *T-Lymphocytes/immunology/metabolism ; CRISPR-Cas Systems/genetics ; Female ; *Lung Neoplasms/therapy/immunology/genetics ; Receptor, Transforming Growth Factor-beta Type II/genetics ; *Neoplasms/therapy/immunology ; Graft vs Host Disease/prevention &amp; control/immunology ; Mice, Inbred NOD ; },
abstract = {Chimeric Antigen Receptor (CAR) T cells have induced remarkable clinical responses in patients with hematological cancers. However, CAR T-cell therapies against solid tumors have not elicited similar outcomes since immunosuppressive barriers in the tumor microenvironment attenuate anti-tumor activity. Here, we describe a multifaceted approach to engineer allogeneic CAR T-cells resistant to both biochemical (hypoxia-adenosinergic) and immunological (PD-L1 and TGF-β) inhibitory signaling using an adenine base editor and a CRISPR-Cas12b nuclease. The resulting EGFR-targeting CAR T-cell product comprised a combination of six gene edits designed to evade allorejection (B2M, CIITA), prevent graft-versus-host disease (CD3E) and overcome biochemical (ADORA2A) and immunological (PDCD1, TGFBR2) barriers in solid tumor microenvironment of subcutaneously grown EGFR[+] human lung tumor xenografts. This combinatorial genetic disruption enhances CAR T cell effector function and anti-tumor efficacy leading to improved tumor elimination and survival in xenograft and humanized mouse solid tumor models. Our strategy confers CAR T cells resistance to multiple clinically relevant inhibitory signaling pathways that are amplified in hypoxic tumor areas and may improve the therapeutic potential of CAR T-cells against solid tumors.},
}
@article {pmid41275110,
year = {2025},
author = {Yadav, B and Sardar, S and Yadav, A and Kumari, A and Gautam, M and Mandlik, R and Arora, S and Kumar, S and Jewaria, PK and Sonah, H and Deshmukh, R and Chinnusamy, V and Ram, H},
title = {A CRISPR-Cas9 library to target putative redundant gene sets facilitates their functional exploration in grain development in rice.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1769},
pmid = {41275110},
issn = {1471-2229},
support = {BT/PR53626/BSA/33/96/2024//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {*Oryza/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Edible Grain/genetics/growth &amp; development ; Gene Library ; *Genes, Plant ; Gene Editing ; Gene Knockout Techniques ; Seeds/growth &amp; development/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Advent of CRISPR-Cas9 library approach has revolutionized the field of high throughput targeted mutagenesis in plants. By identifying an sgRNA spacer that can target multiple paralogous genes in a genome, higher-order knockout plants can be developed. Using this concept, we developed ten CRISPR-Cas9 pool libraries and generated higher-order knockout plants in rice. Towards this, firstly we identified genome-wide sets of genes which are co-expressed and have high sequence similarity and can be targeted by a single sgRNA. Based on the expression pattern, these genes were divided into ten groups, and subsequently ten CRISPR-Cas9 plasmid libraries were developed. One such library designed against seed-expressed genes was transformed into rice and higher-order knockout plants were developed. Genotyping revealed that around 90% T0 plants had editing, and among the edited plants majority of them were higher-order knockouts. Phenotypic analysis in the next generation discovered functions of several seed specific genes in grain length, width, number and 100-grain weight. By analyzing single and double mutants for two Agenet domain-containing proteins, we have discovered an epistatic interaction between them for grain development. Further application of our approach will help to uncover hidden functions of the targeted genes and accelerate functional genomics research in rice. The CRISPR-Cas9 library is a useful approach to generate higher-order knockout mutants and identify functions of the targeted genes in rice.},
}
@article {pmid41272236,
year = {2025},
author = {Pidishetty, D and Damera, SK and Murugavel, M and Susaimanickam, PJ and Chittajallu, SNSH and Kushawah, G and Sarkar, P and Bharadwaj, SR and Mishra, R and Mariappan, I},
title = {Loss of retinal stem cell reserve and lipofuscin accumulation accelerates cone-rod degeneration and replicates Stargardt disease in abca4b null zebrafish.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {44988},
pmid = {41272236},
issn = {2045-2322},
mesh = {Animals ; Zebrafish/genetics ; *Lipofuscin/metabolism ; *Stargardt Disease/genetics/pathology/metabolism ; *ATP-Binding Cassette Transporters/genetics ; Disease Models, Animal ; *Stem Cells/metabolism/pathology ; *Zebrafish Proteins/genetics/metabolism ; *Retina/pathology/metabolism ; Retinal Cone Photoreceptor Cells/metabolism/pathology ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; Retinal Pigment Epithelium/metabolism/pathology ; Gene Knockout Techniques ; *Cone-Rod Dystrophies/pathology/metabolism/genetics ; CRISPR-Cas Systems ; Mutation ; *Macular Degeneration/genetics/pathology/metabolism ; },
abstract = {Mutations in ABCA4 gene causes Stargardt macular degeneration, which manifests with toxic lipofuscin deposits in the outer retina, gradual atrophy of RPE cells, followed by photoreceptor cell loss. The cone-enriched retina, with macula-like 'area-temporalis' of zebrafish are better models than rodents for studying human macular dystrophies. Here, we generated abca4b knockout zebrafish model using CRISPR/Cas9 editing and evaluated the early and late-stage retinal changes. In adult abca4b[-/-] mutants, the RPE cells exhibited hyperpigmentation, altered retinomotor behaviour and lipofuscin accumulation, but they remained viable. However, the photoreceptors underwent progressive degeneration, with a sequential loss of blue and UV cones, followed by red and green cones and finally the rod cells. This triggered the chronic activation and early depletion of retinal stem cells at the ciliary marginal zone of mutants and resulted in accelerated outer-retinal degeneration and severe visual defects, despite them retaining the Müller glia-dependant retinal repair potential.},
}
@article {pmid35173349,
year = {2022},
author = {Shen, Y and Gomez-Blanco, J and Petassi, MT and Peters, JE and Ortega, J and Guarné, A},
title = {Structural basis for DNA targeting by the Tn7 transposon.},
journal = {Nature structural &amp; molecular biology},
volume = {29},
number = {2},
pages = {143-151},
pmid = {35173349},
issn = {1545-9985},
support = {R01 GM129118/GM/NIGMS NIH HHS/United States ; PJT-155941//CIHR/Canada ; },
mesh = {Binding Sites/genetics ; Crystallography, X-Ray ; DNA Transposable Elements/*genetics ; DNA, Bacterial/chemistry/genetics/*metabolism ; DNA-Binding Proteins/chemistry/genetics/metabolism ; Escherichia coli/genetics/metabolism ; Escherichia coli Proteins/chemistry/genetics/metabolism ; Models, Molecular ; Protein Interaction Domains and Motifs ; Protein Structure, Quaternary ; Substrate Specificity ; Transposases/chemistry/genetics/metabolism ; },
abstract = {Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7 family utilize an AAA+ adaptor (TnsC) to coordinate target-site selection with transpososome assembly and to prevent insertions at sites already containing a Tn7 element. Owing to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. TnsC forms an asymmetric ring on target DNA that segregates target-site selection and interaction with the paired-end complex to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites.},
}
@article {pmid41451278,
year = {2025},
author = {Narra, M and Ray, A and Polley, B and Yang, H and Bhowmik, PK},
title = {AI-driven advances in plant biotechnology: sharpening the edge of plant tissue culture and genome editing.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1718810},
pmid = {41451278},
issn = {1664-462X},
abstract = {The advent of artificial intelligence (AI) holds great promise for revolutionizing the fields of plant tissue culture and genome editing. Plant tissue culture is recognized as a powerful tool for rapid multiplication and crop improvement. However, the complex interactions between genetic and environmental factors generate large volumes of data, posing challenges for traditional statistical analysis methods. To address this, researchers are now employing machine learning (ML)-based and artificial neural networks (ANN) approaches to predict and optimize in vitro culture protocols thereby improving precision, sustainability, and efficiency. Integrating AI technologies such as machine learning (ML), artificial neural networks (ANN), and deep learning (DL) can significantly advance the development of data-driven models for CRISPR/Cas9 genome editing. Today, AI-driven methods are routinely applied to enhance precision in predicting on- and off-target sequence locations and editing outcomes. Additionally, predicting protein structures can provide a directed evolution framework that facilitates the creation of improved gene editing tools. However, the application of AI-based CRISPR modeling in plants is not yet fully explored. In this context, we aim to examine representative ML/DL/ANN models of CRISPR/Cas based editing employed in various organisms. This review significantly compiles a diverse set of studies and provides a clear overview of how AI is transforming the fields of plant tissue culture and genome editing. It emphasizes AI's potential to increase the efficiency and precision of biotechnological practices, making them more accessible and cost-effective. While outlining current findings, the paper sets the stage for future research, encouraging further exploration into the integration of AI with plant biotechnology.},
}
@article {pmid41450580,
year = {2025},
author = {Graves, LE and Christina, S and Mullany, KL and Alexander, IE and Falhammar, H},
title = {Exploration of the potential of genomic editing in the treatment of congenital adrenal hyperplasia.},
journal = {Frontiers in endocrinology},
volume = {16},
number = {},
pages = {1719376},
pmid = {41450580},
issn = {1664-2392},
mesh = {*Adrenal Hyperplasia, Congenital/genetics/therapy ; Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Animals ; Steroid 21-Hydroxylase/genetics ; },
abstract = {Despite life-saving glucocorticoids, therapeutic options for congenital adrenal hyperplasia (CAH) remain sub-optimal. Adrenal crisis continues to be the highest cause of mortality in individuals with CAH and even with recommended treatment regimens complications from the disease and treatments themselves persist. These patients have limited treatment options and advanced therapeutics could be a solution. Development of genetic therapies have exponentially increased in recent years. The advent of CRISPR/Cas technology has brought previously inconceivable treatment options to reality. Genomic editing could repair the defective 21-hydroxylase gene and provide a cure for 21-hydroxylase deficiency, the most common CAH variant, eliminating the current need for constant patient intervention. There are a number of technologies within reach for CAH, however, delivery of the genomic editing reagents to the elusive adrenocortical progenitor cells remains challenging. Here we discuss the complexity of CAH genetics, which has implications for choice of genomic editing strategy, and potential future strategies for the development of a cure of CAH.},
}
@article {pmid41449724,
year = {2025},
author = {Plesser, E and Goldenberg, L and Kelly, G and Bdolach, E and Arad, T and Bejerano, E and Masok, O and Carmeli-Weissberg, M and Shaya, F and Sherman, A and Eyal, Y and Carmi, N},
title = {Targeting the "bitterness gene" by genome editing abolishes synthesis of bitter flavanones in citrus; prospects for new varieties and extended climates for cultivation.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {6},
pages = {e70654},
pmid = {41449724},
issn = {1365-313X},
mesh = {*Flavanones/metabolism/biosynthesis ; *Citrus/genetics/metabolism ; *Gene Editing ; Plant Leaves/metabolism/genetics ; Fruit/genetics/metabolism ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Taste/genetics ; Hesperidin/metabolism/analogs &amp; derivatives ; },
abstract = {Bitterness in citrus fruit is conferred by flavanone-neohesperidosides, whose accumulation is catalyzed by a single enzyme flavanone-7-O-glucosides-1,2-rhamnosyltransferase (1,2RhaT), expressed in both leaves and fruit. To eliminate citrus bitterness, we used CRISPR/Cas9 genome editing to inactivate the 1,2RhaT gene in grapefruit (Citrus paradisi) and "Carrizo" citrange (Citrus sinensis × Citrus trifoliata). Edited lines displayed frameshift mutations that introduced premature stop codons, effectively abolishing the synthesis of the bitter neohesperidosides naringin, neohesperidin, and poncirin. Metabolomic analyses in leaves from 1,2RhaT-mutant lines confirmed the absence of bitter flavanone-neohesperidosides and a compensatory increase in the tasteless flavanone-rutinosides hesperidin, didymin, and narirutin. Since 1,2RhaT is encoded by a single gene, our findings in leaves are expected to be identical for fruit and thus demonstrate a strategy for developing non-bitter citrus cultivars while retaining health-benefitting flavonoid levels. Furthermore, cold-hardy citrus species that are currently unacceptably bitter due to high flavanone-neohesperidoside levels may become useful sources for introduction of cold-hardiness following inactivation of the 1,2RhaT gene. This approach thus paves the way for expanding grapefruit markets and breeding cold-hardy, palatable citrus varieties that are better suited to a wider range of climates.},
}
@article {pmid41444327,
year = {2025},
author = {Oh, D and Seok, C and Park, HW and Park, S and Lee, J and Choi, H and Jawad, A and Ham, J and Jang, H and Lee, SC and Oh, BC and Moon, C and Park, KH and Hyun, SH and Kim, D},
title = {Generation and ophthalmological characterization of oculocutaneous albinism type 1 pig models by selection-free genome editing.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {44564},
pmid = {41444327},
issn = {2045-2322},
support = {RS-2025-00518006//National Research Foundation of Korea/ ; 2020R1A2C2101714//National Research Foundation of Korea/ ; RS-2024-00398561 , RS-2024-00399475//Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries/ ; 20023068//Ministry of Agriculture, Food and Rural Affairs/ ; HR22C1363//Korea Health Industry Development Institute/Republic of Korea ; },
mesh = {Animals ; *Albinism, Oculocutaneous/genetics/pathology ; *Gene Editing/methods ; Disease Models, Animal ; Swine ; Monophenol Monooxygenase/genetics ; CRISPR-Cas Systems ; Humans ; Electroretinography ; Phenotype ; Melanins/metabolism ; },
abstract = {Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase (TYR) gene, resulting in melanin deficiency and severe visual impairments. Although mouse models provide insights into OCA1 pathogenesis, they exhibit significant anatomical and physiological differences from humans, particularly in ocular structure and function, thereby limiting their ability to recapitulate human OCA1 phenotypes. Therefore, in this study, we generated a porcine OCA1 model by selection-free genome editing via somatic cell nuclear transfer to characterize ophthalmological features and evaluate their translational relevance to human OCA1. Our approach utilized TYR-targeting CRISPR/Cas9 ribonucleoproteins without the need for single-cell-derived clonal expansion, thus streamlining the generation process. After somatic cell nuclear transfer with TYR knockout donor cells, the embryos demonstrated normal in vitro embryonic development comparable to the control, resulting in four healthy OCA1 piglets that exhibited characteristic OCA1 phenotypes with complete melanin loss in ocular and cutaneous tissues. Comprehensive ophthalmological analyses revealed significant structural abnormalities, including marked reduction in retinal layer thickness and elevated intraocular pressure. Remarkably, electroretinography revealed selective impairment of the rod bipolar pathway with reduced b-wave amplitudes and increased oscillatory potentials, indicating disturbances in synaptic processing. Overall, our study demonstrates the efficiency and reliability of selection-free genome editing for generating porcine OCA1 models. Moreover, the ophthalmological findings provide valuable insights for exploring retinal dysfunction and pigmentation mechanisms and advancing the preclinical evaluation of potential therapeutic interventions for human OCA1.},
}
@article {pmid41441922,
year = {2025},
author = {Govender, P and Ghai, M and Karpoormath, R},
title = {Advances in biosensors for bacterial detection and identification.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {1},
pages = {6},
pmid = {41441922},
issn = {1573-0972},
support = {PMDS2205108850//National Research Foundation/ ; },
mesh = {*Biosensing Techniques/methods ; Humans ; *Bacteria/isolation &amp; purification/genetics/classification ; Artificial Intelligence ; Wearable Electronic Devices ; Bacterial Infections/diagnosis/microbiology ; },
abstract = {Bacterial detection and identification is paramount as it plays a key role in safeguarding human health, food safety and security. Over the past decade, biosensors have emerged as a powerful tool for bacterial detection due to their ability to provide rapid, sensitive, specific and cost-effective monitoring of bacteria. Biosensors rely on the interaction between the target analyte and biological recognition elements, which triggers a measurable signal that can be quantified, thus enabling the detection of bacteria. In recent years, nanoparticles have become a focal point in biosensor research due to their unique physical and chemical properties, enhancing their sensitivity, specificity and functionality. Artificial intelligence, microfluidics and wearable biosensor technologies are shaping the next-generation real-time bacterial monitoring tools. AI-based biosensors interpret complex biological signals and provide automated detection of bacterial pathogens. Similarly, wearable biosensors are emerging as a promising option for non-invasive detection and monitoring of wound infections. Additionally, the integration of CRISPR/Cas systems into biosensing platforms has revolutionized molecular diagnostics by enabling highly specific detection of pathogenic bacteria. In forensic sciences, biosensors are being explored for the identification of body fluids based on their unique bacterial signatures, which can assist in crime scene reconstruction and post-mortem interval estimation. Most studies that have reported on biosensors for detection of bacteria, have targeted a single analyte or bacterial species. Given the growing interest and demand for multiplexed biosensors, future research should focus on developing biosensors capable of detecting multiple bacteria simultaneously, without compromising the accuracy. Biosensors with dual functionality will be instrumental in providing an integrated solution to detect, manage and control bacterial pathogens, thereby mitigating any potential threat to human health.},
}
@article {pmid41390487,
year = {2025},
author = {Carruthers, DN and Kinnunen, PC and Li, Y and Chen, Y and Gin, JW and Yunus, IS and Galliard, WR and Tan, S and Radivojevic, T and Adams, PD and Singh, AK and Sustarich, J and Petzold, CJ and Mukhopadhyay, A and Garcia Martin, H and Lee, TS},
title = {Automation and machine learning drive rapid optimization of isoprenol production in Pseudomonas putida.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11489},
pmid = {41390487},
issn = {2041-1723},
support = {DE-AC0205CH11231//U.S. Department of Energy (DOE)/ ; },
mesh = {*Pseudomonas putida/metabolism/genetics ; *Machine Learning ; *Metabolic Engineering/methods ; *Hemiterpenes/metabolism ; CRISPR-Cas Systems ; Proteomics ; Metabolic Networks and Pathways/genetics ; Automation ; },
abstract = {Advances in genome engineering have improved our ability to perturb microbial metabolic networks, yet bioproduction campaigns often struggle with parsing complex metabolic datasets to efficiently enhance product titers. We address this challenge by coupling laboratory automation with machine learning to systematically optimize the production of isoprenol, a sustainable aviation fuel precursor, in Pseudomonas putida. The simultaneous downregulation through CRISPR interference of combinations of up to four gene targets, guided by machine learning, permitted us to increase isoprenol titer 5-fold in six consecutive design-build-test-learn cycles. Moreover, machine learning enabled us to swiftly explore a vast experimental design space of 800,000 possible combinations by strategically recommending approximately 400 priority constructs. High-throughput proteomics allowed us to validate CRISPRi downregulation and identify biological mechanisms driving production increases. Our work demonstrates that ML-driven automated design-build-test-learn cycles, when combined with rigorous data validation, can rapidly enhance titers without specific biological knowledge, suggesting that it can be applied to any host, product, or pathway.},
}
@article {pmid41085147,
year = {2025},
author = {Liu, WJ and Wang, LY and Ma, F and Zhang, CY},
title = {MnO2 Nanosponge-Accelerated Cas12a Trans-Cleavage: Breaking the Kinetic Barrier for In Vivo RNA Imaging.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {48},
pages = {e11942},
doi = {10.1002/advs.202511942},
pmid = {41085147},
issn = {2198-3844},
mesh = {*Manganese Compounds/chemistry ; Humans ; *Oxides/chemistry ; *CRISPR-Cas Systems/genetics ; Kinetics ; *RNA/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics ; Biosensing Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR/Cas12a system has emerged as a promising tool for in vitro biosensing, but its in vivo applications are hindered by its inefficient intracellular delivery and suboptimal trans-cleavage kinetics. To address these challenges, a Cas12a@MnO2 nanosponge (hMNS) nanoprobe is constructed, in which hMNS as both a degradable carrier and an accelerator of CRISPR/Cas12a system for efficient imaging of RNA in living cells. The Cas12a@hMNS nanoprobe is obtained via a one-step co-assembly process. It not only facilitates synchronous cellular uptake and glutathione (GSH)-responsive release of CRISPR/Cas12a components, but also supplies adequate Mn[2+] cofactors to improve the trans-cleavage activity of Cas12a. This dual-function probe can break the kinetic barrier of conventional CRISPR/Cas12a systems due to its unique characteristics of effective cellular internalization, rapid intracellular release, and accelerated signal gain, enabling sensitive detection of mRNA down to 63.6 pM without pre-amplification. Moreover, the Cas12a@hMNS nanoprobe can profile endogenous mRNA at the single-cell level, discriminate breast cancer tissues from healthy counterparts, and real-time visualize mRNA dynamics in living cells with exceptional spatiotemporal precision. Importantly, the elongation-blocked (EB) activator-modulated CRISPR/Cas12a system can be extended to detect various intracellular biomarkers, holding promising applications in clinical diagnosis, treatment, and surveillance.},
}
@article {pmid41058009,
year = {2025},
author = {Lu, H and Xue, C and Zhao, Y and Sun, J and Zhao, C and Zhang, X and Zhao, G and Liu, Y and Liu, H and Deng, Y and Wang, Y and Zhang, C and Liu, Y and Zeng, L and Yang, Y and Li, B and Ding, S and Zhou, L and Kuang, H and Wang, Z and Ju, W and Lin, H and Lin, J and Guo, Y and Chang, L and Zhao, H and Wang, J and Lin, J and Zheng, L and Chen, YE and Zhang, J},
title = {CRISPR-MI and scRNA-Seq Reveal TREM2's Function in Monocyte Infiltration and Macrophage Apoptosis During Abdominal Aortic Aneurysm Development.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {48},
pages = {e12227},
doi = {10.1002/advs.202412227},
pmid = {41058009},
issn = {2198-3844},
support = {82370490//National Natural Science Foundation of China/ ; 32300958//National Natural Science Foundation of China/ ; 82200536//National Natural Science Foundation of China/ ; 2023A1515010489//Natural Science Foundation of Guangdong Province of China/ ; 20231120142637001//Shenzhen Fundamental Research Program/ ; JCYJ20230807113016034//Science and Technology project of Shenzhen of China/ ; G030410001//Medical Research Innovation Project/ ; HL109946/NH/NIH HHS/United States ; HL153710/NH/NIH HHS/United States ; //Huetwell Endowed Professorship of Cardiovascular Medicine at University of Michigan/ ; 2022JJ40812//Natural Science Foundation of Hunan Province/ ; HL109946/NH/NIH HHS/United States ; HL153710/NH/NIH HHS/United States ; },
mesh = {*Aortic Aneurysm, Abdominal/genetics/metabolism/pathology ; *Monocytes/metabolism ; Animals ; *Receptors, Immunologic/genetics/metabolism ; *Macrophages/metabolism/pathology ; *Membrane Glycoproteins/genetics/metabolism ; Mice ; CRISPR-Cas Systems/genetics ; *Apoptosis/genetics ; Mice, Knockout ; Male ; Mice, Inbred C57BL ; Humans ; Disease Models, Animal ; RNA-Seq/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Gene Expression Analysis ; },
abstract = {Abdominal aortic aneurysm (AAA) is a life-threatening aortic disease without effective medication. The infiltration of monocytes into the aortic wall is critical for AAA development, but the genes and pathways regulating this process remain to be elucidated. A novel method is developed for in vivo genome-wide CRISPR/Cas9 screening of monocyte infiltration (CRISPR-MI). By combining CRISPR-MI with single-cell RNA sequencing (scRNA-Seq), this study finds that Triggering receptor expressed on myeloid cells 2 (Trem2) is a negative regulator of monocyte infiltration into the aortic wall in early AAA induction. Trem2 knockout (KO) increases the expression of adhesion molecules, chemotactic receptors, and cytokines in monocytes. Trem2 KO promotes monocyte adhesion and migration in vitro and increases monocyte infiltration into the aortic wall in vivo. However, Trem2 KO attenuates AAA development because of prominent macrophage death at the late stage. In conclusion, CRISPR-MI is a powerful tool for studying genes underlying monocyte infiltration in disease conditions in vivo. These findings reveal a dichotomous role of Trem2 in monocyte recruitment and macrophage survival during AAA.},
}
@article {pmid41441637,
year = {2025},
author = {Meng, Y and Chen, J and Liu, L},
title = {Functional Coupling and Evolutionary Relationships Between Toxin-Antitoxin Systems and CRISPR-Cas Systems.},
journal = {Toxins},
volume = {17},
number = {12},
pages = {},
pmid = {41441637},
issn = {2072-6651},
support = {20720250095//Fundamental Research Funds for the Central Universities/ ; 20720240046//Fundamental Research Funds for the Central Universities/ ; 32371346//National Natural Science Foundation of China/ ; 32301007//National Natural Science Foundation of China/ ; 2024J011007//Natural Science Foundation of Fujian Province/ ; 2023J01023//Natural Science Foundation of Fujian Province/ ; 2023J05008//Natural Science Foundation of Fujian Province/ ; },
mesh = {*CRISPR-Cas Systems ; *Toxin-Antitoxin Systems/genetics ; *Evolution, Molecular ; *Bacteria/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; *Bacterial Toxins/genetics ; },
abstract = {Bacteria encode a broad range of survival and defence systems, including CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas systems, restriction-modification systems, and toxin-antitoxin (TA) systems, which are involved in bacterial regulation and immunity. The traditional view holds that CRISPR-Cas systems and TA systems are two independent defense lines in prokaryotes. However, groundbreaking studies in recent years have revealed multi-level functional coupling between them. This review systematically elaborates on this mechanism, focusing on three types of TA systems that mediate the core correlation of CRISPR-Cas systems: CreTA maintains the evolutionary stability of CRISPR-Cas systems through an addiction mechanism; CreR enables self-regulation of CRISPR-Cas expression; and CrePA provides herd immunity by triggering abortive infection after the CRISPR-Cas system has been destroyed by Anti-CRISPRS protein. Additionally, we discuss the evolutionary homology between the type III toxin AbiF and the type VI CRISPR effector Cas13, offering a new perspective for understanding the origin of CRISPR-Cas systems. These findings not only reveal the functional coupling of prokaryotic defense systems but also provide a powerful theoretical framework and practical solutions for addressing stability challenges in CRISPR technology applications.},
}
@article {pmid41440302,
year = {2025},
author = {Chen, J and Liu, S and Chen, S and Mai, J and Abudukadi, M and Chen, Y and Lu, J and Li, G and Ge, C},
title = {Rapid Visual Detection of Mycoplasma Hominis Using an RPA-CRISPR/Cas12a Assay.},
journal = {Biosensors},
volume = {15},
number = {12},
pages = {},
pmid = {41440302},
issn = {2079-6374},
mesh = {*Mycoplasma hominis/isolation &amp; purification/genetics ; *Biosensing Techniques ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques ; Humans ; RNA, Ribosomal, 16S/genetics ; },
abstract = {Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor's response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application.},
}
@article {pmid41349356,
year = {2025},
author = {Shahid, N and Hammond, JR},
title = {Characterization of genetically modified human embryonic kidney 293 cells lacking equilibrative nucleoside transporter subtype 2, or both subtypes 1 and 2, and the impact of their loss on sensitivity to chemotherapeutic purine/pyrimidine analogs.},
journal = {Drug metabolism and disposition: the biological fate of chemicals},
volume = {53},
number = {12},
pages = {100203},
doi = {10.1016/j.dmd.2025.100203},
pmid = {41349356},
issn = {1521-009X},
mesh = {Humans ; HEK293 Cells ; *Equilibrative Nucleoside Transporter 1/genetics/metabolism ; *Equilibrative-Nucleoside Transporter 2/genetics/metabolism ; CRISPR-Cas Systems ; *Purines/pharmacology/metabolism ; *Pyrimidines/pharmacology/metabolism ; *Antineoplastic Agents/pharmacology ; Biological Transport ; Gene Knockout Techniques ; },
abstract = {Equilibrative nucleoside transporters (ENTs) 1 and 2 are considered critical to the cellular uptake of purine and pyrimidine analogs used to treat cancer and viral infections. However, a detailed understanding of the discrete and overlapping roles of these ENT subtypes in drug activity remains limited. A significant barrier to progress has been the absence of model systems that enable functional characterization of individual nucleoside transporters in the context of their native environment. To address this, we developed and characterized a panel of CRISPR/cas9-engineered human embryonic kidney 293 cell lines with selective deletion of ENT subtypes: ENT1 knockout, ENT2 knockout, and dual knockout. These models were used to dissect subtype-specific roles of ENT1 and ENT2 in nucleoside/nucleobase analog uptake and cytotoxicity. Our data show that ENT1 and ENT2 in their endogenous environment have a similar affinity for a range of both endogenous and chemotherapeutic nucleoside and nucleobase analogs. Deletion of ENT1 generally enhanced the sensitivity of cells to these drugs, particularly the nucleobase analogs, likely due to reduced nucleoside salvage by the cells via ENT1. Deletion of ENT2, on the other hand, dramatically reduced the ability of a number of the tested drugs to impact cell viability, by mechanisms beyond those related to reduced cellular uptake of the drugs. This study highlights distinctive roles of ENT1 and ENT2 in the actions of nucleoside/nucleobase analog drugs. SIGNIFICANCE STATEMENT: A panel of genetically modified human embryonic kidney 293 cells has been created as a model to screen novel nucleoside transporter inhibitors and substrates. Using these cell lines, it was revealed that ENT2 may play a more functionally significant role in nucleoside analog chemotherapeutic drug activity than previously appreciated.},
}
@article {pmid41440293,
year = {2025},
author = {Safenkova, IV and Kamionskaya, MV and Sotnikov, DV and Biketov, SF and Zherdev, AV and Dzantiev, BB},
title = {Advancing Lateral Flow Detection in CRISPR/Cas12a Systems Through Rational Understanding and Design Strategies of Reporter Interactions.},
journal = {Biosensors},
volume = {15},
number = {12},
pages = {},
pmid = {41440293},
issn = {2079-6374},
support = {25-16-00246//Russian Science Foundation/ ; 1.1.13//State assignment of the State Research Center of Applied Microbiology and Biotechnology/ ; },
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Kinetics ; },
abstract = {CRISPR/Cas12a systems coupled with lateral flow tests (LFTs) are a promising route to rapid, instrument-free nucleic acid diagnostics due to conversion target recognition into a simple visual readout via cleavage of dual-labeled single-stranded DNA reporters. However, the conventional CRISPR/Cas12a-LFT system is constructed in a format where the intact reporter should block nanoparticle conjugate migration and can produce false-positive signals and shows strong dependence on component stoichiometry and kinetics. Here, we present the first combined experimental and theoretical analysis quantifying these limitations and defining practical solutions. The experimental evaluation included 480 variants of LFT configuration with reporters differing in the concentration of interacting components and the kinetic conditions of the interactions. The most influential factor leading to 100% false-positive results was insufficient interaction time between the components; pre-incubation of the conjugate with the reporter for 5 min eliminated these artifacts. Theoretical analysis of the LFT kinetics based on a mathematical model confirmed kinetic constraints at interaction times below a few minutes, which affect the detectable signal. Reporter concentration and conjugate architecture represented the second major factors: lowering reporter concentration to 20 nM and using smaller gold nanoparticles with multivalent fluorescent reporters markedly improved sensitivity. The difference in sensitivity between various LFT configurations exceeded 50-fold. The combination of identified strategies eliminated false-positive reactions and enabled the detection of up to 20 pM of DNA target (the hisZ gene of Erwinia amylovora, a bacterial phytopathogen). The strategies reported here are general and readily transferable to other DNA targets and CRISPR/Cas12a amplification-free diagnostics.},
}
@article {pmid41440288,
year = {2025},
author = {Hwang, SB and Song, YJ and Park, PG},
title = {A Novel Diagnostic Tool for West Nile Virus Lineage 1a and 2 Using a CRISPR-Cas12a System.},
journal = {Biosensors},
volume = {15},
number = {12},
pages = {},
pmid = {41440288},
issn = {2079-6374},
support = {GCU-202502820001//Gachon University/ ; },
mesh = {*West Nile virus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; *West Nile Fever/diagnosis/virology ; Animals ; Humans ; },
abstract = {The West Nile Virus (WNV), transmitted by Culex mosquitoes as a major vector, has been reported worldwide. Also, West Nile neuroinvasive disease (WNND) caused by WNV lineage 1a and 2 neuroinvasive infections has been constantly reported with high fatality rates. Nevertheless, there are no treatments and vaccinations, so diagnosis in the early stages is important. Recently, a molecular diagnostic technique using DNA endonuclease-targeted CRISPR trans reporter (DETECTR) with the CRISPR-Cas12a system integrated with isothermal nucleic acid amplification has newly emerged. In this study, we designed a 2-Step WNV DETECTR with reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and sensitive WNV diagnosis. It successfully detected down to 1.0 × 10[2] RNA copies for both WNV lineage 1a and 2 with demonstrating similar sensitivity to qRT-PCR without cross-reactivity to other viruses. Additionally, we designed a 1-Step WNV DETECTR, incorporating all processing steps into a single tube, capable of detecting down to 1.0 × 10[3] RNA copies for both lineages. Furthermore, we developed a more streamlined method, the 1-Step with Filter WNV DETECTR, which achieved detection limits comparable to the 2-Step method, while reducing the processing time by 5 min. This study also explored the potential of the Punch-it™ NA-Sample Kit as an efficient alternative lysis method by comparing the detection differences across various lysis methods. Through this method, we achieved rapid and simple amplification and detection processes suitable for field diagnostics with high specificity and sufficient sensitivity. Therefore, DETECTR methods presented themselves as promising alternatives to conventional diagnostic tools, potentially overcoming financial and technical constraints in diverse medical settings.},
}
@article {pmid41437670,
year = {2025},
author = {Buendia-Meraz, JA and Silva-Lucero, MD and Padilla-Mendoza, JR and Cardenas-Aguayo, MD},
title = {Basic Science and Pathogenesis.},
journal = {Alzheimer's &amp; dementia : the journal of the Alzheimer's Association},
volume = {21 Suppl 1},
number = {Suppl 1},
pages = {e103956},
doi = {10.1002/alz70855_103956},
pmid = {41437670},
issn = {1552-5279},
mesh = {Humans ; *Alzheimer Disease/genetics/pathology/metabolism ; Cell Differentiation ; *Neural Stem Cells/metabolism/pathology ; Male ; Female ; Mutation/genetics ; Cells, Cultured ; Aged ; },
abstract = {BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder classified as either sporadic or familial (FAD), depending on the genetic component and age of onset. Understanding and applying gene-editing tools like CRISPR-Cas is of great relevance for correcting mutations and/or alterations to reverse the pathological phenotype of neurodegenerative diseases.<br><br>METHOD: To achieve this objective, we performed cell culture, immunodetection via Western blot, immunocytofluorescence, and viral transduction.<br><br>RESULT: Cell culture characterization of control and patient-derived NPCs was performed using immunocytofluorescence and Western blot detection of SOX2, Oct3/4, Nanog, Nestin associated with stem cell stages, as well as cellular maturation states (b-III-Tub, GFAP, Olig2), proliferation (Ki67), and differentiation (MAP2, NeuN). We found that FAD-derived NPCs expressed more mature markers of neuronal differentiation than control individuals' NPCs. Viral transduction with Adeno-associated viruses (AAV-9) carrying the necessary gene sequences for mutation correction in patient-derived cells was successfully performed.<br><br>CONCLUSION: Control patient cells exhibited better characterization towards stem-like phenotypes, while cells from patients with the FAD mutation of interest showed markers of cellular maturation. Viral transduction was successfully carried out; however, further analysis is needed to determine the restoration of the healthy phenotype.},
}
@article {pmid41437291,
year = {2025},
author = {Suárez, A and Melloni, AN and Hyman, BT and Nguyen, L},
title = {Basic Science and Pathogenesis.},
journal = {Alzheimer's &amp; dementia : the journal of the Alzheimer's Association},
volume = {21 Suppl 1},
number = {Suppl 1},
pages = {e102945},
doi = {10.1002/alz70855_102945},
pmid = {41437291},
issn = {1552-5279},
mesh = {Humans ; *Induced Pluripotent Stem Cells ; *Caspase 8/genetics ; *Alzheimer Disease/genetics/pathology ; CRISPR-Cas Systems ; Neurons ; },
abstract = {BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common form of dementia. While various genetic mutations contribute to AD risk, the full genetic landscape of the disease remains unclear. Tandem repeat expansion mutations have been implicated in a subset of neurodegenerative disorders. Recently, a repeat expansion variant in CASP8 (CASP8-GGGAGA-AD-R1) has been associated with an increased risk of AD with odds ratio of 2.2 (p = 3.1 x 10[-5]). These results raise the question of how the CASP8-GGGAGA-AD-R1 sequence variant contributes to AD.<br><br>METHOD: We developed induced pluripotent stem cells (iPSC) patient derived models from AD cases with and without CASP8-GGGAGA-AD-R1 variant and control cases to study CASP8-related pathogenic pathways. We also developed CRISPR/Cas9 editing systems to excise the CASP8-GGGAGA-AD-R1 sequence to generate isogenic cell lines. Neuronal cultures developed from parental and isogenic iPSCs will be studied for disease-relevant molecular and pathogenic phenotypes.<br><br>RESULT: We have generated iPSCs using fibroblast cells derived from 4 CASP8-GGGAGA-AD-R1(+) AD, 5 CASP8-GGGAGA-C-Var(+) AD, and 5 control cases. The iPSC lines show pluripotent markers and normal karyotypes. Repeat primed PCR (RP-PCR) and long-range PCR (LR-PCR) showed the genotypes of CASP8 GGGAGA repeats are consistent with those in fibroblasts. For CRISPR/Cas9 editing of the CASP8 repeat expansion locus, we successfully cloned a plasmid to express Cas 9 protein, a fluorescence marker (mCherry and GFP) and guide RNA (gRNA) that target the upstream and downstream unique sequences of the CASP8 repeat expansion locus. Editing efficiency was tested using HEK293T cells and iPSCs, with GFP and mCherry fluorescence signals detected upon 17 and 30 hours post-transfection, demonstrating a successful expression of Cas9 proteins. LR-PCR using genomic DNA extracted from HEK293 transfected cells show expected cut size upon transfection with Cas9 systems targeting the CASP8 repeat loci.<br><br>CONCLUSION: We successfully developed patient-derived models to investigate the role of the CASP8-GGGAGA-AD-R1 repeat expansion in AD. Through CRISPR/Cas9, we demonstrated efficient excision of the mutation in HEK293 cells. Moving forward, we will apply this system to patient models to assess whether removing the mutation can mitigate disease phenotypes, providing insights into AD mechanisms and potential therapeutic strategies.},
}
@article {pmid41436729,
year = {2025},
author = {Wu, Y and Cai, Z and Cross, D and Noble, JR and Prest, K and Littleboy, J and Cohen, SB and Edlundh, B and Koh, JMS and Xu, R and Noor, Z and Bastami, M and Valentini, S and Richardson, L and Barthorpe, S and Arymanesh, N and Robinson, PJ and Hains, PG and Garnett, MJ and Zhong, Q and Reddel, RR and MacKenzie, KL},
title = {Large-scale drug sensitivity, gene dependency, and proteogenomic analyses of telomere maintenance mechanisms in cancer cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11337},
pmid = {41436729},
issn = {2041-1723},
support = {2017/TPG001, REG171150//Cancer Institute NSW (Cancer Institute New South Wales)/ ; CMP-01//NSW Ministry of Health (NSW Health)/ ; CMP-01//NSW Ministry of Health (NSW Health)/ ; RG24-05//Cancer Council NSW (Cancer Council New South Wales)/ ; IIRS-18-164//National Breast Cancer Foundation (NBCF)/ ; GNT1170739, H2020-SC1-DTH-2018-1//Department of Health | National Health and Medical Research Council (NHMRC)/ ; GNT1170739//Department of Health | National Health and Medical Research Council (NHMRC)/ ; 206194/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Humans ; *Neoplasms/genetics/drug therapy/metabolism ; *Telomere Homeostasis/genetics/drug effects ; Cell Line, Tumor ; *Telomere/metabolism/genetics ; Telomerase/metabolism/genetics ; *Proteogenomics/methods ; CRISPR-Cas Systems ; *Antineoplastic Agents/pharmacology ; Proteomics ; Drug Resistance, Neoplasm/genetics ; },
abstract = {Replicative immortality is a hallmark of cancer, driven by the activation of telomere maintenance mechanisms, that is yet to be therapeutically exploited. To expedite discoveries that will enable the development of therapeutics that target telomere maintenance mechanisms, this study provides a resource of telomere biology metrics for a pan-cancer panel of 976 cell lines. We generate proteomic data from data-independent-acquisition mass spectrometry for most of these cell lines and integrate pre-existing multi-omic, drug sensitivity, and molecular dependency data from CRISPR/Cas9 knock-out screens. The data illustrate a broad range and heterogeneity in telomere biology, including states that diverge from the binary model of telomere maintenance activation involving either telomerase or the Alternative Lengthening of Telomeres mechanism. Using the telomere biology metrics and multi-omic data, we derive proteomic and transcriptomic predictors of Alternative Lengthening of Telomeres and telomerase activity levels. Our investigations also reveal molecular vulnerabilities associated with the Alternative Lengthening of Telomeres mechanism and drug sensitivity correlating with telomerase activity levels. These findings illustrate opportunities for leveraging this resource to realize the potential for telomere biology-directed cancer therapeutics and companion diagnostics.},
}
@article {pmid41434963,
year = {2025},
author = {Ramos, DM and Nelson, MP and Calzada, L and Krishna, S and Neuman, SS and Weller, C and Johnson, N and Nalls, MA and Lara, E and Qi, A and Santiana, M and Pantazis, C and Skarnes, WC and Singleton, A and Cookson, MR and Ward, M},
title = {Basic Science and Pathogenesis.},
journal = {Alzheimer's &amp; dementia : the journal of the Alzheimer's Association},
volume = {21 Suppl 1},
number = {Suppl 1},
pages = {e097245},
doi = {10.1002/alz70855_097245},
pmid = {41434963},
issn = {1552-5279},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism ; *Neurons/metabolism ; *CRISPR-Cas Systems ; *Neurodegenerative Diseases/genetics ; Gene Knockdown Techniques ; },
abstract = {BACKGROUND: The iPSC Neurodegenerative Disease Initiative (iNDI) is the largest-ever induced pluripotent stem cell (iPSC) genome engineering project, modeling over 100 ADRD mutations in high-quality isogenic human iPSCs. iNDI leverages unbiased CRISPRi screens as a powerful tool to identify fundamental mechanisms and modifiers of disease. However, current CRISPRi molecular tools are poorly optimized for use in iPSC-derived neurons (iNeurons). Here we develop a Cre-lox inducible CRISPRi system (CRISPRi-Cre), enabling gene knockdown upon Cre delivery to postmitotic iNeurons, and identification of neuron-specific, disease-relevant modifiers.<br><br>METHOD: We modified a plasmid carrying a potent Zim3-dCas9 transcriptional repressor to include a strong floxed STOP cassette upstream of the Zim3 start codon. We leveraged HaloTag-TDP43 and HaloTag-FUS iSPCs from the iNDI project paired with flow cytometry to validate leakiness and responsiveness to Cre in iPSCs and iNeurons treated with sgRNAs. We then performed a genome-wide CRISPRi survival screen in iNeurons to demonstrate broad functionality of this inducible CRISPRi system with over 20,000 sgRNAs. Finally, we use CRISPRi-Cre to identify neuron-specific regulators of neuronal activity in iNeurons.<br><br>RESULT: We demonstrate that in the absence of Cre, dCas9 is inactive. Delivery of lentivirus-Cre to iNeurons activates dCas9, resulting in potent gene knockdown. In genome-wide CRISPRi screens, we show that CRISPRi-Cre identifies many of the same hits observed in screens using constitutive-active dCas9, and importantly uncovers novel neuron-specific hits not identified in previous CRISPRi screens.<br><br>CONCLUSION: Here, we developed a robust Cre-inducible CRISPRi system that enables post-mitotic gene knockdown in iPSC-derived neurons. Our CRISPRi screens identify neuron-specific hits, demonstrating the utility of our tool to help uncover disease-relevant mechanisms, modifiers, and potential therapeutic targets in relevant cell types.},
}
@article {pmid41432353,
year = {2025},
author = {Tang, X and Ju, D and Hu, H},
title = {A Dual CRISPR-Cas/Cre-loxP Genome Engineering Strategy for Stable Uricase Expression in Food-Grade Probiotics.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00774},
pmid = {41432353},
issn = {2161-5063},
abstract = {The development of robust, food-grade microbial chassis with tailored metabolic functions is critical for advancing synthetic biology applications in health and nutrition. Here, we report a dual genome engineering strategy that integrates CRISPR-Cas9-mediated knock-in with Cre/loxP-driven genome reduction to streamline the genome of Lactococcus lactis NZ9000 and enable stable expression of a high-activity uricase variant. The resulting strain, NZ9000::UA[T]-ΔD6, demonstrated enhanced enzymatic performance in vitro, achieving 2.34 U/mL activity and complete degradation of ∼500 μM urate within 20 h. Beyond improved catalytic output, this dual-system approach established a genetically stable and biosafe probiotic chassis with moderate colonization capacity in the murine gut. The integration of CRISPR-Cas and Cre/loxP techniques in this work is intended to enhance the expression of heterologous genes in the chassis strain, while providing a versatile platform for the rational design of food-grade probiotics and offering a general strategy for constructing living biotherapeutic agents with targeted metabolic activities.},
}
@article {pmid41432281,
year = {2025},
author = {Liu, XL and Liu, L and Cheng, L},
title = {Sequence-independent optical regulation of CRISPR/Cas editing using star-shaped crRNA dendrimers.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc06011g},
pmid = {41432281},
issn = {1364-548X},
abstract = {Precise spatiotemporal control of CRISPR/Cas editing is vital for studying dynamic processes and ensuring therapeutic safety. We present a single-site photolabile crRNA dendrimer platform enabling robust, sequence-independent optical regulation of Cas9 and Cas12a. This simple, universal strategy achieves rapid OFF-to-ON control with minimal leakage, advancing programmable, light-responsive genome editing for biomedical applications.},
}
@article {pmid41417859,
year = {2025},
author = {Bradford, J and Joy, D and Winsen, M and Meurant, N and Wilkins, M and Wilson, LOW and Bauer, DC and Perrin, D},
title = {Democratising high performance computing for bioinformatics through serverless cloud computing: A case study on CRISPR-Cas9 guide RNA design with Crackling Cloud.},
journal = {PLoS computational biology},
volume = {21},
number = {12},
pages = {e1013819},
doi = {10.1371/journal.pcbi.1013819},
pmid = {41417859},
issn = {1553-7358},
mesh = {*Cloud Computing ; *Computational Biology/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Software ; },
abstract = {Organisations are challenged when meeting the computational requirements of large-scale bioinformatics analyses using their own resources. Cloud computing has democratised large-scale resources, and to reduce the barriers of working with large-scale compute, leading cloud vendors offer serverless computing, a low-maintenance and low-cost model that provides ample resources for highly scalable software applications. While serverless computing has broad use, its adoption in bioinformatics remains poor. Here, we demonstrate the most extensive use of high-performance serverless computing for bioinformatics by applying the available technologies to CRISPR-Cas9 guide RNA (gRNA) design. Our adaptation of the established gRNA design tool, named Crackling, implements a novel, cloud-native and serverless-based, high-performance computing environment using technologies made available by Amazon Web Services (AWS). The architecture, compatible with technologies from all leading cloud vendors, and the AWS implementation, contributes to an effort of reducing the barrier to large computational capacity in bioinformatics and for CRISPR-Cas9 gRNA design. Crackling Cloud can be deployed to any AWS account, and is freely available on GitHub under the BSD 3-clause license: https://github.com/bmds-lab/Crackling-AWS.},
}
@article {pmid41284608,
year = {2025},
author = {Zhu, W and Qi, T and Wu, Z and Zhong, W and Yan, L and Feng, J and Jin, F and Chen, W and Cai, Z and Rui, Y and Maria Da Costa, E and Liu, Q and Fu, Q and Zheng, L},
title = {One-Pot Photocontrolled CRISPR-Cas12b Coupled with Loop-Mediated Isothermal Amplification Assay for Point-of-Care Test of Mycoplasma pneumonia.},
journal = {ACS sensors},
volume = {10},
number = {12},
pages = {9323-9334},
doi = {10.1021/acssensors.5c01679},
pmid = {41284608},
issn = {2379-3694},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Mycoplasma pneumoniae/isolation &amp; purification/genetics ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; *Point-of-Care Testing ; Humans ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; DNA, Bacterial/genetics/analysis ; },
abstract = {Mycoplasma pneumoniae (MP) is a highly prevalent respiratory pathogen, making the development of point-of-care testing (POCT) methods for its detection essential. The integration of loop-mediated isothermal amplification (LAMP) with CRISPR-Cas12b systems demonstrates remarkable specificity and offers promising potential for MP POCT application. However, the current one-pot LAMP/CRISPR-Cas12b system (HOLMESv2) faces the challenge of low sensitivity due to the premature cleavage of the template by CRISPR, which limits its practical utility. To address this, this study introduces a photocontrolled HOLMESv2 (pHOLMESv2) assay using gRNA with an NPOM-modified spacer region. This modification prevents full base pairing between the gRNA and MP DNA, thereby keeping the CRISPR-Cas12b system in an inactive condition during the LAMP reaction and avoiding unintended cleavage of the DNA template. After completion of the LAMP reaction, light irradiation eliminates the NPOM group from the gRNA, restoring its activity to cleave the LAMP products, resulting in a fluorescence signal. The pHOLMESv2 assay successfully addresses the issue of premature DNA template cleavage, improving the limit of detection (LoD) by 133-fold (7.5 copies/μL). Additionally, this method enables direct detection of samples treated with nucleic acid release agents, eliminating the need for complex extraction, and features lyophilized reagents for enhanced stability, storage, and transport. The efficacy of pHOLMESv2 was assessed with 160 clinical MP samples, achieving a sensitivity of 99.0% and a specificity of 100.0%. The pHOLMESv2 assay, when combined with the developed smartphone-based amplification reader, provides a highly sensitive, specific, portable, and cost-effective MP detection, indicating its potential for significant diagnostic use.},
}
@article {pmid41218722,
year = {2026},
author = {Madugula, SS and Jayasinghe-Arachchige, VM and Norgan Radler, CR and Wang, S and Liu, J},
title = {Structure-Based Classification of CRISPR/Cas9 Proteins: A Machine Learning Approach to Elucidating Cas9 Allostery.},
journal = {Journal of molecular biology},
volume = {438},
number = {2},
pages = {169538},
doi = {10.1016/j.jmb.2025.169538},
pmid = {41218722},
issn = {1089-8638},
mesh = {*Machine Learning ; Allosteric Regulation ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; Molecular Dynamics Simulation ; Streptococcus pyogenes/genetics/enzymology ; Gene Editing/methods ; Protein Conformation ; },
abstract = {The CRISPR/Cas9 system is a powerful gene-editing tool. Its specificity and stability rely on complex allosteric regulation. Understanding these allosteric regulations is essential for developing high-fidelity Cas9 variants with reduced off-target effects. Here, we used a novel structure-based machine learning (ML) approach to systematically identify long-range allosteric networks in Cas9. Our ML model was trained using all available Cas9 structures, ensuring a comprehensive representation of Cas9's structural landscape. We then applied this model to Streptococcus pyogenes Cas9 (SpCas9) to demonstrate the feature selection process. Using Cα-Cα inter-residue distances, we mapped key allosteric networks and refined them through a two-stage SHAP feature selection (FS) strategy, reducing a vast feature space to 28 critical Lysine-Arginine (Lys-Arg) residue pairs that mediate SpCas9 interdomain communication, stability, and specificity. These Lys-Arg pairs initially shared a 46.5 Å inter-residue distance, but molecular dynamics simulations revealed distinct stabilization behaviors, indicating a hierarchical allosteric network. Further mutational analysis of R78A-K855A (M1) and R765A-K1246A (M2) identified an "electrostatic valley," a stabilizing network where positively charged residues interact with negatively charged DNA to maintain SpCas9's structural integrity. Disrupting this valley through direct (M2) or allosteric (M1) mutations destabilized SpCas9's DNA-bound conformation, leading to distinct pathways for improving SpCas9 specificity. This study provides a new framework for understanding allostery in Cas9, integrating ML-driven structural analysis with MD simulations. By identifying key allosteric residues and introducing the electrostatic valley as a central concept, we offer a rational strategy for engineering high-fidelity Cas9 variants. Beyond Cas9, our approach can be applied to uncover allosteric hotspots in other enzyme regulations and rational protein design.},
}
@article {pmid41037798,
year = {2025},
author = {Park, HJ and Kim, J and Choi, J and Ryou, C and Shin, E and Lee, JY},
title = {Targeted genome editing of ZKSCAN3 mitigates the neurotoxicity caused by mutant HTT (huntingtin) in a Huntington disease animal model and three-dimensional cell culture of Huntington disease.},
journal = {Autophagy},
volume = {21},
number = {12},
pages = {3398-3412},
doi = {10.1080/15548627.2025.2569965},
pmid = {41037798},
issn = {1554-8635},
mesh = {Animals ; *Huntington Disease/genetics/pathology ; Humans ; Disease Models, Animal ; Induced Pluripotent Stem Cells/metabolism ; Autophagy/genetics ; *Huntingtin Protein/genetics/metabolism/toxicity ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Transcription Factors/genetics/metabolism ; Spheroids, Cellular/metabolism ; Mice ; *Mutation/genetics ; Lysosomes/metabolism ; Neurons/metabolism/pathology ; },
abstract = {Huntington disease (HD) is a neurodegenerative disease caused by the expression of a mutant form of HTT (huntingtin; mHTT), caused by an abnormal expansion of polyglutamine in HTT. In HD, macroautophagy/autophagy dysfunction can cause mHTT accumulation. Moreover, the promotion of autophagy is considered a therapeutic strategy for the treatment of HD. ZKSCAN3 (zinc finger with KRAB And SCAN domains 3) has been identified as a transcriptional repressor of TFEB (transcription factor EB), a master regulator of autophagy and lysosomal functions. In this study, we conducted CRISPR-Cas9-based gene ablation to disrupt ZKSCAN3 in HD animal models and HD patient-induced pluripotent stem cell (iPSC) -derived three-dimensional (3D) spheroids. In animal models of HD, targeted in vivo zkscan3 ablation via a single adeno-associated virus (AAV) mediated CRISPR-Cas9 approach resulted in reduced mHTT levels, leading to improvements in both behavioral symptoms and the brain environment. Furthermore, CRISPR-Cas9 mediated ablation of ZKSCAN3 in 3D spheroids from HD patient-derived iPSC resulted in increased autophagy and lysosomal function, along with reduced mHTT accumulation. Specifically, in iPSC-derived neurons from HD patients, ZKSCAN3-depleted neurons demonstrated increased lysosomal function and reduced oxidative stress compared to controls. Additionally, transcriptional analysis of ZKSCAN3-edited neurons revealed an increased expression of genes involved in synaptic function and transporter activity. Taken together, these results suggest that in HD treatment strategies for improving neuronal function and the brain environment, ZKSCAN3 downregulation in neurons by autophagy activation may improve the brain environment through neuronal self-repair.Abbreviations: 2D: two-dimensional; 3D: three-dimensional; 4-HNE: 4-hydroxynonenal; AAV: adeno-associated virus; AD: Alzheimer disease; Aβ: beta-amyloid; DAPI: 4,6-diamidino-2-phenylindole; GFP: green fluorescent protein; HD: Huntington disease; HTT: huntingtin; IXMC: ImageXpress microconfocal high-content imaging system; Indel: insertion or deletion; iPSC: induced pluripotent stem cell; LAMP1: lysosomal-associated membrane protein 1; mHTT: mutant huntingtin; NPCs: neural precursor cells; RBFOX3/NeuN: RNA binding fox-1 homolog 3; PD: Parkinson disease; RNP: ribonucleoprotein; sgRNAs: single guide RNAs; ST: striatum; TFEB: transcription factor EB; TUBB3/Tuj-1: tubulin beta 3 class III; ZKSCAN3: zinc finger with KRAB and SCAN domains 3.},
}
@article {pmid41390513,
year = {2025},
author = {Vermeulen, M and Craig, AW and Babak, T},
title = {Challenges and opportunities for oncology drug repurposing informed by synthetic lethality.},
journal = {NPJ systems biology and applications},
volume = {11},
number = {1},
pages = {143},
pmid = {41390513},
issn = {2056-7189},
support = {PJT 178214//Canadian Institutes of Health Research Project Grant/ ; },
mesh = {Humans ; *Drug Repositioning/methods ; *Synthetic Lethal Mutations/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; *Antineoplastic Agents/pharmacology/therapeutic use ; *Neoplasms/genetics/drug therapy ; Gene Knockout Techniques ; Mutation ; },
abstract = {Although two-thirds of cancers arise from loss-of-function mutations in tumor suppressor genes, there are few approved targeted therapies linked to these alterations. Synthetic lethality offers a promising strategy to treat such cancers by targeting vulnerabilities unique to cancer cells with these mutations. To identify clinically relevant synthetic lethal interactions, we analyzed genome-wide CRISPR/Cas9 knock-out (KO) viability screens from the Cancer Dependency Map and evaluated their clinical relevance in patient tumors through mutual exclusivity, a pattern indicative of synthetic lethality. Indeed, we found significant enrichment of mutual exclusivity for interactions involving cancer driver genes compared to non-driver mutations. To identify therapeutic opportunities, we integrated drug sensitivity data to identify inhibitors that mimic the effects of CRISPR-mediated KO. This approach revealed potential drug repurposing opportunities, including BRD2 inhibitors for bladder cancers with ARID1A mutations and SIN3A-mutated cell lines showing sensitivity to nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. However, we discovered that pharmacological inhibitors often fail to phenocopy KO of matched drug targets, with only a small fraction of drugs inducing similar effects. This discrepancy reveals fundamental differences between pharmacological and genetic perturbations, emphasizing the need for approaches that directly assess the interplay of loss-of-function mutations and drug activity in cancer models.},
}
@article {pmid41205600,
year = {2025},
author = {Saini, M and Castro-Giner, F and Hotz, A and Sznurkowska, MK and Nüesch, M and Cuenot, FM and Budinjas, S and Bilfeld, G and Ildız, ES and Strittmatter, K and Krol, I and Diamantopoulou, Z and Paasinen-Sohns, A and Waldmeier, M and Cássio, R and Kreutzer, S and Kontarakis, Z and Gvozdenovic, A and Aceto, N},
title = {StealTHY: An immunogen-free CRISPR platform to expose concealed metastasis regulators in immunocompetent models.},
journal = {Cell},
volume = {188},
number = {26},
pages = {7591-7609.e32},
doi = {10.1016/j.cell.2025.10.007},
pmid = {41205600},
issn = {1097-4172},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Humans ; *Neoplasm Metastasis/genetics ; Mice ; Cell Line, Tumor ; Immunocompetence ; *Neoplasms/genetics/immunology/pathology ; Mice, Inbred C57BL ; Female ; },
abstract = {CRISPR screens have become standard gene discovery platforms in various contexts, including cancer. Yet commonly available CRISPR-Cas9 tools are increasingly recognized as unfit for in vivo investigations in immunocompetent contexts, due to broad immunogenicity of bacterial nucleases and reporters. Here, we show how conventional CRISPR screens in tumor grafts are systematically jeopardized by immunoediting in syngeneic and humanized immunocompetent hosts, resulting in iatrogenic clonal dropouts and ultimately compromising target identification. To resolve this, we present StealTHY, an immunogen-free CRISPR platform compatible with virtually all immunocompetent designs, enabling preservation of clonal architecture and exposing previously concealed cancer vulnerabilities. Among these, we identify the AMH-AMHR2 axis as a formerly unappreciated metastasis target. Thus, with StealTHY, we provide a new resource to expand the applicability of CRISPR screens to immunocompetent models, including humanized tumor grafts, revealing metastasis regulators of therapeutic relevance.},
}
@article {pmid41430372,
year = {2025},
author = {Kim, GE and Lee, SY and Kang, YJ and Bin Jin, H and Park, HH},
title = {AcrIIA19 binds to the WED domain and inhibits various Cas9 orthologs at multiple stages.},
journal = {Communications biology},
volume = {},
number = {},
pages = {},
doi = {10.1038/s42003-025-09417-6},
pmid = {41430372},
issn = {2399-3642},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; RS-2025-16065724//National Research Foundation of Korea (NRF)/ ; },
abstract = {Anti-CRISPR (Acr) proteins are natural inhibitors of clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems, providing valuable tools for regulating genome editing. Here, we present the crystal structure of AcrIIA19, a plasmid-encoded Type II-A CRISPR-Cas system inhibitor that targets Cas9. AcrIIA19 adopts a previously uncharacterized fold and forms a stable homodimer. Biochemical assays revealed that AcrIIA19 binds selectively to the wedge (WED) domain of Cas9, a conserved structural interface critical for single guide RNA-DNA duplex stabilization and catalysis. This interaction disrupts Cas9 activity at multiple stages, independent of the order of complex assembly. Notably, AcrIIA19 exhibits broad-spectrum inhibition across divergent Cas9 orthologs, including Streptococcus pyogenes and Staphylococcus aureus Cas9, by exploiting a conserved WED domain vulnerability. Our findings establish AcrIIA19 as a versatile Cas9 inhibitor and highlight the WED domain as a strategic target for developing species-agnostic CRISPR regulatory tools in biotechnology and therapeutic applications.},
}
@article {pmid41428734,
year = {2025},
author = {Zhu, C and Xiao, D and Wang, Y and Han, H and Qin, C and Liu, S and Chen, X and Xiao, H and Chen, X and Shi, J and Tang, J and Shen, J and Song, H},
title = {Molecular basis of NFIB-mediated regulation of oncogenic transcription.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41428734},
issn = {1362-4962},
support = {Z2023033//Hunan Health Commission Key Clinical Specialty Major Research Project/ ; NFPS-JJ-501348//Chinese National Key Clinical Specialty Program/ ; 82272508//National Natural Science Foundation of China/ ; 005/2023/SKL//State Key Laboratory of Mechanism and Quality of Chinese Medicine/ ; 0007/2022/AKP, 0068/2023/ITP2, 0143/2025/ITP2//Macau Science and Technology Development Fund/ ; MYRG-GRG2024-00283-ICMS-UMDF, SRG2023-00054-ICMS//University of Macau/ ; 005/2023/SKL//State Key Laboratory of Mechanism and Quality of Chinese Medicine/ ; },
mesh = {Humans ; *NFI Transcription Factors/genetics/chemistry/metabolism ; *Gene Expression Regulation, Neoplastic ; DNA/metabolism/chemistry/genetics ; Cell Line, Tumor ; *Transcription, Genetic ; CRISPR-Cas Systems ; Protein Binding ; *Neoplasms/genetics/pathology/metabolism ; Cell Proliferation/genetics ; Oncogenes ; Models, Molecular ; Cell Movement/genetics ; Transcriptional Activation ; },
abstract = {The Nuclear Factor I (NFI) family of transcription factors orchestrates key regulatory programs in development, differentiation, and metabolism, with dysregulation implicated in diverse pathological conditions, including cancer. Among the paralogs, NFIB has emerged as an oncogenic driver in multiple tumor types, yet the mechanisms through which it engages DNA and directs oncogenic transcriptional programs remain undefined. Here, using cancer cells with high NFIB expression, we demonstrate that NFIB promotes malignant phenotypes, as CRISPR-Cas9 knockout impairs proliferation, migration, and invasion. Transcriptomic profiling reveals that NFIB regulates a cancer-enriched gene network that includes FGFR3 and PDGFRB. Biophysical analyses show that NFIB, including its DNA-binding domain, functions as a monomer and binds DNA with strict 1:1 stoichiometry. High-resolution crystal structures of NFIB DNA-binding domain bound to ChIP-seq-derived DNA motifs reveal a monomeric binding mode mediated by conserved base-specific interactions with the TGGCA sequence, providing an atomic view of NFIB-DNA recognition. Mutational disruption of key DNA-contacting residues abolishes DNA binding and transcriptional activation, linking atomic-level recognition to oncogenic transcriptional regulation. Together, these findings elucidate the structural mechanism underlying NFIB function in cancer and establish a framework for therapeutic strategies targeting NFIB-driven malignancies.},
}
@article {pmid41428733,
year = {2025},
author = {Yang, Z and Yu, M and Li, P and Li, Z and Teng, Y and Zhou, Y and Zhao, M and Liu, C and Zhao, Z and Wang, Z and Li, J and Jing, Y and Li, Y and Zhao, H and Song, W and Bian, C and Zhao, H and Chen, J and Xin, B and Lai, J},
title = {Casδ, an evolutionary transitional CRISPR system enables efficient genome editing across animals and plants.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41428733},
issn = {1362-4962},
support = {//Agriculture Science and Technology/ ; 2023YFD1202900//National Key Research and Development Program of China/ ; PC2023A01004//Pinduoduo-China Agricultural University/ ; //Agriculture Science and Technology/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Genome, Plant ; RNA, Guide, CRISPR-Cas Systems/genetics ; Zea mays/genetics ; Evolution, Molecular ; Oryza/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) adaptive immune systems provide sequence-specific mechanisms for targeting foreign DNA or RNA and have been widely used in genome editing and DNA detection. Type V CRISPR-Cas systems are characterized by a single RNA-guided RuvC domain-containing effector, Cas12. Here, through comprehensive mining of large-scale genomic and metagenomic data from microbial sources, we identified a new Class 2 CRISPR-Cas effector superfamily, designated Casδ, comprising three members with protein sizes ranging from 867 to 936 amino acids. Biochemical analyses revealed that Casδ-1 functions as a single RNA-guided endonuclease with specific recognition of 5'-RYR-3' protospacer-adjacent motifs, where R represents A or G, and Y represents T or C. Casδ-1 exhibits robust double-stranded DNA cleavage activity and target-dependent trans-cleavage activity. Casδ-1 mediates efficient genome editing across species, achieving up to 60% indel rates in human cells while generating homozygous knockout lines in two agriculturally important monocot species (Oryza sativa and Zea mays) through stable transformation. Structural and evolutionary analyses reveal Casδ as an evolutionary transitional nuclease bridging Cas12n and canonical type V systems, featuring a C-terminal loop that is essential for activity. Collectively, Casδ is an evolutionarily distinct, compact (<1000 aa), tracrRNA-free CRISPR system enabling versatile cross-kingdom genome editing.},
}
@article {pmid41428728,
year = {2025},
author = {Gervais, NC and Rogers, RKJ and Robin, MR and Shapiro, RS},
title = {HyperdCas12a-based multiplexed genetic regulation in Candida albicans.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41428728},
issn = {1362-4962},
support = {RGPIN-2018-4914//Natural Sciences and Engineering Research Council of Canada/ ; //NSERC/ ; //Canada Research Chair/ ; RGPIN-2018-4914//NSERC/ ; },
mesh = {*Candida albicans/genetics/metabolism/drug effects ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Fungal ; Ergosterol/biosynthesis ; Fungal Proteins/genetics/metabolism ; Drug Resistance, Fungal/genetics ; },
abstract = {Complex microbial phenotypes involve the combined activity of diverse gene regulatory networks. However, the majority of reverse genetics approaches in microbial pathogenesis research have focused on single-gene perturbation studies, in part due to the lack of available genetic tools in many pathogens. Developing enhanced versions of CRISPR-Cas platforms holds significant promise for improving the scalability of microbial functional genomics research. Here, we demonstrate highly efficient, inducible, and multiplexed activation and repression in the major human fungal pathogen Candida albicans by translating the hyperdCas12a variant to the fungal kingdom. This represents the first application of a CRISPR-Cas12 system in a human fungal pathogen. We profile the effectiveness of our new CRISPR activation and CRISPR interference tools and achieve tunable levels of target modulation. Further, we demonstrate that perturbing combinations of genes in the drug efflux and ergosterol biosynthesis pathways reveals important redundancies and synergistic properties in drug resistance circuitry. Our hyperdCas12a platform is thus an efficient system for the rapid generation of combinatorial mutants that will enable the mechanistic understanding of genetic interactions involved in diverse phenotypes in C. albicans. The enhanced activity with hyperdCas12a in fungi suggests it could be translated to other microbes as a powerful tool for studying genetic interactions.},
}
@article {pmid41428487,
year = {2025},
author = {Deng, C and Hu, J and Chen, Q and Zhou, S and Ni, J},
title = {Expanded global groundwater microbial diversity reveals bioprospecting potential.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116760},
doi = {10.1016/j.celrep.2025.116760},
pmid = {41428487},
issn = {2211-1247},
abstract = {Although the terrestrial subsurface harbors a substantial fraction of Earth's microbial biomass, the genomic diversity of groundwater microbiomes and their potential for bioprospecting remain poorly characterized. Here, we recovered 44,320 bacterial and archaeal genomes from in-house and publicly available metagenomic datasets, establishing a large-scale groundwater microbiota catalog (GWMC) spanning 167 phyla, including four candidate phyla and over 12,000 previously uncharacterized species. This unprecedented phylogenetic diversity was accompanied by a bimodal genome size distribution (0.3-12.8 Mbp), revealing divergent strategies of genomic allocation. By mining extensive genomic resources, we found that small genomes prioritized molecular defense and redox regulation, whereas large genomes frequently harbored greater biosynthetic potential. Notably, we establish the largest selenoprotein catalog to date and highlight groundwater as an overlooked hotspot of microbial selenium metabolism. Overall, this work advances our understanding of microbial diversity in aquifers and uncovers underexplored genomic resources with potential for biotechnology and biomedicine.},
}
@article {pmid41428463,
year = {2025},
author = {Ye, T and Xue, M and Chen, H and Yue, S and Yuan, M and Yu, J and Cao, H and Hao, L and Wu, X and Yin, F and Xu, F},
title = {Allosteric Aptamer CRISPR/Cas Activation Enables Non-competitive ATP Detection and Meat Freshness Assessment.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c12150},
pmid = {41428463},
issn = {1520-5118},
abstract = {CRISPR/Cas-based aptasensors (Cas-aptasensors) hold great promise for detecting non-nucleic acid targets, yet their intrinsic competitive recognition mechanism imposes a trade-off between transduction efficiency and background leakage. In this study, we developed a Cas-aptasensor that employs a non-competitive recognition mechanism. In our design, the aptamer-target interaction accelerates the toehold-mediated strand displacement reaction and exposes a second toehold domain. The CRISPR/Cas system is ultimately activated via a cascade strand-displacement reaction, which is hindered in the absence of the target and, thus, delays activation. We demonstrated the applicability of this non-competitive Cas-aptasensor for the detection of ATP, achieving a detection limit as low as 1.0 nM within 45 min. Furthermore, we successfully applied this method to ATP detection in complex matrices and to assess the freshness of diverse meat products across different storage temperatures. Overall, this work advances the design of Cas-aptasensors and expands their potential applications in food safety monitoring.},
}
@article {pmid41425600,
year = {2025},
author = {Fazeli, A and Ullrich, E and Cathomen, T and Bexte, T},
title = {Engineering with care: safety assessment platforms for CRISPR-modified natural killer cells.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1711414},
pmid = {41425600},
issn = {1664-3224},
mesh = {*Killer Cells, Natural/immunology/metabolism/transplantation ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Immunotherapy, Adoptive/methods/adverse effects ; },
abstract = {CRISPR-based gene editing has become a transformative tool to enhance immune cell therapies. In particular, engineering natural killer (NK) cells with CRISPR/Cas systems has gained traction due to their ability to mediate strong anti-tumor responses in an MHC-unrestricted, non-alloreactive manner. Early trials show the feasibility and safety of allogeneic NK cells, paving the way as scalable "off-the-shelf" products. CRISPR/Cas9 edits genomes by inducing DNA double-strand breaks (DSBs), mainly repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR). While effective, CRISPR carries risks of off-target (OT) activity that may disrupt essential genes, cause chromosomal rearrangements, or trigger oncogenic changes - posing threats to product integrity and patient safety. These concerns intensify with multiplex editing, where multiple loci are modified to improve function, persistence, and immune evasion. Since unmodified NK cells are typically short-lived, many clinical-stage products are engineered to express IL-15 or related constructs, extending their half-life and amplifying risks associated with unintended changes. This underscores the urgent need for robust safety assessments. In this review, we summarize the current landscape of safety assessment platforms for evaluating gene edited NK cells. We highlight predictive in silico tools, biochemical in vitro assays, and emerging cell-based detection systems to identify and quantify CRISPR-induced OT events. Particular attention is given to their suitability, limitations, and practical use in primary NK cells and multiplex editing strategies. Our aim is to support the design of safe, effective editing workflows for NK cell therapies - ensuring rigor as the field advances rapidly toward clinical application.},
}
@article {pmid41424917,
year = {2025},
author = {Sharma, S and Saroha, NK and Sehrawat, A and Tang, G and Singh, D and Teotia, S},
title = {Emerging tools in plant genome editing.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1588089},
pmid = {41424917},
issn = {2673-3439},
abstract = {Plant genome editing has undergone a transformative shift with the advent of advanced molecular tools, offering unprecedented levels of precision, flexibility and efficiency in modifying genetic material. While classical site-directed nucleases such as ZFNs, TALENs and CRISPR-Cas9 have revolutionized genome engineering by enabling targeted mutagenesis and gene knockouts, the landscape is now rapidly evolving with the emergence of novel systems that go beyond the conventional double strand break (DSB)-mediated approaches. Advanced and recent tools include LEAPER, SATI, RESTORE, RESCUE, ARCUT, SPARDA, helicase-based approaches like HACE and Type IV-A CRISPR system, and transposon-based techniques like TATSI and piggyBac. These tools unlock previously inaccessible avenues of genome and transcriptome modulation. Some of these technologies allow DSB-free editing of DNA, precise base substitutions and RNA editing without altering the genomic DNA, a significant advancement for regulatory approval and for species with complex genomes or limited regeneration capacity. While LEAPER, RESCUE and RESTORE are the new advents in the RNA editing tool, SATI allows DSB-free approach for DNA editing, ARCUT offers less off-target and cleaner DNA repairs and Type IV-A CRISPR system induces gene silencing rather than editing. The transposon-based approaches include TATSI, piggyBac and TnpB, and helicases are used in HACE and Type IV-A CRISPR system. The prokaryotic Argonaute protein is used in SPARDA tool as an endonuclease to edit DNA. The transient and reversible nature of RNA editing tools such as RESTORE and LEAPER introduces a new layer of epigenetics-like control in plant systems, which could be harnessed for tissue-specific and environmentally-responsive trait expression. Simultaneously, innovations like ARCUT and SPARDA utilize chemically-guided editing, minimizing reliance on biological nucleases and reducing off-target risks. Their modularity and programmability are enabling gene function studies, synthetic pathway designs and targeted trait stacking. These advances represent a novel synthesis of genome engineering and systems biology, positioning plant genome editing not just as a tool of modification but as a platform for designing adaptive and intelligent crops, tailored to future environmental and nutritional challenges. Although, many of these recent tools remain to be applied on plant systems, they are proven to be effective elsewhere and hold a great potential to be effective in creating climate-resilient crops.},
}
@article {pmid41390669,
year = {2025},
author = {Teske, M and Wertheimer, T and Butz, S and Zwicky, P and Mallona, I and Nopper, SL and Münz, C and Elling, U and Lancrin, C and Becher, B and Grosso, AR and Baubec, T and Schmolka, N},
title = {Targeted CRISPR-Cas9 screening identifies core transcription factors controlling murine haemato-endothelial fate commitment.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11412},
pmid = {41390669},
issn = {2041-1723},
support = {186012//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; FAN//Universit&#xe4;t Z&#xfc;rich (University of Zurich)/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Transcription Factors/genetics/metabolism ; Mesoderm/cytology/metabolism ; Cell Differentiation/genetics ; Cell Lineage/genetics ; *Hematopoietic Stem Cells/cytology/metabolism ; Gene Expression Regulation, Developmental ; Mouse Embryonic Stem Cells/cytology/metabolism ; *Endothelial Cells/cytology/metabolism ; Hematopoiesis/genetics ; },
abstract = {During development, blood generation begins in the yolk sac with the differentiation of haemato-endothelial mesoderm forming haematopoietic progenitors. This study aims to identify the crucial molecular regulators of haemato-endothelial mesoderm formation and to extend our knowledge of the process in an unbiased way. We employ a murine embryonic stem cell model that recapitulates embryonic blood development, and perform targeted CRISPR-Cas9 knock out screens focusing on transcription factors and chromatin regulators. We identify the transcription factors ETV2, LDB1, SMAD1, SIX4 and ZBTB7b as regulators of haemato-endothelial mesoderm commitment. Embryonic stem cells lacking these regulators give rise to mesodermal subsets with a defined lineage differentiation bias, while transcriptome analysis of these cells uncovers the precise impact of each factor on gene expression in the developing mesoderm. Our study reveals molecular pathways governing mesodermal development crucial to allow endothelial and haematopoietic lineage specification and paves the way for future advances in haematopoietic stem cell applications.},
}
@article {pmid41387334,
year = {2025},
author = {Song, N and Tian, G and Li, H and Zhang, L and Wang, Y and Zhao, W and Yao, C and Yang, D},
title = {DNA Nanoflowers Efficiently Encapsulate Photodynamic Agents and CRISPR/Cas9 for Synergistic Pancreatic Cancer Therapy.},
journal = {Nano letters},
volume = {25},
number = {51},
pages = {17693-17701},
doi = {10.1021/acs.nanolett.5c04676},
pmid = {41387334},
issn = {1530-6992},
mesh = {*Pancreatic Neoplasms/drug therapy/pathology/genetics/therapy/metabolism ; *Photochemotherapy/methods ; Humans ; Animals ; *CRISPR-Cas Systems ; Mice ; NF-E2-Related Factor 2/genetics/metabolism ; Cell Line, Tumor ; *Photosensitizing Agents/chemistry/pharmacology/administration &amp; dosage/therapeutic use ; Chlorophyllides ; *DNA/chemistry ; Reactive Oxygen Species/metabolism ; Porphyrins/chemistry/pharmacology/administration &amp; dosage ; Hemin/chemistry ; Apoptosis/drug effects ; Gene Editing ; Ribonucleoproteins/genetics ; G-Quadruplexes ; },
abstract = {Photodynamic therapy (PDT) holds significant promise for treating pancreatic cancer by utilizing photosensitizers to generate reactive oxygen species (ROS) that induce tumor cell death. However, the therapeutic efficacy of PDT is hindered by inadequate ROS accumulation. Herein, we develop a DNA nanoflower that enables the controlled codelivery of Cas9 ribonucleoprotein (RNP), hemin, and chlorin e6 for synergistic PDT. The Cas9 RNP selectively knocks out the antioxidant regulator nuclear factor E2-related factor 2 (Nrf2), thereby increasing cancer cells' sensitivity to ROS. Simultaneously, the G-quadruplex/hemin complex catalyzes the conversion of endogenous H2O2 into O2, alleviating tumor hypoxia and supplying additional oxygen for PDT. This synergistic approach substantially amplifies ROS accumulation by attenuating ROS elimination and enhancing ROS generation, demonstrating high gene editing efficiency, significant Nrf2 down-regulation, elevated apoptosis, and remarkable antitumor efficacy in pancreatic cancer cells and a mouse model, underscoring the potential for precision medicine.},
}
@article {pmid41162706,
year = {2026},
author = {Chong, Z and Hui, S and Qiu, X and Palakurty, S and Sariol, A and Kaszuba, T and Nguyen, MN and Li, P and Raju, S and Hall, PD and Nelson, CA and Baltazar-Perez, I and Price, DA and Rothlauf, PW and Crowe, JE and Whelan, SPJ and Leung, DW and Amarasinghe, GK and Bailey, AL and Fremont, DH and Diamond, MS},
title = {Multiple LDLR family members act as entry receptors for yellow fever virus.},
journal = {Nature},
volume = {649},
number = {8095},
pages = {173-182},
pmid = {41162706},
issn = {1476-4687},
mesh = {Animals ; *Yellow fever virus/physiology/pathogenicity/metabolism ; Humans ; *Virus Internalization ; Mice ; *Receptors, LDL/metabolism/genetics/chemistry/deficiency ; *Yellow Fever/virology/metabolism ; Female ; Low Density Lipoprotein Receptor-Related Protein-1/metabolism/genetics/deficiency/chemistry ; Male ; *Receptors, Virus/metabolism ; Viral Envelope Proteins/metabolism/chemistry ; Protein Domains ; Hepatocytes/metabolism/virology ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Infection by yellow fever virus (YFV), the prototype Orthoflavivirus, induces a febrile syndrome in humans that can progress to liver failure, haemorrhage and death[1]. Despite decades of study, the entry receptors for YFV remain unclear. Here, using a surface protein-targeted CRISPR-Cas9 screen, we identified LRP4, a low-density lipoprotein receptor (LDLR) family member, as a candidate entry receptor for YFV. Genetic ablation of LRP4 impaired YFV infection of cells and, reciprocally, complementation or ectopic expression of LRP4 increased infection. Related viruses in the YFV antigenic complex also showed LRP4-dependent infection. LRP4 promoted YFV entry into cells through LDLR type A (LA) domain binding to domain III of the YFV envelope protein. Soluble LRP4-Fc decoy receptors neutralized YFV infection in cell culture and reduced viral burden in vivo. As we observed residual YFV infection in LRP4-deficient cells, we evaluated whether other LDLR family members promote YFV entry. This approach identified LRP1 and VLDLR as additional receptors for YFV infection in cell culture. LRP1-Fc, LRP4-Fc and VLDLR-Fc decoys protected mice from YFV challenge, and LRP1-Fc decoys inhibited YFV infection and liver pathogenesis in mice engrafted with human hepatocytes. A genetic deficiency of LRP1 in primary human hepatocyte cultures also resulted in reduced YFV infection. Our findings establish a role for multiple LDLR family members in YFV entry, infection and pathogenesis, which has implications for receptor use and countermeasure development for multiple emerging orthoflaviviruses.},
}
@article {pmid40394336,
year = {2026},
author = {Wei, Y and Gao, P and Pan, D and Li, G and Chen, Y and Li, S and Jiang, H and Yue, Y and Wu, Z and Liu, Z and Zhou, M and Chen, Y and Xu, K and Wu, Z and Wang, X},
title = {Engineering eukaryotic transposon-encoded Fanzor2 system for genome editing in mammals.},
journal = {Nature chemical biology},
volume = {22},
number = {1},
pages = {48-57},
pmid = {40394336},
issn = {1552-4469},
mesh = {Animals ; *Gene Editing/methods ; *DNA Transposable Elements/genetics ; Mice ; Humans ; CRISPR-Cas Systems ; Endonucleases/genetics/metabolism ; Mammals/genetics ; HEK293 Cells ; Muscular Dystrophy, Duchenne/genetics/therapy ; },
abstract = {Eukaryotic transposon-encoded Fanzor proteins hold great promise for genome-engineering applications as a result of their compact size and mechanistic resemblance to TnpB. However, the unmodified Fanzor systems show extremely low activity in mammalian cells. Guided by the predicted structure of a Fanzor2 complex using AlphaFold3, we engineered the NlovFz2 nuclease and its cognate ωRNA to create an evolved enNlovFz2 system, with an expanded target-adjacent motif (TAM) recognition scope (5'-NMYG) and a substantially improved genome-editing efficiency, achieving an 11.1-fold increase over the wild-type NlovFz2, comparable to two previously reported IS200 or IS605 transposon-encoded TnpBs and two CRISPR-Cas12f1 nucleases. Notably, enNlovFz2 efficiently mediated gene disruption in mouse embryos and restored dystrophin expression in a humanized Duchenne muscular dystrophy mouse model with single adeno-associated virus delivery. Our findings underscore the potential of eukaryotic RNA-guided Fanzor2 nucleases as a versatile toolbox for both biological research and therapeutic applications.},
}
@article {pmid41424849,
year = {2026},
author = {Liu, M and Fu, X and Zhang, H and Pan, J and Jia, Q and Zhang, C and An, F},
title = {Endothelial KSR2 regulated by genetic variation protects against atherosclerosis through AMPKα1 stabilization.},
journal = {Theranostics},
volume = {16},
number = {5},
pages = {2598-2626},
pmid = {41424849},
issn = {1838-7640},
mesh = {Animals ; *Atherosclerosis/genetics/metabolism/pathology ; Mice ; *AMP-Activated Protein Kinases/metabolism/genetics ; Humans ; Polymorphism, Single Nucleotide/genetics ; Mice, Knockout ; Male ; Endothelial Cells/metabolism ; Diet, High-Fat/adverse effects ; Mice, Inbred C57BL ; Apolipoproteins E/genetics ; Mice, Knockout, ApoE ; Disease Models, Animal ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; Apoptosis/genetics ; CRISPR-Cas Systems ; },
abstract = {Rationale: The single nucleotide polymorphism (SNP) rs11830157 within the scaffold protein kinase suppressor of Ras 2 (KSR2) locus is strongly associated with the incidence of coronary artery disease (CAD), yet its functional role remains undefined. This study aimed to investigate the potential impact of rs11830157 polymorphism on atherosclerosis and to elucidate the underlying molecular mechanisms. Methods: Dual-luciferase reporter assays, chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assays (EMSA), and CRISPR/Cas9 gene-editing techniques were used to investigate the regulatory role of the SNP rs11830157. To assess the role of KSR2 in atherosclerosis, we utilized global KSR2 knockout mice fed a high-fat diet ad libitum, pair-fed global KSR2 and Apoe (Apolipoprotein E) double knockout mice, and mice with endothelial-specific KSR2 overexpression mediated by AAV9-ICAM2. Results: Genetic analyses identified SNP rs12822146, in linkage disequilibrium with rs11830157 and located within an endothelial enhancer, as a regulator of KSR2 expression via differential binding of the transcriptional repressor XBP1s. KSR2 expression was significantly reduced in endothelial cells within atherosclerotic plaques in both humans and mice. Using multiple KSR2 gene-edited mouse models, we demonstrated that endothelial KSR2 protects against atherosclerosis by suppressing inflammation and apoptosis. Mechanistic studies revealed that KSR2 competes with CRBN for binding to the K52 site of AMPKα1, inhibiting CRL4A[CRBN] E3 ubiquitin ligase complex-mediated K48-linked polyubiquitination and proteasomal degradation of AMPKα1. The subsequently activated AMPK signaling pathway maintains glycolytic balance in endothelial cells, ultimately exerting anti-inflammatory and anti-apoptotic effects. Conclusions: Our findings provide the first comprehensive molecular explanation of the rs12822146-KSR2-atherosclerosis axis, with important implications for both primary prevention and secondary treatment of CAD.},
}
@article {pmid41424172,
year = {2025},
author = {Ikram, M and Farhan, M and Derakhshani, B and Kumar, S and Khan, N and Gupta, R and Usman, B and Liu, P},
title = {Machine Learning and CRISPR-Based Validation Elucidate OsWOX13 Involvement in Rice Heat Stress Tolerance and Flowering.},
journal = {Physiologia plantarum},
volume = {177},
number = {6},
pages = {e70714},
doi = {10.1111/ppl.70714},
pmid = {41424172},
issn = {1399-3054},
support = {YSPTZX202206//Special Project for the Academician Team Innovation Center of Hainan Province/ ; ZDYF2022XDNY185//Key Research Program of Hainan Province/ ; },
mesh = {*Oryza/genetics/physiology ; *Machine Learning ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Heat-Shock Response/genetics ; *Flowers/physiology/genetics ; *Thermotolerance/genetics ; CRISPR-Cas Systems/genetics ; Proteomics ; },
abstract = {Rice (Oryza sativa) is a staple food for billions of people globally, but it faces significant yield losses due to heat stress. However, rice responses to heat stress remain understudied as compared to other stress factors. In this study, we combined meta-transcriptomics, machine learning, functional validation, and proteomic analysis to identify the key genes involved in heat stress tolerance in rice. We identified 409 meta-differentially expressed genes (meta-DEGs) between heat-tolerant and susceptible genotypes, which were associated with detoxification, oxidative stress, protein folding, phenylpropanoid biosynthesis, glutathione metabolism, and plant hormone signal transduction. We trained five machine learning models, of which Random Forest (RF) and eXtreme Gradient Boosting (XGBoost) outperformed the others. Using SHAP analysis, the top 14 genes for each model were identified, including the OsWOX13 gene, which was detected simultaneously across both models, indicating a positive regulator and a strong candidate for heat stress tolerance. Functional validation of OsWOX13 via CRISPR/Cas9-mediated knockout (KO) confirmed its positive role in heat stress, with a delay in flowering and survival rate of ~20% compared to ~60% for WT under heat stress. Physiological and antioxidant enzymatic activities showed a significant (p ≤ 0.05) reduction in ABA accumulation levels, increased MDA accumulation, and decreased SOD and POD activities in KO lines compared to WT. The proteomic analysis identified upregulated heat shock proteins (HSF8, BIP1, BIP5, and HSP81-1) and downregulated flowering-associated proteins (ROC6 and 4CL4) in mutant lines in response to heat stress. These results indicate that OsWOX13 enhances heat tolerance by regulating ABA signaling and antioxidant defense mechanisms. Taken together, this study highlights the efficiency of machine learning models in the identification of stress responsive genes and provides OsWOX13 as a strong positive candidate for heat stress tolerance and breeding climate-resilient rice varieties.},
}
@article {pmid41423823,
year = {2025},
author = {Stamilla, A and Recchia, D and Stelitano, G and Maci, L and Marturano, MC and De Rossi, E and Chiarelli, LR and Pasca, MR and Degiacomi, G},
title = {Uncovering Insights Into the Biology of Mycobacterium tuberculosis Using Genetic Tools.},
journal = {MicrobiologyOpen},
volume = {14},
number = {6},
pages = {e70206},
pmid = {41423823},
issn = {2045-8827},
support = {20205B2HZE_003//University of Pavia; Italian Ministry of University and Research/ ; 2022JTPP53//University of Pavia; Italian Ministry of University and Research/ ; PE00000007//NextGenerationEUMUR PNRR/ ; INF-ACT//NextGenerationEUMUR PNRR/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/drug effects/pathogenicity ; Humans ; *Genetic Engineering/methods ; Tuberculosis/microbiology ; Drug Discovery ; Genome, Bacterial ; },
abstract = {Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is one of the most challenging pathogens due to its complex physiology, diverse clinical manifestations, and growing multidrug resistance. The global rise of drug-resistant Mtb strains has prompted the search for innovative genetic and molecular strategies to accelerate drug discovery and vaccine development. Progress in Mtb research has long been hindered by its slow replication rate and impermeable cell envelope, which limit the efficacy of genetic manipulation. This review outlines methodological advances that have transformed the study of Mtb pathogenesis and drug resistance mechanisms. Traditional homologous recombination-based approaches, including allelic exchange and specialized transduction, laid the groundwork for targeted mutagenesis but were limited by low efficiency. The advent of phage-derived recombineering systems, such as the Che9c RecET, has substantially improved the precision and throughput of genetic modification. Hybrid systems such as ORBIT, which combines oligonucleotide-mediated recombineering with Bxb1 integrase, have further enabled rapid and versatile genome engineering across mycobacterial species. Parallel developments in conditional gene expression systems (e.g., the use of TetR/Pip-based promoters) have facilitated the functional analysis of essential genes and the validation of novel drug targets. The advent of CRISPR-Cas technologies has represented a paradigm shift, by enabling programmable, high-fidelity gene regulation and functional genomics even in slow-growing mycobacteria. Together, these genetic innovations are transforming Mtb research by accelerating drug discovery and vaccine design, and shedding light on host-pathogen interactions.},
}
@article {pmid41422267,
year = {2025},
author = {Fair, T and Pavlovic, BJ and Swope, D and Castillo, OE and Schaefer, NK and Pollen, AA},
title = {Mapping cis- and trans-regulatory target genes of human-specific deletions.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11380},
pmid = {41422267},
issn = {2041-1723},
support = {DP2MH122400-01//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; F31 HG011569-01A1//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; Pan troglodytes/genetics ; Animals ; CRISPR-Cas Systems ; *Sequence Deletion/genetics ; Gene Expression Regulation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Pluripotent Stem Cells/metabolism ; Genome, Human ; Chromatin/metabolism/genetics ; Cell Proliferation/genetics ; Brain/metabolism ; },
abstract = {Deletion of functional sequence is predicted to represent a fundamental mechanism of molecular evolution. Comparative genetic studies of primates have identified thousands of human-specific deletions (hDels), and the cis-regulatory potential of short (≤31 base pairs) hDels has been assessed using reporter assays. However, how structural variant-sized (≥50 base pairs) hDels influence molecular and cellular processes in their native genomic contexts remains unexplored. Here, we design genome-scale libraries of single-guide RNAs targeting 7.2 megabases of sequence in 6358 hDels and present a systematic CRISPR interference (CRISPRi) screening approach to identify hDels that modify cellular proliferation in chimpanzee pluripotent stem cells. By intersecting hDels with chromatin state features and performing single-cell CRISPRi (Perturb-seq) to identify their cis- and trans-regulatory target genes, we discovered 20 hDels controlling gene expression. We highlight two hDels, hDel_2247 and hDel_585, with tissue-specific activity in the brain. Our findings reveal a molecular and cellular role for sequences lost in the human lineage and establish a framework for functionally interrogating human-specific genetic variants.},
}
@article {pmid41420495,
year = {2025},
author = {Pan, G and Wang, L and Zhu, H and Wang, H and Zheng, Z},
title = {Utilization of Miniature CRISPR-AsCas12f1 Nuclease for Efficient Genome Editing in Bacillus subtilis.},
journal = {Biotechnology journal},
volume = {20},
number = {12},
pages = {e70168},
doi = {10.1002/biot.70168},
pmid = {41420495},
issn = {1860-7314},
support = {S2023n06020216//Anhui Province Key Research and Development Plan/ ; 2019YFA0904304//National Key Research and Development Program of China/ ; YZJJ2024QN37//Hefei Institutes of Physical Science, Chinese Academy of Sciences Director's Fund/ ; },
mesh = {*Bacillus subtilis/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; *Endonucleases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Bacterial ; },
abstract = {To address the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)9 in Bacillus subtilis, such as low transformation efficiency and strong dependence on specific PAM sequences, this study developed a novel genome-editing tool based on AsCas12f1 nuclease derived from Acidibacillus sulfuroxidans. Using the CRISPR-AsCas12f1 system, we successfully achieved gene knockout and targeted insertion in B. subtilis with a knockout efficiency of up to 100%. We further demonstrated that the length of the donor DNA homology arms and the choice of PAM motifs significantly influenced the editing efficiency. To expand the applicability of this system, gene interference and activation experiments were performed using green fluorescent protein (GFP) as a reporter. The system achieved more than 90% gene knockdown efficiency and effectively activated the reported gene transcription, with a maximum activation fold of 3.20. In conclusion, the CRISPR-AsCas12f1 system established in this study provides an efficient and reliable genome editing tool for the functional gene research and industrial applications of B. subtilis.},
}
@article {pmid41389042,
year = {2025},
author = {Nie, YG and Zhang, HS and Su, M and Zha, CJ and Yang, K and Ying, ZM},
title = {Proximity-Inducible CRISPR/Cas12a Activity by Scaffold RNA Assembly for Sensing Applications.},
journal = {Analytical chemistry},
volume = {97},
number = {50},
pages = {28088-28097},
doi = {10.1021/acs.analchem.5c06530},
pmid = {41389042},
issn = {1520-6882},
mesh = {Humans ; *MicroRNAs/analysis/genetics/blood ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Breast Neoplasms/diagnosis ; *RNA/chemistry/genetics/metabolism ; Adenosine Triphosphate/analysis ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; Female ; *Bacterial Proteins/metabolism ; },
abstract = {The programmability and flexibility of the RNA-directed CRISPR/Cas12a system underpin its utility as a potent tool for diagnostic applications. However, existing engineered crRNA strategies are still limited by a narrow target range, inadequate specificity, and operational complexity. To overcome these challenges, a proximity-assembly and activate (PAA) strategy was developed, employing split dumbbell activators with terminally modified target-binding modules that reassemble on scaffold RNA to reconstruct functional crRNA and activate Cas12a trans-cleavage activity. The design allows universal detection of both nucleic acid and non-nucleic acid targets. Notably, owing to its strict target dependency, the assembled crRNA biosensor significantly reduces background signal and suppresses nonspecific leakage. We demonstrated that the PAA system facilitates rapid and highly specific detection of miRNA-21, ATP, and anti-Dig antibody in complex matrices, enabling single-base discrimination among miRNA variants. Moreover, the platform successfully detected endogenous miRNA-21 in serum and cellular samples from breast cancer patients, clearly distinguishing them from healthy controls. This work presents a modular, plug-and-play, and versatile platform for molecular diagnostics, holding considerable potential for advancing clinical diagnostics and precision medicine.},
}
@article {pmid41383046,
year = {2025},
author = {Zeng, W and Xie, X and Yu, X and Zhang, T and Cai, T and Chen, S and Chen, W and Wang, F and Wang, L and Ma, L},
title = {Combined Use of Engineered PfAGO and LbCas12a for Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {50},
pages = {27941-27949},
doi = {10.1021/acs.analchem.5c05622},
pmid = {41383046},
issn = {1520-6882},
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; Pyrococcus furiosus/enzymology ; Humans ; CRISPR-Cas Systems ; *RNA, Viral/analysis/genetics ; *COVID-19/diagnosis/virology ; Limit of Detection ; *CRISPR-Associated Proteins/genetics/metabolism ; *COVID-19 Nucleic Acid Testing/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The COVID-19 pandemic has underscored the urgent need for rapid, accurate, and accessible nucleic acid detection technologies. Here, we present NAEPLS (Nucleic Acid Detection Based on Engineered PfAgo and LbCas12a with Split crRNA), a novel diagnostic platform that synergistically integrates the engineered DNA-guided nuclease PfAgo (mPfAgo) from Pyrococcus furiosus with CRISPR-Cas12a for ultrasensitive and specific pathogen detection. By leveraging mPfAgo's RNA-cleaving capability and LbCas12a's compatibility with split crRNA, NAEPLS achieved detection of SARS-CoV-2 RNA. Coupling this assay with RT-RPA enabled ultrasensitive detection, achieving a limit of detection (LOD) of 10 copies/mL. The system demonstrates single-nucleotide specificity, robustly discriminating mutations in mocked samples (such as D614G) as well as in clinical isolates (such as S371P). Notably, the compatibility with lateral flow strip visualization allows this method to be implemented as a rapid POCT platform, particularly valuable in resource-limited settings. Validation with 20 clinical samples (Ct 20-39) showed 100% concordance with RT-qPCR, including reliable detection of low viral loads (Ct > 35). This study establishes a transformative paradigm for nucleic acid diagnostics, merging the precision of PfAgo with the amplification power of CRISPR for high-performance, field-deployable pathogen detection.},
}
@article {pmid41381501,
year = {2025},
author = {Padilla, R and Shipman, GA and Horth, C and Gravel, M and Bareke, E and Majewski, J},
title = {H3K36 Methylation as a Guardian of Epigenome Integrity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11371},
pmid = {41381501},
issn = {2041-1723},
support = {PJT-183939//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/ ; P01-CA196539//U.S. Department of Health &amp; Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; },
mesh = {Animals ; *Histones/metabolism/genetics ; Humans ; Mice ; Methylation ; *Epigenome/genetics ; Heterochromatin/metabolism/genetics ; Mesenchymal Stem Cells/metabolism ; Methyltransferases/metabolism/genetics ; Histone-Lysine N-Methyltransferase/genetics/metabolism ; *Epigenesis, Genetic ; Repressor Proteins/metabolism/genetics ; Mice, Knockout ; Chromatin/metabolism ; Euchromatin/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {H3K36 methylation is a key epigenetic mark with critical roles in development and disease. Here, we systematically dissect its functions using CRISPR-engineered mouse mesenchymal stem cells lacking combinations of the five H3K36 methyltransferases, culminating in quintuple knockout cells devoid of H3K36me2/3. We show that H3K36me2 influences enhancer activity, supports the expression of their target genes, and safeguards active genes from encroachment of the repressive marks, H3K27me2/3. In addition, we find that the loss of H3K36me triggers redistribution of large heterochromatic H3K9me3 domains into euchromatin, in part mediated by SUV39H1, leading to global epigenomic remodelling, constitutive heterochromatin erosion, and a collapse of 3D genome organization. Parallel analyses in human HNSCC cells overexpressing the H3K36M oncohistone reveal conserved disruptions to the epigenome and chromatin architecture. Together, these results establish H3K36 methylation as a pivotal regulator of chromatin state and genomic structure.},
}
@article {pmid41370671,
year = {2025},
author = {Gao, Z and Lin, K and Gong, Y and Zhao, Y and Zhang, S},
title = {Core-Shell Tripeptide-Lipid/PEI Nanocarriers Enable Efficient Plasmid-Based CRISPR/Cas9 Editing of VEGFR2.},
journal = {Langmuir : the ACS journal of surfaces and colloids},
volume = {41},
number = {50},
pages = {33716-33730},
doi = {10.1021/acs.langmuir.5c02069},
pmid = {41370671},
issn = {1520-5827},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Polyethyleneimine/chemistry ; *Plasmids/genetics/chemistry ; *Vascular Endothelial Growth Factor Receptor-2/genetics ; Animals ; *Lipids/chemistry ; *Nanoparticles/chemistry ; MCF-7 Cells ; Mice ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a versatile genome editing technology that holds tremendous promise for the treatment of various diseases. Although several delivery technologies such as electroporation, viral vectors, and lipid nanoparticles have already shown promise in preclinical and clinical applications for hematological and neuromuscular genetic disorders, in vivo application is still restricted by the inefficient delivery of CRISPR/Cas9 components. Herein, by employing the tripeptide lipid N,N-ditetradecyloxyamidoethyl trimeric ornithine amide (CDO) and polyethylenimine (PEI), we constructed novel ternary systems (pDNA/PEI/CL) for the delivery of pDNA encoding Cas9 and single-guide RNAs (sgRNAs) targeting the VEGFR2 gene. The pDNA/PEI/CL delivery systems were fabricated by condensing pDNA with PEI, followed by coating with cationic liposomes composed of CDO. This system demonstrated high transfection efficiency, successfully delivering CRISPR/Cas9 to A549 and MCF-7 cells with efficiencies of up to 91.0% (n = 3, P < 0.001), while also exhibiting lower cytotoxicity. Notably, the sgRNA1/P1/C1 complex achieved higher genome editing efficiencies than sgRNA3/P1/C1, with 38.6% vs 31.0% in A549 cells (n = 3, P < 0.01) and 26.45% vs 20.18% in MCF-7 cells (n = 3, P < 0.01). Western blot analysis showed that VEGFR2 expression decreased by 48.1% in A549 and 44.3% in MCF-7 cells, while PI3K levels were reduced by 39.6% and 42.8%, respectively. This suppression of the PI3K/Akt signaling pathway led to cell cycle arrest, thereby inhibiting tumor cell proliferation and migration while promoting apoptosis. Furthermore, animal experiments validated the antitumor efficacy, highlighting the translational potential of this platform in cancer therapy. Collectively, these findings highlight the potential of the ternary complex system as a robust and biocompatible CRISPR/Cas9 delivery strategy, offering a promising avenue for gene therapy in cancer and other genetic diseases.},
}
@article {pmid41360396,
year = {2025},
author = {Jiang, Q and Ramachandran, A and Avaro, AS and Huyke, DA and Santiago, JG},
title = {Reaction Kinetics of CRISPR trans-Cleavage Controlled Using Isotachophoresis.},
journal = {Analytical chemistry},
volume = {97},
number = {50},
pages = {27646-27653},
doi = {10.1021/acs.analchem.5c04301},
pmid = {41360396},
issn = {1520-6882},
mesh = {*Isotachophoresis/methods ; Kinetics ; *CRISPR-Cas Systems ; },
abstract = {CRISPR-based diagnostics are powerful tools for nucleic acid detection due to their high specificity and programmability. However, assay sensitivity is often limited by the slow kinetics of the trans-cleavage reaction, which typically proceeds at a rate of ∼0.1 to 1 turnover per second. Here, we present a reaction-transport model and experimental study that analyze and accelerate this limiting step using electric-field-driven isotachophoresis (ITP). Building on the work of Ramachandran and Santiago, we develop a model that captures the coupling among ITP focusing, mixing, and preconcentration with CRISPR enzymatic reaction kinetics. Our analysis identifies two key regimes in ITP-coupled CRISPR reactions and derives analytical approximations for the limiting behaviors in each. Compared to a standard, well-mixed assay, we predict a 10- to 100-fold reduction in reaction duration using ITP. We validate the model with experiments across a range of target concentrations. Our work offers a quantitative framework for understanding and optimizing CRISPR trans-cleavage dynamics and provides guidance to design assays that use electric-field-mediated transport.},
}
@article {pmid41358836,
year = {2025},
author = {Li, Q and Xu, J and Jiang, J and Gong, L and Mao, X and Wang, F and Yao, P},
title = {Nucleic acid detection method for Chlamydia psittaci based on RPA-CRISPR/Cas12a.},
journal = {Letters in applied microbiology},
volume = {78},
number = {12},
pages = {},
doi = {10.1093/lambio/ovaf138},
pmid = {41358836},
issn = {1472-765X},
support = {BE2023694//Key Research and Development Project of Jiangsu Province/ ; H2023060//Jiangsu Provincial Commission of Health Project/ ; K2024003//Key Project of the Jiangsu Provincial Commission of Health/ ; Ym2023015//Jiangsu Province Preventive Medicine Research Project/ ; Ym2023073//Jiangsu Province Preventive Medicine Research Project/ ; x202339//Jiangsu Provincial Blood Parasite and Endemic Disease Prevention Research Project of China/ ; CE20225041//Changzhou science and technology Foundation/ ; CJ20253132//Changzhou science and technology Foundation/ ; CJ20253133//Changzhou science and technology Foundation/ ; CPHM202401//Nanjing Medical University/ ; CPHM202303//Nanjing Medical University/ ; },
mesh = {*Chlamydophila psittaci/genetics/isolation &amp; purification ; Humans ; *Psittacosis/diagnosis/microbiology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {In recent years, misdiagnosis or delayed diagnosis of Chlamydia psittaci (C. psittaci) infections has led to frequent outbreaks of severe public health events, such as severe pneumonia and respiratory distress, drawing increasing attention. Rapid and simple detection methods are vital for early intervention to reduce severity and mortality. In this study, we designed highly specific RPA primers and crRNA (CRISPR RNA) based on the highly conserved CPSIT_0429 gene in the C. psittaci genome, and preliminarily established a nucleic acid detection method for C. psittaci using the RPA-CRISPR/Cas12a system. In the two-step assay, the combination of the CPSIT_0429-F1/R1 primer pair and CPSIT_0429-crRNA2 achieved a detection limit of 2 × 10° copies/μL. Incorporating 20% glycerol enabled a one-tube assay with a limit of 2 × 102 copies/μL. Furthermore, the method showed no cross-reactivity with common respiratory pathogens such as influenza virus, SARS-CoV-2, and Streptococcus pneumoniae, demonstrating excellent specificity. Both the two-step and one-tube methods were compared with qPCR-verified C. psittaci positive samples. The results indicated that both assays showed high consistency with qPCR results. The RPA-CRISPR/Cas12a detection method is rapid, accurate, highly sensitive, and specific, providing a reliable platform for early diagnosis and clinical management of C. psittaci infections.},
}
@article {pmid41344241,
year = {2026},
author = {Peng, Y and Xu, J and Chen, B and Zeng, D and Yu, X and Chen, W},
title = {Ultrasensitive detection of lead ion in tea samples using a versatile and robust multi-DNAzyme DNA machine mediated CRISPR/Cas12a signal amplification system.},
journal = {Food chemistry},
volume = {499},
number = {},
pages = {147352},
doi = {10.1016/j.foodchem.2025.147352},
pmid = {41344241},
issn = {1873-7072},
mesh = {*DNA, Catalytic/genetics/chemistry ; *Tea/chemistry ; *Lead/analysis ; *Food Contamination/analysis ; CRISPR-Cas Systems ; *Biosensing Techniques/methods/instrumentation ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *Camellia sinensis/chemistry/genetics ; *Endodeoxyribonucleases/genetics/chemistry/metabolism ; Ions/analysis ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Lead ion (Pb[2+]) contamination in tea poses serious threats to food safety. We present a novel detection platform integrating a multifunctional DNAzyme machine with the CRISPR/Cas12a system. Upon target Pb[2+] binding, the multi-DNAzyme activates the DNA machine, initiating multiple isothermal cycles that generate a poly-A sequence. This sequence activates Cas12a, triggering trans-cleavage of the hairpin fluorescent probes and significant signal amplification. The self-sustained amplification of DNA machine eliminates the need for any auxiliary probes, greatly simplifying the assay design. This platform achieves excellent sensing perfromances with detection limit of 67.53 pM, wide linear range (0.01-100 nM), satisfied specificity and high recovery rates above 96 % in real tea samples. This integration of DNAzyme recognition with CRISPR/Cas12a signal enhancement provides a rapid, and cost-effective method for Pb[2+] detection. This strategy offers practical potential for food safety monitoring and environmental assessment, and future research may expand its applicability to other heavy metal contaminants.},
}
@article {pmid41288355,
year = {2025},
author = {Xie, W and Cai, Z and Bao, Z},
title = {Benchmarking the PAM compatibility of Cas12a variants for high-throughput yeast genetic variant engineering.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {12},
pages = {e0161825},
doi = {10.1128/aem.01618-25},
pmid = {41288355},
issn = {1098-5336},
support = {2023YFF1204500//National Key Research and Development Program of China/ ; 22308316//National Natural Science Foundation of China/ ; 226-2025-00043, 226-2022-00214//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Genetic Engineering/methods ; Benchmarking ; Genetic Variation ; },
abstract = {Saccharomyces cerevisiae is an important organism for basic research and applied biotechnology. Genome editing techniques, particularly CRISPR/Cas9 from Streptococcus pyogenes, have greatly facilitated saturation genome editing in yeast to understand mutant functions on a large scale. However, Cas9 is restricted by its targeting preference for G-rich protospacer-adjacent motif (PAM) sequences. To broaden the targeting scope, we established an efficient homology-integrated CRISPR/Cas12a system to install genetic variants through homologous recombination by targeting T-rich PAMs. We benchmarked the PAM compatibility of PAM-relaxed Cas12a variants and identified the improved LbCas12a (impLbCas12a) as the most efficient and PAM-relaxed variant in S. cerevisiae, showing high editing purity and an editing window centering the double-strand break. We show that our system can be used to perform targeted saturation mutagenesis to reveal functional variants not captured previously. By using a homology-integrated CRISPR RNA array, we utilized the multiplexing capability of CRISPR/Cas12a to realize multiplex genetic variant installation. Our system enriches the yeast genetic variant engineering toolbox, complementing the commonly used CRISPR/Cas9 system.IMPORTANCECRISPR/Cas9 has facilitated yeast functional genomics by generating a large number of precise genetic variants in a very short period of time. This enabled the interrogation of reconstituted natural genetic variants across different genetic backgrounds or entirely synthetic mutations to discover novel or improved functions. However, Cas9 only targets a limited genomic sequence space due to its preference for G-rich PAM sequences. In this study, we close this gap by developing a CRISPR/Cas12a-based system to engineer user-defined genetic variants targeting T-rich PAM sequences. Our system adopts a homology-integrated design and the most PAM-relaxed Cas12a characterized in yeast to date. These features collectively enabled the creation of genetic variant libraries and multiplex edited strains. This genome editing tool can be used together with Cas9-based tools to interrogate a greater genomic sequence space.},
}
@article {pmid41195558,
year = {2025},
author = {Mohammadi, R and Sakic, I and Jain, A and Mathur, P and Weiss, T and deMello, AJ and Stavrakis, S and Asghari, M},
title = {Ultra-High-Throughput Viscoelastic Squeezing for Mechanoporation and Efficient Intercellular Delivery.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {51},
pages = {e07981},
doi = {10.1002/smll.202507981},
pmid = {41195558},
issn = {1613-6829},
mesh = {Viscosity ; Humans ; *Elasticity ; *Electroporation/methods ; Microfluidics/methods ; CRISPR-Cas Systems/genetics ; },
abstract = {Cell-based therapies have transformed the treatment landscape for a range of diseases, leveraging both genome modification and cell reprogramming to create targeted treatments. Such therapies rely on the efficient internalization of biomolecules into living cells. Unfortunately, existing cargo delivery methods, such as those based on viral vectors and electroporation, are often compromised by cytotoxicity, poor delivery efficiencies, and low throughput. To overcome these limitations, a viscoelastic squeezing methodology is presented that uses viscoelastic microfluidics to perform mechanoporation in a rapid and contact-free manner. Through the control of the flow rates of a sample stream containing cells and cargo and a surrounding viscoelastic sheath flow, the width of a "virtual channel" formed between the two streams can be regulated. Elastic forces generated within this virtual channel are then used to deform contained cells and internalize user-defined payloads. The effectiveness and utility of the platform are assessed through the delivery of mRNA, plasmid DNA, and clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) ribonucleoprotein complexes into a variety of cell lines. Data confirms that viscoelastic squeezing provides for enhanced delivery efficiencies when compared to conventional poration techniques, whilst maintaining high cell viabilities and throughputs of 20 million cells per minute, and thus represents a powerful tool for cellular engineering.},
}
@article {pmid41071356,
year = {2025},
author = {Asaoka, Y and Tarumoto, S and Hirose, Y and Iwamoto, A and Tokunaga, M and Matsuura, T and Takemoto, Y and Yamashita, H and Furutani-Seiki, M and Sugahara, K},
title = {LOXHD1b knockout alters swimming behavior in zebrafish.},
journal = {Cell and tissue research},
volume = {402},
number = {3},
pages = {243-254},
pmid = {41071356},
issn = {1432-0878},
support = {23H04710//Japanese Ministry of Education, Culture, Sports, Science, and Technology/ ; 20K09733//Japanese Ministry of Education, Culture, Sports, Science, and Technology/ ; },
mesh = {Animals ; *Zebrafish/physiology/genetics ; *Swimming/physiology ; *Zebrafish Proteins/metabolism/genetics ; *Gene Knockout Techniques ; Lateral Line System/metabolism ; Hair Cells, Auditory/metabolism ; CRISPR-Cas Systems/genetics ; Humans ; Larva ; },
abstract = {Since human inner ear hair cells do not regenerate, the current treatments of hereditary deafness depend on hearing aids or cochlear implant. However, uncovering the functions of genes responsible for hereditary hearing loss is not only useful for their diagnosis but also for developing therapies. The pathogenetic mechanism of human non-syndromic deafness DFNB77 without morphological defects in the inner year caused by LOXHD1 mutations is not fully understood. We introduced zebrafish because the lateral line hair cells are structurally and physiologically similar to the human inner ear hair cells and mutations involved in non-symptomatic hearing loss can be assessed by their swimming behavior. The knock-out (KO) of LOXHD1b gene which is expressed in the lateral line hair cells was generated using the CRISPR-Cas9 system in zebrafish, and its morphological and functional changes were evaluated. As with human patients the LOXHD1b KO zebrafish larvae did not exhibit detectable morphological defects, but showed prolonged water flow sensing time. These results suggest that LOXHD1b plays pivotal roles for the hair cell neural activity and its KO zebrafish mutant serves as a useful model for revealing the molecular mechanisms linking LOXHD with hair cell function and for a drug screening to rescue the swimming phenotype.},
}
@article {pmid40402429,
year = {2025},
author = {Shang, Z and Liu, S and Liu, D and Wang, Y and Pei, X and Li, S and He, Y and Tong, Y},
title = {Systematically Investigating CRISPR/Cas12a Fluorescent Biosensor for Sensitive and Specific Single Nucleotide Variants Detection.},
journal = {Journal of fluorescence},
volume = {35},
number = {11},
pages = {11081-11089},
pmid = {40402429},
issn = {1573-4994},
support = {(No. 22004005).//National Natural Science Foundation of China/ ; },
mesh = {*Biosensing Techniques/methods ; *SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide ; Humans ; COVID-19/diagnosis/virology ; Fluorescent Dyes/chemistry ; Fluorescence ; Mutation ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Precise identification and detection of single nucleotide variation (SNV) concomitant with excess wild-type DNA is greatly needed for invasive disease diagnosis, pathogens detection and early prediction of drug responsiveness. Many variants of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), notably the D614G and N501Y mutations, have been shown to significantly increase the infectivity of pandemics. We herein investigated CRISPR/Cas12a integrated three types fluorescent reporters and two crRNAs for SNV detection by taking D614G and N501Y variants of SARS-CoV-2 as model examples. We systematically screened all possible base substitutions from positions 0 to 19 and identified the middle position of crRNA could efficiently increase the specificity from both theoretical and experimental standpoints. With selected mutation location of crRNA, we then investigated the specificity of ssDNA, dsDNA and molecular beacon (MB) fluorescent reporters and proved the MB reporters can efficiently increase the discriminatory factors. Furthermore, we designed an additional mutation site on crRNA to increase the specificity. For user convenience, we engineered the lateral flow strips to present the results visualized with the naked eyes. Results of specific variants from Omicron proved the feasibility of clinical applications. These findings indicated that the proposed method is a powerful tool for monitoring the key mutations in pathogens and allows for modifications to incorporate newer upcoming variants.},
}
@article {pmid41419797,
year = {2025},
author = {Das, IS and Shi, Q and Dreischhoff, S and Polle, A},
title = {Divergent functions of three Kunitz trypsin inhibitor (KTI) proteins in herbivore defense in poplar.},
journal = {BMC plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12870-025-07955-z},
pmid = {41419797},
issn = {1471-2229},
abstract = {BACKGROUND: Climate warming promotes the expansion of insect pests. Among the inducible defense responses activated by attacked plants, Kunitz trypsin protease inhibitors (KTIs) play an outstanding role. KTIs affect food digestion and thereby control the fitness of herbivorous insects. Poplars contain an expanded family of KTIs, whose distinct intrinsic functions are under investigation. Here, we set out to identify KTIs with anti-herbivore activity and assessed the potential growth trade-off incurred by high KTI expression levels.<br><br>RESULTS: Using in-silico database searches, we identified 28 KTIs in the haploid genome of Populus x canescens; 21 of them were responsive to herbivory. The greatest induction by herbivory was observed for KTI_400, KTI_600 and KTI_0882 (P. trichocarpa orthologues Potri.019G124400, Potri.019G124600, Potri.019G088200), whereas a moderate response was found for KTI_53200 (Potri.017G153200 orthologue). Mechanical wounding and methyl-jasmonate treatments resulted in fast and strong induction of KTI_400 and KTI_600 and moderate or lacking responses in KTI_0882 and KTI_53200. Increased KTI expression levels were associated with upregulation of ALLENE OXIDE SYNTHASE, a key enzyme involved in jasmonate biosynthesis. On the contrary, exposure to compounds eliciting ethylene or salicylic acid signaling did not affect KTIs. We generated stable CRISPR-Cas12a-mediated knock-out and p35S-mediated overexpression lines of KTI_400, KTI_600 and KTI_53200 in Populus x canescens. Among the wildtype and transgenic lines, only kti_400&#x2009;+&#x2009;kti_600 double knock-out lines produced greater biomass. Larvae of Helicoverpa armigera, a pest expanding in Europe due to a warmer climate, were allowed to feed on wildtype and transgenic poplar lines. Transgenic poplars overexpressing KTI_400 or KTI_600 resulted in reduced, and their double knockout lines in increased weight gain of the larvae. In contrast, overexpressing or knockout lines of KTI_53200 had no effect on larval weight gain compared with controls.<br><br>CONCLUSION: KTI_400 and KTI_600 are potent, natural in-planta anti-herbivorous agents. Their expression is associated with larval growth reductions. Modulation of KTI_53200 levels had no direct effects on the fitness of leaf-feeding H. armigera or on plant growth. This study sheds light on the potential application of KTI in plant defenses and biocontrol against H. armigera in trees and presents new options to investigate growth-defense theories.},
}
@article {pmid41418951,
year = {2025},
author = {Chen, J and Bian, X and Zheng, X and Peng, B and Li, R and Du, H and Zhou, L and Wen, Y},
title = {The synergistic effect of DNA nanostructures and CRISPR/Cas system for cancer diagnosis and treatment.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {149741},
doi = {10.1016/j.ijbiomac.2025.149741},
pmid = {41418951},
issn = {1879-0003},
abstract = {Cancer remains one of the most formidable global public health challenges, exerting a profound and detrimental impact on human health. Despite substantial advancements in cancer research, the escalating incidence and mortality rates underscore the persistent and growing burden on global healthcare systems. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system, heralded as a revolutionary gene-editing tool, holds immense promise for cancer treatment. However, its efficacy is critically contingent upon developing efficient delivery strategies. DNA nanocarriers, characterized by their programmability, sequence specificity, and design flexibility, emerge as a highly effective vehicle for delivering the CRISPR/Cas system, facilitating the precise transportation of gene-editing tools to the cell nucleus. The integration of DNA nanocarriers with CRISPR/Cas technology provides a new paradigm for precise and controllable gene editing. Through programmable spatial assembly, DNA nanocarriers can protect Cas9 ribonucleoprotein complexs (RNPs), facilitate endosomal escape, and co-localize donor DNA to promote homology-directed repair. These synergistic effects bridge molecular programmability and genetic functionality, paving the way for safer and more efficient genome engineering. This review aims to evaluate the application of DNA nanocarriers in cancer diagnosis comprehensively and to explore their potential utility in cancer therapy when combined with the CRISPR/Cas system, offering novel insights and significant scientific contributions to the field.},
}
@article {pmid41418774,
year = {2025},
author = {Ji, R and Chen, Q and Zhang, Y},
title = {Emerging trends in gene and cell therapy: CRISPR in DNA editing and beyond.},
journal = {Cell reports. Medicine},
volume = {},
number = {},
pages = {102459},
doi = {10.1016/j.xcrm.2025.102459},
pmid = {41418774},
issn = {2666-3791},
abstract = {CRISPR-based gene and cell therapies are rapidly transitioning from experimental platforms to clinical reality, exemplified by the recent approval of CRISPR-derived treatments for β-hemoglobinopathies. This review highlights how advances in genome editing technologies, ranging from CRISPR-Cas nucleases to base and prime editors, are expanding the therapeutic landscape beyond traditional gene knockout approaches. We focus on the clinical translation of these tools, drawing on examples from ongoing and completed human trials to illustrate their potential across diverse disease areas. Furthermore, we discuss critical considerations such as delivery challenges, long-term safety, immune responses, and editing specificity, all of which are critical to the safe and effective integration of CRISPR technologies into modern medicine.},
}
@article {pmid41417901,
year = {2025},
author = {Chammas, P and Xie, SQ and Sepulveda-Rincon, LP and Leeke, BJ and Dore, MH and Dormann, D and Wagner, RT and Chang, N and Jones, PL and McManus, MT and Karimi, MM and Young, G and Percharde, M},
title = {CRISPRa-mediated disentanglement of the Dux-MERVL axis in the 2C-like state, totipotency, and cell death.},
journal = {Science advances},
volume = {11},
number = {51},
pages = {eadu9092},
pmid = {41417901},
issn = {2375-2548},
mesh = {Animals ; Mice ; Cell Death/genetics ; *Homeodomain Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; *Transcription Factors/genetics/metabolism ; *DNA Transposable Elements/genetics ; Zygote/metabolism ; },
abstract = {Transposable elements (TEs) are powerful cis-regulatory drivers of gene expression, particularly during early development when many TEs become de-repressed. MERVL elements are transiently up-regulated in mouse totipotent two-cell (2C) embryos during major zygotic genome activation (ZGA) and 2C-like cells in vitro. One of the most powerful activators of MERVL is the pioneer transcription factor, Dux. However, apparent differences lie in the requirement for Dux versus MERVL activation in embryos. Moreover, sustained Dux activation causes cell toxicity, which may or may not be linked to MERVL. Using a CRISPR activation system, we unpick the relative role of Dux and MERVL in ZGA, totipotent-like characteristics, and cell toxicity. We find that MERVL activation drives a portion of the Dux-dependent transcriptome, sufficient for expanded fate potential, but not other totipotency features. Conversely, Dux-induced pathology is independent of MERVL activation and involves the proapoptotic factor, Noxa. Our study highlights the complexity of the Dux-MERVL transcriptional network and uncovers a previously unknown player in Dux-driven pathology.},
}
@article {pmid41417296,
year = {2025},
author = {Rehman, T and Sharif, A and Khalid, L and Sajid, I},
title = {Whole genome sequencing and genomic characterization of the extensively drug-resistant Acinetobacter baumannii recovered from clinical samples in Lahore, Pakistan.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {217},
pmid = {41417296},
issn = {1573-4978},
mesh = {*Acinetobacter baumannii/genetics/drug effects/isolation &amp; purification/pathogenicity ; Pakistan ; Humans ; *Drug Resistance, Multiple, Bacterial/genetics ; Whole Genome Sequencing/methods ; *Acinetobacter Infections/microbiology/genetics/drug therapy ; Genome, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Virulence Factors/genetics ; Genomics/methods ; Microbial Sensitivity Tests ; },
abstract = {BACKGROUND: Extensively drug-resistant (XDR) Acinetobacter baumannii has turned into a significant nosocomial pathogen often resistant to all available classes of antibiotics. It has been identified as a key public health issue due to its capacity to get resistance determinants, virulence genes, and mobile genetic elements. This study hypothesized that the XDR A. baumannii isolated in Pakistan would harbor some of the key genomic determinants of the resistance and pathogenicity.<br><br>METHODOLOGY: A total of 11 A. baumannii were obtained from the patient samples and were identified using microbiological, biochemical, and genomic analysis. The sensitivity to antibiotics was determined by Kirby-Bauer disc diffusion assay. While the whole genome sequencing (WGS) was performed for the single XDR Acinetobacter isolate TAB-4 and its in-silico genome analysis on resistant genes, virulence factors, plasmids, mobile genetic elements, prophages, CRISPR-Cas and biosynthetic gene clusters was conducted.<br><br>RESULTS: The isolated strains were 10 multidrug resistant (MDR) and one (TAB-4) was XDR Acinetobacter strain. The AST showed resistance to nearly all classes of antibiotics, and with limited susceptibility to tetracyclines and aminoglycosides. The genome of TAB-4 strain comprised of 3.94&#xa0;Mb, 120 contigs, and a GC content of 39.14%. The major resistant determinants found were blaOXA-23, blaOXA-69, blaNDM-1, blaADC-25, aminoglycoside modifying enzymes, and efflux pumps (adeABC, adeFGH, adeIJK). Virulence-associated genes (ompA, bap, csuA/B-E, plc) were identified along with five prophage regions, multiple CRISPR arrays, and a betalactone biosynthetic gene cluster.<br><br>CONCLUSION: This study to the best of our knowledge reports the first detailed WGS-based characterization of an XDR A. baumannii from Lahore, Pakistan. These genomic findings offer significant insights into the resistance and virulence factors underlying this challenging clinical issue. Hence, there is an urgent need to find new or alternative treatment methods against high-risk pathogens like XDR A. baumannii.},
}
@article {pmid41416522,
year = {2025},
author = {Stella, G and Ye, L and Brady, SF and Marraffini, L},
title = {CARF-HAD phosphatase effectors provide immunity during the type III-A CRISPR-Cas response.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41416522},
issn = {1362-4962},
support = {R01GM149834/GF/NIH HHS/United States ; R35GM122559/GF/NIH HHS/United States ; //HHMI/ ; },
mesh = {*CRISPR-Cas Systems ; *Phosphoric Monoester Hydrolases/metabolism/genetics/chemistry ; Protein Domains ; Adenosine Triphosphate/metabolism ; Escherichia coli/genetics/virology ; Adenine Nucleotides/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Bacterial Proteins/metabolism/chemistry/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) systems provide adaptive immunity against phage infection in prokaryotes using an RNA-guided complex that recognizes complementary foreign nucleic acids. Different types of CRISPR-Cas systems have been identified that differ in their mechanism of defense. Upon infection, type III CRISPR-Cas systems employ the Cas10 complex to find phage transcripts and synthesize cyclic oligo-adenylate (cOA) messengers. These ligands bind and activate CARF immune effectors that cause cell toxicity to prevent the completion of the viral lytic cycle. Here, we investigated two proteins containing an N-terminal haloacid dehalogenase (HAD) phosphatase domain followed by four predicted transmembrane helices and a C-terminal CARF domain. We named these proteins Chp for CRISPR-associated HAD phosphatase. We show that, in vivo, Chp localizes to the bacterial membrane and that its activation induces a growth arrest, leads to a depletion of ATP and IMP, and prevents phage propagation during the type III CRISPR-Cas response. In vitro, the CARF domain of Chp binds cyclic tetra-adenylates and the HAD phosphatase domain dephosphorylates dATP, ATP, and IMP. Our findings extend the range of molecular mechanisms employed by CARF effectors to defend prokaryotes against phage infection.},
}
@article {pmid41415454,
year = {2025},
author = {Neupane, S and Pfrender, ME and Wang, L and Xu, S},
title = {Detection of CRISPR-Cas-induced mutations in Daphnia.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2025.12.05.692657},
pmid = {41415454},
issn = {2692-8205},
abstract = {CRISPR-Cas9 has established itself as a robust tool for conducting loss of function gene research in emerging model species including the freshwater zooplankton Daphnia . However, sensitive detection of mutations, especially in genetic mosaic and pooled samples, remains a challenge. In this study we evaluate two of the most widely used mutation screening techniques, the T7 Endonuclease I (T7EI) assay and Fragment Analysis (FA) for their sensitivity, accuracy, and practical use in detecting CRISPR-induced indels in four targeted genes, DNMT3A , DNMT3B , PERIOD2 , and DMRT1 in Daphnia magna . Here, we show that T7EI, although it offers a quick and cost-effective screening method, often produces false positives, especially when examining pooled samples. Conversely, FA facilitates detecting allele size differences at a fine resolution, reproducibility in detecting indels, and distinguishing zygosity and is more reliable as a method to detect mutation. Our comparative analyses convey the importance of carefully selecting the appropriate screening methods depending on research questions.},
}
@article {pmid41415442,
year = {2025},
author = {Sabol, AL and Mengiste, AA and Sreekanth, V and Singh, P and Hendel, SJ and Tran, MTN and Barybin, AM and Chaudhary, S and Harris, RM and Liivak, K and Severance, ZC and Locicero, CM and Kailass, K and Lee, C and Xu, LQ and Butty, VL and Choudhary, A and Shoulders, MD},
title = {Anti-CRISPR-mediated continuous directed evolution of CRISPR-Cas9 in human cells.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2025.12.11.693673},
pmid = {41415442},
issn = {2692-8205},
abstract = {Engineering CRISPR-Cas systems for improved or altered function is central to both research and therapeutic applications. Unfortunately most optimization, especially directed evolution in bacterial hosts, fails to capture the functional requirements of the complex mammalian cellular milieu, where activity is usually required. Robust strategies to enable continuous directed evolution of genome-targeting agents directly in human cells remain lacking. Here, we introduce CRISPR-MACE (Mammalian cell-enabled Adenovirus-assisted Continuous Evolution) as a foundational technology to address this need. CRISPR-MACE integrates virus-based continuous evolution with anti-CRISPR-based tunable selection to generate novel Streptococcus pyogenes Cas9 variants with both increased and decreased DNA binding capacity and nearly 1000-fold-enhanced resistance to AcrIIA4, the strongest known inhibitor of SpCas9. Notably, across independent evolution campaigns the same Cas9 gatekeeper mutation reproducibly emerged first, enabling subsequent adaptive steps along two interdependent axes of Cas9 function. In addition to advancing CRISPR technologies, this work establishes key principles and synthetic circuits for continuously evolving CRISPR-Cas systems directly in human cells.},
}
@article {pmid41414673,
year = {2025},
author = {Chan, J and Wu, Z and Liu, M and Wang, T and Liu, H and Cao, R and Li, X and Li, X and Zhan, S and Cheng, J and Xu, Y and He, M and Feng, Y and Xu, Q and Sun, Y and Chen, L and Hu, P},
title = {Systematic enhancer mapping and functional analysis in zebrafish with optimized CRISPR interference.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41414673},
issn = {1362-4962},
support = {32341061//National Natural Science Foundation of China/ ; 32200414//National Natural Science Foundation of China/ ; 32373113//National Natural Science Foundation of China/ ; 32503168//National Natural Science Foundation of China/ ; 2024M761923//China Postdoctoral Science Foundation/ ; 25ZR1402190//Natural Science Foundation of Shanghai/ ; (32341061//National Natural Science Foundation of China/ ; 32200414//National Natural Science Foundation of China/ ; 32373113//National Natural Science Foundation of China/ ; 32503168//National Natural Science Foundation of China/ ; 2024M761923//China Postdoctoral Science Foundation/ ; 25ZR1402190//Natural Science Foundation of Shanghai/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Enhancer Elements, Genetic ; *CRISPR-Cas Systems ; Promoter Regions, Genetic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Noncoding cis-regulatory elements, particularly enhancers, are crucial for controlling gene expression. However, the in vivo use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi) to study enhancer function has been limited in zebrafish, which is widely used in early development and human disease research. Here, we optimized the CRISPRi system in zebrafish to achieve efficient suppression of tyr expression by fine-tuning component concentrations. Applying this optimized system, we functionally annotated distal enhancers of globin genes. Using Hi-C and histone modification assays, we systematically mapped 434 enhancer-promoter (EP) interactions across the genome. Among these EP loops, CRISPRi perturbation identified previously unreported enhancers with regulatory strengths surpassing known elements, demonstrated by disrupted phenotypes in fin and blood cell development. Additionally, several unreported EP loops were validated, underscoring the robustness of our integrated approach. This study not only provides an optimized CRISPRi system for zebrafish but also introduces a powerful platform that integrates computational and experimental strategies for advancing cis-regulatory element annotation in vertebrate gene regulation.},
}
@article {pmid41414668,
year = {2025},
author = {Semsey, S and Søndberg, E and Røen, M and Hallström, B and Petersen, A&#xd8; and Alfastsen, L and Bosch, BR and Wohl, B and Clube, J and van der Helm, E and Groendahl, C and Mougiakos, I},
title = {Characterization and engineering of a type IV-A3 CRISPR-Cas system for genome editing in Escherichia coli.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41414668},
issn = {1362-4962},
support = {//SNIPR Biome/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Gene Editing/methods ; Plasmids/genetics ; Klebsiella pneumoniae/genetics ; Genome, Bacterial ; DNA Helicases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {CRISPR-Cas systems have revolutionized genome engineering technologies, but type IV CRISPR-Cas systems and their genome engineering potential have been critically underexplored. In this study, we identified a type IV-A3 CRISPR-Cas system from a clinical Klebsiella pneumoniae isolate and characterized its plasmid targeting activity and capacity to suppress chromosomal and plasmid gene expression in Escherichia coli. We revealed the pivotal role of Csf3 (Cas5) and the dispensable roles of Csf1 (Cas8-like) and Csf4 (DinG helicase) subunits in IV-A3 CRISPR-Cas complex formation. The system prevents plasmid propagation via interplay between DinG helicase activity and strategic protospacer positioning relative to plasmid replication and maintenance components. We enabled the IV-A3 CRISPR-Cas system to introduce lethal, sequence-specific double-stranded (ds)DNA breaks in the E. coli chromosome by fusing the nuclease domain of the I-TevI nuclease to the Cas8 N-terminus. Further, we developed a series of base editors, with various editing efficiencies and windows, by fusing the PmCDA1 cytidine deaminase to the Cas8, Cas5, and DinG subunits. Finally, conjugative transfer of the Cas5-PmCDA1 base editor into E. coli deactivated the tryptophan repressor gene, boosting IAA production. Our study provides new insights into type IV-A3 CRISPR-Cas systems and highlights their potential in genome engineering applications.},
}
@article {pmid41414667,
year = {2025},
author = {Klein, N and Sanchez-Londono, M and Kara, MM and Gomes-Filho, JV and Novak, S and Kholeif, KH and Pekarek, L and Caliskan, N and Randau, L},
title = {Type I-Fv and engineered type IV-A1 CRISPR-Cas effectors facilitate genome reduction in Escherichia coli.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41414667},
issn = {1362-4962},
support = {//German Research Foundation/ ; 360987069//DFG/ ; 505997786//DFG/ ; //Microcosm Earth Center/ ; INST 93/1021-1 FUGG//Helmholtz Association/ ; //Philipps-Universit&#xe4;t Marburg/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Gene Editing/methods ; *Genome, Bacterial ; DNA Repair ; CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Class 1 CRISPR-Cas systems utilize multi-subunit effector ribonucleoprotein complexes to identify and target DNA. Upon recognition, type I systems recruit the helicase/nuclease Cas3 for DNA degradation, while type IV-A systems use the helicase CasDinG for transcriptional repression. Here, we developed two recombinant class 1 CRISPR-Cas genome editing tools for inducing large genomic deletions: the compact type I-Fv (also termed I-F2) system from Shewanella putrefaciens and the type IV-A1 system from Pseudomonas oleovorans. In the latter, CasDinG was engineered to include a C-terminal HNH nuclease domain, conferring DNA cleavage activity and enabling analysis of CasDinG processivity. Whole-genome sequencing of Escherichia coli BL21-AI was used to monitor genome reduction and DNA repair mechanisms in response to CRISPR-Cas-induced damage. Small deletions were flanked by microhomologies, consistent with repair via alternative end joining, whereas deletions larger than 10 kb consistently terminated at nearby IS1 elements, implicating these sequences in the repair process. This study introduces compact type I and engineered type IV-A genome editing tools with distinct protospacer-adjacent motif requirements and provides new insights into CasDinG evolution and the DNA repair pathways engaged during CRISPR-Cas-mediated genome editing.},
}
@article {pmid41274609,
year = {2026},
author = {Lau, CH and Li, X and Liang, QL and Guo, R and Ren, X and Xu, Z and Zhu, Y and Lai, Y and Liu, G and Huang, Y and Wu, W and Zhu, H and Chen, J and Zhang, X},
title = {CRISPR technology for diagnosis and treatment of human brucellosis.},
journal = {Journal of microbiological methods},
volume = {240},
number = {},
pages = {107339},
doi = {10.1016/j.mimet.2025.107339},
pmid = {41274609},
issn = {1872-8359},
mesh = {Humans ; *Brucellosis/diagnosis/therapy/drug therapy/microbiology ; Animals ; *Brucella/genetics/isolation &amp; purification/drug effects ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Livestock/microbiology ; Point-of-Care Testing ; Anti-Bacterial Agents/therapeutic use ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The global burden of Brucella infection in livestock and human health is substantial, particularly in developing countries or rural areas. Currently, brucellosis diagnosis primarily relies on PCR, microbiological culture, and serological tests. However, these approaches have several drawbacks such as long experimental duration, lengthy procedure, low positive detection rates, high variability in results, interspecies cross-reactivity, and require expensive equipment and professional operators. Herein, we review how recent emerging CRISPR/Dx technology can address some of these shortcomings to realize field-deployable detection of Brucella in domestic animals and point-of-care testing (POCT) for human brucellosis. CRISPR technology has been successfully used to treat brucellosis by deleting or inactivating the genes associated with the Brucella replication or survival. Therefore, we also discuss how CRISPR technology can be potentially used to treat brucellosis, as antibiotic therapy may lose efficacy when encountering multidrug-resistant Brucella strains and the treatment is long-lasting in infected individuals to prevent relapse. Lastly, we critically discuss the advances, pitfalls, and future perspectives of CRISPR technology for the diagnosis and treatment of brucellosis in humans and livestock. Ultimately, the continued refinement of CRISPR technology will pave the road for field-deployable pathogen diagnostics and home self-tests of brucellosis to mitigate global Brucella infections.},
}
@article {pmid41413028,
year = {2025},
author = {Chen, Q and Jiang, X and Yang, B and Deng, Z and Sun, Y},
title = {Anti-CRISPR protein AcrIIA5 can enhance the activity and security of prime editing.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-025-66237-2},
pmid = {41413028},
issn = {2041-1723},
abstract = {Prime editing (PE) enables the precise installation of intended base substitutions, small deletions or small insertions into the genome of living cells. While the use of Cas9 nickase can avoid DNA double-strand breaks (DSB), undesired insertions and deletions (indels) often accompany the correct edits, particularly when PE activity increased. Here we show that the anti-CRISPR (Acr) protein AcrIIA5 can significantly enhance PE activity by up to 8.2-fold while markedly reducing byproduct indels. Further investigation reveals that AcrIIA5 can promote PE across various approaches (PE2, PE3, PE4, PE5, and PE6), edit types (substitutions, insertions and deletions), and endogenous loci. Mechanistically, AcrIIA5 appears to inhibit the re-nicking activity of PE complex rather than enhancing the core editing machinery itself, suggesting a distinct mode of interaction with Cas9. Overall, we demonstrate that a known "inhibitor" Acr protein can unexpectedly acting as an "enhancer" of CRISPR/Cas-based genome editing, providing an effective strategy to optimize PE specificity and activity.},
}
@article {pmid41412367,
year = {2025},
author = {Jose, J and Hamow, K&#xc1; and Éva, C and Moncsek, B and Kyrpa, T and Reinoso, LG and Bozsó, Z and Bakonyi, J and Balázs, E and Sági, L},
title = {CRISPR/Cas-mediated polyphenol oxidase gene knockout in potato reveals divergent roles in resistance to bacterial wilt and late blight.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {},
number = {},
pages = {112944},
doi = {10.1016/j.plantsci.2025.112944},
pmid = {41412367},
issn = {1873-2259},
abstract = {Polyphenol oxidases (PPOs) play a pivotal role in plant immune responses by catalysing the oxidation of phenolic compounds into cytotoxic quinones and melanin and contributing to the fortification of cell walls. Despite their biological significance, the high expression of PPOs in potatoes is not desirable due to their promotion of tuber browning. This study elucidates the relationship between PPO activity and defense mechanisms against the diverse pathogens Ralstonia solanacearum (Rs) and Phytophthora infestans (Pi) while mitigating enzymatic browning. CRISPR/Cas-mediated editing of the tuber- and root-specific PPO genes in the 'Désirée' and 'Balatoni Rózsa' potato cultivars considerably reduced enzymatic activity and browning. Among four PPO-edited mutant lines, three exhibited increased susceptibility to Rs while responses to Pi remained unchanged, underscoring the importance of PPOs in resistance to Rs. The PPO knockouts resulted in significant shifts in metabolite and hormone profiles characterized by elevated levels of dihydrokaempferol, coniferyl alcohol and taxifolin among other metabolites in the roots of Rs-susceptible mutants. Additionally, reduced PPO activity in these lines correlated with increased concentrations of salicylic acid, jasmonic acid and several antimicrobial compounds and alterations in flavonoid regulation. These findings highlight the complex role of PPOs in plant defense, establishing a positive correlation between PPO activity and resistance to Rs, while offering insights into the trade-offs associated with PPO gene editing in potatoes.},
}
@article {pmid41412287,
year = {2025},
author = {Su, Z and Liang, Z and Wu, Q and Xu, S and Li, C and Zheng, H and Wu, C and Ji, W and Niu, Y and Yang, Z},
title = {Metal-organic frameworks for CRISPR/Cas9 gene editing delivery: Innovations in therapeutic and diagnostic applications.},
journal = {Acta biomaterialia},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.actbio.2025.12.030},
pmid = {41412287},
issn = {1878-7568},
abstract = {CRISPR/Cas gene editing technology demonstrates significant promise in the treatment of various diseases, and a precise, efficient and safe delivery system is a key to realize gene therapy. Although traditional viral vectors can achieve superior transfection efficiency, viruses suffer from low reproduction efficiency and the risk of random gene integration, further limiting their wide application. Notably, metal-organic frameworks (MOFs), with tunable pore structure, easy surface chemical modification, good biocompatibility and physiological stability, have drawn much attention in the domain of targeted delivery of gene editing systems. Compared to lipid nanoparticles (LNPs) and extracellular vesicles (EVs), MOFs offer superior cargo loading (>80 % for proteins) and protect nucleic acids from degradation, while their stimuli-responsive degradation enables controlled release. This review focus on the cutting-edge advances of intelligent-responsive MOFs in delivering gene editing systems to against diseases, including endogenous responses (e.g., ATP, pH, redox microenvironment) and exogenous stimulus responses (e.g., photothermal, ultrasound) in the disease microenvironment, as well as systematically summarize the synergistic therapy of gene editing therapy combined with chemotherapy, chemodynamic therapy, photodynamic therapy, and sonodynamic therapy based on the delivery systems of MOFs. Additionally, we further summarize the research of MOFs-based CRISPR/Cas delivery system as a bio-probe for viral, nucleic acid and RNA examination. This study will help facilitate the clinical translation of MOFs-based CRISPR/Cas delivery systems in the field of therapy and detection of diseases. STATEMENT OF SIGNIFICANCE: This article reviews the cutting-edge advances of intelligent-responsive MOFs in delivering CRISPR/Cas systems to against diseases, including endogenous responses (e.g., pH, ATP, redox microenvironment) and exogenous stimulus responses (e.g., photothermal, ultrasound) in the disease microenvironment, as well as systematically summarize the synergistic therapy of gene editing therapy combined with chemotherapy, chemodynamic therapy, photodynamic therapy, and sonodynamic therapy based on the delivery systems of MOFs. Importantly, the potential applications of MOFs-based CRISPR/Cas delivery system as a bio-probe for viral, nucleic acid and RNA examination also have been discussed. This study will provide insights for the development of MOFs-based CRISPR/Cas delivery systems in the therapy and detection of clinical diseases.},
}
@article {pmid41412110,
year = {2025},
author = {Snell, JC and Nelson, BJ and Matreyek, KA},
title = {DIALing in elevated expression setpoints with promoter shortening.},
journal = {Cell systems},
volume = {16},
number = {12},
pages = {101482},
doi = {10.1016/j.cels.2025.101482},
pmid = {41412110},
issn = {2405-4720},
mesh = {*Promoter Regions, Genetic/genetics ; *Gene Expression Regulation/genetics ; Humans ; CRISPR-Cas Systems/genetics ; },
abstract = {DIAL is a novel framework for temporal control of transcript abundances in engineered cells. Targeted excision of DNA spacers in transgenic promoters permits controlled transitions of protein expression between setpoints. DIAL expands the repertoire of bioengineering tools for controlling protein expression, cell fates, and biological systems in general.},
}
@article {pmid41410934,
year = {2025},
author = {Deora, S and Deora, GS and Nigam, S and Harish, },
title = {Hacking heterocysts: advances in the genetic regulation of heterocyst differentiation.},
journal = {Archives of microbiology},
volume = {208},
number = {1},
pages = {80},
pmid = {41410934},
issn = {1432-072X},
mesh = {Nitrogen Fixation/genetics ; *Gene Expression Regulation, Bacterial ; *Cyanobacteria/genetics/metabolism/growth &amp; development ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Heterocyst differentiation in certain filamentous cyanobacteria is a multifaceted process essential for nitrogen fixation, orchestrated by a sophisticated regulatory network that encompasses several key stages. These include induction, pattern differentiation, commitment, extracellular layer formation, cell-cell communication, and ultimately, nitrogen fixation and metabolism. Key regulators like NtcA and HetR control heterocyst development, while proteins such as PatS, HetN, and PatA modulate pattern formation. Certain non-coding RNAs, such as NsiR1, Yfr1, and NsiR4, also regulate gene expression and contribute to the shutdown of CO2 fixation in differentiating heterocysts. Meanwhile, the heterocysts' unique envelope protects nitrogenase from oxygen, enabling nitrogen fixation. Genetic engineering approaches, including CRISPR-Cas systems, have been employed to increase heterocyst frequency and enhance the production of compounds such as ethanol, butanol and H2. By manipulating genes responsible for heterocyst differentiation, scientists can optimize nitrogen fixation, develop efficient biofertilizers, and unlock opportunities for a more sustainable future in agriculture and biotechnology. This review addresses the current understanding of the regulatory networks and molecular mechanisms that influence the development and function of heterocysts, providing insights into the biology and potential applications of these specialized cells through gene manipulations.},
}
@article {pmid41410807,
year = {2025},
author = {Sarroukh, I and Ibriz, M and Yakkou, L and Lebkiri, N and Fokar, M and Iraqi, D and Gaboun, F and Diria, G and Abdelwahd, R},
title = {The Agrobacterium-mediated genetic transformation: a gateway for efficient CRISPR/Cas9 gene editing in leguminous.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {57},
pmid = {41410807},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Transformation, Genetic/genetics ; *Plants, Genetically Modified/genetics/growth &amp; development ; *Agrobacterium tumefaciens/genetics ; },
abstract = {Climate change enhances the damaging consequences of abiotic and biotic stressors, leading to severe soil fertility loss and ecosystem degradation worldwide. Leguminous have contributed significantly to replenishing soil nitrogen via symbiotic nitrogen fixation, contributing approximately 15% of nitrogen input, which is crucial for soil health and enhancing crop production. There is an increasing integration of new biotechnological interventions, such as genome editing, including the CRISPR/Cas9 system, and transgenesis, in addition to classical breeding, to make agriculture more resilient. In this review, we examine several elements that influence the genetic transformation system employing Agrobacterium tumefaciens strains in leguminous to make it an ideal vehicle for CRISPR/Cas9 component delivery. The variables investigated in our study included the incubation period, co-cultivation duration, bacterial density, selectable marker, concentration, and growth regulators used. In addition, the selection and efficiency of the explant choice for transformation should be considered in future studies. However, there have been parallel recommendations for the gradual application of selectable markers such as kanamycin.},
}
@article {pmid41410797,
year = {2025},
author = {Appolonia, CN and Centore, JT and Shukla, S and Hluck, J and Conlon, RA and Ramakrishnan, P},
title = {A CRISPR/Cas9 assisted strategy for the conditional expression of human NF-kappaB c-Rel cDNA in mouse T cells: design, prospects, and challenges.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {56},
pmid = {41410797},
issn = {1573-9368},
mesh = {Animals ; Humans ; Mice ; *CRISPR-Cas Systems/genetics ; Mice, Transgenic ; *Proto-Oncogene Proteins c-rel/genetics ; Promoter Regions, Genetic ; Mice, Inbred NOD ; Gene Editing ; *T-Lymphocytes/metabolism ; DNA, Complementary/genetics ; *Diabetes Mellitus, Type 1/genetics ; NF-kappa B/genetics ; },
abstract = {Nuclear factor-κB protein c-Rel is a critical regulator of autoimmune diabetes. We found that c-Rel O-GlcNAcylation at serine-350 increases with hyperglycemia, which results in increased transcription of proautoimmune Th1 cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-γ), and decreased transcription of the T regulatory cell transcription factor forkhead box 3 (FOXP3). To further study the translational relevance of c-Rel S350 O-GlcNAcylation in autoimmune diabetes, we sought to generate transgenic non-obese diabetic (NOD) mice conditionally expressing wildtype or mutant S350A human c-Rel cDNA in T cells downstream of the endogenous mouse REL promoter. We used CRISPR-Cas9 gene editing to insert a unique designer cassette containing floxed mouse c-Rel cDNA-STOP sequence to maintain whole body c-Rel expression, followed by a linker and human c-Rel cDNA-STOP sequence. Using comprehensive PCR analyses and high-throughput sequencing, we confirmed successful insertion of the cassette at the mouse REL locus and the expected deletion of the mouse c-Rel cDNA specifically in T cells following CD4-Cre mating. Additional characterization revealed that the knock-in transgenic mice lacked endogenous mouse c-Rel, further confirming desired interference with its natural start codon. Unexpectedly, these mice lacked mouse and human c-Rel protein expression from inserted cDNAs, which mechanistically correlated with increased CpG methylation of the c-Rel promoter region. Thus, our study presents a unique, universal molecular design and method for the generation of conditional knock-in transgenic mice expressing human genes at the endogenous mouse promoter. It also reveals a potential locus-specific challenge that may arise during the development of such novel transgenic mouse models.},
}
@article {pmid41410478,
year = {2026},
author = {Wang, X and Yu, G and Luo, Y and Chen, T and Zhang, X and Ye, L and Yang, C and Chen, Q},
title = {The autophagy-related protein PlAtg26b regulates vegetative growth, reproductive processes, autophagy, and pathogenicity in Peronophythora litchii.},
journal = {Virulence},
volume = {17},
number = {1},
pages = {2606498},
doi = {10.1080/21505594.2025.2606498},
pmid = {41410478},
issn = {2150-5608},
mesh = {*Autophagy ; Virulence ; Plant Diseases/microbiology/parasitology ; *Autophagy-Related Proteins/genetics/metabolism ; Litchi/microbiology/parasitology ; *Oomycetes/pathogenicity/growth &amp; development/genetics ; CRISPR-Cas Systems ; Reproduction ; Plant Leaves/microbiology ; Mitochondria/metabolism ; Gene Knockout Techniques ; Hyphae/growth &amp; development ; },
abstract = {Peronophythora litchii is an oomycete pathogen responsible for litchi downy blight, a significant threat to global litchi production. Autophagy, a conserved degradation pathway crucial for the growth, development, and pathogenicity of phytopathogenic organisms, remains an area of active investigation. In this study, we characterized the function of the Atg26 homolog PlAtg26b in P. litchii. Using the CRISPR/Cas9 genome editing system, we generated PlATG26b knockout mutants and determined that PlAtg26b localizes to mitochondria under stress conditions. Although deletion of PlATG26b did not impair selective autophagy, it markedly reduced Atg8-PE synthesis, vegetative hyphal growth, asexual and sexual reproduction, and zoospore release. Furthermore, PlATG26b-deficient mutants exhibited significantly reduced virulence on litchi fruits and leaves. Collectively, our findings demonstrate that PlAtg26b plays a pivotal role in the biological development and pathogenicity of P. litchii.},
}
@article {pmid41409480,
year = {2025},
author = {Anwar, M and Vinothkanna, A and Jia, AQ},
title = {Fostering plant protection against certain bacterial diseases through quorum-sensing signal molecules: a critical review.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1602573},
pmid = {41409480},
issn = {1664-462X},
abstract = {Quorum sensing (QS) and clustered regularly interspaced short palindromic repeats (CRISPR) systems are envisaged as revolutionary in abating plant bacterial pathogens. Bacterial cell-cell communication and plant pathogen QSSMs (quorum sensing signaling molecules) are dissected for underlying mechanisms in prominent pathogens, viz., Pseudomonas syringae, Erwinia amylovora, and Xanthomonas campestris. Biofilm formation and virulence mechanisms are critically addressed to repurpose potential QS inhibition strategies. CRISPR technologies are combined with CRISPR engineering to produce enhanced disease-resistant varieties, with potential applications. QS-CRISPR interplay for deciphering the key interactive changes in plant health management is prioritized for deliberate future research outcomes. Sustainable agricultural practices are envisaged for successful lab-to-field authentic field trials and large-scale applicability across the globe. Potential technical limitations, the need for stringent agricultural laws, and future innovations are addressed. Moreover, the cost-effectiveness, enhanced crop production, yield, and productivity hindering the above key plant bacterial pathogens are comprehensively addressed against these plant bacterial pathogens. Furthermore, a future outlook characterized by extensive outreach and global implications is substantiated regardless of regional specificity, climate change, and global warming. A decade of research on advancements in adequate plant protection is revisited to incorporate augmented approaches, including artificial intelligence (AI) and machine learning, in sustainable agriculture. The significance of the present review is based on addressing QSSMs and plant protection strategies encompassing modern molecular biological techniques.},
}
@article {pmid41407671,
year = {2025},
author = {Bitew, MA and Paredes-Santos, TC and Maru, P and Krishnamurthy, S and Wang, Y and Sangaré, LO and Duley, S and Yamaryo-Botté, Y and Botté, CY and Saeij, JPJ},
title = {A genome-wide CRISPR screen identifies GRA38 as a key regulator of lipid homeostasis during Toxoplasma gondii adaptation to lipid-rich conditions.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11177},
pmid = {41407671},
issn = {2041-1723},
support = {R01AI173803//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; },
mesh = {*Toxoplasma/genetics/metabolism/pathogenicity/physiology ; Animals ; *Protozoan Proteins/metabolism/genetics ; Homeostasis ; Mice ; *Lipid Metabolism/genetics ; Virulence/genetics ; Phosphatidate Phosphatase/metabolism/genetics ; Toxoplasmosis/parasitology ; Adaptation, Physiological/genetics ; CRISPR-Cas Systems ; Female ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Genome, Protozoan ; },
abstract = {Intracellular parasites like Toxoplasma gondii scavenge host nutrients, particularly lipids, to support their growth and survival. Although Toxoplasma is known to adjust its metabolism based on nutrient availability, the mechanisms that mediate lipid sensing and metabolic adaptation remain poorly understood. Here, we perform a genome-wide CRISPR screen under lipid-rich (10% Fetal Bovine Serum (FBS)) and lipid-limited (1% FBS) conditions to identify genes critical for lipid-responsive fitness. We identify the Toxoplasma protein GRA38 as a lipid-dependent regulator of parasite fitness. GRA38 exhibits phosphatidic acid (PA) phosphatase (PAP) activity in vitro, which is significantly reduced by mutation of its conserved DxDxT/V catalytic motif. Disruption of GRA38 leads to the accumulation of PA species and widespread alterations in lipid composition, consistent with impaired PAP activity. These lipid imbalances correlate with reduced parasite virulence in mice. Our findings identify GRA38 as a metabolic regulator important for maintaining lipid homeostasis and pathogenesis in Toxoplasma gondii.},
}
@article {pmid41407557,
year = {2025},
author = {Tyagi, E and Sachan, A and Bhuyan, R and Kumari, P and Prakash, A},
title = {Next-Gen Biofilm Control: Gene Editing and Computational Approaches.},
journal = {APMIS : acta pathologica, microbiologica, et immunologica Scandinavica},
volume = {133},
number = {12},
pages = {e70122},
doi = {10.1111/apm.70122},
pmid = {41407557},
issn = {1600-0463},
mesh = {*Biofilms/drug effects/growth &amp; development ; *Gene Editing/methods ; Humans ; *Computational Biology/methods ; CRISPR-Cas Systems ; *Bacteria/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Bacterial ; Phage Therapy ; Bacteriophages ; },
abstract = {Biofilms are microbial communities enclosed in an extracellular polymeric substance (EPS), significantly contributing to antimicrobial resistance (AMR) in medical, industrial, and environmental settings. Their matrix enhances microbial survival, inhibits antibiotic penetration, and facilitates horizontal gene transfer, worsening the AMR crisis. Conventional antimicrobial treatments often fail against biofilms, necessitating novel therapeutic strategies. Emerging biofilm-targeted interventions, such as nanotechnology-based antimicrobials, bacteriophage therapy, and CRISPR-Cas9 gene editing, offer promising solutions. Nanoparticles improve drug delivery, bacteriophages selectively lyse resistant bacterial populations, and CRISPR-Cas9 disrupts AMR-related genes and biofilm virulence factors. Additionally, AI and ML are advancing biofilm prediction models and antimicrobial optimization, paving the way for precision-targeted interventions. This review explores biofilm biology and next-generation biofilm control strategies, with a focus on AI-driven bioinformatics. Future research should focus on clinical translation, regulatory standardization, and scalable implementation in healthcare and industrial settings to combat biofilm-associated AMR.},
}
@article {pmid41407085,
year = {2025},
author = {Qi, H and Yang, Y and Hou, X and Chen, Y and Gong, S},
title = {Sensitivity-improving CRISPR-Cas strategies for non-nucleic acid targets detection.},
journal = {Methods (San Diego, Calif.)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymeth.2025.12.004},
pmid = {41407085},
issn = {1095-9130},
abstract = {CRISPR-Cas systems have revolutionized non-nucleic acid targets detection across diverse applications. Nevertheless, the relatively low enzymatic turnover rate of activated Cas nucleases during substrate cleavage remains a critical bottleneck, limiting the sensitivity of such detection methods. To address this challenge, numerous innovative strategies have been proposed to enhance the sensitivity of CRISPR-Cas systems, enabling high-sensitive non-nucleic acid targets detection. This review systematically summarizes the sensitivity-enhancing methodologies for non-nucleic acid targets detection using CRISPR-Cas technologies. We first delineate the working mechanisms of various CRISPR-Cas systems and the signal transduction pathways specific to non-nucleic acid targets. Subsequently, we detail diverse sensitivity-improving approaches, including nucleic acid amplification-facilitated strategies, multimolecular labeling techniques, dual-enzyme cascade methods, and multiplex amplification methodologies. Additionally, the current challenges and future perspectives in this field are discussed, aiming to inspire researchers to develop more ingenious solutions and facilitate real-world applications of CRISPR-Cas system for non-nucleic acid targets detection.},
}
@article {pmid41344099,
year = {2025},
author = {Ryu, YC and Bao, G and Hwang, BH},
title = {Peptide-assisted lipofection enables efficient non-viral delivery of large CRISPR/Cas9 constructs for genome editing applications.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {193},
number = {},
pages = {118838},
doi = {10.1016/j.biopha.2025.118838},
pmid = {41344099},
issn = {1950-6007},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; Animals ; *Transfection/methods ; Plasmids/genetics ; *Gene Transfer Techniques ; *Peptides/chemistry ; Mice ; *Lipids/chemistry ; Genetic Therapy/methods ; Cell Survival ; Genetic Vectors ; },
abstract = {Efficient and safe delivery of large genetic constructs such as CRISPR/Cas9 plasmids remains a critical bottleneck in gene therapy. In this work, the peptide-assisted lipofection (PAL) system was developed as a breakthrough non-viral vector for gene delivery, specifically designed for large plasmids including CRISPR/Cas9 constructs. This system achieved exceptional transfection efficiency up to 98.7 % in HEK293T cells, surpassing conventional delivery methods such as electroporation and standard lipofections. PAL successfully delivered large plasmids up to 29 kb while maintaining high cell viability and achieved 44.1 % indel formation efficiency in gene editing experiments. Fluorescence microscopy verified PAL's efficient endosomal escape and nuclear targeting abilities. The superior performance of the PAL is attributed to its cellular uptake and endosomal escape enhanced by transfection-assisting peptide. In vivo studies in mouse models showed sustained gene expression in liver tissue, demonstrating superior performance compared to naked plasmid delivery. These results establish PAL as a versatile and promising platform for gene therapy and genome editing, offering a safer alternative to viral vectors for large genetic payload delivery.},
}
@article {pmid41329618,
year = {2025},
author = {Udemezue, VC and Shaikh, KM and Vorontsova, M and Valgepea, K},
title = {Optimization of Plasmid Curing from Genetically Engineered Clostridium autoethanogenum.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4967-4972},
doi = {10.1021/acssynbio.5c00456},
pmid = {41329618},
issn = {2161-5063},
mesh = {*Plasmids/genetics ; *Clostridium/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Metabolic Engineering/methods ; Gene Editing/methods ; *Genetic Engineering/methods ; Electroporation ; },
abstract = {Accumulation of greenhouse gases from combustion of fossil fuels drives climate change and threatens biosustainability on Earth. Microbial gas fermentation realizes the capture of CO2 toward biomanufacturing of value-added products. Acetogens are attractive biocatalysts here, as they use CO2 as their sole carbon source with H2. Metabolic engineering of novel cell factories is, however, hampered by the slow and complex genetic engineering workflows. Here, we developed different approaches to optimize plasmid curing from genetically engineered strains of the model acetogen Clostridium autoethanogenum. Interestingly, a CRISPR/Cas9-based curing plasmid (C-plasmid) targeting the origin of replication both in the target editing plasmid and in the C-plasmid did not improve curing over a non-targeting control plasmid. Strikingly, plasmid curing by making cells electrocompetent (ECCs) and by non-transformative electroporation of ECCs or buffer-washed glycerol stocks showed 14-100% curing efficiencies across the approaches for five different genetically engineered C. autoethanogenum strains. The most time-efficient approach with non-transformative electroporation of buffer-washed glycerol stocks also cured an editing plasmid from Escherichia coli, with ∼97% efficiency. This work both improves genetic engineering workflows for C. autoethanogenum by significantly accelerating plasmid curing and offers methods to potentially ease plasmid curing in other microbes.},
}
@article {pmid41285016,
year = {2025},
author = {He, RL and Huang, BC and Wu, J and Sun, D and Li, HH and Liu, JQ and Wu, J and Liu, DF and Li, WW},
title = {One-Step Biological Upcycling of Chitin Wastes into Violacein by Engineered Chromobacterium violaceum.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4733-4743},
doi = {10.1021/acssynbio.5c00472},
pmid = {41285016},
issn = {2161-5063},
mesh = {*Chromobacterium/metabolism/genetics ; *Chitin/metabolism ; *Indoles/metabolism ; *Metabolic Engineering/methods ; Fermentation ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Existing technologies for the valorization of organic wastes have been focused mainly on degradable wastes, while an efficient, low-carbon approach for the upcycling of shell waste is still lacking. Here, we report a one-step chitin biological fermentation process (CBFP), based on the construction of Chromobacterium violaceum engineered strain, for efficiently converting shell waste-derived chitin into high-value violacein. A high-efficiency CRISPR cytosine-base editor (pRK2-BE, 97% editing efficiency) was developed for C. violaceum, which demonstrated cv_4240, cv_1440, and cv_2935 as the major chitin hydrolysis genes and phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) as the major N-acetyl-glucosamine uptake pathway. The engineered strain WT/pBAD-4, co-overexpressing of cv_4240, cv_1440, cv_2935, and vioABCDE, efficiently utilized colloidal chitin and crystalline chitin as the sole carbon and nitrogen source, achieving violacein yields of 159.78 and 120.95 mg·L[-1], respectively. This study provided an economically viable and environmentally sustainable solution for green upcycling of shell waste.},
}
@article {pmid41277095,
year = {2025},
author = {Thornbury, M and Omran, RP and Kumar, L and Knoops, A and Abushahin, R and Whiteway, M and Martin, VJJ},
title = {Tri-Functional CRISPR Screen Reveals Overexpression of QDR2 and QDR3 Transporters Increase Fumaric Acid Production in Kluyveromyces marxianus.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4907-4922},
doi = {10.1021/acssynbio.5c00689},
pmid = {41277095},
issn = {2161-5063},
mesh = {*Fumarates/metabolism ; *Kluyveromyces/metabolism/genetics ; *Fungal Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Metabolic Engineering/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Membrane Transport Proteins/genetics/metabolism ; },
abstract = {Organic acids such as fumaric acid are widely used in the food and beverage industry as acidulants and preservatives, while also serving as versatile precursors for industrially relevant compounds. Fumaric acid is still predominantly produced through petroleum-derived processes. To enhance production efficiency and diversify supply, we are engineering Kluyveromyces marxianus as a biosynthetic platform from renewable feedstocks. In previous work, we have established K. marxianus Y-1190 as a host for lactose valorization based on its high growth rate on lactose and its tolerance for acid conditions. Here, we establish a trifunctional genome-wide library for K. marxianus using CRISPR activation, interference, and deletion to allow identification of gene expression perturbations that enhance tolerance to fumaric acid. We determined that deletion of ATP7, encoding a subunit of the mitochondrial F1F0 ATP synthase, and overexpression of QDR2 and QDR3, two previously uncharacterized members of the 12-spanner H[+] antiporter (DHA1) family in K. marxianus, can enhance fumaric acid tolerance. We also found that integrated overexpression of both QDR2 and QDR3 in a ΔFUM1 background strain improved titers of fumaric acid production from 0.26 to 2.16 g L[-1]. Together, these results highlight roles for membrane transport and mitochondrial function in enabling fumaric acid tolerance and production in K. marxianus.},
}
@article {pmid41273852,
year = {2025},
author = {Gallucci, FP and de Camargo, JA and Viana, NI and Pimenta, RCA and Guimarães, VR and Candido, P and Leite, KRM and Nahas, WC and Dos Reis, ST},
title = {CRISPR/Cas9-mediated MMP-9 silencing inhibits bladder cancer T24 cell invasion and migration in vitro.},
journal = {Clinics (Sao Paulo, Brazil)},
volume = {80},
number = {},
pages = {100842},
pmid = {41273852},
issn = {1980-5322},
mesh = {Humans ; *Urinary Bladder Neoplasms/pathology/genetics/enzymology ; *Matrix Metalloproteinase 9/genetics/metabolism ; *Cell Movement/genetics ; *CRISPR-Cas Systems/genetics ; Cell Proliferation/genetics ; Cell Line, Tumor ; Neoplasm Invasiveness/genetics ; *Gene Silencing ; Apoptosis/genetics ; Blotting, Western ; Flow Cytometry ; Real-Time Polymerase Chain Reaction ; Gene Expression Regulation, Neoplastic ; },
abstract = {PURPOSE: Bladder Cancer (BCa) ranks as the tenth most common cancer worldwide, with high morbidity and mortality. Matrix Metalloproteinases (MMPs), especially MMP-9, are associated with tumor progression and metastasis. This study aimed to evaluate the effects of MMP-9 silencing using CRISPR-Cas9 in T24-luc bladder carcinoma cells.<br><br>METHODS: Guide RNAs (sgRNA1 and sgRNA2) targeting MMP-9 were cloned into pX-330 plasmids and transfected into T24-luc cells. Gene and protein expression were analyzed via RT-qPCR and Western blotting. Functional assays included flow cytometry for proliferation and apoptosis, colony formation, wound healing, and Matrigel&#x2122; invasion assays.<br><br>RESULTS: sgRNA2 significantly reduced MMP-9 gene expression, while both sgRNAs reduced protein expression. Edited cells showed decreased proliferation and colony formation, increased apoptosis, and reduced migration and invasion capacity.<br><br>CONCLUSION: CRISPR-Cas9-mediated silencing of MMP-9 inhibited cell proliferation, migration, invasion, and increased apoptosis in BCa cells, supporting that MMP-9 has an important effect on the progression of bladder cancer.},
}
@article {pmid41267649,
year = {2025},
author = {Matsubara, K and Hirota, M and Kajiwara, K and Senga, H and Matsui, S and Marutani, M and Seki, Y},
title = {Lineage-specific enhancer insertions regulate Prdm14 to drive the rapid transition from naïve to formative pluripotency in rodents.},
journal = {Development (Cambridge, England)},
volume = {152},
number = {24},
pages = {},
doi = {10.1242/dev.204886},
pmid = {41267649},
issn = {1477-9129},
support = {18H02422//Japan Society for the Promotion of Science/ ; //Kwansei Gakuin University/ ; },
mesh = {Animals ; Mice ; *Transcription Factors/genetics/metabolism ; *Enhancer Elements, Genetic/genetics ; Gene Expression Regulation, Developmental ; *Cell Lineage/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; CRISPR-Cas Systems/genetics ; Humans ; Cell Differentiation/genetics ; DNA-Binding Proteins ; RNA-Binding Proteins ; },
abstract = {The network of transcription factors is dynamically reorganized during the transition from naïve- to formative-pluripotency. In mice, Prdm14 is expressed in naïve pluripotent cells but rapidly downregulated upon exit from the naïve state. In contrast, PRDM14 expression persists throughout pluripotency transitions in non-rodent mammals, including pigs and humans. Here, we investigate the molecular mechanisms underlying the rodent-specific expression of Prdm14. Using CRISPR/Cas9-mediated deletions, we demonstrated that POU5F1 and TFCP2L1 recognition sequences within Muroidea-specific cis-regulatory elements located downstream of Prdm14 are essential for its transcriptional upregulation in naïve embryonic stem cells. Loss of these enhancers attenuates the upregulation of Prdm14, leading to reduced Pramel7 induction and impaired degradation of UHRF1, which consequently diminished global DNA demethylation under 2iL conditions. Moreover, deletion of PRDM14-binding motifs in Muroidea-specific enhancers disrupts its negative feedback loop, resulting in a delayed transition from the naïve to formative pluripotent state. Our findings reveal that rodent-specific enhancer insertions endow Prdm14 with a dynamic regulatory architecture, enabling both activation and repression that collectively ensure the timely exit from naïve pluripotency during early embryogenesis.},
}
@article {pmid41257388,
year = {2025},
author = {Gervais, NC and Hendriks, A and Shapiro, RS},
title = {Effective Strategies for Translating CRISPR-dCas Systems to Diverse Microbes.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4624-4635},
doi = {10.1021/acssynbio.5c00537},
pmid = {41257388},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Bacteria/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR-dCas tools have widespread applications for rapidly manipulating and dissecting gene function across the microbial tree of life. However, despite their theoretical suitability for use in a broad range of species, CRISPR-dCas tools that are often initially optimized for use in model cell lines and model organisms still frequently require extensive modifications to enable their application in specific microbial organisms. Here, we review different iterations of CRISPR-dCas in microbes and the application of these techniques. We further discuss common obstacles faced and troubleshooting approaches while developing and applying CRISPR-dCas systems to a microbial organism. Finally, we suggest enhancements that can be made that may help improve the applicability of a CRISPR-dCas tool developed for nonmodel microbial organisms.},
}
@article {pmid41248489,
year = {2025},
author = {Sinan, S and Kooistra, RM and Rajaraman, K and Islam, Z and Madan, D and Nalefski, EA and Finkelstein, IJ},
title = {One-Pot Isothermal Linear Amplification and Cas12a-Based Nucleic Acid Detection.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4714-4722},
doi = {10.1021/acssynbio.5c00463},
pmid = {41248489},
issn = {2161-5063},
mesh = {*Nucleic Acid Amplification Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Human papillomavirus 16/genetics/isolation &amp; purification ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Papillomavirus Infections/diagnosis/virology ; DNA, Viral/genetics ; Uterine Cervical Neoplasms/virology/diagnosis ; DNA, Single-Stranded/genetics ; DNA-Directed DNA Polymerase/metabolism ; },
abstract = {CRISPR-based nucleic acid diagnostics are a promising class of point-of-care tools that could dramatically improve healthcare outcomes for millions worldwide. However, these diagnostics require nucleic acid preamplification, an additional step that complicates deployment to low resource settings. Here, we developed CATNAP (Cas trans-nuclease detection of amplified products), a method that integrates isothermal linear DNA amplification with Cas12a detection in a single reaction. CATNAP uses a nicking enzyme and DNA polymerase to continuously generate single-stranded DNA, activating Cas12a's trans-cleavage activity without damaging the template. We optimized enzyme combinations, buffer conditions, and target selection to achieve high catalytic efficiency. CATNAP successfully distinguished between high- and low-risk HPV strains and detects HPV-16 in crude cell lysates of cervical cancer cells with minimal equipment, offering advantages over PCR-based approaches. We conclude that CATNAP bridges the sensitivity gap in CRISPR diagnostics while maintaining simplicity, making accurate disease detection more accessible in resource-limited settings.},
}
@article {pmid41241768,
year = {2025},
author = {Lotfi, M and Farshchian Yazdi, Z and Hashemi, MR and Abbaszadegan, MR and Sharif, S},
title = {Guarding the genome: strategies for off-target detection and minimization in CRISPR/Cas9 system.},
journal = {Expert review of molecular diagnostics},
volume = {25},
number = {12},
pages = {883-902},
doi = {10.1080/14737159.2025.2581677},
pmid = {41241768},
issn = {1744-8352},
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {INTRODUCTION: CRISPR-based genome editing has revolutionized molecular diagnostics and therapeutic applications, offering precise genetic modifications. However, off-target effects remain a significant challenge, potentially leading to unintended mutations and genomic instability. Addressing these concerns is crucial for ensuring the clinical safety and efficacy of CRISPR technologies.<br><br>AREAS COVERED: This review provides a comprehensive analysis of strategies for detecting and mitigating off-target effects in CRISPR genome editing. We discuss in silico, in vitro, and in vivo detection methods, highlighting their strengths and limitations. Additionally, we examine advancements in guide RNA (gRNA) engineering, improved Cas variants, optimized delivery systems, base and prime editing technologies, and anti-CRISPR proteins as key approaches to enhance genome-editing precision.<br><br>EXPERT OPINION: While significant progress has been made in off-target detection and mitigation, challenges remain in accurately assessing unintended genomic modifications in vivo and ensuring long-term safety in clinical applications. Future research should focus on developing high-fidelity CRISPR variants, refining computational prediction models, and integrating multi-modal detection systems. Advancing delivery mechanisms and regulatory strategies will be essential for translating CRISPR into safe and effective therapeutic interventions.},
}
@article {pmid40107353,
year = {2026},
author = {Huang, J and Liu, F and Xu, ZF and Xiang, HL and Yuan, Q and Zhang, C},
title = {Minichromosome maintenance 4 plays a key role in protecting against acute kidney injury by regulating tubular epithelial cells survival and regeneration.},
journal = {Journal of advanced research},
volume = {79},
number = {},
pages = {277-292},
doi = {10.1016/j.jare.2025.03.032},
pmid = {40107353},
issn = {2090-1224},
mesh = {Animals ; *Acute Kidney Injury/metabolism/pathology/genetics/chemically induced ; Mice ; Cisplatin/toxicity ; Mice, Inbred C57BL ; *Regeneration/genetics ; *Epithelial Cells/metabolism/pathology ; *Minichromosome Maintenance Complex Component 4/metabolism/genetics ; Mice, Knockout ; Reperfusion Injury/metabolism/pathology ; Disease Models, Animal ; Cell Survival/genetics ; *Kidney Tubules/metabolism/pathology/cytology ; Male ; Apoptosis ; Humans ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: Minichromosome maintenance 4 (MCM4), a constituent of the MCM family, playing a pivotal role in DNA replication. Although MCM4 expression has been widely linked to various malignant tumors, its role in kidney diseases is not well-studied. This study primarily investigates the role and underlying mechanism of MCM4 in acute kidney injury (AKI).<br><br>OBJECTIVES: Characterizing a novel target of MCM4 in patients with AKI.<br><br>METHODS: We used CRISPR/Cas9 gene editing to delete MCM4 gene in tubular cells from C57BL/6J mice. Adeno-associated virus 9 harboring MCM4 was administered via intraparenchymal injection into the kidney to enhance MCM4 expression in vivo. These mice were used to established cisplatin- and ischemic reperfusion injury (IRI)-induced AKI mouse models, for detecting the functional role of MCM4 in the pathological process of AKI.<br><br>RESULTS: MCM4 level was increased in the tubules of cisplatin- and IRI-induced AKI mouse models. Compare to wide-type mice, MCM4 knockout mice demonstrated greater degree of histological damage and a higher ratio of apoptotic tubular cells, as well as kidney dysfunction upon cisplatin- and IRI-induced AKI models. Conversely, MCM4 overexpression ameliorated the severity of kidney injury and promoted regenerative capacity of tubular cells during AKI development. Mechanically, loss of MCM4 induced the expression of p53-binding protein 1, activating the p53/p21 pathway and exacerbating AKI progression. Additional, MAD2B, as an upstream molecule of MCM4, regulates the transcription level of MCM4 by affecting the level of E2F1.<br><br>CONCLUSIONS: These findings demonstrate that MCM4 upregulation during AKI development is an adaptive response that preserves tubular cell regenerative capacity and limits the severity of renal injury, thus highlighting the potential value of MCM4 as a biomarker or therapeutic target in patients with AKI.},
}
@article {pmid40054578,
year = {2026},
author = {Zeng, Y and Lu, X and Wang, M and Chen, R and Li, Q and Zhu, J and Su, Z and Lin, F},
title = {Endophyte Acrocalymma vagum establishes the holobiont with rice to attract beneficial microorganisms and promote disease resistance.},
journal = {Journal of advanced research},
volume = {79},
number = {},
pages = {161-177},
doi = {10.1016/j.jare.2025.03.008},
pmid = {40054578},
issn = {2090-1224},
mesh = {*Oryza/microbiology/growth &amp; development ; *Endophytes/physiology ; *Disease Resistance ; *Plant Diseases/microbiology ; Symbiosis ; Plant Roots/microbiology ; Microbiota ; Proteomics ; RNA, Ribosomal, 16S/genetics ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: Endophytic fungi are essential microorganisms in promoting plant health. However, the mechanism of endophytic fungi regulating root microbiota to enhance crop production and resistance remains unclear.<br><br>OBJECTIVES: We aimed i) to explore the microbial alteration driven by endophytic Acrocalymma vagum in developing crop yield and rice resistance; ii) to reveal the mechanism of root-released compound stimulated by A. vagum in recruiting benefit microbes.<br><br>METHODS: The microbiome was applied in a culture-dependent and culture-independent method to study the microbial communities of the A. vagum-rice holobiont using 16S rRNA and ITS gene metabarcoding. Non-target metabolome identified distinct metabolites responsible for community variations. Label-free proteomic analyses investigated the association between primary genes related to the holobiont formation. CRISPR/Cas9 technique and homologous recombination replacement were used to characterize the functions of putative genes.<br><br>RESULTS: A. vagum enhanced cultivated rice yield by 5.73&#xa0;&#xb1;&#xa0;1.76&#xa0;% and induced 83.24&#xa0;&#xb1;&#xa0;9.86&#xa0;% control efficiency against rice blast. We discovered that A. vagum simplified rice microbial structure based on co-occurrence networks, by lowering the proportion of potentially pathogenic predominant Burkholderia and driving rice to recruit beneficial Lactobacillus, Sarocladium and Nigrospora to promote rice growth with the increases of 44.41&#xa0;&#xb1;&#xa0;5.10&#xa0;% shoot height and 70.21&#xa0;&#xb1;&#xa0;9.57&#xa0;% shoot biomass. Moreover, the holobiont released coumaric and trans-ferulic acids to attract beneficial microbes. 206 rice proteins were notably up-regulated in the holobiont, particularly the OsPrxs. CRISPR/Cas9-edited mutants of OsPRX70 and OsPRX95 reduced the promotion effect of A. vagum on rice growth. Furthermore, the pathways of 39 overexpressed proteins in A. vagum were enriched in invading the host and inducing resistance. The knockouts of AvGH3, AvGH7, AvMFS1, and AvCBA transformed A. vagum role from endophyte to pathogen.<br><br>CONCLUSIONS: The A. vagum-rice holobiont releases recruitment signals and improves the rice community structure. We provide ecological and molecular evidence to confirm the mutualism of endophyte-plant-promoting growth and disease resistance.},
}
@article {pmid41409641,
year = {2024},
author = {Zhang, Z and Ding, S},
title = {Gene editing and reprogramming of human fibroblast cells (hFBs) to human pluripotent stem cells (hiPSCs).},
journal = {Neuromethods},
volume = {210},
number = {},
pages = {39-59},
pmid = {41409641},
issn = {0893-2336},
support = {R01 NS123023/NS/NINDS NIH HHS/United States ; },
abstract = {Predictive disease models play significant roles in advancing our knowledge of the pathology of human disease. In this field, animal models have been extensively employed and have provided crucial insights into the pathophysiological mechanisms of human disease. However, they often fail to fully capture many human phenotypes due to significant species differences in genomic responses. Human induced pluripotent stem cells (hiPSCs) are genetically reprogrammed cells that exhibit qualities remarkably similar to those of embryonic stem cells (ESC) and have emerged as a promising source for cell therapy and fundamental research in pathology. The ability to reprogram human fibroblast cells (hFBs) to iPSCs provides an opportunity to model human diseases. However, even hiPSCs from different persons have different genetic background, thus generation of isogenic unaffected control hiPSCs is necessary to study model human disease. Here, we describe methods to generate isogenic hFBs using CRISPR/Cas9 gene editing method, and subsequently reprogram them into iPSCs using commercially available Sendai virus vectors. Specifically, using the CRISPR/Cas9 system and Sendai virus vector, isogenic iPSC lines can be generated. This protocol provides a structured approach for obtaining multiple isogeneic hiPSC lines, which facilitate the modeling of various human diseases.},
}
@article {pmid41406894,
year = {2025},
author = {Heinemann, JA and Hiscox, TC and Zanatta, CB and Kurenbach, B and Walker, S and McCabe, AW and Hoepers, AM and Agapito-Tenfen, SZ},
title = {Genome editing outside of controlled facilities: A review of plausible futures and risks.},
journal = {Ecotoxicology and environmental safety},
volume = {309},
number = {},
pages = {119565},
doi = {10.1016/j.ecoenv.2025.119565},
pmid = {41406894},
issn = {1090-2414},
abstract = {Vectors for delivering proteins and/or nucleic acids into the cells of whole organisms, from single to multicellular, are rapidly advancing. Common cargos are nucleic acids needed to express the components of a genome editing reaction, or ribonucleoproteins (RNP) that can act immediately upon delivery. In only 20 years, improvements in associated formulation technologies have decreased the dependence of genome editing on the need for a laboratory or trained personnel, allowing for genome editing outside of controlled facilities. As this happens, both target and non-target organisms may be exposed to active biological agents, necessitating a new framework for risk assessment. Some scientists deny developments for gene editing in uncontrolled environments, leading to scientifically unjustified dismissals of risk.},
}
@article {pmid41405812,
year = {2025},
author = {Wang, L and Ren, S and Behan, AA and Buzdar, JA and Arain, MA and Li, Y},
title = {Promising Future of Engineered Probiotics for Antimicrobial Peptides and Protein Production: Prospects and Industrial Challenges.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41405812},
issn = {1867-1314},
}
@article {pmid41404798,
year = {2025},
author = {Yang, F and Xu, C and Li, C and Xiang, X and Zhao, Y and Hu, C and Rong, H and He, Y and Li, J and Wang, Y and Tang, C and Liu, X and Li, R and Deng, F and Xiang, T},
title = {Amplification-free cancer diagnosis based on inhibition of Cas12a activity by site-specific 5mC-modified cfDNA.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41404798},
issn = {1362-4962},
support = {82172619//National Natural Science Foundation of China/ ; YXQN202421//Outstanding Young Talents Program of Chongqing/ ; 2023nlts007//Enhancing Scientific Research Capabilities of Chongqing University Cancer Hospital/ ; 2024MSXM136//Chongqing Municipal Science and Health Joint Medical Research/ ; },
mesh = {Humans ; *DNA Methylation ; *CRISPR-Cas Systems/genetics ; *Cell-Free Nucleic Acids/genetics/blood/chemistry ; *Neoplasms/diagnosis/genetics/blood ; *CRISPR-Associated Proteins/metabolism/antagonists &amp; inhibitors/genetics ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Bacterial Proteins ; },
abstract = {DNA methylation detection holds significant value for cancer diagnosis and recurrence monitoring. However, current methods are often time-consuming, costly, and necessitate specialized techniques. The CRISPR-Cas system, particularly Cas12a, presents a precise and user-friendly platform for disease diagnosis. We developed the CRISPR-Methylated DNA Detection Test (CRISPR-MeDNA Test), a Cas12a-based method for detecting methylation in plasma cell-free DNA (cfDNA). The results reveal that 5mC-modified DNA significantly suppresses the trans-cleavage activity of Cas12a, depending on the methylation site, number, and interval spacing. Simultaneously, methylation of the non-target strand (NTS) suppresses Cas12a activity more strongly than methylation of the target strand (TS), as the NTS plays a critical role in R-loop formation, which is essential for Cas12a cleavage target DNA. Mechanistically, 5mC-modified DNA was found to trigger conformational rearrangements in the Cas12a complex, as predicted by AlphaFold3 modeling and corroborated by FRET assays. Notably, the combination of Cas12a with multiplexed guide RNAs enables effective discrimination between cfDNA from healthy donors and cancer patients without the need for pre-amplification, based on the inhibitory effects of methylated DNA on the Cas12a trans-cleavage activity. This work provides a Cas12a-based detection for a rapid, cost-effective, low-complexity method for 5mC-modified cfDNA in liquid biopsies.},
}
@article {pmid41404502,
year = {2025},
author = {Gao, H and Gao, S and Kan, G and Valentovich, LN and An, Y},
title = {Research progress of base editing and prime editing tools based on the CRISPR/Cas system.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102771},
pmid = {41404502},
issn = {2162-2531},
abstract = {The base editor (BE) and prime editing guide RNA (pegRNA)-based prime editor (PE) technologies relying on the CRISPR/Cas system are very efficient gene editors that have been developed in recent years. The BEs include cytosine base editors (CBEs) that mediate the conversion of C to T, adenine base editors (ABEs) that mediate the conversion of A to G, glycosylase base editors (GBEs) that mediate the conversion of C to G, and the dual-base editors (DBEs) that mediate the simultaneous conversion of C to T and A to G. The BEs and PEs have been successfully applied for genome editing of various animals, plants, and microorganisms due to their advantages of high efficiency and independence of DNA double-strand breaks or donor DNA. The development process and characteristics of various BEs and PEs and their effectiveness of application are systematically introduced to provide a reference for selecting appropriate genome editing technologies. Moreover, the urgent issues that need to be addressed for more efficient and precise editing are summarized and prospected.},
}
@article {pmid41404500,
year = {2025},
author = {Hundal, T and Luo, Y and Qie, Y and Gadd, ME and Brim, AD and Vazquez-Rosario, I and Guo, S and Kharfan-Dabaja, MA and Qin, H},
title = {Novel allogeneic CAR T-cell platform involving microhomology-mediated end joining repair and low off-targeting potential.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102778},
pmid = {41404500},
issn = {2162-2531},
abstract = {Several allogeneic chimeric antigen receptor (CAR) T-cell therapies in clinical trials rely on CRISPR-Cas genome editing, but the enzyme's random repair mechanism increases the risk of undesired off-target effects, challenging safe CAR T-cell generation. To address this, we developed a novel CRISPR RNA (crRNA) targeting the T-cell receptor beta constant (TRBC) gene. Combined with AsCas12a Ultra, this crRNA edits primary human T-cells via a predictable microhomology-mediated end joining (MMEJ) DNA repair pathway, significantly lowering off-target risks. During evaluation, we sequestered a unique T-cell subset with disrupted T-cell receptor (TCR), retained CD3 expression, and no in vivo alloreactivity. Termed CD3-retained, allogeneically functioning T-cells (CRAFT-cells), these cells exhibited growth kinetics comparable to unedited T-cells. When engineered with CD19- or BAFF-R-targeted CARs, CRAFT CAR T-cells showed strong antigen-specific cytotoxicity and significant ex vivo expansion compared to conventional CD3-disrupted CAR T-cells. Moreover, CRAFT CAR T-cells effectively served as effector cells for bispecific T-cell engagers (BiTEs), enabling CD3-dependent tumor cell killing. Our CRAFT crRNA platform offers a novel strategy to generate safer allogeneic CAR T-cells. The distinct properties of CRAFT CAR T-cells, combined with BiTE therapy, represent a promising and potentially more durable approach for next-generation allogeneic CAR T-cell therapies in clinical applications.},
}
@article {pmid41404141,
year = {2025},
author = {Shilpha, J and Kang, WH},
title = {Molecular and genomic insights into viral resistance in Capsicum spp.: pathogenesis, defense mechanisms, and breeding innovations.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1716114},
pmid = {41404141},
issn = {1664-462X},
abstract = {Plant viruses represent a major challenge to agricultural systems, threatening global food security amid a rising population. Specifically, pepper cultivation (Capsicum annuum L.) is often hindered by various viral diseases, with more than 60 viruses identified as affecting pepper plants. The most efficient strategy for controlling viral diseases is the development of resistant cultivars of peppers. A comprehensive understanding of complex interactions between plant defense mechanisms and the strategies employed by viruses to evade these defenses, coupled with host factors that facilitate viral replication and movement, is essential for developing resistant cultivars. Natural antiviral defense mechanisms in plants are well characterized and include resistance genes, RNA silencing, autophagy-mediated degradation, translational repression, and resistance to viral movement. Recent advances in next-generation sequencing (NGS), genome-wide association studies (GWAS), high-density genotyping platforms and gene-editing tools such as CRISPR/Cas have accelerated the identification of resistance loci and key host factors involved in viral pathogenesis. This review summarizes current molecular and genomic insights into virus-host interactions in Capsicum spp., highlighting their role in advancing marker-assisted selection (MAS) and genomic-assisted breeding. The integration of molecular markers and genome editing into breeding pipelines offers new opportunities for developing durable, broad-spectrum viral resistance in peppers, ultimately supporting sustainable crop production and agricultural resilience.},
}
@article {pmid41403730,
year = {2025},
author = {Khari, M and Jain, N and Kaul, S and Pandey, M and Sharma, N},
title = {siRNA and mRNA-Based Preventive and Therapeutic Strategies for HPV-Induced Cervical Cancer.},
journal = {Advanced pharmaceutical bulletin},
volume = {15},
number = {3},
pages = {552-573},
pmid = {41403730},
issn = {2228-5881},
abstract = {Human papillomavirus (HPV), specifically types 16 and 18, is the main cause of cervical cancer and a significant cause of death among women. Specifically, HPV E6 and E7 oncogenes hinder the normal cell cycle regulation, resulting in uncontrolled cell growth and cervical cancer. The available therapy options include surgery, radiotherapy, and chemotherapy, which show success but also demonstrate notable complications. SiRNA (small interfering RNA) and mRNA (messenger RNA) therapies have emerged as precise and effective tools to silence the HPV E6 and E7 oncogenes and stimulate the immune system to fight against HPV infection, respectively, presenting a targeted therapy approach and overcoming the available therapy challenges. Nanoparticles and Pegylated liposomes are the delivery systems that increase the efficacy and safety of siRNA and mRNA therapies. This review critically appreciates the effective targeting of siRNA and mRNA-based therapies by highlighting their key advantages and limitations. Despite being a target-specific and effective approach, there are certain challenges like scale-up, cost-effectiveness, and developing stable delivery systems, which are required to be discussed. In addition, other precision medicine approaches, such as CRISPR/CAS-9, antisense oligonucleotides, or immunotherapy, have also been included as compared to siRNA/mRNA therapies. Their preclinical, patent, and clinical translations have also been discussed exhaustively.},
}
@article {pmid41402770,
year = {2025},
author = {Jiang, M and Zhang, K and Wang, Z and Gao, M and Su, S and He, J and Xu, H and Bo, Z and Jiang, Z and Zhang, C and Hui, JH and Wei, R},
title = {Nanomaterials in gene therapy and genome editing: challenges and emerging directions.},
journal = {Journal of nanobiotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12951-025-03895-w},
pmid = {41402770},
issn = {1477-3155},
abstract = {Nanomaterials are redefining the landscape of gene and genome editing, yet their translation to clinical reality remains constrained by multiple unresolved challenges. While they provide structural and functional advantages for delivering nucleic acids and CRISPR/Cas systems across biological barriers, their behavior within living systems is often unpredictable, leading to issues such as off-target editing, immune activation, and inconsistent biodistribution. The design of nanocarriers, whether lipid-based, polymeric, inorganic, must therefore balance efficiency with safety, integrating physicochemical precision with biological adaptability. Recent advances in ionizable lipid nanoparticles demonstrate how fine-tuning charge, surface chemistry, and degradation kinetics can enhance endosomal escape and target specificity, but reproducibility and large-scale manufacturing continue to limit broader application. Moreover, polymeric and exosome-inspired systems promise modularity and targeted reuse, yet they demand clearer understanding of long-term biocompatibility and regulatory acceptance. The future of nanomaterial-enabled genome engineering depends not only on optimizing delivery vehicles but also on establishing predictive models of nano-bio interactions, harmonizing ethical oversight, and developing standardized evaluation pipelines that link nanoscale design to therapeutic outcomes.},
}
@article {pmid41402634,
year = {2025},
author = {Oliynyk, RT and Mahas, A and Karpinski, E and Church, GM},
title = {Plasmid2MC: efficient cell-free generation of high-purity minicircle DNA for genome editing in mammalian cells.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1778},
pmid = {41402634},
issn = {2399-3642},
mesh = {*Gene Editing/methods ; *Plasmids/genetics ; Humans ; HEK293 Cells ; Animals ; *DNA, Circular/genetics ; Mice ; CRISPR-Cas Systems ; Cell-Free System ; Mouse Embryonic Stem Cells/metabolism ; },
abstract = {DNA plasmids are widely used for delivering proteins and RNA in genome editing. However, their bacterial components can lead to inactivation, cell toxicity, and reduced efficiency compared to minicircle DNA (mcDNA), which lacks such bacterial sequences. Existing commercial kits that recombine plasmids into mcDNA within proprietary bacterial strains are labor-intensive, yield inconsistent results, and often produce endotoxin-contaminated low-quality mcDNA. To address this challenge, we developed Plasmid2MC, a novel cell-free method utilizing ΦC31 integrase-mediated recombination to efficiently excise the bacterial backbone from conventionally prepared plasmids, followed by digestion of the bacterial backbone and all other DNA contaminants, resulting in highly pure and virtually endotoxin-free mcDNA. We demonstrated the application of mcDNA to express CRISPR-dCas9 for base editing in HEK293T cells and mouse embryonic stem cells, as well as for homology-independent targeted insertion (HITI) genome editing. The method's ease of preparation, high efficiency, and the high purity of the resulting mcDNA make Plasmid2MC a valuable tool for applications requiring bacterial backbone-free circular DNA.},
}
@article {pmid41390734,
year = {2025},
author = {Ren, J and Yao, J and Cao, Q and Li, Y and Li, Y and Zhang, Z and Ge, X and Wang, S and Zhang, Y and Wang, X and Zhang, X},
title = {Protein-nucleic acid language model-assisted design of precise and compact adenine base editor.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11207},
pmid = {41390734},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; Humans ; HEK293 Cells ; *Adenine/metabolism/chemistry ; Mice ; Genetic Therapy/methods ; Nanoparticles/chemistry ; CRISPR-Cas Systems/genetics ; *Nucleic Acids/genetics/chemistry ; Proprotein Convertase 9/genetics/metabolism ; Mutation ; },
abstract = {Adenine base editors (ABEs) are powerful tools for gene therapy. However, efficient version of ABEs (e.g. ABE8e) always induce excessive bystander and off-target editing events and are large in size, hindering their potential in clinical disease treatment. Here, we develop a pre-trained Protein-Nucleic Acid Constrained Language Model to design ABE8e with high activity, reduced editing window and decreased size. By further engineering, the smallest ABE8e- PNLM-pcABE- with a 27% size reduction, exhibits high activity, precise 3-nt editing window, and reduced off-target events near background level in HEK293T cells. Compared to ABE8e, PNLM-pcABE has up to 133.5-fold precision improvement in pathogenic mutation correction. By PNLM-pcABE, the albino mouse model carrying desired base mutation is nearly 100% obtained via zygotes microinjection and the expression of PCSK9 substantially decreases in mice receiving in vivo delivery with lipid nanoparticle (LNP), indicating their great potential in gene therapy and disease modeling.},
}
@article {pmid41381416,
year = {2025},
author = {Cheng, AS and Li, LX and Zhou, JX and Harris, PC and Calvet, JP and Li, X},
title = {In vivo base editing rescues ADPKD in a humanized mouse model.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11212},
pmid = {41381416},
issn = {2041-1723},
support = {DK129241//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; DK126662//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; DK059597//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; DK058816//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; PR221810//U.S. Department of Defense (United States Department of Defense)/ ; },
mesh = {Animals ; *Polycystic Kidney, Autosomal Dominant/genetics/therapy/pathology ; *Gene Editing/methods ; Disease Models, Animal ; Humans ; Mice ; Dependovirus/genetics ; TRPP Cation Channels/genetics ; Kidney/pathology/metabolism ; CRISPR-Cas Systems/genetics ; Mutation ; *Genetic Therapy/methods ; Liver/pathology/metabolism ; Male ; Promoter Regions, Genetic ; Female ; },
abstract = {Autosomal dominant polycystic kidney disease (ADPKD) is a genetic kidney disease, caused by mutations of the PKD1 and PKD2 genes, characterized by the development of renal cysts and extrarenal complications, such as cardiac hypertrophy. Recently, a revolutionary approach, adeno-associated virus (AAV) delivered CRISPR-Cas9 gene editing, has been developed to treat inherited diseases. However, the use of this technology in kidney diseases in vivo is challenged. In this study, we adapt one of the gene editing systems, adenine base editor (ABE9), to develop a broadly expressed and a kidney-specific promoter mediated base editors, and test the effects of these two systems delivered by AAV9 on preventing disease in humanized Pkd1[RC/RC] mice carrying an arginine (R) to cystine (C) mutation that mimics a mutation in ADPKD patients. We show that one dose of the broadly expressed dual ABE9-AAV9 treatment corrects the pathogenic variant in kidneys, hearts and livers, and result in delaying cyst growth, decrease heart hypertrophy and improve liver function. To confirm the specificity of the base editor system in kidneys, we show that one dose of the kidney specific promoter mediated dual-ABE9-AAV9 treatment corrects the Pkd1 gene mutation in the kidney, and not in the heart, resulting in delaying cyst growth in Pkd1[RC/RC] kidneys, supporting a promising strategy of using base editor to target specific organs. Treatment with ABE9 base editors mediated by either the broadly expressed or kidney specific promoter increased the survival rate of Pkd1[RC/null] mice. These preclinical studies support a potential that single-dose genetic therapies may be through the correction of pathogenic variants to prevent ADPKD development in the clinic.},
}
@article {pmid41352695,
year = {2026},
author = {Pradhan, K and Anoop, S},
title = {CRISPR 2.0: Expanding the genome engineering Toolbox for epigenetics, RNA editing, and molecular diagnostics.},
journal = {Gene},
volume = {979},
number = {},
pages = {149938},
doi = {10.1016/j.gene.2025.149938},
pmid = {41352695},
issn = {1879-0038},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Epigenesis, Genetic ; *RNA Editing/genetics ; *Pathology, Molecular/methods ; Epigenomics/methods ; Animals ; Genetic Engineering/methods ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Non-canonical CRISPR systems adaptation has led to genome editing through nucleases, and the development of transcriptional and epigenetic regulation, transcriptome editing, and molecular diagnostics has resulted in a diversified set of tools-CRISPR 2.0. In this review, the author summarizes the mechanisms and recent engineering advances of (i) dCas9-based epigenetic effectors, (ii) RNA-targeting Cas13 systems and engineered RNA editors, (iii) DNA base editors and prime editors, and (iv) CRISPR-powered diagnostic platforms and their translational readiness. There is a critical comparison of the various approaches (e.g., RNAi/ASO versus Cas13-based methods; base editing versus prime editing) along with practical translational considerations such as delivery technologies, safety (off-target/edit windows, mosaicism), and regulatory pathways which are evaluated. Three concise case studies refer to map laboratory evidence to clinical or near-clinical outcomes and the ethical and governance discussion is widened to include global access, intellectual property and equity in deployment. Finally, the authors classify technologies according to their level of readiness - diagnostics and some ex-vivo therapeutic approaches are already in or very close to clinical use, chosen in-vivo editing methods are undergoing early trials, and AI-assisted nuclease design is still mostly theoretical but is getting better fast. This comprehensive viewpoint is intended to help researchers and physicians understand which CRISPR tools are most likely to be translated soon and where more validation is required.},
}
@article {pmid41344486,
year = {2026},
author = {Huang, Y and Li, L and Do, CW and Luo, Q and Zheng, Z and Xiong, W},
title = {Lipid nanoparticle-mediated CRISPR/Cas9 delivery enables efficient trabecular meshwork gene editing in mice.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {389},
number = {},
pages = {114499},
doi = {10.1016/j.jconrel.2025.114499},
pmid = {41344486},
issn = {1873-4995},
mesh = {Animals ; *Trabecular Meshwork/metabolism ; *Gene Editing/methods ; *Nanoparticles/administration &amp; dosage/chemistry ; *CRISPR-Cas Systems ; *Lipids/chemistry/administration &amp; dosage ; Mice, Inbred C57BL ; Mice ; RNA, Messenger/administration &amp; dosage/genetics ; *Gene Transfer Techniques ; Humans ; Calcium-Binding Proteins/genetics ; Green Fluorescent Proteins/genetics ; Extracellular Matrix Proteins/genetics ; Male ; CRISPR-Associated Protein 9/genetics ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) enable efficient mRNA delivery, yet their potential for ocular gene editing remains largely unexplored. Here, we systematically evaluated three LNP formulations containing distinct ionizable lipids, DLin-MC3-DMA, ALC0315, and SM102, for gene delivery to ocular tissues. Among them, SM102-based LNP encapsulating GFP mRNA (SM102-GFP) exhibited the highest transfection efficiency across three cultured ocular cells in vitro. Following intravitreal injection in mice, SM102-GFP achieved selective and robust expression in the trabecular meshwork (TM) without detectable retinal transfection. GFP expression in TM peaked at one week post-injection, declined by three weeks, and could be effectively re-induced by a second dosing of the same vector. Compared with adeno-associated viral (AAV) and adenoviral (Ad) vectors, SM102-GFP showed superior TM specificity and reduced retinal inflammation. Co-delivery of SpCas9 mRNA and sgRNA via SM102-based LNPs enabled efficient CRISPR-mediated knockout of Matrix Gla Protein (Mgp), a key inhibitor of TM calcification. Mgp knockout induced sustained intraocular pressure elevation and anterior chamber deepening with open angles, recapitulating features of primary open-angle glaucoma. Chronic ocular hypertension further led to Müller gliosis and ganglion cell complex thinning, indicative of progressive retinal stress. These findings establish SM102-based LNPs as a safe and efficient platform for TM-targeted gene editing and glaucoma modeling.},
}
@article {pmid41286485,
year = {2025},
author = {Zhao, Y and Liang, Y and Ni, Z and Sun, Q and Zong, Y and Wang, Y},
title = {Advances and prospects of large DNA fragment editing in plants.},
journal = {Nature plants},
volume = {11},
number = {12},
pages = {2461-2475},
pmid = {41286485},
issn = {2055-0278},
mesh = {*Gene Editing/methods ; *Genome, Plant/genetics ; *Crops, Agricultural/genetics ; *DNA, Plant/genetics ; *Plants/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; },
abstract = {Structural variations drive plant genome evolution and shape agronomic traits. Manipulating structural variations has great potential to improve complex plant traits and enhance agricultural sustainability. Genome editing technologies have evolved from gene knockouts and base editing to the modification of short DNA fragments, and are now advancing towards the precise manipulation of large DNA fragments. This advancement facilitates targeted, large-scale genomic changes such as deletions, insertions, replacements, inversions, translocations and duplications. In this Review, we summarize recent advances in developing technologies for large DNA fragment editing and highlight their key applications in plants as well as their potential to accelerate crop improvement. Finally, we discuss the current challenges and future prospects for these technologies in plant science.},
}
@article {pmid41265646,
year = {2026},
author = {Bao, W and Ji, P and Xi, W and Chimed, G and Liu, Z and Ping, Y},
title = {Genome editing of Spp1 by inhalable CRISPR/Cas9 formulation for treating pulmonary fibrosis.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {389},
number = {},
pages = {114424},
doi = {10.1016/j.jconrel.2025.114424},
pmid = {41265646},
issn = {1873-4995},
mesh = {Animals ; *Gene Editing/methods ; *Osteopontin/genetics ; *CRISPR-Cas Systems ; Polylactic Acid-Polyglycolic Acid Copolymer/chemistry ; Administration, Inhalation ; Nanoparticles/chemistry/administration &amp; dosage ; Humans ; Mice ; Mice, Inbred C57BL ; *Pulmonary Fibrosis/therapy/genetics ; RNA, Guide, CRISPR-Cas Systems/administration &amp; dosage/genetics ; *Idiopathic Pulmonary Fibrosis/therapy/genetics ; Bleomycin ; Male ; Genetic Therapy/methods ; Lung/metabolism/pathology ; Calcium Phosphates ; },
abstract = {Secreted phosphoprotein 1 (Spp1) encoding osteopontin (OPN), a matrix cell protein with pro-inflammatory and pro-necrotic tissue properties, plays a crucial role in the onset and progression of idiopathic pulmonary fibrosis (IPF). In order to treat IPF by taking advantage of Spp1, we herein developed an inhalable system composed of calcium phosphate/ poly (lactic-co-glycolic acid) (PLGA) core-shell nanoparticles which are loaded with CRISPR/Cas9 system targeting Spp1 to investigate its therapeutic potential. Specifically, the plasmid encoding Cas9 and single-guide RNA (sgRNA) selectively targeting Spp1 gene was first condensed by calcium phosphate to form Cas9 complexes, which was then encapsulated by PLGA to formulate into a gene-editing inhalable delivery system (termed CaP/Cas9/PLGA). Interestingly, the aerosolized inhaled delivery of CaP/Cas9/PLGA nanoparticles results in the effective traverse of mucosal barriers to fibrotic lungs, where they are internalized by lung cells without inducing noticeable cytotoxicity. Following endo/lysosomal escape and gene expression of CRISPR system, the disruption of Spp1 gene by Cas9/sgRNA induces the mutation frequency exceeding 30 %, resulting in efficient down-regulation of OPN level. In a bleomycin-induced pulmonary fibrosis mouse model, the inhalation of aerosolized CaP/Cas9/PLGA complexes significantly attenuates fibrosis development and improves lung function with undetectable systemic toxicity. This current study defines an innovative inhalable gene-editing formulation and offers a promising gene therapy modality for treating IPF.},
}
@article {pmid41222266,
year = {2025},
author = {Monahan, CC and Held, K and Yang, H and Sotelo, G and Grieshaber, N and Grieshaber, S and Omsland, A},
title = {Ectopic overexpression and CRISPRi-based knockdown of Chlamydia trachomatis ObgE inhibits RB replication and EB reformation.},
journal = {Journal of bacteriology},
volume = {207},
number = {12},
pages = {e0028225},
doi = {10.1128/jb.00282-25},
pmid = {41222266},
issn = {1098-5530},
support = {R01AI130072/NH/NIH HHS/United States ; },
mesh = {*Chlamydia trachomatis/genetics/metabolism/growth &amp; development/physiology ; *Bacterial Proteins/genetics/metabolism ; Humans ; Gene Expression Regulation, Bacterial ; Gene Knockdown Techniques ; HeLa Cells ; Chlamydia Infections/microbiology ; CRISPR-Cas Systems ; },
abstract = {Chlamydia trachomatis is an obligate intracellular bacterium of major clinical significance. While untreated sexually transmitted infections can result in pelvic inflammatory disease and infertility, ocular infections can cause the blinding disease trachoma. During infection of host cells, C. trachomatis transitions between the non-replicative, infectious elementary body (EB) and the replicative, non-infectious reticulate body (RB). ObgE is a GTPase that can promote morphological differentiation in some bacteria. In C. trachomatis, obgE is maximally expressed from 16 to 24hpi, a timeframe that is associated with logarithmic growth and the onset of production of infectious progeny; therefore, ObgE is predicted to have significance during Chlamydia replication and/or morphological transitions. To determine the role of ObgE during the C. trachomatis developmental cycle, we assessed the effects of ObgE ectopic overexpression and CRISPRi-based knockdown of obgE on RB replication and EB formation. When ectopic overexpression of ObgE was induced, we observed a significant decrease in infectious progeny but no changes in bacterial ultrastructure. These data suggest that during ectopic overexpression of ObgE, RBs can transition into EBs; however, EBs are diminished in their ability to establish new infections. CRISPRi-based knockdown of obgE resulted in a 2-log decrease in bacterial yield and infectious progeny. Ultrastructural analysis revealed that knockdown of obgE resulted in small, underdeveloped inclusions with few cells inside. In total, while ectopic overexpression of ObgE negatively affects production of infectious EBs, CRISPRi-based knockdown of obgE severely affects RB replication, inclusion development, and generation of EBs.IMPORTANCEThe pathogenesis of C. trachomatis is reliant on the transition between the non-replicative, infectious elementary body (EB) and the replicative, non-infectious reticulate body (RB). Therefore, understanding the molecular determinants of Chlamydia developmental transitions is of the utmost importance. ObgE has been shown to regulate morphological transitions in other bacteria and is thus predicted to have relevance during regulation of the Chlamydia developmental cycle. Using both ectopic overexpression and CRISPRi-based knockdown of ObgE/obgE, we identify the significance of balanced ObgE expression for RB replication and the formation of infectious EBs. Our findings further expand our knowledge of how developmental transitions in Chlamydia are regulated.},
}
@article {pmid41221676,
year = {2025},
author = {Petersen, F and Westermann, S and Smialkovska, V and Mathony, J and Feldmann, A and Niopek, D},
title = {Engineering the Link: From Genome Interaction Maps to Functional Insight.},
journal = {Advanced biology},
volume = {9},
number = {12},
pages = {e00525},
doi = {10.1002/adbi.202500525},
pmid = {41221676},
issn = {2701-0198},
support = {//Baden-W&#xfc;rttemberg Stiftung/ ; VH-NG-1604//Helmholtz Association/ ; 453202693//Deutsche Forschungsgemeinschaft/ ; //Carl-Zeiss-Stiftung/ ; //Aventis Foundation/ ; //Health + Life Science Alliance Heidelberg Mannheim/ ; //Hans und Ria Messer Stiftung/ ; ERC-STG 101115812/ERC_/European Research Council/International ; },
mesh = {Humans ; *Chromatin/genetics/metabolism ; *Genetic Engineering/methods ; Animals ; *Genome ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Gene Editing/methods ; },
abstract = {The 3D organization of the genome constitutes a spatial layer of information processing that helps govern gene expression and thus cell function. Advances in chromosome conformation capture sequencing have enabled detailed assessment of chromatin architecture, from enhancer-promoter loops to topological domains and higher-order contacts, across cell types and developmental states. While the ability to investigate genome conformation is maturing, the field faces a central challenge: The link between chromatin interactions and cellular function remains largely correlative, leaving their causality unresolved. This review explores how recent developments in genome engineering enable the targeted manipulation of 3D chromatin architecture - specifically DNA loops - to illuminate causal links between genome structure and function. Synthetic strategies are introduced that rewire enhancer-promoter communication through engineered chromatin loops, leveraging programmable DNA-binding platforms such as zinc fingers, transcription activator-like effectors (TALEs), and CRISPR-Cas9. The current limitations of these approaches related to efficiency, scalability, and specificity are also highlighted, and the strategies to address them are outlined. As these systems mature, programmable 3D genome engineering is emerging as a transformative pillar of synthetic biology, complementing sequence-based editing as a core modality for both understanding and ultimately reprogramming genome function.},
}
@article {pmid41214365,
year = {2025},
author = {Tzou, FY and Hong, CL and Chen, KH and Vaughen, JP and Lin, WS and Hsu, CH and Rivas-Serna, IM and Hsu, KY and Ho, SM and Panganiban, MR and Hsieh, HT and Li, YJ and Hsiao, Y and Yeh, HC and Yu, CY and Tang, HW and Chou, YH and Wu, CL and Lo, CC and Mazurak, VC and Clandinin, MT and Huang, SY and Chan, CC},
title = {Functional profiling and visualization of the sphingolipid metabolic network in vivo.},
journal = {EMBO reports},
volume = {26},
number = {24},
pages = {6380-6417},
pmid = {41214365},
issn = {1469-3178},
support = {113-2320-B-002-022-MY3//National Science and Technology Council (NSTC)/ ; 114-2311-B-002-006-//National Science and Technology Council (NSTC)/ ; 112-2311-B-001-036-MY3//National Science and Technology Council (NSTC)/ ; EX114-11228NI//National Health Research Institutes (NHRI)/ ; NHRI-13A1-CG-CO-08-2325-2//National Health Research Institutes (NHRI)/ ; 114L8522//National Taiwan University ()/ ; 114L891303//National Taiwan University ()/ ; 114L910203//National Taiwan University ()/ ; 113-L3003//National Taiwan University Hospital (NTUH)/ ; },
mesh = {*Sphingolipids/metabolism ; Animals ; *Metabolic Networks and Pathways ; Brain/metabolism ; Mice ; Humans ; Sphingomyelin Phosphodiesterase/metabolism/genetics ; Membrane Microdomains/metabolism ; CRISPR-Cas Systems ; Ethanolamines/metabolism ; Sphingomyelins ; },
abstract = {Sphingolipids govern diverse cellular processes; their dysregulation underlies numerous diseases. Despite extensive characterizations, understanding the orchestration of the sphingolipid network within living organisms remains challenging. We established a versatile genetic platform of CRISPR-engineered reporters of 52 sphingolipid regulators, recapitulating endogenous gene activity and protein distribution. This platform further allows conditional protein degradation for functional characterization. In addition, we developed the biosensor OlyA[w] to detect ceramide phosphoethanolamine and visualize membrane raft dynamics in vivo. Using this platform, we established comprehensive profiles of the sphingolipid metabolic network in the brain at the transcriptional and translational levels. The highly heterogeneous patterns indicate extensive coordination between distinct cell types and regions, suggesting the brain functions as a coherent unit to execute specific steps of sphingolipid metabolism. As a proof-of-concept application, we showed cell type-specific requirements of sphingomyelinases, including CG6962/dSMPD4 and CG3376/aSMase, degrading distinct subcellular pools of ceramide phosphoethanolamine to maintain brain function. These findings establish a foundation for future studies on brain sphingolipid metabolism and showcase the utilization of this genetic platform in elucidating in vivo mechanisms of sphingolipid metabolism.},
}
@article {pmid40977075,
year = {2026},
author = {Heu, CC and Schutze, IX and LeRoy, DM and Wang, YH and DeGain, BA and Kerns, DD and Abdelgaffar, H and Jurat-Fuentes, JL and Matzkin, LM and Carrière, Y and Tabashnik, BE and Fabrick, JA},
title = {Knockout of chitin synthase gene confers resistance to Bt toxin Vip3Aa in Helicoverpa zea.},
journal = {Pest management science},
volume = {82},
number = {1},
pages = {911-919},
doi = {10.1002/ps.70248},
pmid = {40977075},
issn = {1526-4998},
support = {//U.S. Department of Agriculture/ ; //USDA National Institute of Food and Agriculture NC-246/ ; },
mesh = {Animals ; *Insecticide Resistance/genetics ; *Moths/genetics/drug effects/growth &amp; development/enzymology ; *Bacterial Proteins/pharmacology ; *Chitin Synthase/genetics/metabolism ; Gene Knockout Techniques ; *Insecticides/pharmacology ; Larva/growth &amp; development/genetics/drug effects ; *Insect Proteins/genetics/metabolism ; Bacillus thuringiensis ; CRISPR-Cas Systems ; Bacillus thuringiensis Toxins ; },
abstract = {BACKGROUND: Genetically engineered crops that produce insecticidal proteins from Bacillus thuringiensis (Bt) have many benefits and are used globally to manage key insect pests, including Helicoverpa zea (Lepidoptera: Noctuidae), a major pest of crops in the Americas. However, pests of at least 11 species, including H. zea, have evolved resistance to Bt crops, diminishing their effectiveness and benefits. For H. zea in the United States, practical resistance to Bt corn and cotton producing crystalline (Cry) Bt proteins is widespread and early warning of resistance to the vegetative insecticidal protein Vip3Aa has been reported. Thus, a better understanding of the genetic basis of resistance to Vip3Aa is needed to monitor, manage and counter resistance. In some strains of lepidopteran pests, resistance to Vip3Aa is associated with disruptive mutations in the chitin synthase 2 (CHS2) gene but this association had not been investigated previously in H. zea.<br><br>RESULTS: Here, we show that mutations introduced by CRISPR/Cas9 editing of the CHS2 gene can cause resistance to Vip3A in H.&#x2009;zea. Disruptive mutations in CHS2 facilitated the creation of strain CHS2-KO that had 29&#x2009;000-fold resistance to Vip3Aa relative to its unedited parental susceptible strain. Resistance to Vip3Aa in CHS2-KO was autosomal, recessive, and did not cause cross-resistance to Cry1Ac or Cry2Ab.<br><br>CONCLUSION: Results of this study indicate that CHS2 plays an important role in Vip3Aa intoxication in H.&#x2009;zea. It will be important to determine if mutations in CHS2 contribute to field-evolved resistance to Vip3Aa in H.&#x2009;zea and other pests. &#xa9; 2025 Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.},
}
@article {pmid40964909,
year = {2026},
author = {Wang, P and Liu, Z and Kang, Q and Liao, C and Zou, L and Mao, K and Yao, H and Li, Y and Xiao, Y},
title = {Functional loss of CHS2 confers high-level resistance to Bacillus thuringiensis Vip3Aa in Spodoptera exigua and Agrotis ipsilon.},
journal = {Pest management science},
volume = {82},
number = {1},
pages = {714-720},
doi = {10.1002/ps.70226},
pmid = {40964909},
issn = {1526-4998},
support = {CAAS-CSCB-202303//Innovation Program of Chinese Academy of Agricultural Sciences/ ; KQTD20180411143628272//Shenzhen Science and Technology Program/ ; //Biological Breeding-National Science and Technology Major Project/ ; CAAS-ZDRW202412//Agricultural Science and Technology Innovation Program/ ; 32202352//National Natural Science Foundation of China/ ; //The Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences/ ; 2022ZD04021//Biological Breeding-National Science and Technology Major/ ; //Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences/ ; },
mesh = {Animals ; *Insecticide Resistance/genetics ; *Bacterial Proteins/pharmacology ; *Spodoptera/genetics/drug effects/growth &amp; development ; *Bacillus thuringiensis/chemistry ; *Chitin Synthase/genetics/metabolism ; *Moths/genetics/drug effects/growth &amp; development ; *Insecticides/pharmacology ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Larva/growth &amp; development/genetics/drug effects ; Pest Control, Biological ; },
abstract = {BACKGROUND: Bacillus thuringiensis (Bt) crops, which produce insecticidal proteins such as Vip3Aa and Cry toxins, have revolutionized pest management by reducing reliance on chemical pesticides. However, the evolution of resistance in target pests has prompted investigation into the underlying mechanisms. A recent study identified a mutation in the chitin synthase gene (SfCHS2) as a key factor in Vip3Aa resistance in Spodoptera frugiperda. Here, we examined the role of CHS2 in resistance in two additional lepidopteran species: Spodoptera exigua and Agrotis ipsilon.<br><br>RESULTS: Using a CRISPR/Cas9 gene-editing approach, we generated CHS2 knockout strains in both species. The mutants exhibited high-level resistance to Vip3Aa, surviving the highest tested concentration (800&#x2009;&#x3bc;g/cm[2]), with resistance ratios exceeding 33&#x2009;333-fold in S. exigua and 11&#x2009;268-fold in A. ipsilon. Additionally, knockout strains lack the peritrophic matrix (PM), whereas the resistant Sfru_R3 strain retained its PM.<br><br>CONCLUSIONS: These findings further validate the essential role of the CHS2 gene-and the PM it produces-in Vip3Aa toxicity. Complete knockout confers high resistance but imposes severe fitness costs, suggesting that such alleles are unlikely to persist in natural populations. This study advances our understanding of the molecular mechanisms behind resistance to Vip3Aa and provides insights for developing effective resistance management strategies in Bt crop management. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid40441291,
year = {2026},
author = {Kim, G and Siprashvili, Z and Yang, X and Meyers, JM and Ji, A and Khavari, PA and Ducoli, L},
title = {In Vivo CRISPR Interference Screen Reveals Long Noncoding RNA Portfolio Crucial for Cutaneous Squamous Cell Carcinoma Tumor Growth.},
journal = {The Journal of investigative dermatology},
volume = {146},
number = {1},
pages = {223-235.e6},
pmid = {40441291},
issn = {1523-1747},
support = {I01 BX001409/BX/BLRD VA/United States ; R01 AR045192/AR/NIAMS NIH HHS/United States ; R01 AR076965/AR/NIAMS NIH HHS/United States ; },
mesh = {*RNA, Long Noncoding/genetics/metabolism ; *Skin Neoplasms/genetics/pathology ; Humans ; *Carcinoma, Squamous Cell/genetics/pathology ; Mice ; Animals ; Cell Proliferation/genetics ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Biomarkers, Tumor/genetics ; Keratinocytes ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Xenograft Model Antitumor Assays ; },
abstract = {Cutaneous squamous cell carcinoma (cSCC) accounts for 20% of all skin cancer mortality globally, making it the second-highest subtype of skin cancer. The high prevalence of cSCC in humans highlights the need to uncover alternative actors and mechanisms influencing skin cancer development. Significant advances have been made to better understand some key factors in cSCC growth. However, little is known about the role of noncoding RNAs, particularly of a specific subclass termed long noncoding RNA (lncRNA). By performing pseudobulk analysis of single-cell sequencing data from normal and cSCC human skin tissues, we determined a global portfolio of lncRNAs specifically expressed in keratinocyte subpopulations. Integration of CRISPR interference screens in vitro and the xenograft model identified several lncRNAs impacting the growth of cSCC cancer lines both in vitro and in vivo. Among these, we further validated LINC00704 and LINC01116 as proliferation-regulating lncRNAs in cSCC lines and potential biomarkers of cSCC growth. Taken together, our study provides a comprehensive signature of lncRNAs with roles in regulating cSCC growth.},
}
@article {pmid41402624,
year = {2025},
author = {Sinkunas, T and Tamulaitiene, G},
title = {A DNA mimic jams the Cas9 scissors.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.70374},
pmid = {41402624},
issn = {1742-4658},
support = {S-MIP-20-39//Lietuvos Mokslo Taryba/ ; },
abstract = {Anti-CRISPR (Acr) proteins are small protein inhibitors that block the RNA-guided nucleic acid (DNA or RNA) targeting activity of CRISPR-Cas enzymes. Despite their shared function, Acr proteins display minimal sequence or structural similarity and employ diverse mechanisms to block nuclease activity. Lee and Park characterized the previously undescribed AcrIIA13b protein, which inhibits Cas9 protein. Structural, biochemical, and mutational analyses revealed that AcrIIA13b acts as a DNA mimic, thereby disabling the Cas9 complex from binding to the DNA target.},
}
@article {pmid41402279,
year = {2025},
author = {Wang, D and Ritz, C and Luo, Y and Suresh, A and Pierce, A and Veo, B and Brunt, B and Dahl, N and Serkova, N and Venkataraman, S and Danis, E and Kus, K and Mazan, M and Rzymski, T and Vibhakar, R},
title = {Transcriptional regulation of protein synthesis by mediator kinase represents a therapeutic vulnerability in MYC-driven medulloblastoma.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11152},
pmid = {41402279},
issn = {2041-1723},
mesh = {*Medulloblastoma/genetics/metabolism/pathology/drug therapy ; Humans ; *Proto-Oncogene Proteins c-myc/metabolism/genetics ; *Cyclin-Dependent Kinase 8/metabolism/genetics/antagonists &amp; inhibitors ; *Protein Biosynthesis/genetics ; Animals ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Mice ; *Cerebellar Neoplasms/genetics/metabolism/pathology/drug therapy ; Transcription, Genetic ; TOR Serine-Threonine Kinases/metabolism/antagonists &amp; inhibitors ; RNA Polymerase II/metabolism ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {MYC-driven medulloblastoma (MB) is a highly aggressive brain tumor with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening, we identify the Mediator-associated kinase CDK8 as a critical regulator of MYC-driven MB. Both genetic loss and pharmacological inhibition of CDK8 impair MB tumor growth. Moreover, we find that CDK8 cooperates with MYC to sustain the MYC-mediated translational program, as CDK8 depletion induces pronounced transcriptional changes in translation-associated gene sets, reduces ribosome biogenesis, and impairs protein synthesis. Mechanistically, CDK8 regulates the occupancy of RNA polymerase II at specific chromatin loci, facilitating epigenetic alterations that promote the transcription of ribosomal genes. Furthermore, combined inhibition of CDK8 and mTOR synergistically enhances therapeutic efficacy in vivo, leading to more pronounced tumor growth suppression. Overall, our findings establish a functional link between CDK8-mediated transcriptional regulation and mRNA translation, suggesting a promising therapeutic approach targeting protein synthesis for MYC-driven MB.},
}
@article {pmid41399500,
year = {2025},
author = {Swartjes, T and Bouzetos, E and Adiego-Pérez, B and Pool, VD and Staals, RHJ and van der Oost, J and Wu, WY},
title = {Base editing both DNA strands in distinct editing windows with small CRISPR-associated effector Cas12f1.},
journal = {iScience},
volume = {28},
number = {12},
pages = {114033},
pmid = {41399500},
issn = {2589-0042},
abstract = {CRISPR-associated base editors have been established as genome editing tools that enable base conversions in targeted DNA sequences, without generating double-strand breaks. Here, we describe the development of new base editors based on CRISPR-Cas12f1, a miniature Cas protein of only 422 amino acids. Chimeric constructs have been generated by fusing a catalytically inactive dCas12f1, to either a cytosine deaminase or an adenine deaminase. Using these synthetic fusion proteins, systematic analyses have been performed on base editing of a target sequence on a plasmid in Escherichia coli. Interestingly, apart from the previously described base editing of the displaced non-target DNA strand, we also observed efficient editing of the target DNA strand. This effect was not observed for Un1Cas12f1 BEs. In addition to the small size of AsCas12f1 base editors, its unique editing profile makes it a valuable addition to the CRISPR-Cas toolbox.},
}
@article {pmid41399197,
year = {2026},
author = {Singh, K and Sharma, S and Kalia, A and Manchanda, P},
title = {Advancement in Mushroom Transformation: From Conventional Techniques to Modern Genetic Engineering.},
journal = {Journal of basic microbiology},
volume = {66},
number = {1},
pages = {e70132},
doi = {10.1002/jobm.70132},
pmid = {41399197},
issn = {1521-4028},
mesh = {*Genetic Engineering/methods ; *Agaricales/genetics/growth &amp; development ; *Transformation, Genetic ; CRISPR-Cas Systems ; Gene Editing ; Agrobacterium/genetics ; },
abstract = {Mushrooms have long been valued for their nutritional, pharmaceutical, and culinary benefits. Recent studies showcased mushrooms as bio-factories for protein production, and as a source of value-added products by employing genetic manipulation and molecular transformation techniques. Advancements in molecular tools and transformation methods have enhanced the efficiency of genetic improvements in mushrooms by both conventional and modern genetic engineering techniques, paving the way for their use in various industrial applications. Genetic transformation in mushrooms involves transferring genes within and across species to understand gene functions and improve mushroom qualities. The techniques involved in transformation includes Agrobacterium-mediated transformation, hybridization, mutation breeding, particle bombardment, protoplast fusion, and CRISPR/Cas9. This review outlines the life cycle of mushrooms, major difficulties in mushroom transformation, various transformation techniques, their history, efficiency, and success rate. It also highlights the potential of genetic engineering to revolutionize mushroom cultivation and their applications.},
}
@article {pmid41398311,
year = {2025},
author = {Kang, YJ and Ha, HJ and Jin, HB and Lee, SY and Park, HH},
title = {Structural basis of dimerization and cascade formation by Cas5.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-025-32766-5},
pmid = {41398311},
issn = {2045-2322},
support = {2025//Chung-Ang University/ ; RS-2025-02316334//National Research Foundation of Korea/ ; },
abstract = {CRISPR-Cas systems are essential for prokaryotic adaptive immune mechanisms; however, the structural details of many subtype-specific components remain unclear. Herein, we report the crystal structure and biophysical characterization of Cas5 from Moraxella bovoculi (MboCas5), a component of the type I-C CRISPR-Cas system. We found that M. bovoculi encodes both type I-C and type III-B systems, and that MboCas5 forms a dimer that is stabilized by key interactions, including a salt bridge between R72 and D167. Structural comparisons with other Cas5 homologs and AlphaFold 3 predictions further validated the unique dimer configuration, suggesting that it is conserved across species. Additionally, structural comparison revealed a highly flexible loop region, which likely undergoes conformational changes upon Cascade assembly and might mediate interactions with Cas8 and crRNA. Overall, the findings provided structural and mechanistic insights into Cas5 function and could potentially contribute to our understanding of the assembly of type I-C Cascade complexes.},
}
@article {pmid41397585,
year = {2025},
author = {Hsu, CY and Polatova, D and Hamad, RH and Patel, PN and Akram, M and Singh, G and Arora, V and Nayak, PP and Kadhem, M and Hamzah, HF},
title = {Phage Therapy in Cancer Treatment: Mechanisms, Emerging Innovations, and Translational Progress.},
journal = {Critical reviews in oncology/hematology},
volume = {},
number = {},
pages = {105085},
doi = {10.1016/j.critrevonc.2025.105085},
pmid = {41397585},
issn = {1879-0461},
abstract = {Bacteriophage therapy has re-emerged as a rapidly advancing field in oncology, bridging antimicrobial precision with tumor-targeted biotherapy. Beyond infection control, phages are now recognized as programmable biological systems capable of eradicating multidrug-resistant (MDR) pathogens, modulating tumor-associated microbiota, activating immune responses, and delivering therapeutic genes or drugs. Preclinical evidence shows that phages can selectively eliminate Fusobacterium nucleatum in oral squamous cell carcinoma, restore microbial balance in colorectal cancer, and enhance immune infiltration via cytokine or antigen display. Engineered constructs including GM-CSF-expressing and MAGE-A1-displaying phages, λ-phage ASPH vaccines, and PEGylated nanocarriers delivering MEG3 or TRAIL have demonstrated strong anti-tumor efficacy across melanoma, hepatocellular, and colorectal cancer models. Additionally, CRISPR-Cas-armed phages precisely remove resistance genes such as bla-CTX-M and mecA, while AI-driven selection pipelines enable data-guided design of personalized phage cocktails. These advances represent a paradigm shift from empirical antibacterial use toward mechanistically engineered, multifunctional phage platforms that integrate microbiome modulation, immune activation, and nanocarrier-mediated gene delivery. Although challenges such as immune clearance, bacterial resistance, and regulatory complexity remain, the convergence of AI, CRISPR, and synthetic biology is accelerating the evolution of phage therapy into a clinically viable precision-oncology strategy. In this context, bacteriophages emerge not merely as antibacterial agents but as intelligent, patient-specific nanomedicines poised to redefine therapeutic boundaries in cancer treatment.},
}
@article {pmid41396047,
year = {2025},
author = {Matsuoka, T and Kano, S},
title = {Impact of patent-granting differences between Japan and the United States on patent protection for medical methods: insights from genome editing patents.},
journal = {Expert opinion on therapeutic patents},
volume = {},
number = {},
pages = {},
doi = {10.1080/13543776.2025.2605318},
pmid = {41396047},
issn = {1744-7674},
abstract = {INTRODUCTION: Securing patents in multiple countries has become essential for the development of global medical products. However, differences in national patent systems result in varying patentability standards. Although global claim construction strategies have been applied in practice, these approaches have not yet been systematically organized.<br><br>AREA COVERED: This study examines how the patent scope for patent families of international applications related to genome editing technologies filed in 2013, differs between Japan, where medical method patents are prohibited, and the United States, where such patents are permitted.<br><br>EXPERT OPINION: For CRISPR-Cas system patents, claim structures varied significantly, even among the corresponding family patents. To navigate these differences, the following strategies were proposed for filing patents in countries that prohibit medical method patents such as Japan:- Convert medical method claims in the U.S. into composition claims that include product inventions, as this process ensures that such claims allow for the enforcement of rights against the suppliers of infringing products.- Clearly define the scope of the claimed use-inventions when specifying the characteristics of the product based on its effects.- Explicitly describe cells produced by a specific manufacturing method within the claimed rights.},
}
@article {pmid41395656,
year = {2025},
author = {Lin, Z and Pu, Z and Wu, J and Zeng, J and Dou, Q and Mao, M and Zhang, Y},
title = {A Versatile CRISPR/Cas12a Autocatalytic Cascade System via Structure-Switching V-Type Split Probe for Highly Sensitive DNA Diagnostics.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c06488},
pmid = {41395656},
issn = {1520-6882},
abstract = {The rapid detection of pathogen nucleic acids is critical for controlling infectious disease outbreaks and providing timely treatment. However, current molecular diagnostic applications, including sensitive CRISPR/Cas-based detection systems, rely on target preamplification, which often requires expensive equipment and strict adherence to sometimes complex workflows. Here, we describe a rapid, simple, and amplification-free CRISPR/Cas-based diagnostic system that employs a structure-switching V-shaped DNA probe with a Cas12a recognition sequence split by an ssDNA loop to establish a positive feedback loop and a signal amplification cascade. This approach exhibited an ultralow background signal, rapid production of an exponential signal, and atto-molar sensitivity. It was incorporated into microfluidic and lateral flow assay applications for multiplex detection of distinct papillomavirus strains and point-of-care detection of monkeypox virus infections, respectively. The approach thus has significant potential for rapid and sensitive detection of specific pathogen-derived DNA targets in both clinical laboratory and point-of-care applications.},
}
@article {pmid41395238,
year = {2025},
author = {Zou, Y and Yao, ZW and Xiao, T and Ma, YR and He, J and Chen, LM and Chen, XQ and Chen, N},
title = {Emerging Trichomonad Infections in Companion Animals: Rapid Visual Detection of Pentatrichomonas hominis and Tritrichomonas foetus Using an RPA-CRISPR/Cas12a Assay.},
journal = {Transboundary and emerging diseases},
volume = {2025},
number = {},
pages = {9995679},
pmid = {41395238},
issn = {1865-1682},
mesh = {Animals ; Cats ; *Protozoan Infections, Animal/diagnosis/parasitology ; *Tritrichomonas foetus/isolation &amp; purification/genetics ; Dogs ; *Trichomonadida/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Cat Diseases/parasitology/diagnosis ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Pets ; *Dog Diseases/diagnosis/parasitology ; },
abstract = {Pentatrichomonas hominis (P. hominis) and Tritrichomonas foetus (T. foetus) are prevalent intestinal protozoa. P. hominis is associated with chronic diarrhea in humans and animals, whereas T. foetus causes gastrointestinal disease in companion animals and reproductive-tract infection in cattle. Rapid and accurate identification of these infections at the point-of-care (POC) is crucial for the diagnosis and effective management of zoonotic diseases. In this study, we developed two novel recombinase polymerase amplification (RPA) assays coupled with CRISPR/Cas12a detection. The dual-species assay, using a lateral-flow format, targeted species-specific regions of the 18S rRNA gene of P. hominis and T. foetus, and under ideal conditions, delivered visual results within 40 min for a single sample at 37°C. P. hominis-specific assay: To differentiate P. hominis in mixed infections with T. foetus, a second assay targeted the highly conserved Spo11-1 gene of P. hominis. Optimal crRNA-412 and RPA primers were selected for maximal Cas12a cleavage efficiency. Analytical sensitivity and specificity were compared with conventional nested polymerase chain reaction (PCR) and Sanger sequencing. The results showed that The dual-species assay detected as few as 50 DNA copies/µL of either parasite with no cross-reactivity to Giardia lamblia, Cystoisospora canis, Cryptosporidium spp., Toxoplasma gondii, Toxocara canis, and Toxascaris leonina. Among 70 fecal samples of companion animal (48 dogs and 22 cats), 14 (29.2%) dogs tested positive for P. hominis, and eight cats (36.4%) tested positive for T. foetus by nested PCR. Due to financial and logistical constraints, we selected a smaller subset for subsequent analysis with the RPA-CRISPR/Cas12a lateral-flow strip (LFS) assay, which showed 100% diagnostic concordance with PCR. The Spo11-1 assay achieved a limit of detection of 20 DNA copies/µL and specifically recognized P. hominis among a panel that included seven non-target protozoa and helminths. Validation on 10 additional canine and feline samples (four positives and six negatives) showed complete agreement with nested-PCR results. In conclusion, this CRISPR-based diagnostic approach significantly enhances the efficiency and accuracy of Trichomonads detection, offering a practical, cost-effective solution particularly suitable for veterinary and potentially human healthcare diagnostics in resource-limited settings.},
}
@article {pmid41394966,
year = {2025},
author = {Punde, A and Dey, S and Pandire, R and Bhattacharjee, A and Patra, C},
title = {Expanding the CRISPR/Cas toolkit: applications in proteomics and theranostics.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {13},
number = {},
pages = {1713700},
pmid = {41394966},
issn = {2296-4185},
abstract = {Conventional methods available for genome editing have proven non-specific, labour-intensive, and time-consuming. In this context, CRISPR/Cas technology represents a significant breakthrough. It is derived from a sophisticated microbial defence system consisting of clustered regularly interspaced short palindromic repeats, or CRISPR, and the RNA-guided DNA endonuclease Cas. Beyond its original role in genome editing, CRISPR continues to play a major role in the field of proteomics, functional genomics, and molecular therapy. Animal models, including mice, Drosophila, zebrafish, etc., have substantially benefited from CRISPR in uncovering protein function through reverse genetics approaches, including knock-in, knockout, CRISPRi, and indel mutation strategies. On the clinical front, CRISPR gene therapy has also seen successes, including applications in sickle cell disease, hypercholesterolemia, and cancer immunotherapy. However, notable challenges remain, including in vivo packaging and delivery efficiency, toxicity, and genomic off-target effects. Ongoing efforts to overcome these include the development of novel delivery formulations (e.g., nanoparticles, exosomes), artificial intelligence-guided experimental design, and miniaturization of Cas proteins. This review focuses on CRISPR/Cas gene editing mechanisms and explores its state-of-the-art applications in the field of proteomics and theranostics.},
}
@article {pmid41392542,
year = {2025},
author = {Qiao, Z and Choi, S and Chen, Z and Rodriguez, RM and Wang, Q and Yang, Z and Theuerkauf, SA and Nabhan, JF and Hensch, TK and Buchholz, CJ and Lu, Q},
title = {Targeted Intracellular Delivery via Precision Programming of ARRDC1-Mediated Microvesicles.},
journal = {Journal of extracellular vesicles},
volume = {14},
number = {12},
pages = {e70199},
pmid = {41392542},
issn = {2001-3078},
support = {//Vesigen Therapeutics/ ; P42ES030990/NH/NIH HHS/United States ; R01ES029097/NH/NIH HHS/United States ; R01HL139496/NH/NIH HHS/United States ; },
mesh = {Animals ; Mice ; CD8-Positive T-Lymphocytes/metabolism ; Humans ; *Cell-Derived Microparticles/metabolism ; Neurons/metabolism ; *Drug Delivery Systems/methods ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; },
abstract = {Efficient and cell-specific delivery remains a major barrier to realising the full therapeutic potential of modalities such as mRNA and CRISPR-based gene editors. Here, we report a versatile delivery platform based on engineered ARRDC1-mediated microvesicles (ARMMs) capable of delivering cargo to defined cell populations. By decorating ARMMs with engineered Nipah virus (NiV)-derived fusion and attachment proteins conjugated to cell-specific ligands, we enable selective binding and membrane fusion-mediated cargo release. ARMMs functionalized with anti-CD8 single-chain variable fragment (scFv) delivered protein, mRNA, or CRISPR-Cas9 base editor selectively to CD8[+] T cells. Similarly, ARMMs displaying a designed ankyrin repeat protein (DARPin) targeting the GluA4 receptor enabled delivery to parvalbumin-positive (PV[+]) neurons. In vivo, administration of targeted ARMMs resulted in functional delivery to CD8[+] splenocytes and PV[+] cortical neurons in mice. These findings establish surface-engineered ARMMs as a programmable and modular system for precision delivery of therapeutic macromolecules, with broad applicability in gene and RNA-based medicine.},
}
@article {pmid41381532,
year = {2025},
author = {Zaada, DSY and Toren, O and Krsticevic, F and Haber, DA and Gildman, D and Galpaz, N and Häcker, I and Schetelig, MF and Marois, E and Arien, Y and Papathanos, PA},
title = {Mosquito sex separation using complementation of selectable traits and engineered neo-sex chromosomes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11175},
pmid = {41381532},
issn = {2041-1723},
support = {3-1679//Ministry of Science, Technology and Space/ ; 1833/7-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 101059523//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; ANR-23-CE35-0003//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {Animals ; Female ; Male ; *Aedes/genetics/physiology ; *Sex Chromosomes/genetics ; CRISPR-Cas Systems ; Genetic Engineering ; *Sex Determination Processes/genetics ; Animals, Genetically Modified ; Pigmentation/genetics ; Phenotype ; Insect Proteins/genetics ; },
abstract = {Effective sex separation remains a critical challenge for mosquito genetic control. Genetic sexing strains (GSS) address this by linking maleness with selectable traits, enabling efficient female removal. Here, we present a versatile platform for GSS development in the invasive Aedes albopictus mosquito that integrates CRISPR-engineered selectable phenotypes with sex conversion via nix, the male-determining factor. As a proof-of-concept, we disrupt the yellow pigmentation gene and restore its function in males using nix-containing transgenes, producing a stable strain with yellow females and dark males. Beyond serving as a vivid marker, yellow confers added advantages: GSS females pupate later than wild females, enhancing protandry-based sorting, and lay desiccation-sensitive eggs, lowering accidental female release risk. The strain is compatible with size-based separation, improving sexing accuracy through the integration of natural and engineered dimorphisms. To our knowledge, this represents the first engineered sex-linked selectable trait in mosquitoes based on endogenous genes, establishing a foundation for scalable GSS development.},
}
@article {pmid41380779,
year = {2026},
author = {Hill, AC and Schank, MB and Zhang, Y and Sun, N and Wang, L and Zhao, J and Banik, P and Pyburn, JS and Orfield, H and Lightner, JW and Leshaodo, TO and Wu, XY and Ning, S and El Gazzar, M and Moorman, JP and Guo, H and Yao, ZQ},
title = {Suppression of HBV replication and expression by CRISPR/Cas9 ribonucleoproteins.},
journal = {Antiviral research},
volume = {245},
number = {},
pages = {106326},
doi = {10.1016/j.antiviral.2025.106326},
pmid = {41380779},
issn = {1872-9096},
mesh = {*Hepatitis B virus/genetics/physiology/drug effects ; *Virus Replication/drug effects ; Humans ; *CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/pharmacology/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; DNA, Viral/genetics ; Antiviral Agents/pharmacology ; Hep G2 Cells ; Genetic Therapy/methods ; Hepatitis B/therapy/virology ; Cell Line ; CRISPR-Associated Protein 9 ; },
abstract = {HBV infection is a global public health problem. The current treatment using nucleotide analogues (NA) can suppress viral replication but cannot eliminate HBV infection due to the persistence of covalently closed circular DNA (cccDNA), which sustains HBV replication and integration into the host cell genome and is refractory to NA treatment. CRISPR/Cas9 has been used to disrupt integrated HBV DNA and minichromosomal cccDNA for HBV suppression, but its expression and delivery require viral or non-viral vectors, which pose safety concerns for human application. We have previously reported the use of synthetic guide RNA (gRNA)/Cas9 ribonucleoprotein (RNP) as a non-viral formulation for HBV gene editing and viral suppression. To formulate highly effective CRISPR/Cas9 modalities for HBV gene therapy, here we designed additional gRNA/Cas9 RNPs and compared their antiviral efficacy in HBV-transfected as well as -infected cells. We found that two selected gRNA/Cas9 RNPs (gRNA5/Cas9, gRNA9/Cas9, and particularly their combinations) elicited the most potent antiviral efficacy, as evidenced by the significant inhibition of HBV DNA, RNA, and protein productions. DNA sequencing of the treated cells revealed moderate to high rates of insertion and deletion (indel) or knock-out (KO) mutations at the HBV target genes. Gene alignment analysis showed a high level of conservation for both gRNA5 and gRNA9 target sequences across major HBV genotypes, indicating that these CRISPR-based gene editing therapeutics have the potential to target different HBV strains worldwide. Thus, these synthetic gRNA/Cas9 RNPs represent promising novel therapeutics that can be developed and utilized for HBV gene disruption and viral eradication.},
}
@article {pmid41365143,
year = {2025},
author = {Queffeulou, M and Fakhfakh, R and Fani, F and Dos Santos, A and Reis Ferreira, G and Bigot, S and Godin, C and Leprohon, P and Papadopoulou, B and Ouellette, M},
title = {CRISPR-Cas13b mediated gene knockdowns in Leishmania infantum.},
journal = {International journal for parasitology. Drugs and drug resistance},
volume = {29},
number = {},
pages = {100629},
doi = {10.1016/j.ijpddr.2025.100629},
pmid = {41365143},
issn = {2211-3207},
mesh = {*Leishmania infantum/genetics/drug effects ; *CRISPR-Cas Systems/genetics ; *Gene Knockdown Techniques/methods ; Antiprotozoal Agents/pharmacology ; Phosphorylcholine/analogs &amp; derivatives/pharmacology ; Protozoan Proteins/genetics ; Antimony/pharmacology ; Luciferases, Firefly/genetics ; RNA, Messenger/genetics ; },
abstract = {Chemotherapy is an effective means to control infections caused by the protozoan parasite Leishmania. However, available treatments are limited, expensive, and associated with considerable toxicity. Genomic strategies have contributed to a better understanding of Leishmania's response to drugs and in the characterization of drug targets. Nonetheless, there is no knockdown system operational for Leishmania. In this study, we show that the CRISPR-Cas13 system can be an effective strategy to knockdown expression levels of both exogenous and endogenous transcripts. We succeeded in effectively knocking down the expression of the firefly luciferase gene integrated in the genome of L. infantum. This Cas13-mediated decrease in mRNA was paralleled with a significant reduction in both the luciferase protein level and its activity. Furthermore, we tested the effectiveness of the Cas13 system to target the endogenous miltefosine transporter (MT) and the aquaglyceroporin 1 (AQP1) genes. Knockdown was effective and parasites with less MT or AQP1 mRNA levels exhibited reduced susceptibility to miltefosine or antimonials, respectively. While further optimization is warranted, this knockdown system has the potential to facilitate numerous studies related to various aspects of Leishmania biology.},
}
@article {pmid41363172,
year = {2025},
author = {Wen, TT and Xu, L and Jin, R and Liu, Z and Liu, MQ and Dong, CH and Sun, L and Wang, HY and Sun, WJ and Cui, FJ},
title = {Functional Characterization of a β-1,3-Glucanosyltransferase CmGel4 in Cordyceps militaris Using a Precise CRISPR-Cas9 Genome-Editing System.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {50},
pages = {32078-32093},
doi = {10.1021/acs.jafc.5c08653},
pmid = {41363172},
issn = {1520-5118},
mesh = {CRISPR-Cas Systems ; *Cordyceps/genetics/enzymology/chemistry ; Gene Editing ; *Fungal Proteins/genetics/metabolism/chemistry ; beta-Glucans/metabolism ; Amino Acid Sequence ; Glucan Endo-1,3-beta-D-Glucosidase ; },
abstract = {Cordyceps militaris polysaccharides, especially β-glucans, have presented significant antitumor, hypoglycemic, and immunomodulatory activities. However, the enzymes involved in the branching formation of C. militaris β-glucans remain to be elucidated. In the present study, a 1.69-kb β-1,3-glucanosyltransferase CmGel4 gene putatively involved in β-glucan branching was cloned from C. militaris mycelia and bioinformatically analyzed. The encoded 54.12 kDa CmGel4p consisted of 515 amino acid residues and contained a typical GH72[+] structural characteristic of a signal peptide (1-19aa), a GH72 conserved domain (20-334aa), a GPI-anchor site (485aa), and a CBM43/X8 domain (382-458aa). Using the established CRISPR-Cas9 genome-editing system, the full length of 1.69-kb CmGel4 was precisely inserted at a genomic safe-harbor site CmSh1, and the GH72 conserved domain of CmGel4 was successfully deleted in C. militaris genome for the first time. By comparing the mycelial growth and fermentation performance of WT, control, and CmGel4-overexpressed/knockout mutants, β-1,3-glucanosyltransferase gene CmGel4 was shown to play key roles in cell growth and branching of exo-polysaccharides of C. militaris, accompanied by the transcriptional changes of genes such as CmGel4, CmUgp, and CmPgm. These findings provided the proof of β-1,3-glucanosyltransferases vital for formatting cell walls and maintaining cellular integrity, and a fine regulation strategy for precisely remodeling the β-1,3-glucan with high-branched structures in edible fungi.},
}
@article {pmid41361999,
year = {2025},
author = {Cheng, Y and Zhang, X and Zhao, P and Zhu, D and Meng, YH and Fu, X and Wang, X},
title = {Enhanced Mannan Production of Saccharomyces cerevisiae by CRISPR/Cas9 and Mannoproteins Characteristics on Wine Astringency Modulation.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {50},
pages = {32195-32208},
doi = {10.1021/acs.jafc.5c10790},
pmid = {41361999},
issn = {1520-5118},
mesh = {*Saccharomyces cerevisiae/metabolism/genetics ; *Wine/analysis/microbiology ; *Mannans/metabolism/chemistry ; *Membrane Glycoproteins/metabolism/genetics/chemistry ; CRISPR-Cas Systems ; Taste ; Humans ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; Mannose/metabolism/analysis ; },
abstract = {Mannoproteins are critical in modulating wine astringency, yet the specific impacts of their monosaccharide ratio and side-chain structure remain insufficiently explored. This study employed CRISPR/Cas9 to engineer yeast strains producing mannoproteins with either a high mannose-to-glucose ratio (high-yield-mannan strain BSFA12) or a nonbranched N-glycan structure (BY4741-ΔMNN2). The resulting mannoproteins (MPBSFA, MPBY2) were compared against controls (MPBY extracted from Saccharomyces cerevisiae BY4741, a commercial product MP60) using physicochemical analyses (fluorescence quenching, dynamic light scattering, and isothermal titration calorimetry) and sensory evaluation. Both engineered variants demonstrated superior astringency reduction in model and red wines. Our results establish that an increased mannose-to-glucose ratio and reduced N-glycosylation significantly enhance the astringency-mitigating effect, providing a foundation for the industrial-scale production and application of mannoprotein additives.},
}
@article {pmid41346237,
year = {2025},
author = {Pandit, B and Hanson, E and Dagci, H and Yang, Q and Yigit, MV and Royzen, M},
title = {Effects of N[6]-Methyladenosine (m[6]A) and 5-Methylcytosine (m[5]C) Modifications in the Guide Region of CRISPR RNA on Cas12a Nuclease Activity.},
journal = {Bioconjugate chemistry},
volume = {36},
number = {12},
pages = {2551-2556},
doi = {10.1021/acs.bioconjchem.5c00498},
pmid = {41346237},
issn = {1520-4812},
mesh = {*5-Methylcytosine/chemistry/metabolism ; *Adenosine/analogs &amp; derivatives/chemistry/metabolism ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry ; *CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism ; *Bacterial Proteins/metabolism ; },
abstract = {CRISPR-Cas12a is a versatile biosensing platform that detects sequence-specific DNA or RNA targets via a CRISPR RNA (crRNA) guide. While Cas12a's specificity is dictated by its crRNA, chemical modifications within the crRNA can influence nuclease performance. Here, we examined the effects of two well-known RNA modifications, N[6]-methyladenosine (m[6]A) and 5-methylcytosine (m[5]C), introduced into the different positions of the guide region of a crRNA. Melting temperature (Tm) analysis showed that m[6]A had a minimal impact on RNA-DNA duplex stability. In contrast, the incorporation of m[5]C residues stabilized the duplex. Using a fluorescence recovery assay, we found that both modifications preserved Cas12a's nuclease activity, indicating that small thermodynamic shifts in duplex formation are insufficient to disrupt its catalytic function. Despite the greater Tm increase with m[5]C, m[6]A incorporation led to a faster fluorescence recovery rate than that with m[5]C.},
}
@article {pmid41266327,
year = {2025},
author = {Corda, L and Volpe, E and Dallali, H and Di Tommaso, E and Colantoni, A and Guarracino, A and Chittoor, SS and Capulli, M and Tassone, E and Giunta, S},
title = {Cell line-matched reference enables high-precision functional genomics.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11194},
pmid = {41266327},
issn = {2041-1723},
support = {#101078838//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; #25189//Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)/ ; },
mesh = {Humans ; *Genomics/methods/standards ; *Genome, Human/genetics ; Cell Line ; Haplotypes ; Centromere/genetics ; Gene Editing ; CRISPR-Cas Systems ; Kinetochores/metabolism ; },
abstract = {Comparative analyses of newly available human genome assemblies highlight extensive variation that peaks at centromeres. Reliance on a single generic reference genome can thus hinder whole-genome analysis of sequencing data derived from laboratory cell lines and limit their accurate genomic manipulation. Here, we demonstrate that using an "isogenomic" diploid reference genome - specific for the experimental cell line - substantially improves the accuracy of genomic, epigenomic, transcriptomic analyses and genome editing compared to a non-matched reference. Using our recently generated reference genome of the widely used diploid human cell line RPE-1, we uncover haplotype-specific genetic and epigenetic divergence across all centromeres. Mapping quality of RPE-1 data - DNA- and RNA-seq reads, improves both genome-wide and at highly divergent loci when using the matched RPE1v1.1 reference, resolving haplotype-specific enrichment. For genome engineering experiments, centromeric CRISPR guide RNA efficiency and chromosome specificity are best achieved using the RPE-1 reference. Leveraging high-confidence CUT&amp;RUN read mapping using the matched reference, we define the site of the human kinetochore and identify a wide variation in the position, size and structural organization between haplotypes and chromosomes. This work establishes matched-reference genomics as a powerful framework for high-precision cell biology, calling for the systematic assembly of experimentally relevant cell line genomes.},
}
@article {pmid41250994,
year = {2025},
author = {Bereiter, R and Manjunath, A and Beal, PA},
title = {Clickable RNA via 4'-C-Ethynyl Cytidine─A Novel Design for Metabolically Stable Guide RNAs in RNA Editing.},
journal = {Bioconjugate chemistry},
volume = {36},
number = {12},
pages = {2637-2644},
doi = {10.1021/acs.bioconjchem.5c00480},
pmid = {41250994},
issn = {1520-4812},
mesh = {*Cytidine/chemistry/analogs &amp; derivatives ; Click Chemistry ; Humans ; *RNA Editing ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; Alkynes/chemistry ; Cycloaddition Reaction ; Triazoles/chemistry ; },
abstract = {Chemical modifications in RNA therapeutics have addressed major challenges by enhancing metabolic stability, cellular uptake, and biological activity─regardless of their mechanism of action. Here, we report on the synthesis of 4'-C-ethynyl cytidine (4'-C-EthC) and its 2'-O-methylated derivative (4'-C-EthC-2'-OMe) as phosphoramidite building blocks and their subsequent incorporation into oligonucleotides. These ribose C4-terminal alkyne modifications provide a click handle directly within oligonucleotides. The novel modification is accessible via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and serves as a universal 4'-C-ribose modifier on the oligonucleotide level. We identified both aromatic and aliphatic triazole residues that increase the thermodynamic stability in A-form RNA duplexes. Furthermore, 4'-C-triazole-modified oligonucleotides exhibit high resistance to nuclease-mediated degradation in metabolic stability assays. Finally, we introduced the novel modification and its substituted triazoles into guide RNAs (gRNAs) for site-directed A-to-I editing in mammalian cells and compared their performance with phosphorothioate-modified gRNAs.},
}
@article {pmid41391580,
year = {2025},
author = {Zarei, S and Hosseiniara, SM and Zijoud, SSH and Hosseiniara, R},
title = {Electrochemical MicroRNA biosensors for kidney Cancer: From biomarker discovery to point-of-care diagnostics.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {},
number = {},
pages = {120786},
doi = {10.1016/j.cca.2025.120786},
pmid = {41391580},
issn = {1873-3492},
abstract = {Kidney cancer, particularly clear cell renal cell carcinoma (ccRCC), presents a significant clinical burden due to late-stage detection and limited effectiveness of current diagnostic modalities. Minimally invasive strategies, such as liquid biopsy, have emerged as promising alternatives, with microRNAs (miRNAs) gaining attention as stable, disease-specific biomarkers detectable in biofluids. miRNAs function as oncogenes or tumor suppressors, offering advantages over conventional protein biomarkers in early cancer detection and prognostic assessment. Electrochemical biosensors provide a highly sensitive, rapid, and cost-effective platform for miRNA detection, enabling potential point-of-care applications. Recent advances include the integration of nanomaterials, enzymatic and isothermal amplification methods, and CRISPR-Cas systems to enhance specificity and signal sensitivity. Prototype sensors targeting RCC-relevant miRNAs, multiplexed detection for biomarker panels, and smartphone-compatible platforms demonstrate the feasibility of translating these technologies into clinical practice. Despite challenges in assay standardization, pre-analytical variability, and regulatory pathways, electrochemical miRNA biosensors hold transformative potential for non-invasive RCC diagnostics, treatment monitoring, and precision oncology. Continued innovation and clinical validation may establish these platforms as integral tools for personalized patient management.},
}
@article {pmid41390996,
year = {2026},
author = {Wang, AJ and Du, C and Liu, H and Wang, HL},
title = {Effect of the sdc4 gene knockdown on muscle development in zebrafish.},
journal = {Comparative biochemistry and physiology. Part B, Biochemistry &amp; molecular biology},
volume = {281},
number = {},
pages = {111171},
doi = {10.1016/j.cbpb.2025.111171},
pmid = {41390996},
issn = {1879-1107},
mesh = {Animals ; *Zebrafish/genetics/growth &amp; development/metabolism ; *Syndecan-4/genetics/metabolism ; *Muscle Development/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; MicroRNAs/genetics/metabolism ; *Muscle, Skeletal/metabolism/growth &amp; development ; Wnt Signaling Pathway ; CRISPR-Cas Systems ; },
abstract = {The growth and regeneration of skeletal muscle are closely related to syndecan-4 (Sdc4), which is a type I transmembrane heparan sulfate proteoglycan belonging to the syndecan family. However, it remains unclear how the sdc4 gene affects fish muscle development. Therefore, an sdc4 knockdown zebrafish line (sdc4[-/-]) was generated by CRISPR/Cas9 technology in this study, and its phenotypes were analyzed. The results revealed that sdc4[-/-] zebrafish exhibited reduced body length and weight compared to the wild-type (WT) at 90 days post fertilization (dpf). Furthemore, sdc4[-/-] zebrafish also showed a significantly larger number of muscle fibers, and significantly reduced individual muscle fiber cross-sectional area. The mRNA expression levels of genes associated with myogenic regulatory factors (MRFs) and the wnt/β-catenin pathway were all significantly downregulated. Based on dual luciferase reporter assays, sdc4 gene expression was regulated by the transcription factor myocyte enhancer factor 2aa (Mef2aa) and miR-141-3p, which bind to its promoter and 3' untranslated region (UTR), respectively. Additionally, the reduced average swimming speed and distance observed in sdc4[-/-] zebrafish at 90 dpf were concomitant with a significant downregulation of mitochondrial respiratory chain complex-related genes and a reduction in ATP concentration. This research aids understanding of sdc4 function in fish and may provide a new perspective for studying the molecular mechanisms of muscle growth and development.},
}
@article {pmid41389662,
year = {2025},
author = {Ma, J and Zhao, CF and Liu, X},
title = {Advances in targeted therapeutics and smart delivery systems based on precision nano-oncology.},
journal = {International immunopharmacology},
volume = {169},
number = {},
pages = {115946},
doi = {10.1016/j.intimp.2025.115946},
pmid = {41389662},
issn = {1878-1705},
abstract = {The convergence of nanotechnology and precision oncology is revolutionizing cancer treatment by enabling highly specific, minimally invasive, and personalized therapeutic strategies. This review explores recent breakthroughs in nano-therapeutics and their pivotal role in overcoming the limitations of conventional cancer therapies. Emphasis is placed on the design and function of nanocarriers that facilitate targeted drug delivery via both passive (EPR effect) and active ligand-mediated mechanisms. Special attention is given to stimuli-responsive systems that release therapeutic agents in response to pH, enzymes, temperature, or redox environments, enhancing spatiotemporal control. The article further discusses the integration of nanotechnology with emerging modalities including immunotherapy, photothermal and photodynamic therapies, gene editing tools (e.g., CRISPR/Cas systems), and multifunctional theranostic platforms. While these innovations offer transformative potential, the review also addresses persistent challenges such as tumor heterogeneity, immune clearance, off-target effects, large-scale manufacturing, and regulatory complexity. By highlighting both promise and hurdles, this article provides a comprehensive lens into the future of precision cancer nanomedicine.},
}
@article {pmid41387770,
year = {2025},
author = {Ge, L and Li, W and Dou, Y and Ma, Y and Sun, M and Chen, X and Feng, X and Li, Y and Yu, Q},
title = {Callus and endosperm green fluorescence reporter-assisted selection system in maize CRISPR/Cas9 gene editing.},
journal = {BMC plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12870-025-07429-2},
pmid = {41387770},
issn = {1471-2229},
support = {2022SZX13//Science&amp;Technology Specific Projects in Agricultural High-tech Industrial Demonstration Area of the Yellow River Delta/ ; ZR2024MC067//Natural Science Foundation of Shandong Province/ ; },
abstract = {BACKGROUND: Genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has emerged as a promising approach for functional gene analysis and genetic improvement. Since stable transformation remains the primary method for implementing this system, the ultimate goal in crop breeding programs would require the selection of transgene-free plants with the CRISPR/Cas expression cassette removed.<br><br>RESULTS: In this study, we developed an endosperm-specific fluorescence reporter-assisted selection system for CRISPR/Cas9 gene editing (pAZS22-eGFP/CRISPR/Cas9) in maize (Zea mays L.), utilizing enhanced green fluorescent protein (eGFP) expressed specifically in the endosperm to facilitate the easy identification of transgenic and transgene-free plants from the T1 generation on. In addition, the 22&#xa0;kDa alpha zein (z1C1_10) promoter from maize, employed in this system, has been shown to be active in both callus and endosperm, thereby being able to enhance the accuracy of transformant identification during the tissue culture process by reducing false positives compared to the traditional selective media methods. Our studies targeting the ZmSnRK2.1 or Dwarf1 (D1) genes demonstrated a reasonable editing efficiency, with rates ranging from 56.3% for T0 plants targeting ZmSnRK2.1, to 87.5% and 100% for T1 plants targeting D1 and ZmSnRK2.1, respectively. In addition, we successfully identified 1 transgene-free homozygous d1 mutant in the T1 generation and 7 transgene-free homozygous snrk2.1 mutants in the T2 generation.<br><br>CONCLUSIONS: The pAZS22-eGFP/CRISPR/Cas9 system provides an efficient tool for gene editing, transformant selection and transgene status identification in maize breeding.},
}
@article {pmid41385994,
year = {2025},
author = {Luo, Y and Wang, X and Yang, F and Zhao, Y and Hu, S and Liu, S and Li, S and Luo, G and Sun, Q},
title = {Construction and validation of a rapid semen identification system based on SHERLOCK technology.},
journal = {Forensic science international. Genetics},
volume = {82},
number = {},
pages = {103410},
doi = {10.1016/j.fsigen.2025.103410},
pmid = {41385994},
issn = {1878-0326},
abstract = {This study developed a rapid detection system for semen-specific mRNA based on CRISPR/Cas13a system to meet the timeliness requirements of forensic on-site body fluid identification. Specific primers and CRISPR RNA (crRNA) short fragments on semen specific mRNA genes were designed and screened, to establish a SHERLOCK detection method based on technology principles of CRISPR/Cas. Furthermore, nucleic acid rapid release agents for treating samples were screend to construct a new detection method in combination with SHERLOCK, and the specificity and sensitivity of the method were tested. The method can rapidly detect the presence of semen from unknown body fluid samples, and the relative fluorescence unit (RFU) value of the semen sample is significantly higher than those of non-semen samples (P < 0.0001), with a sample detection sensitivity of down to 0.25 μL. The construction of the rapid semen detection method using rapid extraction and SHERLOCK reduces operation time, significantly reduces instrument dependence, and provides an innovative solution for forensic on-site rapid body fluid identification.},
}
@article {pmid41284810,
year = {2025},
author = {Park, S and Kim, MM},
title = {Mutation of the Collagen Type 1α Gene in Human Fibrosarcoma Cells Using the CRISPR/Cas9 System.},
journal = {Biochemistry},
volume = {64},
number = {24},
pages = {4794-4804},
doi = {10.1021/acs.biochem.5c00394},
pmid = {41284810},
issn = {1520-4995},
mesh = {Humans ; Collagen Type I, alpha 1 Chain ; *Fibrosarcoma/genetics/metabolism/pathology ; *Collagen Type I/genetics/metabolism ; *CRISPR-Cas Systems ; *Mutation ; Cell Line, Tumor ; Signal Transduction ; Gene Editing ; },
abstract = {Collagen in the connective tissue plays a key role in the expression the aging phenotypes. While collagen production decreases with aging, collagenase expression increases, resulting in collagen breakdown. The purpose of this study is to investigate the change in the expression of proteins and genes related to the collagen signaling pathway, cell cycle, and aging phenotypes of cells with the collagen type 1 α (COL1A1) gene edited by the CRISPR/Cas9 system. The mutation of the COL1A1 gene was induced by the CRISPR/Cas9 system. Sanger DNA sequencing and Indel analyses, Sanger DNA sequencing analysis and Swiss protein modeling analysis were used to verify the induction of mutation. Aging phenotypes in the mutated cells were evaluated by collagen staining assay, SA-β-galactosidase staining assay, RT-PCR assay, Western blot analysis, gelatin zymography, and immunofluorescent staining assay. Sanger DNA sequencing analysis demonstrated that human fibrosarcoma cells with COL1A1 gene mutations were successfully established in this study. Swiss protein modeling analysis displayed the altered structure of COL1A1 in the edited cells. In addition, while collagen production was decreased, the SA-β-galactosidase staining level was increased in the edited cells. It was also found that the expression levels of CDC2, CDk2, and cyclin D were increased by down-regulating p53 and p21 levels through the increased expression of MDM2 in the edited cells. Moreover, the expression levels of MMP-1, MMP-2, MMP-9, AKT, and p-mTOR were reduced in the edited cells. These findings could provide a crucial clue in elucidating the close relationship between collagen production and senescence.},
}
@article {pmid41284414,
year = {2025},
author = {Iyer, KA and Tenchov, R and Lotti Diaz, LM and Jain, P and Thite, T and Deng, Y and Zhou, QA},
title = {CRISPR Technology: Transforming the Future of Medicine and Diagnostics.},
journal = {Biochemistry},
volume = {64},
number = {24},
pages = {4628-4660},
doi = {10.1021/acs.biochem.5c00480},
pmid = {41284414},
issn = {1520-4995},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods/trends ; *Genetic Therapy/methods/trends ; Neoplasms/therapy/genetics/diagnosis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; },
abstract = {In this report, we examine the extensive research landscape of CRISPR with an emphasis on CRISPR therapeutics and showcase our results from an in-depth analysis of the most up-to-date scientific information consisting of more than 53,000 publications encompassing academic journal articles and patents, spanning nearly three decades, extracted from the CAS Content Collection. Our analysis indicates that cancer and infectious diseases are the most explored in the context of CRISPR. Identified gene targets associated with CRISPR-related publications are led by TP53, c-myc, and hemoglobin beta subunit (HBB). Among the many delivery methods, adeno-associated vectors (AAVs) appear to be highly explored. With >140 CRISPR-based therapeutics in the clinical development pipeline and billions of dollars in investment, the field of CRISPR continues to evolve rapidly. We also briefly discuss the ethical implications of CRISPR technology. While some fundamental challenges persist, the future of CRISPR is undoubtedly bright.},
}
@article {pmid41264498,
year = {2025},
author = {Guo, Y and Guo, W and Wu, Z and Xu, H and Zhang, X and Zou, X and Sun, Z},
title = {A Microfluidic Chip-Based Electrochemical Biosensor Coupled with CRISPR/Cas12a for Simultaneous Detection of Foodborne Pathogens.},
journal = {Analytical chemistry},
volume = {97},
number = {49},
pages = {27266-27275},
doi = {10.1021/acs.analchem.5c05232},
pmid = {41264498},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/instrumentation ; *Staphylococcus aureus/isolation &amp; purification/genetics ; Gold/chemistry ; *Salmonella/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Food Microbiology ; Metal Nanoparticles/chemistry ; *CRISPR-Associated Proteins/metabolism/genetics ; *Lab-On-A-Chip Devices ; Limit of Detection ; Food Contamination/analysis ; Pyrroles/chemistry ; *Bacterial Proteins/genetics/metabolism ; Endodeoxyribonucleases ; Polymers ; },
abstract = {Staphylococcus aureus (S. aureus) and Salmonella frequently cocontaminate food products, posing significant health threats. We developed a microfluidic electrochemical biosensor that enables simultaneous detection of both pathogens within 65 min. The chip integrates sample loading, recombinase polymerase amplification (RPA), and CRISPR/Cas12a-based recognition into the upper layer. Reaction products enter a detection chamber with a single-walled carbon nanohorns-polypyrrole-gold nanoparticles (SWCNHs-ppy-AuNPs)-modified three-electrode system, where ssDNA probes act as signal reporters. Upon target recognition, Cas12a cleaves the probes, releasing electroactive molecules and reducing the current. The sensor exhibits linear responses for S. aureus (1.06 × 10[1]-1.06 × 10[7] CFU/mL) and Salmonella (1.04 × 10[1]-1.04 × 10[7] CFU/mL), with detection limits of 3 CFU/mL. This platform offers a rapid, sensitive, and accurate tool for on-site detection of foodborne pathogens in food products.},
}
@article {pmid41218606,
year = {2025},
author = {Shevade, K and Yang, YA and Feng, K and Mader, K and Sevim, V and Parsons, J and Arora, G and Elfawy, H and Mace, R and Federman, S and Esanov, R and Shafer, S and Chow, ED and Przybyla, L},
title = {Simultaneous capture of single cell RNA-seq, ATAC-seq, and CRISPR perturbation enables multiomic screens to identify gene regulatory relationships.},
journal = {Cell reports methods},
volume = {5},
number = {12},
pages = {101222},
doi = {10.1016/j.crmeth.2025.101222},
pmid = {41218606},
issn = {2667-2375},
mesh = {Humans ; *Single-Cell Analysis/methods ; *Gene Regulatory Networks/genetics ; *RNA-Seq/methods ; Induced Pluripotent Stem Cells/metabolism ; *Chromatin Immunoprecipitation Sequencing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Dasatinib/pharmacology ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Chromatin/metabolism/genetics ; Transposases/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Here, we introduce CRISPR and transcriptomics-assay for transposase-accessible chromatin (CAT-ATAC), a technique that adds CRISPR guide RNA (gRNA) capture to the existing 10× Genomics Multiome assay, generating linked transcriptome, chromatin accessibility, and perturbation identity data from the same individual cells. We demonstrate up to 77% capture rate for both arrayed and pooled delivery of lentiviral gRNAs in induced pluripotent stem cells (iPSCs) and cancer cell lines. This capability allows us to construct gene regulatory networks (GRNs) in cells under drug and genetic perturbations. By applying CAT-ATAC, we identified a GRN associated with dasatinib resistance, indirectly activated by the HIC2 gene. Using loss-of-function experiments, we further validated that ZFPM2, a component of the predicted GRN, also contributes to dasatinib resistance. CAT-ATAC can thus be used to generate high-content multidimensional genotype-phenotype maps to reveal gene and cellular interactions and functions.},
}
@article {pmid41202713,
year = {2026},
author = {Wang, Y and Xiang, L and Su, Z},
title = {CRISPR/Cas9 gene editing in muscle-related genetic disorders: Restoring function and exercise capacity.},
journal = {Tissue &amp; cell},
volume = {98},
number = {},
pages = {103217},
doi = {10.1016/j.tice.2025.103217},
pmid = {41202713},
issn = {1532-3072},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Genetic Therapy ; *Muscular Diseases/genetics/therapy ; *Muscular Dystrophies/genetics/therapy ; },
abstract = {Due to their progressive and debilitating character and lack of curative therapies, muscle-related hereditary illnesses such as muscular dystrophies (MDs), myopathies, and motor neuron diseases need immediate care. A pressing need exists for novel therapeutics that can accurately and persistently correct the underlying mutations in muscle-related genetic disorders, as existing treatments are mostly palliative rather than addressing the underlying genetic cause. Traditional therapies for genetic diseases, such as hereditary myopathies and MDs, are often insufficient; however, the advent of CRISPR/Cas9 technology has altered this trajectory. While other evaluations have compiled the potential of gene editing, this one compiles the latest findings on CRISPR/Cas9 applications for musculoskeletal disorders. With an emphasis on the repair of recessive and dominant-negative mutations, we provide a critical evaluation of the development of new therapeutic vectors and in vivo editing techniques that have gone from conceptual models to preclinical triumphs. More specifically, we address the issues of efficient delivery, off-target effects, and immunological responses specific to muscle tissue, all of which are ongoing challenges. By facilitating targeted correction of pathogenic mutations, CRISPR/Cas9 holds promise for restoring muscle integrity and functional performance, potentially translating into improved exercise capacity. This study offers a prospective view on the near-term therapeutic promise of CRISPR/Cas9 for treating crippling muscle diseases by combining recent advances with an evaluation of the translational route.},
}
@article {pmid41101973,
year = {2025},
author = {Ishizuka, T and Tsuboyama, K and Tomari, Y},
title = {Comprehensive identification and functional analysis of fully disordered proteins essential for cell survival.},
journal = {RNA (New York, N.Y.)},
volume = {32},
number = {1},
pages = {61-70},
doi = {10.1261/rna.080626.125},
pmid = {41101973},
issn = {1469-9001},
mesh = {*Intrinsically Disordered Proteins/genetics/metabolism/chemistry ; Cell Survival/genetics ; Humans ; Genes, Essential ; Introns ; CRISPR-Cas Systems ; },
abstract = {Proteins have traditionally been understood through their tertiary structures, with well-defined conformations considered essential for biological function. This classical structure-function paradigm implies that proteins with high intrinsic disorder would be less critical for cellular survival. Recent discoveries have suggested that some intrinsically disordered proteins or even fully disordered proteins without any apparent tertiary structures are essential. However, the biological significance of such disordered proteins is not comprehensively understood. Here, using genome-wide CRISPR screening, we demonstrated that highly or fully disordered proteins show comparable essentiality to well-folded proteins. We found that the proportion of essential proteins is comparable across proteins of varying disorder levels, although structured proteins are more prevalent among essential genes. Focusing on FAM32A, one of the essential, fully disordered proteins identified in our screen, we show that its depletion leads to increased intron retention and downregulation of many other essential genes. These findings reshape our understanding of the structure-function paradigm, highlighting that fully disordered proteins can be essential for cellular viability.},
}
@article {pmid40865518,
year = {2025},
author = {Hong, D and Lyu, Y and Nayak, R and Becker, JS and Booker, MA and Masuzawa, K and Devos, Z and Wang, T and Saito, S and Liu, Q and Li, Y and Li, Z and Knelson, EH and Thai, T and Duplaquet, L and Laimon, YN and Roberti De Oliveira, G and Signoretti, S and Doench, JG and Barbie, DA and Tolystorukov, MY and Qi, J and Bernstein, BE and Ge, Y and Oser, MG},
title = {Loss of NOTCH2 creates a TRIM28-dependent vulnerability in small cell lung cancer.},
journal = {Developmental cell},
volume = {60},
number = {24},
pages = {3462-3479.e13},
doi = {10.1016/j.devcel.2025.07.023},
pmid = {40865518},
issn = {1878-1551},
mesh = {*Receptor, Notch2/metabolism/genetics ; *Tripartite Motif-Containing Protein 28/metabolism/genetics ; *Small Cell Lung Carcinoma/genetics/metabolism/pathology ; Animals ; Humans ; *Lung Neoplasms/metabolism/genetics/pathology ; Mice ; Cell Line, Tumor ; CRISPR-Cas Systems ; Signal Transduction ; },
abstract = {Small cell lung cancer (SCLC) is a highly aggressive malignancy that lacks effective targeted therapies, in part due to frequent loss-of-function mutations in tumor suppressors and the absence of recurrent oncogenic drivers. Approximately 15% of SCLCs harbor inactivating mutations in NOTCH1 or NOTCH2, and most neuroendocrine-high SCLCs exhibit low NOTCH activity. Using CRISPR-Cas9 screening in primary cell lines derived from NOTCH1/2-isogenic SCLC genetically engineered mouse models, we identified TRIM28 as a synthetic lethal dependency in NOTCH2-inactivated SCLCs. Loss of TRIM28 in this context robustly induced expression of endogenous retroviruses (ERVs), activated viral sensing pathways, and triggered a type I interferon response. Mechanistically, NOTCH2 inactivation increased reliance on TRIM28-mediated ERV silencing, creating a hyperdependence on TRIM28 via the STING-MAVS-TBK1 axis. Notably, TRIM28 was essential for tumor growth only in the setting of NOTCH2 loss. These findings identify TRIM28 as a potential therapeutic target in NOTCH2-deficient or low-NOTCH2-expressing SCLC.},
}
@article {pmid40681864,
year = {2025},
author = {Ronda, C and Perdue, T and Schwanz, L and Rivera Gelsinger, D and Brockmann, L and Kaufman, A and Huang, Y and Sternberg, SH and Wang, HH},
title = {Precise virulence inactivation using a CRISPR-associated transposase for combating Enterobacteriaceae gut pathogens.},
journal = {Nature biomedical engineering},
volume = {9},
number = {12},
pages = {2017-2027},
pmid = {40681864},
issn = {2157-846X},
support = {1R01EB031935//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 2R01AI132403//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1R01DK118044//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1R21AI146817//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; DP2HG011650//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R21AI68976//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01EB031935//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; S-168-4X5-001//U.S. Department of Defense (United States Department of Defense)/ ; 1016691//Burroughs Wellcome Fund (BWF)/ ; 527896//Simons Foundation/ ; },
mesh = {Animals ; *Transposases/genetics/metabolism ; Mice ; *Enterobacteriaceae/pathogenicity/genetics ; Virulence/genetics ; *CRISPR-Cas Systems/genetics ; *Gastrointestinal Microbiome/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Shiga Toxin/genetics ; *Enterobacteriaceae Infections/microbiology ; Humans ; },
abstract = {Targeted gene manipulation in a complex microbial community is an enabling technology for precise microbiome editing. Here we introduce BACTRINS, an in situ microbiome engineering platform designed for efficient and precise genomic insertion of a desired payload and simultaneous knockout of target genes. This system leverages conjugation-mediated delivery of CRISPR-associated transposases to achieve RNA-guided genomic integration, allowing precise insertion of a therapeutic payload while neutralizing pathogen virulence without causing cell death. When applied against an Enterobacteriaceae Shiga toxin-producing pathogen in the gut, this system delivers a CRISPR-associated transposase by bacterial conjugation for site-specific inactivation of the Shiga toxin gene and integration of a nanobody therapeutic payload to disrupt pathogen attachment. A single dose of this therapy results in high-efficiency Shiga gene inactivation and improved survival in a murine infection model of Shiga-producing pathogen. This work establishes a new type of live bacterial therapeutic capable of reducing gut infections by transforming toxigenic pathogens into commensal protectors.},
}
@article {pmid41385544,
year = {2025},
author = {Fu, YZ and Luo, FF and Yang, L and Zhang, YX and Li, JY and Wang, SY and Zhang, Y and Wang, YY},
title = {SPNS1 is an essential cellular factor for EV-A71 by acting as a transporter of viral pocket factor.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {50},
pages = {e2510020122},
doi = {10.1073/pnas.2510020122},
pmid = {41385544},
issn = {1091-6490},
support = {2023YFC2306100//The National Key R&amp;D Program China/ ; 82372230//The National Natural Science Foundation of China/ ; U23A20168//The National Natural Science Foundation of China/ ; No.2022338//The Youth Innovation Promotion Association CAS/ ; },
mesh = {Humans ; Animals ; *Enterovirus A, Human/physiology/metabolism/genetics ; Virus Replication ; *Enterovirus Infections/virology/metabolism/genetics ; Lysosomes/metabolism ; Mice ; Capsid Proteins/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; Endosomes/metabolism ; Receptors, Scavenger ; Lysosomal Membrane Proteins ; },
abstract = {Human enterovirus A71 (EV-A71) is a major cause of hand, foot and mouth disease. Cellular factors critical for EV-A71 infection remain enigmatic. Here, we performed CRISPR/Cas9 screens and identified sphingolipid transporter 1 (SPNS1) as an essential factor for EV-A71. SPNS1 deficiency inhibits infection of EV-A71 and 9 of 11 examined enteroviruses. Mechanistically, the endo/lysosomal localization of SPNS1 and the acidification of the endo/lysosomes are essential for SPNS1 to support EV-A71 infection. SPNS1 deficiency inhibits EV-A71 genomic RNA replication, but barely affects replication of EV-A71 RNA directly transfected into the cytoplasm. SPNS1 interacts with the EV-A71 capsid protein VP1 and entry receptor SCARB2 in the endo/lysosomes, where it acts as a transporter to release the viral pocket factor into the cytosol, leading to uncoating. Animal experiments show that SPNS1 deficiency results in reduced viral loads, pathological effects, and lethality following EV-A71 infection. Our findings collectively identified SPNS1 as a transporter of the EV-A71 viral pocket factor.},
}
@article {pmid41385323,
year = {2025},
author = {Zahm, AM and Cranney, CW and Gormick, AN and Rondem, KE and Schmitz, B and Himes, SR and English, JG},
title = {ConSeqUMI, an error-free nanopore sequencing pipeline to identify and extract individual nucleic acid molecules from heterogeneous samples.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41385323},
issn = {1362-4962},
support = {1DP2GM146247-01/GM/NIGMS NIH HHS/United States ; //NIH/ ; },
mesh = {*Nanopore Sequencing/methods ; Humans ; SARS-CoV-2/genetics ; Spike Glycoprotein, Coronavirus/genetics ; COVID-19/virology ; High-Throughput Nucleotide Sequencing/methods ; CRISPR-Cas Systems ; *Sequence Analysis, DNA/methods ; Dependovirus/genetics ; *Nucleic Acids/genetics/isolation &amp; purification ; Genome, Viral ; },
abstract = {Nanopore sequencing has revolutionized genetic analysis by offering linkage information across megabase-scale genomes. However, the high intrinsic error rate of nanopore sequencing impedes the analysis of complex heterogeneous samples, such as viruses, bacteria, complex libraries, and edited cell lines. Achieving high accuracy in single-molecule sequence identification would significantly advance the study of diverse genomic populations, where clonal isolation is traditionally employed for complete genomic frequency analysis. Here, we introduce ConSeqUMI, an innovative experimental and analytical pipeline designed to address long-read sequencing error rates using unique molecular indices for precise consensus sequence determination. ConSeqUMI processes nanopore sequencing data without the need for reference sequences, enabling accurate assembly of individual molecular sequences from complex mixtures. We establish robust benchmarking criteria for this platform's performance and demonstrate its utility across diverse experimental contexts, including mixed plasmid pools, recombinant adeno-associated virus genome integrity, and CRISPR/Cas9-induced genomic alterations. Furthermore, ConSeqUMI enables detailed profiling of human pathogenic infections, as shown by our analysis of severe acute respiratory syndrome coronavirus 2 spike protein variants, revealing substantial intra-patient genetic heterogeneity. Lastly, we demonstrate how individual clonal isolates can be extracted directly from sequencing libraries at low cost, allowing for post-sequencing identification and validation of observed variants. Our findings highlight the robustness of ConSeqUMI in processing sequencing data from UMI-labeled molecules, offering a critical tool for advancing genomic research.},
}
@article {pmid41384994,
year = {2025},
author = {Zhang, J and Liu, J and Bayani, A},
title = {Phage therapy and the microbiome in hematologic malignancies: opportunities, mechanisms, and early evidence.},
journal = {Journal of cancer research and clinical oncology},
volume = {152},
number = {1},
pages = {8},
pmid = {41384994},
issn = {1432-1335},
mesh = {Humans ; *Hematologic Neoplasms/therapy/microbiology/immunology ; *Phage Therapy/methods ; Animals ; *Bacteriophages ; *Gastrointestinal Microbiome ; *Microbiota ; },
abstract = {Hematologic malignancies remain among the most difficult cancers to treat, challenged by profound heterogeneity, treatment-induced immune dysfunction, and the frequent emergence of drug resistance. Beyond tumor-intrinsic mechanisms, dysbiosis of the gut microbiome is increasingly recognized as a critical determinant of therapeutic outcomes, shaping hematopoiesis, immune responses, and drug metabolism. Bacteriophage (phage) therapy has re-emerged as a precision tool capable of selectively eradicating pathogenic taxa while preserving commensal short-chain fatty acid-producing communities. Preclinical and early human studies demonstrate that phages can recalibrate microbial ecosystems, disrupt antibiotic-tolerant biofilms, and enrich metabolites such as butyrate that support mucosal integrity and immune balance. Mechanistically, phage DNA enriched with CpG motifs engages Toll-like receptor 9, activating dendritic cells and enhancing cytotoxic T lymphocyte responses, suggesting dual benefits in infection control and anti-tumor immunity. Emerging applications extend further, with engineered phages serving as vectors for CRISPR-Cas gene editing, targeted cytokine delivery, and nanocarrier platforms for leukemia therapy. Despite translational promise, major hurdles persist, including immunogenicity, horizontal gene transfer, resistance evolution, and regulatory uncertainty. Addressing these challenges through GMP-compliant manufacturing, metagenomics-guided personalization, and AI-optimized cocktail design could establish phage therapy as a microbiome-informed adjunct to overcome drug resistance in blood cancers. However, direct clinical evidence of phage therapy efficacy in hematologic malignancies remains limited, and current data are largely derived from preclinical and compassionate-use contexts.},
}
@article {pmid41381927,
year = {2025},
author = {Yousuf, F and Solanki, M and Singh, SS and Ch, SR and Neeraja, CN and Sundaram, RM and Mangrauthia, SK},
title = {Tissue culture optimization and genome editing for yield improvement of an Indian rice landrace Chittimuthyalu.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {54},
pmid = {41381927},
issn = {1573-9368},
support = {ICAR-EFC Sub-Scheme 10: Enhancing climate resilience and ensuring food security with genome editing tools//Indian Council of Agricultural Research/ ; },
mesh = {*Oryza/genetics/growth &amp; development/drug effects ; *Gene Editing/methods ; *Tissue Culture Techniques/methods ; Plant Breeding ; India ; *Plants, Genetically Modified/genetics/growth &amp; development ; Purines/pharmacology ; CRISPR-Cas Systems ; Phenylurea Compounds ; Thiadiazoles ; },
abstract = {Chittimuthyalu, a rice landrace from Southern India, is known for its pleasant aroma, rich nutritive value, and excellent cooking qualities. However, it has a poor plant type (tall and weak stem prone to lodging) and is low yielding. The efforts to improve such valuable rice accessions with existing cross-breeding or random mutagenesis often result in undesirable traits due to linkage drag or untargeted mutations in large numbers. Genome editing, the most precise breeding tool, offers a viable solution to address such issues. In this study, we developed an efficient tissue culture protocol for callus induction, transformation, and regeneration of Chittimuthyalu. The highest callus induction frequency was achieved on L3 basal media enriched with 2.5 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D) and 600 mg/l of both proline and glutamine. For regeneration, a combination of Thidiazuron (TDZ), 6-Benzylaminopurine (BAP), and kinetin yielded an optimal regeneration frequency. The optimized tissue culture protocol was utilized to transform a multiplex gene editing construct developed by combining the four guide RNAs designed from yield and disease resistance-associated genes OsDEP1, OsTB1, OsCKX2, and OsSWEET14. The OsDEP1genome-edited rice plants exhibit thicker culm, enhanced grain size, ~ 100% increase in the thousand-grain weight, and ~ 50% increase in total grain yield per plant. The optimized tissue culture protocol and development of further edits in the remaining genes will pave the way for improving the agronomic traits of Chittimuthyalu. This study also highlights much-needed efforts to develop efficient tissue culture and genome editing methods for wild rice species and landraces, which will help bring these hardy, climate-resilient, and nutrient-rich accessions into mainstream cultivation.},
}
@article {pmid41381092,
year = {2025},
author = {Lane, KR and Jones, SE and Osborne, TH and Geller-McGrath, D and Nwaobi, BC and Chen, L and Thomas, BC and Hudson-Edwards, KA and Banfield, JF and Santini, JM},
title = {Bioleaching Microbial Community Metabolism and Composition Driven by Copper Sulphide Mineral Type.},
journal = {Environmental microbiology reports},
volume = {17},
number = {6},
pages = {e70261},
pmid = {41381092},
issn = {1758-2229},
support = {NE/L002485/1//Natural Environment Research Council/ ; BB/N012674/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //Hellenic Coppers Mines Ltd/ ; },
mesh = {*Copper/metabolism/chemistry ; *Bacteria/metabolism/genetics/classification/isolation &amp; purification ; *Archaea/metabolism/genetics/classification/isolation &amp; purification ; *Sulfides/metabolism/chemistry ; *Microbial Consortia ; Metagenomics ; *Minerals/metabolism/chemistry ; *Microbiota ; Plasmids/genetics ; },
abstract = {Copper bioleaching is a green technology for the recovery of copper from chalcopyrite (CuFeS2) and chalcocite (Cu2S) ores. Much remains to be learned about how mineral type and surface chemistry influence microbial community composition. Here, we established a microbial consortium from a copper bioleaching column in Cyprus on chalcopyrite and then sub-cultured it to chalcocite to investigate how the community composition shifts due to changes in mineral structure and the absence of mineral-derived Fe. The solution chemistry was determined and microbial communities characterised by genome-resolved metagenomics after 4 and 8 weeks of cultivation. Acidithiobacillus species and strains, a Rhodospirilales, Leptospirillum ferrodiazotrophum and Thermoplasmatales archaea dominated all enrichments, and trends in abundance patterns were observed with mineralogy and surface-attached versus planktonic conditions. Many bacteria had associated plasmids, some of which encoded metal resistance pathways, sulphur metabolic capacities and CRISPR-Cas loci. CRISPR spacers on an Acidithiobacillus plasmid targeted plasmid-borne conjugal transfer genes found in the same genus, likely belonging to another plasmid, evidence of intra-plasmid competition. We conclude that the structure and composition of metal sulphide minerals select for distinct consortia and associated mobile elements, some of which have the potential to impact microbial activity during sulphide ore dissolution.},
}
@article {pmid41380995,
year = {2025},
author = {Roy, S and Nandy, S and Morita, D and Nandy, RK and Veeraraghavan, B and Walia, K and Das, S and Basu, S},
title = {Genomic analysis of a novel high-risk ST5217/ExoU+/O11 clone of carbapenem-resistant OXA-181- and VIM-2-producing Pseudomonas aeruginosa in India.},
journal = {Journal of global antimicrobial resistance},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgar.2025.12.002},
pmid = {41380995},
issn = {2213-7173},
abstract = {OBJECTIVES: Studies of carbapenem-resistant Pseudomonas aeruginosa (CRPA)-harbouring OXA-48-like carbapenemases are rare. The study aimed to report the emergence and characterization of a novel high-risk clone of CRPA-harbouring OXA-48-like from India.<br><br>METHODS: Identification, AST, phenotypic detection of carbapenemases and WGS using Ion-Torrent-S5 platform were carried out. Analyses included ResFinder, VFDB, MLST, PAst, Phastest and CRISPR/Cas. SNP-based phylogenetic analysis with global OXA-48-like-harbouring CRPA genomes was carried out by CSI Phylogeny and iTOL for visualization.<br><br>RESULTS: The clinical strain of CRPA AMRIR00655 belonged to a novel sequence type ST5217 and serotype O11. The strain was MDR. Phenotypic tests followed by WGS revealed the presence of dual carbapenemases, OXA-181 (serine-carbapenemase) and VIM-2 (zinc-carbapenemase), both located on chromosome. 4,261 bp of blaOXA-181-bearing contig-DNA showed 100% homology to K. pneumoniae plasmid pKP3-A. ISEcp1 was present on upstream and on downstream, &#x25b3;lysR, &#x25b3;ereA and repA genes were detected. blaVIM-2 was located within class 1 integron along with aacC6-II, dfrB5, aac(3)-Id, tniC in surrounding regions. Presence of other ARGs (blaPAO, blaOXA-488,aph(3'')-Ib, aph(6)-Id, crpP, catB7, fosA, sul2) and efflux-pump genes might explain its MDR phenotype. Virulence factors including T3SS (PscF, PopB, PopD, PcrV) and its effectors (ExoT, ExoU, ExoY) indicated the pathogenic potential of ST5217. Core genome analysis showed that ST5217 was closest with other high-risk clones ST1339 and ST773-harbouring blaOXA-48-like.<br><br>CONCLUSIONS: To the best of our knowledge, this is the first report of blaOXA-181-harbouring novel high-risk clone of CRPA ST5217/ExoU+/O11 in India which emphasises the spread of OXA-181 among bacteria other than Enterobacteriaceae-family and warrant close monitoring.},
}
@article {pmid41377346,
year = {2025},
author = {Abedin, ZU and Waggan, AI and Khan, E and Suleman, MU and Tabassum, SN},
title = {Letter to the Editor: CRISPR-based gene editing for cardiac protection in Barth syndrome.},
journal = {Annals of medicine and surgery (2012)},
volume = {87},
number = {12},
pages = {9163-9164},
pmid = {41377346},
issn = {2049-0801},
abstract = {Barth syndrome is a rare X-linked mitochondrial disorder caused by mutations in the Tafazzin (TAZ) gene. These mutations make it hard for cardiolipin to remodel and mitochondria to work properly. This condition is characterized by growth retardation, neutropenia, skeletal myopathy, and dilated cardiomyopathy, frequently leading to significant morbidity and mortality, with numerous patients necessitating heart transplants. There are no treatments available at this time to fix the genetic problem. Recent progress in gene editing, especially CRISPR-based methods, holds great promise for fixing TAZ mutations. Research utilizing patient-derived cardiomyocytes has demonstrated that the rectification of TAZ mutations reinstates mitochondrial efficiency and enhances cellular functionality. Animal models, including TAZ-knockout mice, have exhibited substantial enhancements in cardiac function, survival rates, and diminished fibrosis subsequent to gene replacement therapy.},
}
@article {pmid41285673,
year = {2025},
author = {Munusamy, S and Jahani, R and Zheng, H and Chen, J and Kong, J and Zhao, Y and Guan, L and Zhou, S and Guan, X},
title = {Enhanced CRISPR-Cas12a Fluorescent Assay for Detecting Trace Levels of Procalcitonin.},
journal = {ACS applied bio materials},
volume = {8},
number = {12},
pages = {10818-10826},
doi = {10.1021/acsabm.5c01535},
pmid = {41285673},
issn = {2576-6422},
mesh = {*Procalcitonin/analysis/blood ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Biocompatible Materials/chemistry/chemical synthesis ; Gold/chemistry ; Materials Testing ; Metal Nanoparticles/chemistry ; Particle Size ; Fluorescence ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and accurate detection of procalcitonin (PCT), a major biomarker for bacterial infections and sepsis, remains a pressing need in clinical diagnostics because sepsis progresses rapidly and may initially present with nonspecific or even subtle symptoms. Herein, we report a CRISPR-Cas12a-based fluorescence biosensing platform for ultrasensitive detection of PCT. The platform employs antibody-functionalized magnetic beads (MBs) for specific protein enrichment and antibody- and oligonucleotide- dual-functionalized gold nanoparticles (AuNPs) for high-density DNA payload. After sandwich complex formation with the target PCT, a programmed ssDNA strand is released by thermal denaturation, which then activates Cas12a collateral cleavage, thereby generating a fluorescence signal. Thorough physicochemical characterizations, including zeta potential, dynamic light scattering, UV-vis spectroscopy, and TEM, were carried out to confirm the successful functionalization of MBs and AuNPs. The developed PCT sensor was highly sensitive with a limit of detection (LOD) reaching 3 pg/mL. Moreover, the biosensor exhibited an excellent specificity toward PCT against clinically relevant interferents such as C-reactive protein (CRP), interleukin-2β (IL-2β), interleukin-6 (IL-6), human serum albumin (HSA), and bovine serum albumin (BSA), and simulated serum sample analysis was successfully carried out with the recoveries ranging from 108 to 122%. The PCT sensing technique developed in this work offers the potential to be expanded to construct a multiplexing platform for simultaneous detection of multiple biomarker species for early and accurate disease diagnosis.},
}
@article {pmid41272938,
year = {2025},
author = {Salvador, PJ and Lin, S and Chinn, MM and Jauregui-Matos, V and Manjunath, A and Yang, I and Jacobsen, CS and Beal, PA},
title = {Discovery and Tuning of RNA Editing Guides via High-Throughput Screening and Chemical Modification.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {26},
number = {24},
pages = {e202500735},
doi = {10.1002/cbic.202500735},
pmid = {41272938},
issn = {1439-7633},
mesh = {Humans ; *RNA Editing ; *High-Throughput Screening Assays ; HEK293 Cells ; Adenosine Deaminase/metabolism/genetics ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Structure-Activity Relationship ; RNA-Binding Proteins/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; },
abstract = {Adenosine deaminases acting on RNA (ADAR) catalyze the deamination of adenosine to inosine in double-stranded RNA. Because inosine is read as guanosine during translation, this process enables programmable A-to-G recoding at the transcript level. ADARs can be harnessed for therapeutic correction of pathogenic mutations through site-directed RNA editing with guide RNAs. To expand the design space of editing-enabling guides, we applied EMERGe, a high-throughput screening platform, to identify motifs targeting a premature termination codon in the MeCP2 transcript associated with Rett syndrome. This uncovered a guide RNA motif that supported efficient ADAR2-mediated editing in vitro, featuring a 5'-GUG-3' sequence predicted to form an asymmetric loop. To enable therapeutic application, structure-activity relationship studies and chemical optimization were performed, yielding a fully modified guide RNA with 2'-O-methyl, 2'-fluoro, and phosphorothioate linkages. This stabilized guide retained the activity of unmodified RNA and showed enhanced nuclease resistance. The optimized guide induces dose-dependent editing at two MECP2 loci in reporter assays in HEK293T cells, demonstrating that EMERGe-selected motifs can be rendered viable in cells through targeted chemical modification. These findings highlight the utility of EMERGe as a discovery platform and establish a pipeline for identifying and optimizing editing-enabling guide RNA features beyond traditional design rules.},
}
@article {pmid41266370,
year = {2025},
author = {Giddins, M and Kratz, AF and De Los Santos, MB and Forget, A and Tiwari, R and Jang, G and Blazejewski, T and Qin, C and Huang, Y and Lao, YH and Falconer, T and Leong, KW and Krogan, N and Staller, M and Wang, H and Wei, L and Chavez, A},
title = {Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11114},
pmid = {41266370},
issn = {2041-1723},
support = {HR0011-19-2-0009//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; DP2NS131566-01//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1R01EB031935//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; U54 CA274502/CA/NCI NIH HHS/United States ; U54 CA274502/CA/NCI NIH HHS/United States ; },
mesh = {*Protein Engineering/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; HEK293 Cells ; Protein Domains ; Recombinant Fusion Proteins/genetics/metabolism ; },
abstract = {Current protein engineering methods are inadequate to explore the combinatorial potential offered by nature's vast repertoire of protein domains-limiting our ability to create optimal synthetic tools. To overcome this barrier, we develop an approach to create and test thousands of chimeric proteins and employ it to probe an expansive combinatorial landscape of over 15,000 multi-domain CRISPR activators. Our findings indicate that many activators produce substantial cellular toxicity, often unrelated to their capacity to regulate gene expression. We also explore the biochemical features of activation domains and determine how their combinatorial interactions shape activator behavior. Finally, we identify two potent CRISPR activators, MHV and MMH, and demonstrate their enhanced activity across diverse targets and cell types compared to the gold-standard MCP activator, synergistic activation mediator (SAM).},
}
@article {pmid41102841,
year = {2025},
author = {Li, X and Lv, H and Wu, C and Li, H and Yi, W and Niu, X and Peng, Q and Meng, C and Yuan, Y and Tian, S and Wang, Z and Fang, R and Zhou, Z},
title = {Corynebacterium pseudotuberculosis phospholipase D targets mitochondrial sphingomyelin and induces NLRP3-GSDMD axis-mediated pyroptosis in macrophages to promote infection.},
journal = {Veterinary research},
volume = {56},
number = {1},
pages = {198},
pmid = {41102841},
issn = {1297-9716},
support = {2021YFD1800800//National Key Research and Development Program of China/ ; CSTC2021JCYJ-MSXMX0884//Natural Science Foundation of Chongqing/ ; CSTB2024NSCQ-MSX1262//Natural Science Foundation of Chongqing/ ; XDJK2018C054//Fundamental Research Funds for the Central Universities/ ; XDJK2020B016//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Phospholipase D/metabolism ; *Sphingomyelins/metabolism ; *Corynebacterium Infections/enzymology/pathology/veterinary ; Mitochondria/physiology ; Pyroptosis/physiology ; *Macrophages/microbiology/physiology ; Bacterial Zoonoses/enzymology/pathology ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism ; Gasdermins/metabolism ; Phosphate-Binding Proteins/metabolism ; Mice, Inbred C57BL ; Animals ; Mice/microbiology ; CRISPR-Cas Systems ; *Corynebacterium pseudotuberculosis/enzymology/physiology ; Animals, Outbred Strains ; },
abstract = {Infection by Corynebacterium pseudotuberculosis (Cp), a facultative intracellular bacterium, causes mainly purulent inflammation and chronic granulomas in animals and humans. Pyroptosis is a proinflammatory form of programmed cell death that is important in response to pathogen infection. The role of pyroptosis in Cp infection and the related mechanisms are still unclear. Here, we reveal that Cp infection induces pyroptosis in macrophages. The inhibition of pyroptosis by disulfiram (DSF) or dimethyl fumarate (DMF) decreases the pathogenicity of Cp in mice and prevents the escape of this pathogen from infected macrophages. In contrast, LPS treatment achieves the opposite results in mice and macrophages infected with Cp. In addition, we revealed that phospholipase D (PLD) is a key virulence factor that induces pyroptosis and subsequently promotes Cp escape and spread in macrophages and confirmed that mutation of the enzyme active site at D66S, G80I, K114N, and G242P weakens PLD-induced pyroptosis. Furthermore, the indispensable role of the NLRP3-GSDMD axis in PLD-induced pyroptosis was confirmed using Nlrp3 knockout (Nlrp3[-/-]) and Gsdmd knockout (Gsdmd[-/-]) macrophages. Mechanistically, the PLD of Cp targets mitochondrial sphingomyelins within macrophages, induces cardiolipin externalization to the outer mitochondrial membrane, and releases mtROS, leading to pyroptosis. In conclusion, our data indicate that Cp infection induces mitochondrial dysfunction in macrophages through the secretion of PLD, which targets mitochondrial sphingomyelins and induces NLRP3-GSDMD axis-dependent pyroptosis. Inducing pyroptosis is one of the mechanisms underlying the pathogenesis and transmission of Cp, and inhibiting pyroptosis may be an important strategy for controlling this pathogen.},
}
@article {pmid40991373,
year = {2025},
author = {Karimzadeh, A and Kim, R and Garcia, V and Florea, M and Peacker, BL and Kobayashi, S and Watkins, D and Messemer, K and Zeng, J and Bauer, DE and Serwold, T and Wagers, AJ},
title = {In situ gene editing of hematopoietic stem cells via AAV-delivered CRISPR guide RNAs.},
journal = {Blood advances},
volume = {9},
number = {24},
pages = {6563-6574},
doi = {10.1182/bloodadvances.2025016775},
pmid = {40991373},
issn = {2473-9537},
mesh = {*Gene Editing/methods ; *Hematopoietic Stem Cells/metabolism/cytology ; *Dependovirus/genetics ; Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Mice ; *CRISPR-Cas Systems ; Mice, Transgenic ; Genetic Vectors/genetics ; Humans ; },
abstract = {Hematopoietic stem cells (HSCs) are self-renewing, multipotent, and engraftable precursors of all blood cells. Efficient delivery of therapeutic gene products and gene editing machinery to correct disease-causing gene variants in endogenous HSCs while they remain in the body holds exciting potential to leverage HSC potency for the treatment of monogenic blood disorders. Toward this goal, we used adeno-associated virus (AAV) to deliver CRISPR guide RNAs (gRNAs) to edit HSC genomes in situ in Ai9;SpCas9-EGFP transgenic mice carrying a Cas9-activatable Lox-STOP-Lox-tdTomato reporter cassette together with a constitutive SpCas9-2A-EGFP. Using a variety of conditions and vector designs, we tested whether systemic administration to these mice of AAVs carrying SpCas9-compatible gRNAs designed to cut DNA upstream and downstream of the STOP cassette would induce tdTomato expression in HSCs. Our findings identify self-complementary AAVs (scAAVs) and increased ratio of guide to Cas9 as parameters facilitating higher editing efficiency. Of note, we find preserved multilineage output and engraftability of HSCs upon scAAV-gRNA editing. In an example application of this technology, we explore the potential for in situ HSC gene editing by dual AAV-CRISPR delivery and demonstrate robust gene modification, concurrent with induction of therapeutic fetal hemoglobin, in a sickle cell disease mouse model modified to express SpCas9. In summary, this work offers a sensitive and adaptable platform that allows robust modification of HSC genomes in situ.},
}
@article {pmid40542106,
year = {2025},
author = {Wu, X and Luo, S and Guo, C and Zhao, Y and Zhong, J and Hu, R and Yang, X and Liu, C and Zhang, Q and Zhuang, S and Chen, Y and Liu, Y and Zhang, X},
title = {LbuCas13a directly targets DNA and elicits strong trans-cleavage activity.},
journal = {Nature biomedical engineering},
volume = {9},
number = {12},
pages = {2141-2154},
pmid = {40542106},
issn = {2157-846X},
support = {2024A1515011877//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; 22104048//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *DNA/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Leptotrichia/enzymology/genetics ; Cytochrome P-450 CYP2C19/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {Traditionally perceived as an RNA-specific nuclease, Cas13a has been used primarily for RNA detection. We discover the ability of Leptotrichia buccalis Cas13a (LbuCas13a) to directly target DNA without the restrictions of protospacer flanking sequence and protospacer adjacent motif sequences, coupled with robust trans-cleavage activity. Contrary to conventional understanding, LbuCas13a does not degrade DNA targets. Our study reveals an enhancement in the single-nucleotide specificity of LbuCas13a against DNA compared to RNA. This heightened specificity is attributed to the lower affinity of CRISPR RNA (crRNA) towards DNA, raising the crRNA-DNA binding energy barrier. We introduce a molecular diagnostic platform called superior universal rapid enhanced specificity test with LbuCas13a (SUREST) for high-resolution genotyping. SUREST is capable of detecting DNA concentrations of CYP2C19 (rs4986893) as minute as 0.3 aM (0.18 cps µl[-1]). We also apply SUREST to human genotyping scenarios, indicating that SUREST performs well across a broad range of mutations and sequence contexts. SUREST represents an advancement in real-time nucleic acid detection, making it a useful tool for pathogen identification and mutation analysis in clinical diagnostics.},
}
@article {pmid41375334,
year = {2025},
author = {Lai, CM and Xiao, XS and Liu, LW and Lin, XD and Dou, DL and Cai, HY and Mei, ZF and Yang, F and Cheng, Y and Qin, Y},
title = {Nanotechnology Strategies in Plant Genetic Engineering: Intelligent Delivery and Precision Editing.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {23},
pages = {},
pmid = {41375334},
issn = {2223-7747},
support = {2024NZ029029//Major Science and Technology Project of Fujian Province/ ; },
abstract = {Plant genetic engineering is crucial for enhancing crop yield, quality, and resilience to both abiotic and biotic stresses, thereby promoting sustainable agriculture. Agrobacterium-mediated, biolistic bombardment, electroporation, and poly (ethylene glycol) (PEG)-mediated genetic transformation systems are widely applied in plant genetic engineering. However, these systems have limitations, including species dependency, destruction of plant tissues, low transformation efficiency, and high cost. Recently, gene-delivery methods based on nanotechnology have been developed for plant genetic transformation. This nanostrategy demonstrates remarkable transformation efficiency, excellent biocompatibility, effective protection of exogenous nucleic acids, and the potential for plant regeneration. However, the application of nanomaterial-mediated gene-delivery systems in plants is still in its early stages and faces numerous challenges for widespread adoption. Herein, the conventional genetic transformation techniques utilized in plants are succinctly examined. Subsequently, the advancements in nanomaterial-based gene-delivery systems are reviewed. The applications of CRISPR-Cas-mediated genome editing and its integration with plant nanotechnology are also examined. The innovations, methods, and practical applications of nanomaterial-mediated genetic transformation summarized herein are expected to facilitate the progress of plant genetic engineering in modern agriculture.},
}
@article {pmid41375204,
year = {2025},
author = {Li, W and Shi, Y and Li, D and Wang, Y and Sun, Y and Li, H and Han, Y},
title = {A CRISPR Powered Immobilization-Free, Amplification-Free Carbon-Nanotube Field-Effect Transistor (FET) Sensor for Influenza A Virus (IAV).},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {23},
pages = {},
pmid = {41375204},
issn = {1420-3049},
support = {2023YFC2605101//National Key Research and Development Program of China/ ; },
mesh = {*Influenza A virus/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; Transistors, Electronic ; *Nanotubes, Carbon/chemistry ; Humans ; *RNA, Viral/genetics/analysis ; CRISPR-Cas Systems ; Limit of Detection ; *Influenza, Human/diagnosis/virology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The epidemic of infectious diseases, such as influenza A, has imposed a severe health burden on the population. Early detection, diagnosis, reporting, isolation, and treatment are crucial for the prevention, control, and management of infectious diseases. Nucleic acid testing represents a vital approach for the rapid diagnosis of pathogenic microorganism types. However, current nucleic acid detection methods face notable bottlenecks: traditional CRISPR fluorescence assays require time-consuming pre-amplification of target nucleic acids, while existing carbon-nanotube field-effect transistor (FET)-based platforms, though amplification-free, often necessitate complex chip surface modification and probe immobilization, and suffer from non-reusable chips, all limiting their utility in point-of-care testing (POCT) and large-scale screening. This study reports a CRISPR-based amplification-free RNA detection platform (CRISPR-FET) for the rapid identification of influenza A virus. The CRISPR-FET platform described herein enables the detection of viral RNA without amplification within 20 min, with a limit of detection as low as 1 copy/μL. Secondly, a reporter RNA conjugated with gold particles is used to achieve signal amplification in FET detection; meanwhile, the method eliminates probe immobilization, thereby omitting this step and simplifying chip modification to reduce complex work-flows and pre-treatment costs. The chip's reusability further enhances cost-effectiveness. Additionally, streptavidin-modified magnetic bead adsorption minimizes background errors from excessive reporter RNA and non-target nucleic acids. Finally, validation with 24 clinical samples confirmed the platform's efficacy. By integrating rapidity, simplicity, and high sensitivity, alongside cost advantages from reusable chips, this CRISPR-FET platform meets the critical need for early influenza A diagnosis and holds promise for advancing POCT and large-scale epidemiological screening.},
}
@article {pmid40968311,
year = {2025},
author = {Park, H and Yu, S and Koo, T},
title = {Gene editing in cancer therapy: overcoming drug resistance and enhancing precision medicine.},
journal = {Cancer gene therapy},
volume = {32},
number = {12},
pages = {1293-1302},
pmid = {40968311},
issn = {1476-5500},
mesh = {Humans ; *Gene Editing/methods ; *Precision Medicine/methods ; *Neoplasms/therapy/genetics ; *Drug Resistance, Neoplasm/genetics ; CRISPR-Cas Systems ; *Genetic Therapy/methods ; Animals ; },
abstract = {The CRISPR system has revolutionized cancer gene therapy, offering unparalleled precision in genetic manipulation for targeted oncogene disruption, mutation correction, and immune system modulation. This breakthrough tool has demonstrated remarkable potential in overcoming drug resistance, enhancing chemotherapy sensitivity, and improving immunotherapy strategies such as CRISPR-engineered CAR-T cells. Additionally, oncolytic virus-mediated CRISPR delivery has emerged as a novel approach for tumor-specific gene editing, minimizing off-target effects. The rapid transition of CRISPR-based cancer therapeutics from preclinical research to clinical trials underscores its therapeutic potential. This review explores the latest advancements in CRISPR applications for cancer therapy, including gene knockout, base editing for mutation correction, and integration with immune and viral therapies. Despite significant progress, challenges such as off-target effects, immune responses, and delivery limitations remain key hurdles. We discuss current strategies to enhance CRISPR safety and efficacy, emphasizing its potential for personalized cancer treatment.},
}
@article {pmid40753800,
year = {2025},
author = {Behrouzian Fard, G and Ahmadi, MH and Gholamin, M and Hosseini Bafghi, M},
title = {CRISPR-Cas9: a prominent genome editing tool in the management of inherited blood disorders and hematological malignancies.},
journal = {Current research in translational medicine},
volume = {73},
number = {4},
pages = {103531},
doi = {10.1016/j.retram.2025.103531},
pmid = {40753800},
issn = {2452-3186},
mesh = {Humans ; *Gene Editing/methods/trends ; *CRISPR-Cas Systems/genetics ; *Hematologic Neoplasms/therapy/genetics ; Genetic Therapy/methods ; *Hematologic Diseases/therapy/genetics ; },
abstract = {Several hematologic diseases with genetic defects, like sickle cell disease and β-thalassemia can be treated with allogeneic hematopoietic stem cell transplantation (HSCT) from healthy donors. However, suitable tissue-matched donors are often unavailable, and HSCT involves risks such as graft-versus-host disease and potential disease relapse. Due to the genetic heterogeneity of blood disorders and the complexity of the hematopoietic system, identifying effective genes for managing and treating both benign and malignant conditions remains a significant challenge. The genome editing field is rapidly expanding and is essential for identifying genetic factors in pathological processes. These developments highlight the importance of using ex vivo gene therapy approaches for autologous hematopoietic stem cells. Also, gene editing technologies are gaining significant interest in engineered cell therapies for hematological malignancies . Today, various programmable nucleases are available for genome editing, with the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system standing out due to its high efficiency, low cytotoxicity, cost-effectiveness, and precision. This system can serve as a genomic modification tool for treating blood disorders, including hereditary diseases and immunotherapy for cancer using chimeric antigen receptor T cells (CAR-T cells). Advancements in CRISPR-Cas9 are expected to significantly impact medical research and clinical applications. However, challenges such as off-target effects and immunogenicity must be addressed. This review summarizes the mechanism and delivery strategies of CRISPR-Cas9, discusses its applications in treating inherited blood disorders such as sickle cell disease, β-thalassemia, and fanconi anemia, as well as hematological malignancies, and highlights the associated challenges.},
}
@article {pmid41374373,
year = {2025},
author = {Sambo, CN and Skepu, A and Nxumalo, NP and Polori, KL},
title = {Diagnostic Advances and Public Health Challenges for Monkeypox Virus: Clade-Specific Insight and the Urgent Need for Rapid Testing in Africa.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {15},
number = {23},
pages = {},
pmid = {41374373},
issn = {2075-4418},
support = {B1B0741B-9691-4333-B77E-3B8D42A7B5FF//Technology Innovation Agency/ ; },
abstract = {Background: Monkeypox (MPX), caused by the Monkeypox virus (MPOX) of the Orthopoxvirus genus, has re-emerged as a significant global health threat. Once confined to Central and West Africa, the 2022-2025 multi-country outbreaks, predominantly caused by Clade IIb, demonstrated sustained human-to-human transmission and global spread. Objective: This review summarizes current knowledge on MPX virology, epidemiology, clinical presentation, and diagnostic technologies, with a focus on innovations supporting rapid and field-deployable detection in resource-limited settings. Methods: The recent literature (2019-2025), including peer-reviewed studies, WHO and Africa CDC reports, and clinical guidelines, was critically reviewed. Data were synthesized to outline key developments in diagnostic methodologies and surveillance approaches. Results: MPX comprises two genetic clades: Clade I (Congo Basin) and Clade II (West African), which differ in virulence and transmission. Clade IIb is associated with sexual and close-contact transmission during recent outbreaks. Clinical manifestations have shifted from classic disseminated rash to localized anogenital lesions and atypical or subclinical infections. RT-PCR remains the diagnostic gold standard, while emerging assays such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and CRISPR/Cas-based platforms show promise for rapid point-of-care (POC) testing. Complementary serological tools, including ELISA and lateral flow assays, enhance surveillance and immune profiling. Conclusions: The resurgence of MPX highlights the urgent need for accessible, sensitive, and specific diagnostic platforms to strengthen surveillance and outbreak control, especially in endemic and resource-constrained regions.},
}
@article {pmid41053217,
year = {2025},
author = {Schwämmle, T and Noviello, G and Kanata, E and Froehlich, JJ and Bothe, M and Martitz, A and Altay, A and Scouarnec, J and Feng, VY and Mallie, H and Vingron, M and Schulz, EG},
title = {Reporter CRISPR screens decipher cis-regulatory and trans-regulatory principles at the Xist locus.},
journal = {Nature structural &amp; molecular biology},
volume = {32},
number = {12},
pages = {2465-2475},
pmid = {41053217},
issn = {1545-9985},
mesh = {Animals ; *RNA, Long Noncoding/genetics ; Mice ; Female ; X Chromosome Inactivation ; Transcription Factors/metabolism/genetics ; *CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; Octamer Transcription Factor-3/metabolism/genetics ; Otx Transcription Factors/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genes, Reporter ; },
abstract = {Developmental genes are controlled by an ensemble of cis-acting regulatory elements (REs), which in turn respond to multiple trans-acting transcription factors (TFs). Understanding how a cis-regulatory landscape integrates information from many dynamically expressed TFs has remained a challenge. Here we develop a combined CRISPR screening approach using endogenous RNA and RE reporters as readouts. Applied to the murine Xist locus, which is crucial for X-chromosome inactivation in females, this method allows us to comprehensively identify Xist-controlling TFs and map their TF-RE wiring. We find a group of transiently upregulated TFs, including ZIC3, that regulate proximal REs, driving the binary activation of Xist expression. These basal activators are more highly expressed in cells with two X chromosomes, potentially governing female-specific Xist upregulation. A second set of developmental TFs that include OTX2 is upregulated later during differentiation and targets distal REs. This regulatory axis is crucial to achieve high levels of Xist RNA, which is necessary for X-chromosome inactivation. OCT4 emerges as the strongest activator overall, regulating both proximal and distal elements. Our findings support a model for developmental gene regulation, in which factors targeting proximal REs drive binary on-off decisions, whereas factors interacting with distal REs control the transcription output.},
}
@article {pmid40907203,
year = {2025},
author = {Tabibian, M and Motevaseli, E and Ghafouri-Fard, S},
title = {CRISPR-mediated modulation of EGFR signaling in lung cancer.},
journal = {Cancer treatment and research communications},
volume = {45},
number = {},
pages = {100992},
doi = {10.1016/j.ctarc.2025.100992},
pmid = {40907203},
issn = {2468-2942},
mesh = {Humans ; *Lung Neoplasms/genetics/therapy/drug therapy/pathology/metabolism ; ErbB Receptors/genetics/metabolism/antagonists &amp; inhibitors ; Signal Transduction/genetics/drug effects ; *CRISPR-Cas Systems ; Gene Editing/methods ; Protein Kinase Inhibitors/pharmacology/therapeutic use ; Mutation ; },
abstract = {Lung cancer is among the most common cancers and the leading source of cancer death. Inhibition of EGFR signaling by small-molecule tyrosine kinase inhibitors and monoclonal antibodies has provided new opportunities for treatment of this type of cancer. However, prognosis remained unfavorable due to the incidence of intrinsic or attained resistance. The advent of CRISPR/Cas9 technology has offered additional chances for cancer genome editing. This technology has been applied in the context of lung cancer research in order to minimize the effects of activating EGFR mutations. In the current manuscript, we address the application of CRISPR/Cas9 method in the modulation of EGFR signaling and its consequence in the treatment of lung cancer.},
}
@article {pmid40840103,
year = {2025},
author = {Watts, A and Raipuria, RK and Chauhan, M and Mehta, K and Annamalai, M and Abbas, AZ and Bhattacharya, R and Watts, A and Singh, N},
title = {CRISPR/Cas9-mediated knockout of TRANSPARENT TESTA 8 downregulates flavonoid biosynthetic pathway in seeds of Brassica juncea.},
journal = {Plant physiology and biochemistry : PPB},
volume = {229},
number = {Pt A},
pages = {110330},
doi = {10.1016/j.plaphy.2025.110330},
pmid = {40840103},
issn = {1873-2690},
mesh = {*CRISPR-Cas Systems/genetics ; *Seeds/metabolism/genetics ; *Flavonoids/biosynthesis/genetics ; *Mustard Plant/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Knockout Techniques ; Gene Expression Regulation, Plant ; *Biosynthetic Pathways/genetics ; Down-Regulation ; Gene Editing ; Proanthocyanidins ; },
abstract = {Accumulation of proanthocyanidin (PA) in the seed coat determines black to brown seed color in Brassicaceae members. Consequently, yellow-seed coat results from mutation in the regulatory or biosynthetic genes of the flavonoid pathway which perturb PA accumulation. We identified two homeologs of Transparent Testa 8 (BjTT8) gene in the allotetraploid genome of Brassica juncea with BjTT8A homeolog exhibiting higher expression than BjTT8B. To investigate whether targeted knockout of BjTT8 can result into yellow seed coat color, a CRISPR/Cas9-based genome editing vector was constructed using the binary vector pORE O4 backbone. A single-guide RNA (sgRNA) was designed to precisely target the second exon of both the BjTT8 homeologs. Successful knockout of both BjTT8A and BjTT8B genes deploying pORE O4-CRISPR/Cas9 vector resulted in yellow seed coat color. The mutations were stably inherited over the successive generations, giving consistent yellow seed color including in the lines segregated free from T-DNA bound CRISPR/Cas9 cassette. Comparative transcriptome and gene expression analysis revealed that, a subset of flavonoid pathway genes was downregulated in the Bjtt8 edited lines. Flavonoid profiling demonstrated that, the flavan-3-ol monomer (viz., catechin/epicatechin), the precursor of PA biosynthesis was completely absent in the Bjtt8 edited lines. More significantly, the seeds of Bjtt8 edited lines exhibited higher oil content as compared to their parental Varuna line. In this study, we developed a new yellow-seeded line through CRISPR/Cas9-mediated knockout of BjTT8 genes, providing molecular insights of seed coat color regulation in B. juncea.},
}
@article {pmid39966655,
year = {2025},
author = {Xia, C and Colognori, D and Jiang, XS and Xu, K and Doudna, JA},
title = {Single-molecule live-cell RNA imaging with CRISPR-Csm.},
journal = {Nature biotechnology},
volume = {43},
number = {12},
pages = {2023-2030},
pmid = {39966655},
issn = {1546-1696},
support = {X-0001//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; D-0001//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R35 GM149349/GM/NIGMS NIH HHS/United States ; D-0001//Howard Hughes Medical Institute (HHMI)/ ; K99 GM151484/GM/NIGMS NIH HHS/United States ; },
mesh = {*In Situ Hybridization, Fluorescence/methods ; Humans ; *Single Molecule Imaging/methods ; *CRISPR-Cas Systems/genetics ; RNA, Messenger/genetics/metabolism ; *RNA/genetics/metabolism ; Animals ; Single-Cell Analysis/methods ; Receptor, Notch2/genetics/metabolism ; Mice ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Understanding the diverse dynamic behaviors of individual RNA molecules in single cells requires visualizing them at high resolution in real time. However, single-molecule live-cell imaging of unmodified endogenous RNA has not yet been achieved in a generalizable manner. Here, we present single-molecule live-cell fluorescence in situ hybridization (smLiveFISH), a robust approach that combines the programmable RNA-guided, RNA-targeting CRISPR-Csm complex with multiplexed guide RNAs for direct and efficient visualization of single RNA molecules in a range of cell types, including primary cells. Using smLiveFISH, we track individual native NOTCH2 and MAP1B transcripts in living cells and identify two distinct localization mechanisms including the cotranslational translocation of NOTCH2 mRNA at the endoplasmic reticulum and directional transport of MAP1B mRNA toward the cell periphery. This method has the potential to unlock principles governing the spatiotemporal organization of native transcripts in health and disease.},
}
@article {pmid39820813,
year = {2025},
author = {Zhang, F and Chow, RD and He, E and Dong, C and Xin, S and Mirza, D and Feng, Y and Tian, X and Verma, N and Majety, M and Zhang, Y and Wang, G and Chen, S},
title = {Multiplexed inhibition of immunosuppressive genes with Cas13d for combinatorial cancer immunotherapy.},
journal = {Nature biotechnology},
volume = {43},
number = {12},
pages = {2054-2067},
pmid = {39820813},
issn = {1546-1696},
support = {R33 CA281702/CA/NCI NIH HHS/United States ; R01 CA231112/CA/NCI NIH HHS/United States ; DP2 CA238295/CA/NCI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; F30 CA250249/CA/NCI NIH HHS/United States ; R33 CA225498/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *Immunotherapy/methods ; Mice ; Tumor Microenvironment/genetics/immunology ; *Neoplasms/therapy/immunology/genetics ; Humans ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Gene Silencing ; Dependovirus/genetics ; Mice, Inbred C57BL ; Female ; },
abstract = {The complex nature of the immunosuppressive tumor microenvironment (TME) requires multi-agent combinations for optimal immunotherapy. Here we describe multiplex universal combinatorial immunotherapy via gene silencing (MUCIG), which uses CRISPR-Cas13d to silence multiple endogenous immunosuppressive genes in the TME, promoting TME remodeling and enhancing antitumor immunity. MUCIG vectors targeting four genes delivered by adeno-associated virus (AAV) (Cd274/Pdl1, Lgals9/Galectin9, Lgals3/Galectin3 and Cd47; AAV-Cas13d-PGGC) demonstrate significant antitumor efficacy across multiple syngeneic tumor models, remodeling the TME by increasing CD8[+] T-cell infiltration while reducing neutrophils. Whole transcriptome profiling validates the on-target knockdown of the four target genes and shows limited potential off-target or downstream gene alterations. AAV-Cas13d-PGGC outperforms corresponding shRNA treatments and individual gene knockdown. We further optimize MUCIG by employing high-fidelity Cas13d (hfCas13d), which similarly showed potent gene silencing and in vivo antitumor efficacy, without weight loss or liver toxicity. MUCIG represents a universal method to silence multiple immune genes in vivo in a programmable manner, offering broad efficacy across multiple tumor types.},
}
@article {pmid41373623,
year = {2025},
author = {Petrova, IO and Smirnikhina, SA},
title = {Ex Vivo Gene and Cell Therapy in Hematopoietic Stem Cells.},
journal = {International journal of molecular sciences},
volume = {26},
number = {23},
pages = {},
pmid = {41373623},
issn = {1422-0067},
support = {not applicable//Ministry of Education and Science of Russia/ ; },
mesh = {Humans ; *Genetic Therapy/methods ; *Hematopoietic Stem Cells/metabolism/cytology ; *Cell- and Tissue-Based Therapy/methods ; Animals ; *Hematopoietic Stem Cell Transplantation/methods ; Genetic Vectors/genetics ; Lentivirus/genetics ; },
abstract = {Ex vivo cell and gene therapy is a prospective approach to treatment of genetic diseases. To date, one of the most prevalent examples of genetically engineered cell therapies is hematopoietic stem/progenitor cells (HSPCs). This mini review is focused on HSPC therapy methods that have been approved for medical use. Most gene therapy methods rely on the lentiviral integration of the gene into the target cell genome, as lentiviruses are extremely effective, particularly in transduction of non-dividing cells. In this constantly evolving field, it is important to find the balance between safety concerns and efficiency. Analyzing cases of several diseases, for which ex vivo gene therapy was developed, we strive to understand which factors are crucial to success and what the potential drawbacks are. Although in general, viral gene integration demonstrates a considerable therapeutic effect, it has oncogenic potential. Development of self-inactivating vectors was a breakthrough in regard to safety, but the possibility of oncogenesis remains, and strict analysis of integration sites is required.},
}
@article {pmid41373550,
year = {2025},
author = {Koller, F},
title = {The Potential of NGTs to Overcome Constraints in Plant Breeding and Their Regulatory Implications.},
journal = {International journal of molecular sciences},
volume = {26},
number = {23},
pages = {},
pmid = {41373550},
issn = {1422-0067},
support = {3522840500//Federal Agency for Nature Conservation/ ; },
mesh = {*Plant Breeding/methods ; CRISPR-Cas Systems ; Genome, Plant ; *Plants/genetics ; *Genomics/methods ; Gene Editing/methods ; },
abstract = {Conventional plant breeding relies on the occurrence of chromosomal crossover and spontaneous or non-targeted mutations in the genome induced by physical or chemical stressors. However, constraints exist concerning the number and variation of genotypes that can be achieved in this way, as the occurrence and combination of mutations are not equally distributed across the genome. The underlying mechanisms and causes of reproductive constraints can be considered the result of evolution to maintain the genomic stability of a species while at the same time allowing necessary adaptations. A continuous horizon scan was carried out to identify plants derived from new genomic techniques (NGTs), which show that CRISPR/Cas is able to circumvent at least some of these mechanisms and constraints. The reason for this is the specific mode of action: While physico-chemical mutagens such as radiation or chemicals merely cause a break in DNA, recombinant enzymatic mutagens (REMs), such as CRISPR/Cas, additionally interfere with cellular repair mechanisms. More recently developed REMs even expand the capabilities of NGTs to introduce new genetic variations within the target sequences. Thus, NGTs introduce genetic changes and combinations that are unknown in the current breeding pool and that are also unlikely to occur as a result of any previously used breeding methods. The resulting genotypes may need to be considered as 'new to the environment'. The technical potential of NGTs should also be taken into account in regulatory provisions. Previously unknown genotypes and phenotypes may negatively impact plant health, ecosystems, biodiversity, and plant breeding. It must further be acknowledged that the different outcomes of NGTs and conventional breeding are not always evident at first sight. As a starting point, within a process-oriented approval process, molecular characterization can inform the following steps in risk assessment and guide requests for further data.},
}
@article {pmid41373007,
year = {2025},
author = {Song, J and Yang, D and Kong, L and Tsai, LK and Zhang, J and Chen, YE and Tsai, RY and Xu, J},
title = {Development of a high-yield Rabbit line for enhanced animal pharming.},
journal = {Biological research},
volume = {58},
number = {1},
pages = {73},
pmid = {41373007},
issn = {0717-6287},
support = {R41GM110822/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Rabbits/genetics ; *Animals, Genetically Modified/genetics ; *Recombinant Proteins/biosynthesis/genetics ; *Milk/chemistry ; Gene Knock-In Techniques ; CRISPR-Cas Systems/genetics ; *Caseins/genetics ; Female ; Promoter Regions, Genetic/genetics ; },
abstract = {Animal pharming involves producing recombinant protein drugs using transgenic animals. The United States Food and Drug Administration (FDA) has approved certain drugs produced in the milk of transgenic Rabbits. Traditionally, these pharming Rabbits have been developed using conventional transgenic technology, which often results in an unpredictable success rate, uncontrollable transgene insertion sites, varying copy numbers, and generally low recombinant protein yields, typically 1-2 g/L or lower. We hypothesized that utilizing the promoter of a native major milk protein gene to drive transgene expression could significantly enhance yield. To test this, we developed a rabbit line that expresses tdTomato under the control of the CSN2 gene promoter, responsible for encoding β-casein, the most abundant protein in Rabbit milk. We successfully generated knock-in founder Rabbits using CRISPR/Cas9-mediated knock-in technology, augmented by the homology-directed repair (HDR)-promoting small molecule RS-1. These founder Rabbits were able to transmit the knock-in allele to their offspring, producing both heterozygous and homozygous tdTomato knock-in Rabbits. Remarkably, the recombinant protein yield reached 15-20 g/L in the milk of homozygous animals. Our work demonstrates a promising strategy to enhance recombinant protein production in Rabbit pharming.},
}
@article {pmid41372233,
year = {2025},
author = {Macak, D and Kanis, P and Riesenberg, S},
title = {Repurposing clinically safe drugs for DNA repair pathway choice in CRISPR genome editing and synthetic lethality.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11077},
pmid = {41372233},
issn = {2041-1723},
mesh = {Humans ; *Gene Editing/methods ; *Synthetic Lethal Mutations/drug effects ; *Drug Repositioning/methods ; DNA End-Joining Repair/drug effects ; Induced Pluripotent Stem Cells/drug effects/metabolism ; *DNA Repair/drug effects ; Recombinational DNA Repair/drug effects ; *CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded/drug effects ; Tumor Suppressor p53-Binding Protein 1/metabolism/genetics ; },
abstract = {We evaluate the effect of most FDA-approved drugs (>7,000 conditions) on double-strand DNA break repair pathways by analyzing mutational outcomes in human induced pluripotent stem cells. We identify drugs that can be repurposed as inhibitors and enhancers of repair outcomes attributed to non-homologous and microhomology-mediated end joining (NHEJ, MMEJ), and homology-directed repair (HDR). We also identify functions of the proteins estrogen receptor 2 (ESR2) and aldehyde oxidase 1 (AOX1), affecting several key DNA repair proteins, such as ATM and 53BP1. Silencing of ESR2 can have a synergistic effect on increasing HDR when combined with NHEJ inhibition (mean 4.6-fold increase). We further identify drugs that induce synthetic lethality when NHEJ or HDR is blocked and may therefore be candidates for precision medicine. We anticipate that the ability to modulate the DNA repair outcomes with clinically safe drugs will help disease modeling, gene therapy, chimeric antigen receptor immunotherapy, and cancer treatment.},
}
@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.},
}
@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 = {},
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.},
}
@article {pmid41344296,
year = {2025},
author = {Landi, E and Zondag, R and Dehnen, JA and Albert, S and Dickman, MM and LaPointe, VLS and van Bokhoven, H},
title = {Biallelic excision of the CTG18.1 expansion in two Fuchs endothelial corneal dystrophy-derived iPSC lines and one control (SCTCi046-A-1, SCTCi047-A-1 and SCTCi041-A-1) using an episomal vector-based CRISPR/Cas9 approach.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103881},
doi = {10.1016/j.scr.2025.103881},
pmid = {41344296},
issn = {1876-7753},
mesh = {Humans ; *Fuchs' Endothelial Dystrophy/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *CRISPR-Cas Systems/genetics ; *Trinucleotide Repeat Expansion/genetics ; Cell Line ; Transcription Factor 4/genetics ; Alleles ; Plasmids/genetics ; },
abstract = {An expanded CTG repeat in intron 2 of the transcription factor 4 (TCF4) gene is the main cause of Fuchs endothelial corneal dystrophy (FECD), a complex corneal disease. The prevailing paradigm is that the expanded repeat exerts toxic effects, resulting in corneal endothelium degeneration. Here we explored the use of CRISPR/Cas9-mediated, non-homologous end-joining (NHEJ) for disease-modeling purposes, by performing a biallelic excision of the CTG18.1 expansion in two FECD- and one control-derived induced pluripotent stem cell lines (iPSCs). The three Δ/Δ CTG18.1 lines generated by this study provide a platform to investigate the CTG18.1 contribution to FECD pathogenesis.},
}
@article {pmid41330274,
year = {2025},
author = {Macklin, BL and Runyon, WV and Feliciano, CM and Dierks, PH and Kelly, KR and Watry, HL and Judge, LM and Conklin, BR},
title = {Generation of WTD, a control human iPSC line for genetic research.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103872},
doi = {10.1016/j.scr.2025.103872},
pmid = {41330274},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; Mutation ; },
abstract = {The establishment of well characterized control iPSC lines is essential for robust, reproducible research across laboratories. We used CRISPR/Cas9 to derive an isogeneic control line from a patient-derived iPSC line carrying a mutation in the NEFL gene (E396K). After correction of the E396K mutation, UCSFi003-A (WTD) exhibits multi-lineage differentiation potential, a normal karyotype, no large genomic abnormalities, and has consents for public distribution of cells and genomic data.},
}
@article {pmid41308567,
year = {2025},
author = {Downton, P and Bates, N and Woods, S and Adamson, A and Sergouniotis, PI},
title = {Genome editing of a low-penetrance albinism-associated variant in TYR in patient-derived pluripotent stem cells.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103855},
doi = {10.1016/j.scr.2025.103855},
pmid = {41308567},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Monophenol Monooxygenase/genetics/metabolism ; *Gene Editing/methods ; *Albinism/genetics/pathology ; CRISPR-Cas Systems ; Cell Differentiation ; Male ; },
abstract = {TYR encodes tyrosinase, the enzyme catalysing the initial steps of melanin biosynthesis in melanocytes and retinal pigment epithelia (RPE). TYR c.1205G>A (p.Arg402Gln) is a common genetic variant associated with several pigmentation traits. Notably, when this variant is encountered in specific haplotypic backgrounds in the homozygous state, it predisposes to albinism. We generated an induced pluripotent stem cell (iPSC) line from an affected individual carrying such a homozygous genotype (UMANi255-A), and then used CRISPR-Cas9 to correct the TYR c.1205G>A variant (UMANi255-A-1). The resulting iPSC lines demonstrate capacity for multi-lineage differentiation, providing a useful in vitro model for studying pigmentation biology.},
}
@article {pmid41275813,
year = {2026},
author = {Zhang, Z and Zhang, T and Li, Z and Zeng, Z},
title = {CRISPR/Cas13a-based colorimetric biosensing platform for point-of-care detection of viral nucleic acids.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {348},
number = {Pt 1},
pages = {127221},
doi = {10.1016/j.saa.2025.127221},
pmid = {41275813},
issn = {1873-3557},
mesh = {*Colorimetry/methods ; *SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Dengue Virus/genetics/isolation &amp; purification ; *COVID-19/diagnosis/virology ; *Point-of-Care Systems ; *RNA, Viral/analysis/genetics ; G-Quadruplexes ; Limit of Detection ; Point-of-Care Testing ; DNA, Catalytic/chemistry ; Sensitivity and Specificity ; },
abstract = {Rapid and accurate diagnosis is important in preventing and effectively combating infectious disease outbreaks. The CRISPR/Cas13a-based Specific High-sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) platform possesses the advantages of high efficiency, good specificity and sensitivity, and it has been widely adopted in molecular diagnostics. However, the traditional SHERLOCK platform requires dual-labeled RNA probes for fluorescence detection or lateral flow assay, which entail tedious modification procedures and sophisticated optical instruments, limiting its broad applications. Herein, we developed a rapid, sensitive, and label-free point-of-care (POC) platform for colorimetric assays of dengue virus (DV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with the SHERLOCK method. The adoption of the SHERLOCK-mediated guanine-quadruplex (G4)/hemin DNAzyme-based colorimetric strategy produced cascade signal amplification detection with improved analytical performance. Moreover, it exhibited high sensitivity and specificity for detection in cell-cultured DV samples, and DV and SARS-CoV-2 clinical samples, as well as accurate identification of the four DV serotypes. Hence, the proposed colorimetric biosensing platform has great potential for rapid, accurate, and specific POC detection of viral infections in field-deployable assay.},
}
@article {pmid41265249,
year = {2025},
author = {Papadopoulou, M and Ramachandran, H and Binder, S and Hildebrandt, B and Rossi, A and Krutmann, J},
title = {CRISPR/Cas9-mediated editing of XPA in induced pluripotent stem cells: A model for investigating Xeroderma Pigmentosum and NER dysfunction.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103869},
doi = {10.1016/j.scr.2025.103869},
pmid = {41265249},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *CRISPR-Cas Systems/genetics ; *Xeroderma Pigmentosum/genetics/pathology/metabolism ; *Xeroderma Pigmentosum Group A Protein/genetics/metabolism ; *DNA Repair/genetics ; *Gene Editing ; Cell Differentiation ; Cell Line ; },
abstract = {Xeroderma pigmentosum group A (XPA) is caused by defects in the nucleotide excision repair (NER) pathway, which is essential for repairing UV-induced DNA damage. Mutations in XPA impair lesion recognition and repair, resulting in mutation accumulation, genomic instability, and a high risk of skin cancers. In this study, we generated a CRISPR/Cas9-engineered human induced pluripotent stem cell (iPSC) line, WTSIi018-B-30, carrying a homozygous single nucleotide variant in exon 3 of XPA. The edited iPSCs retained normal morphology, expressed pluripotency markers, and differentiated into all three germ layers. This mutant iPSC line provides a robust isogenic model to dissect the molecular consequences of XPA deficiency and to explore therapeutic strategies for XPA-associated diseases.},
}
@article {pmid41240694,
year = {2026},
author = {Gao, Y and Dong, P and Lin, H and Tian, J},
title = {A circular crRNA-triggered CRISPR/Cas12a fluorescent platform for detection of myeloperoxidase activity.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {348},
number = {Pt 1},
pages = {127149},
doi = {10.1016/j.saa.2025.127149},
pmid = {41240694},
issn = {1873-3557},
mesh = {*Peroxidase/metabolism/analysis ; *CRISPR-Cas Systems/genetics ; Humans ; *RNA, Circular/chemistry ; Limit of Detection ; Spectrometry, Fluorescence/methods ; *CRISPR-Associated Proteins/metabolism ; *Biosensing Techniques/methods ; Animals ; Hypochlorous Acid/metabolism ; *Enzyme Assays/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Myeloperoxidase (MPO) is a key enzymatic biomarker for the diagnosis and therapeutic monitoring of acute leukemia. Here, we developed a CRISPR/Cas12a-based fluorescent sensing platform using a circular phosphorothioate-modified crRNA (crRNA-PS) for selective MPO activity detection. In this system, MPO catalyzes hypochlorous acid (HClO) generation, which oxidizes and linearizes the circular crRNA-PS, activating Cas12a-mediated trans-cleavage of a fluorescent reporter. The assay exhibited high sensitivity with detection limits of 0.79 ng/mL for MPO and 0.21 μM for HClO, along with excellent selectivity against reactive oxygen/nitrogen species and biomolecular interferents. Reliable performance was achieved in 1 % fetal bovine serum with recoveries of 92.87-112.54 %. The sensor also maintained stable responses over 8 weeks, indicating strong structural integrity of the circular crRNA-PS. Furthermore, the system was applied for inhibitor screening, yielding IC50 values of 127.2 μM for SHA and 0.81 μM for 4-ABAH. This strategy provides a rapid, sensitive, and robust platform for MPO detection with promising clinical potential.},
}
@article {pmid41238027,
year = {2026},
author = {Zhang, Z and Tang, D and Dong, L and Hu, Y and Li, S and Wang, D and Wang, M and Wang, Q},
title = {Functional divergence of Msp porins in substrate uptake and stress resistance in Mycobacterium smegmatis.},
journal = {Microbial pathogenesis},
volume = {210},
number = {},
pages = {108185},
doi = {10.1016/j.micpath.2025.108185},
pmid = {41238027},
issn = {1096-1208},
mesh = {*Mycobacterium smegmatis/genetics/metabolism/growth &amp; development/drug effects ; *Porins/genetics/metabolism ; *Stress, Physiological ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Anti-Bacterial Agents/pharmacology ; Mutation ; CRISPR-Cas Systems ; Amino Acids/metabolism ; Drug Resistance, Bacterial ; Muramidase/pharmacology ; Hydrogen-Ion Concentration ; *Bacterial Outer Membrane Proteins/genetics/metabolism ; Carbon/metabolism ; },
abstract = {Mycobacterium smegmatis acquires external nutrients primarily through its outer membrane porins, notably the Msp proteins. Although the bacterium encodes four Msp porins (MspA, MspB, MspC, and MspD), previous work has identified MspA as the most abundant and functionally dominant porin, while regarding others as backup porins sharing considerable functional redundancy with MspA; however, their specific physiological roles and transcriptional dynamics remain poorly characterized. To dissect the individual function of each porin, we constructed a precise M. smegmatis Msp quadruple mutant using CRISPR-Cas9, and subsequently generated a panel of isogenic strains expressing individual porins at controlled levels. These strains were assessed in growth assays for their capacity to utilize various nutrients, including carbon sources, amino acids, and ions, as well as for their resistance to antibiotics, low pH, and lysozyme. We also employed Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) to examine the transcriptional responses of each msp gene in the wild-type strain under the tested conditions. Our results demonstrated distinct substrate preferences among the four Msp porins and revealed their differential contributions to resistance against environmental stressors; the transcriptional changes of each Msp porin in the wild-type strain correlated well with their expected roles. Furthermore, the engineered M. smegmatis quadruple mutant provides a robust platform for investigating the functions of other channel proteins in mycobacteria.},
}
@article {pmid41207276,
year = {2026},
author = {Luo, N and Li, X and Tian, S and Li, X and Zou, L},
title = {Label-free fluorescent aptasensor for sensitive detection of oxytetracycline based on CRISPR/Cas12a and G-quadruplex DNAzyme with rhodamine B reporting.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {348},
number = {Pt 2},
pages = {127141},
doi = {10.1016/j.saa.2025.127141},
pmid = {41207276},
issn = {1873-3557},
mesh = {*Rhodamines/chemistry ; *Oxytetracycline/analysis ; *G-Quadruplexes ; *Aptamers, Nucleotide/chemistry ; Limit of Detection ; *Biosensing Techniques/methods ; *DNA, Catalytic/chemistry/metabolism ; Milk/chemistry ; *CRISPR-Cas Systems ; Spectrometry, Fluorescence/methods ; Animals ; Fluorescent Dyes/chemistry ; *Anti-Bacterial Agents/analysis ; *Endodeoxyribonucleases/metabolism ; Rivers/chemistry ; *CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; },
abstract = {Oxytetracycline (OTC), a widely used broad-spectrum antibiotic, frequently persists in food and the environment due to overuse, posing risks of resistance, allergy, and toxicity. The World Health Organization has set strict limits for OTC residues, highlighting the need for rapid and reliable detection. Conventional assays, though accurate, remain costly and technically demanding, underscoring demand for simpler alternatives. We report a label-free fluorescent aptasensor integrating CRISPR/Cas12a with G-quadruplex (G4) DNAzyme catalysis, using rhodamine B (RhB) as a reporter. In the absence of OTC, the aptamer binds the activator strand, allowing G4 DNAzyme formation that quenches RhB fluorescence. OTC binding liberates the activator, triggering Cas12a cleavage of G4 DNA, thereby preventing DNAzyme formation and preserving fluorescence. This switch provides efficient signal amplification with low background. The assay achieved a detection limit of 0.3 nM and enabled accurate quantification of OTC in river water and milk samples. This platform offers a cost-effective, label-free strategy for antibiotic residue monitoring, eliminating dependence on nanomaterials or external labeling. Its validated sensitivity and robustness in real samples underscore its potential for practical applications in food safety and environmental surveillance.},
}
@article {pmid41192264,
year = {2025},
author = {Raabe, J and Fuchs, S and Augustin, C and Hammerschmidt, A and Krämer, E and Orthey, E and Ehler, E and Cuello, F},
title = {Generation of a NRAP-overexpressing mutant from a human iPSC line.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103863},
doi = {10.1016/j.scr.2025.103863},
pmid = {41192264},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Myocytes, Cardiac/metabolism/cytology ; Cell Differentiation ; Cell Line ; *Mutation ; CRISPR-Cas Systems ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; },
abstract = {Cardiomyopathies are a major contributor to cardiovascular mortality and are frequently linked to abnormalities in intercalated discs, which coordinate mechanical and electrical signaling between cardiomyocytes. The Nebulin-Related Anchoring Protein (NRAP), a key component of these structures, is essential for myofibril formation and force transmission. In various cardiac diseases such as cardiomyopathies with differing genetic mutations, NRAP protein abundance is increased, yet the functional consequences of this expression change remain insufficiently characterized. To investigate the outcome of NRAP-overexpression (NRAP-OE) on cardiac development and disease, we established a human induced pluripotent stem cell (hiPSC) line with stable and specific NRAP-OE in cardiomyocytes using CRISPR-Cas9-based genome editing. The resulting line was rigorously validated for chromosomal integrity, pluripotency markers, absence of off-target effects and mycoplasma contamination, as well as its capacity for trilineage differentiation. This NRAP-OE model offers a novel platform for investigating how increased NRAP levels influence cardiomyocyte structure and function, and may provide insight into its role in the pathogenesis of cardiomyopathy.},
}
@article {pmid41187652,
year = {2025},
author = {Masser-Mitchell, BE and McLoughlin, HS},
title = {Generation of an ITPR1[+/-] and isogenic control induced pluripotent stem cell line for SCA15/16 model development.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103858},
doi = {10.1016/j.scr.2025.103858},
pmid = {41187652},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Spinocerebellar Ataxias/pathology/genetics/metabolism ; *Inositol 1,4,5-Trisphosphate Receptors/genetics/metabolism ; Cell Line ; Cell Differentiation ; CRISPR-Cas Systems ; },
abstract = {Spinocerebellar ataxia type 15/16 (SCA15/16) is a rare neurodegenerative disorder caused by heterozygous deletions of ITPR1, leading to haploinsufficiency of the encoded endoplasmic reticulum membrane calcium channel. Patients present with progressive gait disturbances, abnormal eye movements, difficulties with speech and swallowing, and tremors associated with atrophy of the cerebellum. Using CRISPR/Cas9 technology, we generated ITPR1[+/-] and isogenic control induced pluripotent stem cell (iPSC) lines from PGP1 iPSCs for SCA15/16 model development. The clones were genotyped, karyotyped, and assessed for pluripotency and differentiation potential.},
}
@article {pmid41167048,
year = {2025},
author = {Wei, S and Ma, R and Zhang, T and Wen, L},
title = {Targeted large-fragment genomic deletion in human pluripotent stem cells (hPSCs) via CRISPR/Cas9.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103859},
doi = {10.1016/j.scr.2025.103859},
pmid = {41167048},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Large genomic deletions are increasingly recognized as contributors to human disease. Here, we describe a CRISPR/Cas9-based protocol to induce targeted large-fragment deletions in human pluripotent stem cells (hPSCs). Two sgRNAs flanking the target region were designed, synthesized, and co-delivered with Cas9 protein into single-cell hPSCs via electroporation. Deletions were identified using junction PCR. Successfully edited cell pools were expanded as single-cell colonies and assessed for genomic deletion, stem cell identity, karyotype integrity, and tri-lineage differentiation capacity. This approach provides a robust method for modeling genomic deletions in hPSCs for disease research and functional genomics.},
}
@article {pmid41138562,
year = {2025},
author = {Marteau, S and Duboscq-Bidot, L and Aizawa, T and Hamlin, M and Villard, E and Guicheney, P and Fontaine, V},
title = {Generation of a FAM189A2/ENTREP1 knockout human induced pluripotent stem cell line using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103857},
doi = {10.1016/j.scr.2025.103857},
pmid = {41138562},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Line ; *Membrane Proteins/genetics/metabolism ; *Gene Knockout Techniques ; },
abstract = {FAM189A2/ENTREP1 encodes a transmembrane protein, EREP1, ubiquitously expressed, with higher levels in thyroid gland, skeletal muscle, heart and glial cells, suggesting tissue-specific roles. It is regulated in various cancers and genetic studies suggested that it could be associated with cardiac and cardiometabolic diseases. EREP1 was reported to activate ITCH, an E3 ubiquitin ligase, involved in endosomal dynamics and protein trafficking. We generated by CRISPR/Cas9 technology a human induced pluripotent stem cells (hiPSCs) line carrying two compound heterozygous deletions leading to gene invalidation, with the aim of further exploring the poorly understood function of this protein.},
}
@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.},
}
@article {pmid41067151,
year = {2025},
author = {Li, C and Meng, C and Wang, X and Yuan, C and Chen, Q and Yang, Y and Zhang, Z and Duan, Y and Dong, Q and Sun, X},
title = {CRISPR/Cas9-targeted mutagenesis of the PpTCP4 gene increased tiller number in Poa pratensis.},
journal = {Plant physiology and biochemistry : PPB},
volume = {229},
number = {Pt C},
pages = {110588},
doi = {10.1016/j.plaphy.2025.110588},
pmid = {41067151},
issn = {1873-2690},
mesh = {*CRISPR-Cas Systems/genetics ; *Poa/genetics/growth &amp; development ; *Mutagenesis ; Gene Editing ; Plants, Genetically Modified/genetics ; *Plant Proteins/genetics/metabolism ; *Genes, Plant ; },
abstract = {Poa pratensis, commonly known as Kentucky bluegrass, is a widely used cool-season grass species for turf in lawns and recreational areas worldwide. However, the lack of genome editing platforms has impeded research into enhancing forage crops suitable for land unsuitable for other crops. In this study, we successfully established an Agrobacterium-mediated genetic transformation system for P. pratensis. Furthermore, we successfully produced PpTCP4-edited plants using the CRISPR/Cas9 system. This research represents a significant advancement in the genetic transformation and gene editing of P. pratensis, providing a foundational technology for future investigations into gene function and breeding in P. pratensis.},
}
@article {pmid41045726,
year = {2025},
author = {Rhode, J and Hagenau, L and Edwards, S and Buettner, FFR and Tzvetkova, A and Jensen, LR and Kuss, AW},
title = {Three modified human IPSC lines containing mutations in the distal DEHMBA associated locus of the SRCAP gene.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103847},
doi = {10.1016/j.scr.2025.103847},
pmid = {41045726},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; *Mutation/genetics ; CRISPR-Cas Systems ; Cell Differentiation ; Frameshift Mutation ; },
abstract = {We modified an existing human iPSC line (MHHi001-A), using CRISPR/Cas9, to introduce heterozygous frameshift mutations in a locus of the SRCAP gene that is associated with the DEHMBA disease (OMIM 619595). The modified iPSCs express several stem cell markers and are able to differentiate into cells originating from all three embryonic germ layers. No additional modifications or chromosomal defects were detected. The modified cells can serve as a model for the investigation of the involvement of SRCAP in DEHMBA (Developmental delay, Hypotonia, Musculoskeletal defects, and Behavioral Abnormalities) disease and/or its molecular functions in different cell types.},
}
@article {pmid41016181,
year = {2025},
author = {Shibuya, K and Nomizu, T and Morimoto, H and Satou, K},
title = {CRISPR/Cas9-mediated LhNAP mutagenesis extends flower longevity in lily.},
journal = {Plant physiology and biochemistry : PPB},
volume = {229},
number = {Pt C},
pages = {110551},
doi = {10.1016/j.plaphy.2025.110551},
pmid = {41016181},
issn = {1873-2690},
mesh = {*Flowers/genetics/physiology ; *CRISPR-Cas Systems/genetics ; *Lilium/genetics/physiology ; *Plant Proteins/genetics/metabolism ; *Mutagenesis ; Plants, Genetically Modified ; *Transcription Factors/genetics/metabolism ; Gene Editing ; Oryza/genetics ; },
abstract = {Flower longevity is an important trait determining the ornamental plant quality. NAM/ATAF1,2/CUC2 (NAC) transcription factors regulate leaf and petal senescence in several plants. To extend the longevity of lily flowers, in this study we used the CRISPR/Cas9 technology for the targeted mutagenesis of LhNAP encoding the NAC transcription factor in the Oriental hybrid lily (Lilium spp.) 'Acapulco'. Filament-derived calli were transformed with binary vectors containing guide RNA expression cassettes targeting one or two regions of LhNAP and the codon-optimized Cas9 for Oryza sativa driven by Zea mays ubiquitin promoter. Mutant lines harbouring biallelic mutations at the LhNAP locus exhibited clearly delayed tepal senescence and abscission compared to the wild-type plants. Overall, our results highlight the potential of LhNAP-targeted genome editing to produce lilies with extended flower longevity and reveal the crucial role of LhNAP in regulating tepal senescence and abscission in lilies.},
}
@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.<br><br>OBJECTIVE: We intended to identify regulatory factors driving monocyte/macrophage differentiation.<br><br>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.<br><br>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.<br><br>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.},
}
@article {pmid40976167,
year = {2025},
author = {Park, S and Lee, H and Song, J and Kim, EH and Lim, CJ and Oh, J and Lee, SB and Kim, JA and Kim, BG},
title = {Redirecting flavonoid flux in purple Chinese cabbage via Cas9-mediated BrDFR knockout.},
journal = {Plant physiology and biochemistry : PPB},
volume = {229},
number = {Pt B},
pages = {110534},
doi = {10.1016/j.plaphy.2025.110534},
pmid = {40976167},
issn = {1873-2690},
mesh = {*Flavonoids/metabolism/biosynthesis ; *Alcohol Oxidoreductases/genetics/metabolism ; Gene Knockout Techniques ; *CRISPR-Cas Systems ; Anthocyanins/metabolism/biosynthesis ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; *Brassica/metabolism/genetics ; *Brassica rapa/genetics/metabolism ; },
abstract = {Purple varieties of Chinese cabbage (Brassica rapa subsp. pekinensis) predominantly accumulate cyanidin-based anthocyanins. Although dihydroflavonol 4-reductase (DFR) is a key enzyme in anthocyanin biosynthesis, the function of the putative B. rapa DFR gene (Bra027457) remained unverified. We isolated and sequenced the coding region of Bra027457 from four B. rapa inbred lines with either green or purple phenotypes and detected no sequence variation. Bra027457 expression correlated with anthocyanin accumulation, and in vitro assays confirmed its ability to reduce all three dihydroflavonol substrates. Using CRISPR/Cas9, we knocked out Bra027457 in the purple line 8267 and obtained transgene-free, homozygous BrDFR-KO plants. These exhibited a green phenotype due to complete anthocyanin loss, verifying Bra027457 as the authentic BrDFR gene. Metabolite profiling of BrDFR-KO heads revealed significant increases in quercetin (Q), isorhamnetin (IR), and dihydroquercetin (DHQ). LC/MS analysis further identified five flavonol glycosides and one DHQ glycoside, of whch Q 3,7-di-O-glucoside and IR 3-O-(2‴-O-feruloyl)sophoroside-7-O-glucoside were predominant. These findings advance our understanding of flavonoid biosynthesis in Brassica species and provide valuable genetic resources for Chinese cabbage improvement.},
}
@article {pmid40857358,
year = {2025},
author = {Sharma, A and Locatelli, F and Bhatia, M and Molinari, L and Mapara, MY and Liem, RI and Dedeken, L and Wall, D and Eckrich, MJ and Kuo, KHM and Smith, W and Imren, S and Kohli, P and Li, N and Liu, T and Rubin, J and Hobbs, W and Grupp, SA and Frangoul, H},
title = {Improvements in health-related quality of life in patients with severe sickle cell disease after exagamglogene autotemcel.},
journal = {Blood advances},
volume = {9},
number = {24},
pages = {6481-6490},
doi = {10.1182/bloodadvances.2025016701},
pmid = {40857358},
issn = {2473-9537},
mesh = {Humans ; *Anemia, Sickle Cell/therapy ; *Quality of Life ; Adolescent ; Male ; Female ; Adult ; Young Adult ; Patient Reported Outcome Measures ; Severity of Illness Index ; Gene Editing ; Treatment Outcome ; CRISPR-Cas Systems ; *Genetic Therapy/methods ; Hematopoietic Stem Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Severe sickle cell disease (SCD), an inherited blood disorder characterized by recurrent vaso-occlusive crises (VOCs), significantly impairs health-related quality of life (HRQoL). Exagamglogene autotemcel (exa-cel), a one-time, ex vivo CRISPR/Cas9 gene-edited cell therapy, eliminated VOCs in 97% of participants with severe SCD in the phase 3 CLIMB SCD-121 trial. Here, we describe changes in patient-reported outcomes (PROs) in 30 adults and 12 adolescents with ≥16 months follow-up. Adult PROs include EuroQol Quality of Life Scale-5 dimensions-5 levels of severity (EQ-5D-5L), Functional Assessment of Cancer Therapy Bone Marrow Transplant (FACT-BMT), Adult Sickle Cell Quality of Life Measurement Information System (ASCQ-Me), and 11-point pain Numerical Rating Scale (NRS). In adolescents, EuroQol Quality of Life Scale-5 dimensions-youth (EQ-5D-Y), Pediatric Quality of Life Inventory (PedsQL), and pain NRS were used. Adult EQ-5D-5L US and UK healthy utility index and EQ visual analog scale (VAS) scores, which were lower than general population norm at baseline, substantially improved by month 6 after exa-cel infusion and were sustained through month 36, with similar improvements in FACT-General (FACT-G) total and bone marrow transplant subscale. All FACT-G (physical, social/family, emotional, functional well-being) and ASCQ-Me (emotional, social functioning, stiffness, sleep impact) subscales showed clinically meaningful improvement, including ASCQ-Me pain subscales (impact, episode frequency, severity), with substantial decreases in pain episode frequency by month 6. Mean pain NRS score decreased by month 6 and was sustained. Consistent with adults, adolescents had improvements in mean EQ-5D-Y VAS score, PedsQL score, and pain NRS. Exa-cel led to broad and clinically meaningful HRQoL benefits in adults and adolescents with SCD. The CLIMB SCD-121 and CLIMB-131 trials were registered at www.clinicaltrials.gov as #NCT03745287 and #NCT04208529, respectively.},
}
@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.},
}
@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.},
}
@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.},
}
@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.},
}
@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.},
}
@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 &amp; 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.},
}
@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.},
}
@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.},
}
@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 &amp; purification/genetics ; *Nucleopolyhedroviruses/isolation &amp; 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.},
}
@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 &amp; dosage ; Polyethylene Glycols/chemistry ; *Polymers/chemistry ; Mice, Inbred BALB C ; Deoxycholic Acid/chemistry/administration &amp; dosage ; *Nanoparticles/chemistry/administration &amp; 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.},
}
@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 &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; P50CA097186 PNW Prostate Cancer SPORE Career Enhancement Program//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; 1F32CA236347-01//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; CA230516-02S1//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; 1R01CA221969-01//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; 1R01CA244550//U.S. Department of Health &amp; 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 &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; 1R01CA227025//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; P50CA186786//U.S. Department of Health &amp; 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//Vetenskapsr&#xe5;det (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 &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01GM131641//U.S. Department of Health &amp; 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.},
}
@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 &amp; Components Technology Development Program/ ; },
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Phlebovirus/isolation &amp; 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.},
}
@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.<br><br>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.},
}
@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.},
}
@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).},
}
@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 &amp; 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 &amp; 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.},
}
@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.},
}
@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.},
}
@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.},
}
@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.},
}
@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 &amp; 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.},
}
@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.},
}
@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.<br><br>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.<br><br>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.<br><br>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.},
}
@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.},
}
@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.},
}
@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.},
}
@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.},
}
@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.},
}
@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.},
}
@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.},
}
@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.},
}
@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 &amp; 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.},
}
@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.},
}
@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.},
}
@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.},
}
@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.},
}
@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.<br><br>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/&#x3bc;L for MUC1-positive exosomes within 60&#xa0;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.<br><br>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.},
}
@article {pmid41330004,
year = {2025},
author = {Coşar, B and Kılıç, P and İşeri, &#xd6;D},
title = {The intersection of CAR-T immunotherapy with emerging technologies.},
journal = {Cytokine &amp; 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.},
}
@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.},
}
@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.},
}
@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 &amp; 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.},
}
@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 &amp; 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.<br><br>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.<br><br>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.<br><br>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.},
}
@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 &amp; 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.},
}
@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.},
}
@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 &amp; 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.},
}
@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.},
}
@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.<br><br>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 (&#x394;S&#xa0;=&#xa0;-862, -813, and -615&#xa0;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.<br><br>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.},
}
@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 &amp; development ; *Plant Breeding/methods ; *Plant Proteins/genetics/metabolism ; Fruit/genetics/growth &amp; 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.},
}
@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.},
}
@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.},
}
@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.},
}
@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.},
}
@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 &amp; purification ; *Viral Vaccines/immunology/genetics/isolation &amp; 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.},
}
@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 &amp; 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.},
}
@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&amp;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.},
}
@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.<br><br>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.<br><br>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.<br><br>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.},
}
@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 &amp; 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.},
}
@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.},
}
@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.},
}
@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 &amp; 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 &amp; 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.},
}
@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.},
}
@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.},
}
@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 &amp; bioelectronics},
volume = {294},
number = {},
pages = {118241},
doi = {10.1016/j.bios.2025.118241},
pmid = {41285661},
issn = {1873-4235},
mesh = {Humans ; Female ; *MicroRNAs/genetics/isolation &amp; purification/analysis ; *Uterine Cervical Neoplasms/diagnosis/genetics ; *Biosensing Techniques ; *Telomerase/isolation &amp; 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.},
}
@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 &amp; 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.},
}
@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.},
}
@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.<br><br>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&#x202f;&#xd7;&#x202f;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.<br><br>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&#x202f;min (IQR: 41.25-98.75&#x202f;min).<br><br>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.<br><br>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.<br><br>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. NF&#x3ba;B, 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.<br><br>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.<br><br>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.},
}
@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.},
}
@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.},
}
@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.},
}
@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.<br><br>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.<br><br>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.<br><br>CONCLUSION: We explored the possibility of obtaining Cas9 mutants with high HDR efficiency via this screening system.},
}
@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.<br><br>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.<br><br>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.},
}
@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.},
}
@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.},
}
@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 &amp; 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.<br><br>AIM: This study aimed to develop a rapid, accurate, and field-deployable molecular diagnostic assay for T. equi.<br><br>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.<br><br>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.<br><br>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.},
}
@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 &amp; purification/genetics ; *Influenza A Virus, H1N1 Subtype/isolation &amp; 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.},
}
@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 &amp; 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.},
}
@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.},
}
@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 &amp; 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.},
}
@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.},
}
@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.},
}
@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.},
}
@article {pmid40684770,
year = {2025},
author = {Ali, A and Roy, B and Schott, MB and Grove, BD},
title = {AKAP12 Variant 1 Knockout Enhances Vascular Endothelial Cell Motility.},
journal = {Journal of vascular research},
volume = {62},
number = {6},
pages = {312-329},
pmid = {40684770},
issn = {1423-0135},
support = {P20 GM104360/GM/NIGMS NIH HHS/United States ; P20 GM113123/GM/NIGMS NIH HHS/United States ; P30 GM103329/GM/NIGMS NIH HHS/United States ; U54 GM128729/GM/NIGMS NIH HHS/United States ; },
mesh = {*A Kinase Anchor Proteins/genetics/metabolism ; *Cell Movement ; Humans ; *Cell Cycle Proteins/genetics/metabolism ; *Endothelial Cells/metabolism ; Cells, Cultured ; *Human Umbilical Vein Endothelial Cells/metabolism ; Intercellular Junctions/metabolism/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: Previous work indicates that AKAP12 is expressed in endothelial cells as two variants and may play a role in cell motility. However, the role of each variant in cell motility is unknown; therefore, this study investigated the role of AKAP12 in endothelial cell motility with a specific focus on AKAP12 variants, AKAP12v1 and AKAP12v2.<br><br>METHODS: AKAP12 expression levels in cultured endothelial cells were determined by Western blotting and immunofluorescence microscopy. AKAP12 knockdown and AKAP12 variant knockout were done using antisense oligonucleotide and siRNA treatment and CRISPR/Cas9 knockout, respectively. The effect of AKAP12 variant knockout was further analyzed by RNA-seq.<br><br>RESULTS: AKAP12 expression was cell density-dependent, with the highest expression in subconfluent cultures and lowest in confluent cultures. AKAP12 expression was also elevated in cells at the wound edge of wounded endothelial cell monolayers. Knockdown of both variants inhibited cell migration, but CRISPR/Cas9 knockout of AKAP12v1 enhanced migration. RNA-seq revealed that loss of AKAP12v1 affected genes associated with cell migration and intercellular junctions.<br><br>CONCLUSION: We propose that AKAP12v1 and AKAP12v2 play distinct yet complementary roles in endothelial cell migration and likely work together in controlling the signaling events associated with vascular repair and development.},
}
@article {pmid41330932,
year = {2025},
author = {Van Vu, T and Thi Nguyen, N and Kim, J and Sung, YW and Chung, WS and Kim, JY},
title = {The evolving landscape of precise DNA insertion in plants.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10428},
pmid = {41330932},
issn = {2041-1723},
support = {RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome, Plant ; Plants, Genetically Modified/genetics ; *DNA, Plant/genetics ; *Plants/genetics ; *Mutagenesis, Insertional/methods ; Gene Targeting/methods ; },
abstract = {Precise DNA insertion into plant genomes is central to advancing crop improvement and synthetic biology. CRISPR-Cas systems have enabled programmable DNA integration using tools such as gene targeting (GT), prime editing (PE), and recombinase- or transposase-based platforms. These tools are transitioned from theoretical concepts to practical applications, supporting applications like in-locus protein tagging, regulatory element engineering, and multi-gene stacking. Key challenges persist, such as inefficient large-fragment insertion, delivery barriers, and regulatory hurdles. This review traces the evolution from random to CRISPR-Cas-based systems, analyzes current limitations, and discusses emerging solutions paving the way for predictable DNA insertion in modern plant biotechnology.},
}
@article {pmid41330849,
year = {2025},
author = {Boob, AG and Zhang, C and Pan, Y and Zaidi, A and Whitaker, RJ and Zhao, H},
title = {Discovery, characterization, and application of chromosomal integration sites in the hyperthermophilic archaeon Sulfolobus islandicus.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.11.003},
pmid = {41330849},
issn = {1879-3096},
abstract = {Sulfolobus islandicus, an emerging archaeal model organism, offers unique advantages for metabolic engineering and synthetic biology applications owing to its ability to thrive in extreme environments. Although several genetic tools have been established for this organism, the lack of well-characterized chromosomal integration sites has limited its potential as a cellular factory. Here, we systematically identified and characterized 13 artificial CRISPR RNAs targeting eight integration sites in S. islandicus using the CRISPR-COPIES pipeline and a multi-omics-informed computational workflow. We leveraged the endogenous CRISPR-Cas system to integrate the reporter gene lacS and validated heterologous expression through a β-galactosidase assay, revealing significant positional effects. As a proof of concept, we utilized these sites to genetically manipulate lipid ether composition by overexpressing glycerol dibiphytanyl glycerol tetraether (GDGT) ring synthase B (GrsB). This study expands the genetic toolbox for S. islandicus and advances its potential as a robust platform for archaeal synthetic biology and industrial biotechnology.},
}
@article {pmid41330674,
year = {2026},
author = {Hou, L and Ruan, F and Zhao, K and Li, B},
title = {Boosting split-crRNA CRISPR/Cas12a activity by 3'-end extension of DNA activator for direct microRNA sensing.},
journal = {Analytica chimica acta},
volume = {1382},
number = {},
pages = {344841},
doi = {10.1016/j.aca.2025.344841},
pmid = {41330674},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics/blood ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Humans ; *DNA/chemistry/metabolism/genetics ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: The unique trans-cleavage activity of CRISPR/Cas12a has been extensively utilized in the domain of biosensing. Nevertheless, the detection of miRNAs using the traditional CRISPR/Cas12a system requires nucleic acid amplification or reverse transcription to convert miRNA into DNA, which increases reaction time and the risk of contamination.<br><br>RESULTS: This study presents a split-crRNA CRISPR/Cas12a-based biosensing for direct detection of miRNA. The target miRNA-375 was utilized as the spacer region of the crRNA, facilitating its binding to the truncated scaffold RNA, thereby resulting in the formation of a complete crRNA. More importantly, we discovered that the cleavage activity of split-crRNA CRISPR/Cas12a was significantly enhanced by extending sequences at the 3'-end of the DNA activator. Compared with the conventional split-crRNA CRISPR/Cas12a system, the split-crRNA CRISPR/Cas12a with 24-nucleotide random sequence extension at the 3'-end of the DNA activator exhibited a 6.4-fold increase in activity. The enhancement mechanism of 3'-end extension of DNA activator was discussed. This proposed split-crRNA CRISPR/Cas12a system was applied to detect miRNA-375 with a linear range of 5 pM-1&#xa0;nM, and the detection limit was estimated to be 0.6 pM (3&#x3c3;). Furthermore, this system was used to detect miRNA-375 in 10&#xa0;% diluted human serum, achieving satisfactory recovery rate (98&#xa0;%-106&#xa0;%).<br><br>SIGNIFICANCE: This finding indicates that it is feasible to enhance the activity of the split-crRNA CRISPR/Cas12a by extending the 3'-end of the DNA activator, thereby achieving highly sensitive direct detection of miRNA. It is a simple yet effective strategy for enhancing the sensitivity of direct miRNA detection.},
}
@article {pmid41330665,
year = {2026},
author = {Bao, Y and Ding, W and Zhang, L and Wang, W and Liu, J and Qu, Y and Zhu, L and Zhang, K and Zhong, G and Han, R and Shen, Q and Wang, B and Gu, X and Cao, Y and Sun, W},
title = {A novel multiplex RPA/CRISPR-Cas12a integrated biosensor for on-site detection of high-risk HPV genotypes.},
journal = {Analytica chimica acta},
volume = {1382},
number = {},
pages = {344830},
doi = {10.1016/j.aca.2025.344830},
pmid = {41330665},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Genotype ; *Nucleic Acid Amplification Techniques/methods ; *Papillomaviridae/genetics/isolation &amp; purification ; *Recombinases/metabolism ; Papillomavirus Infections/virology/diagnosis ; DNA, Viral/genetics ; },
abstract = {Accurate genotyping of high-risk human papillomavirus (HR-HPV) at the point of care is critical for global cervical cancer elimination, but its application remains limited by the need for complex equipment and specialized procedures, particularly in resource-limited regions. Here, we develop H-MRC12a-an integrated platform based on degenerate primers and type-specific crRNAs that combines multiplex recombinase polymerase amplification (RPA) with CRISPR-Cas12a trans-cleavage activity-for ultrasensitive detection of eight key HR-HPV genotypes (16, 18, 31, 33, 52, 53, 58, 66). By introducing a degenerate primer strategy coupled with type-specific crRNAs, the system overcomes interference from primer dimer formation and achieves single-copy sensitivity within 50 min under isothermal conditions (37 °C). Clinical validation demonstrated 100 % concordance with qPCR and identified three additional low viral-load positives (Ct > 35) that were missed by conventional methods. Crucially, the platform enables visual readout under UV light and eliminates the need for specialized instruments. This 'broad-spectrum capture and precision typing' paradigm establishes a versatile framework for multiplexed pathogen detection, advancing accessible molecular diagnostics for global health equity.},
}
@article {pmid41288387,
year = {2025},
author = {Kim, B and Yuk, M and Park, M and Ryu, H and Park, J and Yu, H and Park, S and Hong, JT and Lim, KH and Han, SB and Song, N and Park, H},
title = {CRISPR editing of miR-33 restores ApoE lipidation and amyloid-β metabolism in ApoE4 sporadic Alzheimer's disease.},
journal = {Brain : a journal of neurology},
volume = {148},
number = {12},
pages = {4400-4415},
doi = {10.1093/brain/awaf244},
pmid = {41288387},
issn = {1460-2156},
support = {//National Research Foundation of Korea/ ; 2021R1C1C1006551//Korean government/ ; //Bio &amp; Medical Technology Development Program/ ; //National Research Foundation/ ; RS-2024-00440787//Korean government/ ; },
mesh = {*MicroRNAs/genetics/metabolism ; *Alzheimer Disease/genetics/metabolism/pathology ; Animals ; Humans ; Mice ; *Gene Editing/methods ; *Apolipoprotein E4/genetics/metabolism ; *Lipid Metabolism/genetics ; *Amyloid beta-Peptides/metabolism ; Male ; CRISPR-Cas Systems ; Female ; Astrocytes/metabolism ; Mice, Transgenic ; *Apolipoproteins E/metabolism/genetics ; Aged ; Disease Models, Animal ; },
abstract = {Sporadic Alzheimer's disease (sAD) is marked by dysregulated lipid metabolism, prominently involving apolipoprotein E (ApoE). MicroRNA-33 (miR-33) has emerged as a key regulator of lipid homeostasis, yet its role in sAD remains unclear. This study investigated miR-33 dysregulation in APOE ε4 allele (ApoE4)-associated sAD and explored its therapeutic potential using clustered regulatory interspaced short palindromic repeats (CRISPR)-mediated gene editing. Elevated miR-33 expression was observed in both AD patients, particularly those with ApoE4-associated sAD, and in the ApoE4 mouse model, implicating its role in AD pathology. Using CRISPR/Cas9, we modulated miR-33 expression in astrocytes to regulate ApoE lipidation and ameliorate AD-related pathology. Our results show that targeted miR-33 regulation in astrocytes via CRISPR/Cas9 restores ApoE lipidation and mitigates AD pathology in both in vitro and in vivo AD mice. Additionally, applying this gene therapy approach in ApoE4 sAD patient cell lines highlights its translational potential for therapeutic intervention. In conclusion, our findings elucidate miR-33's role in AD pathogenesis and underscore the therapeutic promise of CRISPR-mediated miR-33 targeting for restoring lipid homeostasis and ameliorating AD pathology. This study provides valuable insights into developing miRNA-based gene therapy strategies for treating sAD.},
}
@article {pmid41284724,
year = {2025},
author = {da Silva, GLA and Damasceno, JD and Black, JA and Lapsley, C and McCulloch, R and Tosi, LRO},
title = {ATR, a DNA damage kinase, modulates DNA replication timing in Leishmania major.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011899},
doi = {10.1371/journal.pgen.1011899},
pmid = {41284724},
issn = {1553-7404},
mesh = {*Leishmania major/genetics ; *Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; DNA Damage/genetics ; *DNA Replication/genetics ; CRISPR-Cas Systems ; *DNA Replication Timing/genetics ; *Protozoan Proteins/genetics/metabolism ; },
abstract = {All cells possess mechanisms to maintain and replicate their genomes, whose integrity and transmission are constantly challenged by DNA damage and replication impediments. In eukaryotes, the protein kinase Ataxia-Telangiectasia and Rad3-related (ATR), a member of the phosphatidylinositol 3-kinase-like family, acts as a master regulator of the eukaryotic response to DNA injuries, ensuring DNA replication completion and genome stability. Here we aimed to investigate the functional relevance of the ATR homolog in the DNA metabolism of Leishmania major, a protozoan parasite with a remarkably plastic genome. CRISPR/cas9 genome editing was used to generate a Myc-tagged ATR cell line (mycATR), and a Myc-tagged C-terminal knockout of ATR (mycATRΔC-/-). We show that the nuclear localisation of ATR depends upon its C-terminus. Moreover, its deletion results in single-stranded DNA accumulation, impaired cell cycle control, increased levels of DNA damage, and delayed DNA replication re-start after replication stress. In addition, we show that ATR plays a key role in maintaining L. major's unusual DNA replication program, where larger chromosomes duplicate later than smaller chromosomes. Our data reveals loss of the ATR C-terminus promotes the accumulation of DNA replication signal around replicative stress fragile sites, which are enriched in larger chromosomes. Finally, we show that these alterations to the DNA replication program promote chromosome instability. In summary, our work shows that ATR acts to modulate DNA replication timing, limiting the plasticity of the Leishmania genome.},
}
@article {pmid41233591,
year = {2025},
author = {Li, L and Gao, J and Yi, D and Sheft, AP and Schimenti, JC and Ding, X},
title = {A primordial germ cell-like-cell platform enables CRISPRi screen for epigenetic fertility modifiers.},
journal = {EMBO reports},
volume = {26},
number = {23},
pages = {6044-6078},
pmid = {41233591},
issn = {1469-3178},
support = {R01 HD082568/HD/NICHD NIH HHS/United States ; 1P50HD096723//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; CO29155//New York State Stem Cell Science (NYSTEM)/ ; C30293GG//New York State Stem Cell Science (NYSTEM)/ ; R01 HD082568/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Germ Cells/metabolism/cytology ; *Epigenesis, Genetic ; Male ; Positive Regulatory Domain I-Binding Factor 1/genetics/metabolism ; Cell Differentiation/genetics ; Nanog Homeobox Protein/genetics/metabolism ; *Fertility/genetics ; Transcription Factor AP-2/genetics/metabolism ; Transcription Factors/genetics/metabolism ; *CRISPR-Cas Systems ; Female ; Repressor Proteins/genetics/metabolism ; RNA-Binding Proteins/genetics ; DNA-Binding Proteins/genetics/metabolism ; },
abstract = {Primordial germ cells (PGCs) are the precursors of gametes, and the ability to derive PGC-like cells (PGCLCs) from pluripotent stem cells has transformed germline research. A key limitation remains producing PGCLCs in sufficient numbers for large-scale applications. Here, we show that overexpression of Nanog plus three PGC master regulators - Prdm1, Prdm14, and Tfap2c - in mouse epiblast-like cells and formative embryonic stem cells yields abundant and highly enriched PGCLCs without costly recombinant cytokines. Nanog enhances the PGC regulatory network, suppresses somatic differentiation, and stabilizes PGCLC fate. Transcriptomically, these PGCLCs are developmentally more advanced than cytokine-induced counterparts and can be sustained long-term or differentiated into spermatogonia-like cells. Using this platform, we conduct a CRISPRi screen of 701 epigenetic genes to identify those needed for PGCLC formation. Downregulation of Ncor2, a histone deacetylase (HDAC) recruiter, has the greatest impact. Additionally, the HDAC inhibitors valproic acid and sodium butyrate suppress PGCLC formation and sperm counts of in utero-exposed animals. This work establishes a scalable system for functional screening of genes that influence germline development.},
}
@article {pmid41214358,
year = {2025},
author = {Liu, XY and Li, YF and Zhou, JZ and Huang, JQ and Wang, LJ and Yang, L and Li, CY and Fang, X and Chen, XY},
title = {Gamma-selinene synthase catalyzes the first step of dihydroagarofuran sesquiterpene alkaloid biosynthesis in Tripterygium.},
journal = {Science China. Life sciences},
volume = {68},
number = {12},
pages = {3696-3710},
pmid = {41214358},
issn = {1869-1889},
mesh = {*Tripterygium/genetics/enzymology/metabolism ; *Sesquiterpenes/metabolism/chemistry ; *Alkaloids/biosynthesis ; *Plant Proteins/metabolism/genetics ; Biosynthetic Pathways ; Phylogeny ; CRISPR-Cas Systems ; },
abstract = {Tripterygium wilfordii and T. hypoglaucum, which belong to family Celastraceae, are perennial vine shrubs with medicinal uses in treating rheumatoid arthritis, cancer, and male contraception. Among the bioactive ingredients, the macrocyclic dihydroagarofuran sesquiterpene alkaloids (DASAs), also serving as chemotaxonomic markers of Celastraceae, are well-known for cytotoxicity and insecticidal properties. Despite intensive phytochemical investigations over the last half-century, the DASAs biosynthetic pathway remains unsolved. Here, we mined multi-omics data of Tripterygium species and identified 14 sesquiterpene synthase genes. Detailed characterization revealed that a group of enzymes (TwTPS5, TwTPS16, ThTPS5) catalyzed the key step committed to the biosynthesis of DASAs in Tripterygium, as evidenced by CRISPR/Cas9 knockout of TwTPS5 in T. wilfordii. Our biochemical assay showed that these enzymes converted FPP to gamma-selinene. Homologues in other Celastraceae species (EaTPS3, CaTPS5, and CaTPS6) also produced gamma-selinene, suggesting functional conservation in this DASAs-producing lineage. Notably, knocking out TwTPS5 resulted in a reduction of DASAs and enhanced accumulation of pharmaceutically valuable compounds, generating a safer and innovative medicinal plant resource for future applications.},
}
@article {pmid41139756,
year = {2025},
author = {Kang, CM and Jeong, RD},
title = {Rapid and visual detection of Lentinula edodes spherical virus using CRISPR/Cas12a-based RT-RPA.},
journal = {Virus genes},
volume = {61},
number = {6},
pages = {724-732},
pmid = {41139756},
issn = {1572-994X},
mesh = {*Shiitake Mushrooms/virology/genetics ; *CRISPR-Cas Systems ; *Fungal Viruses/genetics/isolation &amp; purification ; Republic of Korea ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {Lentinula edodes (shiitake) is a globally important edible mushroom, but viral infections can hinder mycelial growth, reduce yield, and degrade quality, leading to economic losses. This study examined viral infections in L. edodes cultivated in South Korea. We developed a CRISPR/Cas12a-based reverse transcription recombinase polymerase amplification (RT-RPA) assay for the rapid and accurate detection of L. edodes spherical virus (LeSV), the predominant mycovirus-infecting L. edodes in Korea. The optimized RT-RPA-CRISPR/Cas12a assay can be completed within 20 min at 34 °C and demonstrated 100 times higher sensitivity than RT-PCR. Field sample verification further confirmed its superior detection rate over RT-PCR. The RT-RPA-CRISPR/Cas12a method described herein has the potential to facilitate early diagnosis of LeSV infections and support disease management in L. edodes cultivation.},
}
@article {pmid41016102,
year = {2026},
author = {Guo, X and Zhang, J and Han, K and Li, Y and Wang, D},
title = {Aptamer-based PSA biosensing technology: From nanomaterials to CRISPR-diagnostics technology.},
journal = {Talanta},
volume = {298},
number = {Pt A},
pages = {128871},
doi = {10.1016/j.talanta.2025.128871},
pmid = {41016102},
issn = {1873-3573},
mesh = {*Prostate-Specific Antigen/analysis/blood ; Humans ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide/chemistry ; *Nanostructures/chemistry ; *Prostatic Neoplasms/diagnosis ; Male ; *CRISPR-Cas Systems ; },
abstract = {Prostate cancer is a leading cause of cancer-related mortality in men, making early detection crucial for improving patient outcomes. Prostate-specific antigen (PSA) is a key biomarker for prostate cancer. Traditional PSA detection methods, such as ELISA and CLIA, are widely used but have limitations including complex sample preparation and long assay times. Aptamer-based biosensors offer a promising alternative due to their high affinity, specificity, and ease of customization. Recent advancements in aptamer-based PSA biosensors, including the use of novel nanomaterials and dual-modal detection technologies, have significantly enhanced sensitivity and reliability. The integration of CRISPR-Dx technology has further improved the accuracy of PSA detection in complex biological samples. These innovations hold great potential for developing portable point-of-care testing (POCT) devices for early screening and personalized treatment. This review examines the latest developments in aptamer-based biosensing technology for PSA detection from 2019 to 2025, focusing on nanomaterial innovation, CRISPR-Dx integration, and dual-modal sensing. It aims to provide a comprehensive overview of the strengths and limitations of current technologies and to outline future directions for more efficient and clinically valuable PSA detection methods.},
}
@article {pmid40985613,
year = {2025},
author = {Chen, S and Chen, X and Peng, Y and Li, Q and Zhou, J and Li, J and Du, G and Chen, J and Zhang, G},
title = {CRISPR-DNA Polymerase Assisted Targeted Mutagenesis for Regulable Laboratory Evolution.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {45},
pages = {e11448},
doi = {10.1002/advs.202511448},
pmid = {40985613},
issn = {2198-3844},
support = {2024YFA0918300//National Key Research and Development Program of China/ ; 32172153//National Natural Science Foundation of China/ ; BK20202002//Natural Science Foundation of Jiangsu Province/ ; },
mesh = {*DNA-Directed DNA Polymerase/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Mutagenesis/genetics ; Escherichia coli/genetics ; *Directed Molecular Evolution/methods ; Mutation/genetics ; },
abstract = {Targeted hypermutation tools are useful for engineering proteins and pathways, and exploring the evolutionary landscapes. However, existing targeted hypermutation tools for genomic loci mostly exhibit restricted mutation windows and limited mutational types. Here, by integrating mutagenic, high-processivity bacteriophage T5 or T7 DNA polymerases (DNAPs) with CRISPR-Cas9, the study develops an in vivo mutagenesis system that enables all possible types of nucleotide substitutions and an expanded mutation window of up to 2 kilobases, achieving a maximum mutation rate 1.1 × 10[6]-fold higher than wild-type Escherichia coli. Through MS2-mediated recruitment of T5 or T7 DNAP for co-localization with nickase nCas9, off-target rate is reduced by up to 96.8% without compromising on-target rate. Further benefiting from the dTnpB-based transcriptional repression system, the mutagenesis process can be properly regulated during continuous evolution. Finally, the CRISPR-TDNAP-assisted targeted mutagenesis for regulable laboratory evolution (CTRLE) confers cellular triple-antibiotic resistance in 8 days, and enhances the efficiency of the twin-arginine translocation pathway by over threefold in 6 days. Furthermore, CTRLE proves effective in Bacillus subtilis and Kluyveromyces lactis, yielding targeted mutation rates 1.2 × 10[5]-fold and 5 × 10[7]-fold higher than host backgrounds, respectively. Collectively, CTRLE provides an efficient and universal way to accelerate the continuous evolution of different microbial cells.},
}
@article {pmid40961712,
year = {2026},
author = {Zheng, Y and Li, Y and Zhu, J and Yi, J and Li, T and Tang, H},
title = {Synergistic UCNP/CRISPR-Cas12a enhanced near-infrared photoelectrochemical biosensor for quantitative Kanamycin detection.},
journal = {Talanta},
volume = {298},
number = {Pt A},
pages = {128857},
doi = {10.1016/j.talanta.2025.128857},
pmid = {40961712},
issn = {1873-3573},
mesh = {*Kanamycin/analysis ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Milk/chemistry ; Zinc Oxide/chemistry ; *Food Contamination/analysis ; Animals ; *Anti-Bacterial Agents/analysis ; Infrared Rays ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; Sulfides/chemistry ; *Nanoparticles/chemistry ; Cadmium Compounds/chemistry ; *Bacterial Proteins/metabolism/genetics ; Electrodes ; Ytterbium/chemistry ; Photochemical Processes ; Yttrium/chemistry ; Endodeoxyribonucleases ; Zinc Compounds ; CRISPR-Associated Proteins ; },
abstract = {Antibiotic residues in food pose significant health risks, and sensitive on-site detection remains challenging in complex matrices. We report a near-infrared photoelectrochemical (NIR-PEC) biosensor with CRISPR-Cas12a signal amplification for sensitive detection of kanamycin in food. A ZnO/CdS heterojunction photoanode boosts visible-light charge separation, producing a photocurrent of 4.6 mA-96 % higher than ZnO alone. Upconversion nanoparticles (NaYF4:Yb[3+], Er[3+]) convert 980 nm light into 542 nm emission to drive the heterojunction under NIR illumination. In the presence of kanamycin, activator DNA is displaced from an aptamer, triggering Cas12a to cleave ssDNA probes on the electrode and reduce photocurrent. The sensor shows a linear response from 10 to 1000 nM, a detection limit of 0.284 nM and less than 2.8 % cross-reactivity with related compounds. Average recovery in spiked milk samples averages 97 % (RSD = 2.2 %). This platform offers high specificity and sensitivity for antibiotic residue analysis in complex food matrices.},
}
@article {pmid40956287,
year = {2025},
author = {Shen, Y and Lu, B and Ma, B and Ding, X},
title = {Enhanced One-Pot Dual-CRISPR-Based Assay Lyophilized on a 3D-Printed Disc for Field-Deployable Multiplex Bacteria Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {45},
pages = {e09355},
doi = {10.1002/advs.202509355},
pmid = {40956287},
issn = {2198-3844},
support = {2023YFD2402800//National Key Research and Development Program of China for Young Scientists/ ; 2022YFF1102600//National Key Research and Development Program of China for Young Scientists/ ; 82373629//National Natural Science Foundation of China/ ; BE2023725//Jiangsu Provincial Key Research and Development Program/ ; 4060692202/023//Medical Foundation of Southeast University/ ; },
mesh = {*Printing, Three-Dimensional ; Freeze Drying/methods ; *Nucleic Acid Amplification Techniques/methods ; *Bacteria/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; },
abstract = {One-pot CRISPR-based detection combining recombinase polymerase amplification (RPA) enables rapid and accurate nucleic acid testing but faces challenges in performance, multiplexing, and field-ready lyophilization. Here, an enhanced one-pot, helicase-assisted RPA (hRPA)-combined, dual-CRISPR/uAsCas12a (EOD-CRISPR) assay is described which can be lyophilized on a 3D-printed microfluidic disc to achieve field-deployable multiplex bacteria detection. In EOD-CRISPR reactions, the reaction speed, sensitivity, and fluorescence signal are significantly enhanced due to the synergistic effect of bovine serum albumin, hRPA, and uAsCas12a nuclease. The 3D-printed disc features four central chambers encircled by eight outer chambers, permitting detecting four targets simultaneously. For stable lyophilization on disc chambers, glassfiber membranes are inserted as substrates to adsorb the EOD-CRISPR reagents containing a protectant of 5% trehalose and 1% glycine. Toward point-of-need testing, EOD-CRISPR-lyophilized discs are applied to build an onsite detection platform. Through detecting synthetic food samples contaminated by four foodborne bacteria (i.e., Bacillus cereus, Salmonella enterica, Staphylococcus aureus, and Escherichia coli O157:H7), the onsite detection platform is validated and the sensitivity (80%-88.9%) and specificity (92.3%-100%) are comparable to those of standard PCR methods. Therefore, the field-deployable multiplex EOD-CRISPR assays holds great potentials for onsite bacteria detection and beyond.},
}
@article {pmid40946480,
year = {2026},
author = {Zhang, Q and Liu, Z and Sheng, E and Liu, Z and Zhao, R and Li, M},
title = {Glucosemeter-based aptasensor with an efficient and green system driven by CRISPR/Cas12a for ZEN detection.},
journal = {Talanta},
volume = {298},
number = {Pt A},
pages = {128844},
doi = {10.1016/j.talanta.2025.128844},
pmid = {40946480},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Zearalenone/analysis ; *Aptamers, Nucleotide/chemistry/genetics/metabolism ; Limit of Detection ; *CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {To address the challenge of readily available and cost-effective monitoring, a novel aptasensor platform was proposed relying on the clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a)-driven and glucosemeter-assisted system for zearalenone (ZEN). The magnetic bead (MB)-based probes of MB@Apt-cDNA and MB@invertase were prepared to provide specific recognition and efficient conversion. When ZEN presented, the released cDNA activated the cutting function of CRISPR/Cas12a for ssDNA, releasing numerous invertase to convert the environmentally friendly sucrose. Then, the generated glucose signals were positively related to ZEN levels, which quantified by a glucosemeter. The proposed aptasensor displayed an excellent sensitivity, achieving the limit of detection (LOD) and detection range of 0.218 ng/mL and 0.218-109.89 ng/mL, respectively. The platform also exhibited high specificity (with cross-reactivity values below 6.81 % for other analytes) and high accuracy for ZEN detection. The satisfactory reliability and practicability had been demonstrated through its application in real agricultural byproduct samples. The area under the curve was 0.979, with an optimal operating point being (0.125, 1.000). This study can provide an alternative pathway for improving the detection performance and accessibility to public in limited situation, suggesting the efficient CRISPR/Cas12a and green glucosemeter detection strategies for other pollutant monitoring.},
}
@article {pmid40939440,
year = {2026},
author = {Xiang, Q and Zhou, W and Li, D},
title = {Dumbbell-shaped DNA topology drives self-sustaining CRISPR/Cas12a exponential amplification for ultrasensitive monitoring of DNA methyltransferase activity.},
journal = {Talanta},
volume = {298},
number = {Pt A},
pages = {128841},
doi = {10.1016/j.talanta.2025.128841},
pmid = {40939440},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems ; *DNA/chemistry/metabolism ; DNA Methylation ; Nucleic Acid Conformation ; Humans ; *Enzyme Assays/methods ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {DNA methylation is an essential epigenetic mechanism, and abnormal methylation has been linked to the onset and progression of many diseases, representing a potential threat to health. Monitoring DNA methyltransferase (MTase) activity is essential for understanding DNA methylation regulation and developing MTase-targeted inhibitors. To address this challenge, we developed a dumbbell-shaped DNA topology that drives self-sustaining (autocatalytic) CRISPR/Cas12a system for exponential signal amplification, enabling ultrasensitive fluorescent detection of DNA MTase activity. In this strategy, a DNA dumbbell topological structure (DDTS) is designed, in which two double-strand DNA (dsDNA) loops effectively block the activity of CRISPR/Cas12a. Upon Dam MTase presence, DpnI endonuclease cleaves the methylated recognition sites in the DNA probe, disrupting the DDTS topology to generate linear dsDNA activators. These activators restore the trans-cleavage of CRISPR/Cas12a, which further cleaves the single-stranded DNA (ssDNA) domain in DDTS probes to produce additional activators, creating an exponential amplification loop through autocatalysis. The system achieves a detection limit of 6.37 × 10[-4] U/mL for Dam MTase, with a linear range of 1 × 10[-3] to 15 U/mL, and shows excellent selectivity over other MTases and nucleases. It also enables inhibitor screening, with the half-maximal inhibitory concentration (IC50) value of 1.84 μM for 5-fluorouracil. Therefore, the method has great potential for application in the early diagnosis of diseases and drug discovery.},
}
@article {pmid40898619,
year = {2025},
author = {Noh, B and Gopalappa, R and Lin, H and Gee, HY and Choi, JY and Kim, HH and Jung, J},
title = {Engineered virus-like particles for in vivo gene editing ameliorate hearing loss in murine DFNA2 model.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {12},
pages = {6449-6462},
doi = {10.1016/j.ymthe.2025.08.049},
pmid = {40898619},
issn = {1525-0024},
mesh = {Animals ; Mice ; *Gene Editing/methods ; Disease Models, Animal ; *Genetic Therapy/methods ; *Hearing Loss/therapy/genetics ; CRISPR-Cas Systems ; Hair Cells, Auditory, Outer/metabolism ; Genetic Vectors/genetics ; Gene Transfer Techniques ; KCNQ Potassium Channels/genetics ; *Virion/genetics ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Although gene editing therapy is applicable to human diseases, its efficiency and safety require further investigation. Further, non-virus-mediated gene editor delivery is challenging in the inner ear. Here, engineered virus-like particles (eVLPs) were used for inner ear delivery of SpCas9 and single-guided RNA to delete the Kcnq4 dominant-negative mutant allele, which causes progressive hearing loss in a non-syndromic hearing loss murine model. eVLP-delivered SpCas9 was administered to the inner ears of Kcnq4[W277S/+] mice to target the Kcnq4-expressing outer hair cells (OHCs). Hearing loss was significantly alleviated 7 weeks after eVLP administration. OHC survival improved significantly, and OHC-innervating neurite (connected to type II spiral ganglion neuronal body) loss was ameliorated. Finally, OHC membrane potential was hyperpolarized with eVLP gene editor treatment in Kcnq4-mutant mice, indicating that their OHCs were healthier and more stable than those of uninjected mice. Our findings suggest that eVLPs are feasible inner ear gene editor deliverers to treat hearing loss.},
}
@article {pmid40885667,
year = {2025},
author = {Liu, W and Duan, W and Peng, Z and Liao, Y and Wang, X and Liu, R and Jing, Q and Jiang, H and Fan, Y and Ge, L and Huang, L and Xing, Y},
title = {Highly efficient prime editors for mammalian genome editing based on porcine retrovirus reverse transcriptase.},
journal = {Trends in biotechnology},
volume = {43},
number = {12},
pages = {3253-3278},
doi = {10.1016/j.tibtech.2025.07.029},
pmid = {40885667},
issn = {1879-3096},
mesh = {Animals ; *Gene Editing/methods ; Swine ; *RNA-Directed DNA Polymerase/genetics/metabolism ; Humans ; *Endogenous Retroviruses/genetics/enzymology ; CRISPR-Cas Systems ; Swine, Miniature ; HEK293 Cells ; },
abstract = {Prime editing is a versatile and precise genome-editing tool. Most prime editors (PEs) rely on reverse transcriptase (RT) derived from Moloney murine leukemia virus (MMLV). Here, we established a PE, pvPE, using a RT derived from a porcine endogenous retrovirus (PERV) from a Bama mini-pig. Through various optimization strategies, including RT engineering, structural modifications, and La protein fusion, we gradually upgraded to pvPE-V4. This version achieved 24.38-101.69-fold higher efficiency compared with pvPE-V1 and up to 2.39-fold higher efficiency than another upgraded PE, PE7, with significantly fewer unintended edits across multiple mammalian cell lines. We further show that nocodazole (Noc) significantly enhanced pvPE efficiency by 2.25-fold on average. Using our pvPE system, we efficiently modified three genes simultaneously in porcine fibroblasts and subsequently generated cloned pigs that could serve as valuable models for Alzheimer's disease (AD) in humans. Our results highlight the broad application prospects of pvPE systems in mammalian genome editing.},
}
@article {pmid40878354,
year = {2025},
author = {Shan, S and Pisias, MT and Zhang, Z and Mavrodiev, EV and Gitzendanner, MA and Hauser, BA and Grover, CE and Barbazuk, WB and Soltis, PS and Yang, B and Soltis, DE},
title = {Development of an efficient CRISPR-mediated genome editing platform in the diploid-polyploid model system Tragopogon (Asteraceae).},
journal = {Journal of experimental botany},
volume = {76},
number = {22},
pages = {6700-6713},
doi = {10.1093/jxb/eraf380},
pmid = {40878354},
issn = {1460-2431},
support = {IOS-1923234//National Science Foundation/ ; DEB-2043478//National Science Foundation/ ; },
mesh = {*Gene Editing/methods ; *Polyploidy ; *CRISPR-Cas Systems ; *Tragopogon/genetics ; *Genome, Plant ; Diploidy ; Alcohol Oxidoreductases/genetics/metabolism ; },
abstract = {Polyploidy or whole-genome duplication (WGD) is a significant evolutionary force. However, the mechanisms governing polyploid genome evolution remain unclear, limited largely by a lack of functional analysis tools in organisms that best exemplify the earliest stages of WGD. Tragopogon (Asteraceae) includes an evolutionary model system for studying the immediate consequences of polyploidy. In this study, we significantly improved the transformation system and obtained genome-edited T. porrifolius (2x) and T. mirus (4x) primary generation (T0) individuals. Using CRISPR/Cas9, we knocked out the dihydroflavonol 4-reductase (DFR) gene, which controls anthocyanin synthesis, in both species. All transgenic allotetraploid T. mirus individuals had at least one mutant DFR allele, and 71.4% had all four DFR alleles edited. The resulting mutants lacked anthocyanin, and these mutations were inherited in the T1 generation. This study demonstrates a highly efficient CRISPR platform, producing genome-edited Tragopogon individuals that have completed the life cycle. The approaches used and challenges faced in building the CRISPR system in Tragopogon provide a framework for building similar systems in other non-genetic models. Genome editing in Tragopogon paves the way for novel functional biology studies of polyploid genome evolution and the consequences of WGD on complex traits, holding enormous potential for both basic and applied research.},
}
@article {pmid40744808,
year = {2025},
author = {Luo, G and Trinh, MDL and Falkenberg, MKD and Chiurazzi, MJ and Najafi, J and Nørrevang, AF and Correia, PMP and Palmgren, M},
title = {Unlocking in vitro transformation of recalcitrant plants.},
journal = {Trends in plant science},
volume = {30},
number = {12},
pages = {1306-1321},
doi = {10.1016/j.tplants.2025.07.001},
pmid = {40744808},
issn = {1878-4372},
mesh = {*Gene Editing/methods ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; *Plants/genetics ; },
abstract = {Genome editing offers powerful opportunities for crop improvement by enabling precise and targeted mutagenesis. Tools such as CRISPR-associated protein 9 and single-guide RNAs can be introduced into plant cells via in vitro transformation, which, despite the rise of in planta methods, remains an important method because it is highly effective when successful. However, transformation-induced stress is a critical and underexplored barrier to successful in vitro transformation, especially in recalcitrant plants. In this opinion article, we discuss in vitro methods for transforming recalcitrant plants, the challenges encountered, and potential solutions through the conceptual lens of stress biology. Reducing cellular stress, transiently weakening the immune response and optimizing regeneration protocols may be essential for expanding the transformation capacity across a broader range of plant species.},
}
@article {pmid41328758,
year = {2026},
author = {Plewnia, A and Hoenig, BD and Lötters, S and Heine, C and Erens, J and Böning, P and Bending, GD and Krehenwinkel, H and Williams, MA},
title = {The Emergence of a CRISPR-Cas Revolution in Ecology: Applications, Challenges, and an Ecologist's Overview of the Toolbox.},
journal = {Molecular ecology resources},
volume = {26},
number = {1},
pages = {e70086},
pmid = {41328758},
issn = {1755-0998},
support = {//University of Warwick/ ; NE/S010270/1//Natural Environment Research Council/ ; },
mesh = {*CRISPR-Cas Systems ; *Ecology/methods ; *Gene Editing/methods ; },
abstract = {CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated nucleases) systems allow researchers to detect, capture, and even alter parts of an organism's genome. However, while the use of CRISPR-Cas has revolutionised many fields in the life sciences, its full potential remains underutilised in ecology and biodiversity research. Here we outline the emerging applications of CRISPR-Cas in ecological contexts, focusing on three main areas: nucleic acid detection, CRISPR-enhanced sequencing, and genome editing. CRISPR-based nucleic acid detection of environmental DNA samples is already reshaping species monitoring, providing highly sensitive and non-invasive tools for both scientists and the public alike, with reduced costs and minimal experience required. Further, CRISPR-enhanced sequencing, including Cas-mediated target enrichment, enables efficient recovery of ecologically relevant loci and supports diverse applications such as amplification-free metagenomics. Finally, while genome editing on wild species remains largely theoretical in ecology, these tools are already being used in controlled settings to study adaptation and resilience in the face of ongoing global stressors. Together, the applications of CRISPR-Cas are paving the way for more affordable, accessible, and impactful applications for species conservation, and promise to improve our ability to tackle the ongoing global biodiversity crisis.},
}
@article {pmid41328592,
year = {2025},
author = {Geng, Y and Jiang, C and Zhang, H and Yang, H and Peng, Y and Chen, Y and Hu, C and Liu, H and Li, S and Chen, H and Xie, S and Guo, A},
title = {Genome-scale CRISPR screen identifies host factors associated with bovine parainfluenza virus 3 infection.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2589554},
doi = {10.1080/21505594.2025.2589554},
pmid = {41328592},
issn = {2150-5608},
mesh = {Animals ; Cattle ; *Parainfluenza Virus 3, Bovine/physiology/genetics ; CRISPR-Cas Systems ; Virus Replication ; *Host-Pathogen Interactions/genetics ; Cell Line ; *Respirovirus Infections/virology/veterinary/genetics ; Wnt-5a Protein/genetics/metabolism ; *Cattle Diseases/virology ; Virus Internalization ; Gene Knockout Techniques ; },
abstract = {Bovine parainfluenza virus type 3 (BPIV-3) is a major pathogen associated with the bovine respiratory disease complex. However, the limited understanding of host factors crucial for BPIV-3 replication has hindered the development of effective preventive and therapeutic strategies. To tackle this critical issue, we constructed a bovine genome-wide CRISPR/Cas9 knockout library in Madin-Darby bovine kidney cells, which was then used to systematically identify and characterize the host genes essential for BPIV-3a replication. Subsequently, 10 genes were validated using both RT-qPCR and viral titration assays. Furthermore, through gene knockout or knockdown and rescue experiments, we identified three key genes required for BPIV-3a replication: Wnt family member 5A (WNT5A), solute carrier family 16 member 13 (SLC16A13), and selenoprotein N (SELENON). However, their effects on viral adhesion and internalization varied. WNT5A was involved in both processes, SLC16A13 participated solely in internalization, while SELENON had no significant impact on either. Beyond BPIV-3a, these three genes were also found to be essential for the infection of BPIV-3c and Bovine enterovirus. In conclusion, this study offers novel insights into the molecular mechanisms governing the replication and pathogenesis of BPIV-3a, BPIV-3c, and bovine enterovirus within host cells, thereby providing a foundation for identifying potential targets in the development of novel antiviral strategies.},
}
@article {pmid41328409,
year = {2026},
author = {Zhang, H and Song, Y and Liu, W and Zheng, X and An, X and Li, C and Chen, W and Wang, H and Zhang, Y},
title = {Defect-complementation homologous recombination: A novel strategy for precise genome engineering of virulent phages.},
journal = {Synthetic and systems biotechnology},
volume = {12},
number = {},
pages = {59-70},
pmid = {41328409},
issn = {2405-805X},
abstract = {Engineered bacteriophages (phages) have been developed to overcome the limitations of natural phage therapies and serve as precision-targeted agents against drug-resistant bacterial infections. However, their application has been constrained by the low efficiency of existing genome-editing tools, largely because of the absence of effective selection markers. This study proposed a novel strategy, termed defect-complementation homologous recombination (DCHR), for precise phage genome editing. In this approach, CRISPR-Cas9 cleaves a donor plasmid in host cells to release a linear donor template carrying homology arms, an essential phage gene used as a selection marker, and two lox sites. The donor template undergoes homologous recombination with the genome of essential gene-deficient phage, thereby enabling targeted genome modifications. Using DCHR, we successfully generated large genomic deletions (1.48-kb gp0.4-0.7 and 1.02-kb gp4.3-4.7), achieved gene insertion (3.08-kb lacZ), and introduced a single-base substitution (TGA to TAA) in the stop codon of gp9 within the same T7 phage genome, all with 100 % accuracy. The significant advantages of DCHR are as follows: (i) High-efficiency screening: Only progeny phages derived from successful homologous recombination retain viability and replicative capacity, thereby greatly simplifying recombinant isolation. (ii) Editing flexibility: Unlike CRISPR-Cas systems, DCHR cannot be constrained by protospacer adjacent motif dependence and allows modifications across diverse genomic loci. (iii) High recombination efficiency: DCHR can achieve a recombinant phage titer of 3.1 × 10[5] PFU mL[-1] (plaque-forming units per mL) without relying on exogenous homologous recombination systems. In summary, DCHR demonstrates potential as a precise and efficient general genome-editing tool that facilitates design of engineered phages and advances functional genomic studies.},
}
@article {pmid41328347,
year = {2026},
author = {Li, Q and Bao, Q and Zhao, S and Wu, F and Li, Y and Wang, K and Li, W and Gao, H},
title = {Advancements in CRISPR-based therapies for ocular pathologies: from disease mechanisms to intervention strategies.},
journal = {Theranostics},
volume = {16},
number = {1},
pages = {156-192},
pmid = {41328347},
issn = {1838-7640},
mesh = {Humans ; *Genetic Therapy/methods ; *Eye Diseases/therapy/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {Eye diseases caused by genetic mutations affect over 2.2 billion people worldwide. The development of CRISPR technology has opened exciting possibilities for how we diagnose and treat these conditions. However, designing effective CRISPR systems, managing potential risks, and considering the ethical questions around gene therapy in clinical practice are major challenges. To move forward successfully, it's important to evaluate how practical CRISPR-based treatments are for eye diseases from a clinical perspective, while also understanding how CRISPR systems work. In this review, we start by covering the basic principles behind CRISPR technology and explore its different types. Next, we look at various ways CRISPR is being used in eye research and treatments, from early studies to new clinical approaches. Lastly, we address the regulatory environment and ethical issues involved, discussing existing rules, safety concerns, and guidelines for genetic modifications in medical settings. Our goal is to share new insights into innovative treatments for eye diseases and to support the safe use of CRISPR in clinical eye care. This review aims to be a helpful resource for researchers, doctors, and regulators working on CRISPR-based therapies.},
}
@article {pmid41326076,
year = {2026},
author = {Niu, RC and Zeng, QH and Wang, WJ and Hu, J and Liu, TX and Zhang, SZ},
title = {Multi-omics analyses identify the modulators COX6A1 and NAL as regulators of silk cocoon formation in Plutella xylostella.},
journal = {Pesticide biochemistry and physiology},
volume = {216},
number = {Pt 1},
pages = {106764},
doi = {10.1016/j.pestbp.2025.106764},
pmid = {41326076},
issn = {1095-9939},
mesh = {Animals ; *Moths/genetics/metabolism/growth &amp; development ; *Silk/biosynthesis/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Larva/metabolism/genetics/growth &amp; development ; Proteomics ; CRISPR-Cas Systems ; *Electron Transport Complex IV/genetics/metabolism ; Transcriptome ; Multiomics ; },
abstract = {The diamondback moth (DBM) is a major global pest of cruciferous crops. Silk production, essential for DBM larval locomotion and pupal attachment, is governed by fibroin heavy chain (FibH), fibroin light chain (FibL), and fibrohexamerin (P25). However, the regulatory mechanisms and downstream key genes involved in silk production in DBM remain poorly understood. To address this, we integrated transcriptomic and proteomic data from CRISPR/Cas9 generated PxFibH, PxFibL, and PxP25 mutants to investigate the impact of silk gene deletions in the silk gland and identify modulators of silk formation. In the transcriptomic analysis, we identified 1994, 913, and 1266 differentially expressed genes (DEGs) in the three mutant strains, respectively. GO and KEGG enrichment analysis revealed significant involvement in pathways such as oxidation-reduction process, transmembrane transport, enzyme activity, and extracellular matrix (ECM) receptor interaction. At the proteomic level, 604, 210, and 266 differentially expressed proteins (DEPs) were identified in the three mutants, respectively. GO and KEGG enrichment analysis of these DEPs consistently highlighted energy metabolism, hydrolase activity, and catalytic activity pathways. Integrated multi-omics analyses identified three conserved regulator genes: cytochrome c oxidase subunit 6A1 (COX6A1), N-acetylneuraminate lyase (NAL), and protein phosphatase 1 regulatory subunit 14B (PPP1R14B). CRISPR/Cas9 knockout of PxCOX6A1 resulted in incomplete cocoon formation, along with increased larval mortality, prolonged development, and reduced oviposition. PxNAL knockout was lethal, while heterozygotes exhibited decreased cocoon formation, pupal weight, and fecundity. This study reveals FibH/FibL/P25-dependent metabolic networks regulating silk production and identifies COX6A1 and NAL as novel targets for environmentally sustainable pest control strategies.},
}
@article {pmid41325825,
year = {2025},
author = {Zhang, Y and Deng, S},
title = {Geminivirus vectors: From gene silencing to synthetic biology.},
journal = {Biotechnology advances},
volume = {87},
number = {},
pages = {108771},
doi = {10.1016/j.biotechadv.2025.108771},
pmid = {41325825},
issn = {1873-1899},
abstract = {Geminiviruses, the largest plant DNA virus family, cause devastating diseases in crops worldwide. These viruses possess distinctive features, such as the stem-loop structure and replication protein (Rep), which enable the creation of functional geminiviral replicons (GVRs) in plants. Over three decades, geminiviruses have been developed into vectors for virus-induced gene silencing (VIGS), high-level protein expression, and genome editing. This review introduces the genomic structure, Rep protein domains and functions, as well as the historical applications of geminiviruses, then highlights their prominent roles in VIGS and synthetic biology. As VIGS vectors, bipartite geminiviruses utilize AV1 gene replacement, while monopartite species rely on satellite DNAs to insert target sequences, enabling gene silencing in diverse plants. In synthetic biology, GVRs facilitate high-level protein expression through autonomous replication and enhance CRISPR/Cas genome editing efficiency in crops. Additionally, gene regulatory elements, including tissue-specific promoters and gene expression enhancement sequences from geminiviral genomes or satellite DNA expand their utility in genetic engineering. Finally, this review provides an outlook on the future development of geminivirus vectors. GVRs can work as plasmid-like DNAs for supporting diverse and creative designs in plant synthetic biology. The stem-loop structure and Rep are not unique to geminiviruses, a fact that suggests potential cross-kingdom applications of GVRs beyond plants. Vast viral resources enable further acceleration of GVR applications through resource mining and optimization. Moreover, attenuated or engineered geminiviral strains hold promise as "plant vaccines" via cross-protection. Collectively, geminivirus vectors bridge fundamental viral research with practical innovations in crop improvement, biomanufacturing, and synthetic biology.},
}
@article {pmid41261131,
year = {2025},
author = {Pierce, SE and Erwood, S and Oye, K and An, M and Krasnow, N and Zhang, E and Raguram, A and Seelig, D and Osborn, MJ and Liu, DR},
title = {Prime editing-installed suppressor tRNAs for disease-agnostic genome editing.},
journal = {Nature},
volume = {648},
number = {8092},
pages = {191-202},
pmid = {41261131},
issn = {1476-4687},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *RNA, Transfer/genetics/metabolism ; Mice ; Codon, Nonsense/genetics ; Cystic Fibrosis/genetics/therapy ; Codon, Terminator/genetics ; Female ; Male ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; HEK293 Cells ; },
abstract = {Precise genome-editing technologies such as base editing[1,2] and prime editing[3] can correct most pathogenic gene variants, but their widespread clinical application is impeded by the need to develop new therapeutic agents for each mutation. For diseases that are caused by premature stop codons, suppressor tRNAs (sup-tRNAs) offer a more general strategy. Existing approaches to use sup-tRNAs therapeutically, however, require lifelong administration[4,5] or show modest potency, necessitating potentially toxic overexpression. Here we present prime editing-mediated readthrough of premature termination codons (PERT), a strategy to rescue nonsense mutations in a disease-agnostic manner by using prime editing to permanently convert a dispensable endogenous tRNA into an optimized sup-tRNA. Iterative screening of thousands of variants of all 418 human tRNAs identified tRNAs with the strongest sup-tRNA potential. We optimized prime editing agents to install an engineered sup-tRNA at a single genomic locus without overexpression and observed efficient readthrough of premature termination codons and protein rescue in human cell models of Batten disease, Tay-Sachs disease and cystic fibrosis. In vivo delivery of a single prime editor that converts an endogenous mouse tRNA into a sup-tRNA extensively rescued disease pathology in a model of Hurler syndrome. PERT did not induce detected readthrough of natural stop codons or cause significant transcriptomic or proteomic changes. Our findings suggest the potential of disease-agnostic therapeutic genome-editing approaches that require only a single composition of matter to treat diverse genetic diseases.},
}
@article {pmid41201289,
year = {2025},
author = {Liu, T and Su, C and Yan, X and Zhang, Y and Wang, Y and Duan, K},
title = {Creating Hypoallergenic and Low Antinutritional Soybeans through CRISPR/Cas12a-Mediated Multiplex Gene Editing.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {48},
pages = {30922-30932},
doi = {10.1021/acs.jafc.5c08935},
pmid = {41201289},
issn = {1520-5118},
mesh = {*Glycine max/genetics/immunology/chemistry/metabolism ; Animals ; *Gene Editing ; *CRISPR-Cas Systems ; Mice ; *Soybean Proteins/genetics/immunology ; *Plants, Genetically Modified/genetics/immunology/metabolism/chemistry ; *Allergens/immunology/genetics ; Humans ; Food Hypersensitivity/immunology ; Female ; },
abstract = {Soybean protein is a valuable plant-based nutrition source for food. However, allergens and antinutritional factors like Gly Bd 30k (P34), Kunitz trypsin inhibitor (KTI), and soybean agglutinin (LE) pose risks. In this study, we employed the CRISPR/Cas12a system to generate hypoallergenic and low antinutritional soybean lines without agronomic traits penalties. The edited lines exhibited markedly reduced levels and enzymatic activity of trypsin inhibitors, as well as soybean agglutinin content. Feeding mice with these edited soybeans, deficient in P34, KTI, and LE proteins, significantly alleviated allergenic responses. This research provides a promising resource of hypoallergenic and low antinutritional soybeans for applications in food production, animal feed, and immunotherapy.},
}
@article {pmid40715744,
year = {2025},
author = {Zhang, K and Yuan, X and Lu, S and Shu, Y and Wang, C and Cen, J and Wu, B and Hui, L},
title = {Expansion of human hepatocytes and their application in three-dimensional culture and genetic manipulation.},
journal = {Nature protocols},
volume = {20},
number = {12},
pages = {3722-3754},
pmid = {40715744},
issn = {1750-2799},
support = {92168202//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32370793//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32221002//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Hepatocytes/cytology/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; *Cell Culture Techniques, Three Dimensional/methods ; Lentivirus/genetics ; *Cell Culture Techniques/methods ; },
abstract = {Hepatocytes are one of the most important cell types in the liver, carrying out key functions. They are essential for hepatocyte-based therapy, disease modeling and drug development. However, the availability of primary human hepatocytes (PHHs) is limited by a shortage of donors. It is therefore of great value to expand PHHs in large quantities. Here we provide a detailed protocol for the large-scale expansion of PHHs (proliferating human hepatocytes, ProliHHs) derived from healthy donors and patients with inherited liver diseases, which can be rematured in a three-dimensional culture system. Moreover, we provide a protocol for the genetic manipulation of ProliHHs, including lentivirus transduction and CRISPR-Cas9-mediated knockout and knock-in. The protocol described here will help to realize the full potential of ProliHH-based therapy, organoid-based liver disease modeling and drug screening. The protocol to expand PHHs takes ~1-2 months, the protocol to establish the 3D-cultured ProliHHs takes ~8 d and the protocol to perform gene editing takes ~3 d. Personnel with basic scientific training can conduct these protocols.},
}
@article {pmid40715743,
year = {2025},
author = {Guruprasad, P and Ramasubramanian, R and Nason, S and Carturan, A and Liu, S and Paruzzo, L and Hornet, V and Plesset, J and Patel, RP and Bhoj, V and Beatty, GL and Ruella, M},
title = {Manufacturing of CRISPR-edited primary mouse CAR T cells for cancer immunotherapy.},
journal = {Nature protocols},
volume = {20},
number = {12},
pages = {3629-3654},
pmid = {40715743},
issn = {1750-2799},
support = {R00CA212302//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01-37-CA262362-03//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; Mice ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Receptors, Chimeric Antigen/genetics/immunology ; *T-Lymphocytes/immunology/metabolism ; *Neoplasms/therapy/immunology ; *Immunotherapy, Adoptive/methods ; Humans ; Tumor Microenvironment/immunology ; },
abstract = {Editing chimeric antigen receptor (CAR) T cells by using CRISPR-Cas9 has become a routine strategy to improve their antitumor function or safety profile. Xenograft tumor models in immunodeficient mice are often used to evaluate the function of CRISPR-edited human CAR T cells. These models, however, lack functional immune systems and thus fail to recapitulate barriers such as the immunosuppressive tumor microenvironment (TME) that CAR T cells will encounter in patients. Thus, genetically modifying mouse CAR T cells for use in immune-intact models is an attractive approach to explore the impact of a given gene deletion on CAR T cells within a natural TME. Here, we describe a protocol to perform CRISPR-Cas9 editing in primary mouse T cells, thereby enabling studies of gene-edited CAR T within the TME and in the presence of a functional immune system. This protocol is integrated into a standard mouse CAR T manufacturing workflow, a process that typically spans ~5-6 days. The first stage of this protocol involves isolating mouse T cells, electroporating them with a ribonucleoprotein complex and activating them by using magnetic bead stimulation. The second stage involves transducing the CAR gene and expanding these cells, and the third stage focuses on validating knockout efficiency and the functionality of gene-edited mouse CAR T cells. This procedure requires a proficiency in aseptic cell culture techniques and a basic understanding of T cell biology. We anticipate that efficient and reliable genetic modification of mouse T cells will have wide-ranging applications for cancer immunotherapies and related fields.},
}
@article {pmid40640356,
year = {2025},
author = {Ma, Y and Xiong, Y and Xu, J and Xu, H and Fu, Z and Zhao, GR and Wu, Y and Yuan, YJ},
title = {Assembly and delivery of large DNA via chromosome elimination in yeast.},
journal = {Nature protocols},
volume = {20},
number = {12},
pages = {3755-3782},
pmid = {40640356},
issn = {1750-2799},
support = {[32471483]//National Natural Science Foundation of China (National Science Foundation of China)/ ; [23JCYBJC00220]//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; },
mesh = {*Saccharomyces cerevisiae/genetics ; CRISPR-Cas Systems ; *Chromosomes, Fungal/genetics ; Haploidy ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *DNA/genetics ; *DNA, Fungal/genetics ; },
abstract = {Manipulation of large-scale genetic information provides a powerful approach to deciphering and engineering complex biological functions. However, the manipulation of large DNA, such as assembly and delivery, remains complex and difficult. Here we describe the experimental design strategy and protocol for a chromosome elimination-mediated large DNA assembly and delivery method (HAnDy), which enables efficient Mb-scale DNA assembly and delivery in yeast conveniently. This protocol is divided into three parts: (1) CRISPR-Cas9-mediated elimination of chromosome, which includes design and integration of a synthetic single-guide RNA (sgRNA) site near the centromere, activation of chromosome elimination by mating, and verification of the chromosome elimination. It can be used to eliminate multiple chromosomes, achieving haploidization in yeast. (2) Haploidization-mediated DNA assembly, which includes the design and construction of initial assembly strains, DNA assembly by programmed haploidization and verification of the assembled clones. (3) Haploidization-mediated DNA delivery, which includes the design and construction of inducible haploidization strains, DNA delivery by programmed haploidization and verification of the delivered clones. Users can utilize this protocol entirely or selectively depending on their needs. With the use of this protocol, it takes 10 d to achieve chromosome elimination and 7-11 d to achieve a standard cycle of haploidization-mediated DNA assembly or delivery. This protocol provides an efficient approach that is useful for the elimination, assembly and delivery of large DNA in yeast, requiring basic molecular biology skills.},
}
@article {pmid40541508,
year = {2025},
author = {Woodward, MJ and Dallaire, A and Paszkowski, U and Kokkoris, V},
title = {Is genetic manipulation of arbuscular mycorrhizal fungi possible?.},
journal = {Trends in microbiology},
volume = {33},
number = {12},
pages = {1331-1343},
doi = {10.1016/j.tim.2025.06.002},
pmid = {40541508},
issn = {1878-4380},
mesh = {*Mycorrhizae/genetics ; CRISPR-Cas Systems ; Transformation, Genetic ; Gene Editing/methods ; Protoplasts ; },
abstract = {Unlike many fungi, arbuscular mycorrhizal (AM) fungi have proven recalcitrant to genetic manipulation due to their obligate biotrophic lifestyle and multinucleate, coenocytic cellular structure. In this review, we examine past attempts at AM fungal transformation, we identify key biological and technical barriers and explore recent advances to overcome them. We focus on techniques never before applied in AM fungi, including CRISPR/Cas9, microinjection, and protoplast-based transformation, and we explore how they provide viable strategies for achieving this elusive goal. We conclude by outlining guidelines for future research, distinguishing between established approaches that are readily applicable to AM fungi and others that first require addressing key outstanding questions in AM fungal cell biology and genetics to ensure success.},
}
@article {pmid41325824,
year = {2025},
author = {Uddin, N and Ullah, MW and Zhu, D and Li, X and Yang, S and Xin, X},
title = {Engineering lignin pathway and plant cell wall modification and genome editing for advanced renewable bioenergy and material applications.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108772},
doi = {10.1016/j.biotechadv.2025.108772},
pmid = {41325824},
issn = {1873-1899},
abstract = {Lignin biosynthesis and plant cell wall engineering are central to plant structural integrity and biomass utility. Recent advances in molecular and synthetic biology have opened opportunities to tailor lignin contents, composition, and polymer structure for renewable bioenergy and sustainable biomaterial applications. This review provides an integrative perspective on biosynthesis, regulation, and engineering of lignin. It summarizes the current progress in understanding the genetic, transcriptional, epigenetic, and metabolic networks that control lignin formation, with a focus on emerging tools such as CRISPR/Cas genome editing, synthetic promoters, and metabolic rewiring. Beyond cataloguing current knowledge, it critically analyzes the trade-offs involved in lignin modification for biomaterials, addressing unresolved challenges such as monolignol transport, metabolic flux control, and species-specific regulatory divergence. Engineered lignin and modified plant cell walls hold significant potential for biorefineries, advanced polymers, pharmaceuticals, and carbon sequestration, yet their translation from the laboratory to the field remains limited. Engineered lignin offers real-world applications across diverse industries, including bioenergy, bioplastics, carbon fiber composites, pharmaceuticals, and sustainable construction materials, thereby reinforcing its pivotal role in advancing a circular bioeconomy. The review further proposes future research directions that integrate multi-omics, single-cell technologies, machine learning, and field-based validation to enable precision lignin engineering. Strategic advances in this field will support next-generation bioenergy systems, advanced biomaterials, and the transition to a circular bioeconomy.},
}
@article {pmid41325351,
year = {2025},
author = {Jansen van Vuuren, J and Matthews, MC and Robène, I and Rozsasi, S and Campa, M and Burger, J and Viljoen, A and Mostert, D},
title = {Combined Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Detecting Fusarium oxysporum f. sp. cubense Tropical Race 4.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {225},
pages = {},
doi = {10.3791/68841},
pmid = {41325351},
issn = {1940-087X},
mesh = {*Fusarium/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Recombinases/genetics/chemistry/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Plant Diseases/microbiology ; DNA, Fungal/genetics/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Regular and accurate surveillance stands central to the efficient management of plant diseases. It can indicate which course of action is most appropriate, and whether prevention, eradication, or no action is required. Surveillance based on symptomology in host plants alone is often not reliable due to similarities in the symptoms caused by biotic and abiotic stresses. Laboratory-based molecular methods such as polymerase chain reaction (PCR) and quantitative (q)PCR are the most commonly and reliably used for plant pathogen detection, but rely on expensive equipment and skilled operators. Here, we describe a protocol combining a simplified DNA extraction, recombinase polymerase amplification (RPA), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (RPA-Cas12a) for the detection of the invasive pathogen, Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). The technique provides a simple single-tube detection alternative that is analytically robust with improved specificity compared to available molecular detection assays and negates the need for expensive and sophisticated laboratory equipment.},
}
@article {pmid41324747,
year = {2025},
author = {Priyanka, SS and Iqbal, G and Nidarshan, NC and Kumari, K and Vanjre, S and Rasal, K and Sonwane, A and Brahmane, M and Goswami, M},
title = {Avenues of genome editing for color trait improvement in ornamental fishes: current status and future perspectives.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {51},
pmid = {41324747},
issn = {1573-9368},
mesh = {Animals ; *Gene Editing/methods ; *Fishes/genetics/growth &amp; development ; *Pigmentation/genetics ; CRISPR-Cas Systems/genetics ; Animals, Genetically Modified/genetics/growth &amp; development ; },
abstract = {Ornamental fish industries are growing sectors contributing significantly to livelihood, trade and export, driven by the worldwide demand for colourful and unique species. Pigmentation is the focal point of the visual appeal of ornamental fish, market value, and species-specific interaction. The pigment cells are called chromatophores, derived from neural crest cells and controlled by sophisticated genetic mechanisms, conferring these fish with distinctive colours and patterns. Historically, selective breeding and dietary pigment supplementation have been applied to enhance colouration. Such traditional practices, however, are prone to disadvantages such as slow development, genetic ambiguity, and unforeseen consequences. With the invention of genome editing, such as CRISPR-Cas9, researchers now have a sensitive and powerful tool to control pigmentation traits at the genetic level. Central pigmentation gene manipulation, such as Tyr, Mc1r, and Slc45a2, can allow researchers to introduce stable and uniform color changes. Such enhancements confer unparalleled control of fish colour, a promising avenue in the ornamental fish industry. This review discusses the genetic nature of fish pigmentation. It reports recent advances in CRISPR-based modifications and describes their possible applications and implications for future ornamental fish breeding.},
}
@article {pmid41324601,
year = {2025},
author = {Bhattacharya, S and Goyal, K and Satpati, P},
title = {Thermodynamics of PAM Recognition by Cas9 of Streptococcus pyogenes.},
journal = {Journal of chemical information and modeling},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jcim.5c01934},
pmid = {41324601},
issn = {1549-960X},
abstract = {The CRISPR/Cas9 system from Streptococcus pyogenes (SpCas9) requires a canonical 5'-NGG-3' PAM sequence in target DNA for effective genome editing. Base-specific interactions between the guanines (second and third position) and arginine dyad (R1333 and R1335) ensured specificity. We evaluated the PAM recognition strength of SpCas9 by using alchemical free energy calculations, revealing the energetics that influence genome editing accuracy. SpCas9 does not discriminate at the first position of the NGG sequence, but it penalizes mutations in the second and third positions. SpCas9 imposes a higher penalty for guanine mutation in the third PAM position compared to the second due to the greater conformational rigidity of R1335 in relation to R1333. Conformational rigidity of R1335 prevents side-chain readjustment for new protein-DNA interactions in noncanonical PAMs. A guanine-to-cytosine substitution in either the second or third position of canonical PAM disrupts direct protein-PAM interactions and leads to solvent exposure. This happens due to strong electrostatic repulsion between the arginine dyad's guanidinium groups and the amine group of cytosine. Interestingly, the strength of SpCas9 in disfavoring a single cytosine substitution (by >10 kcal/mol) is comparable to that of disfavoring double base substitutions in the NGG sequence. The ability of SpCas9 to differentiate between noncanonical and canonical PAMs (ΔΔG) is directly related to the number of direct interactions between SpCas9 and the PAM sequence, as well as the degree of solvent exposure. Loss of direct interactions and increased solvent exposure enhance ΔΔG. The calculated ΔΔG adequately explains the observed differences in DNA cleavage activity of SpCas9 across various DNA substrates with different PAM sequences. This study connects thermodynamics, structures, and activity to elucidate PAM selectivity in SpCas9 and may also apply to other CRISPR/Cas systems, offering valuable insights for the rational design of Cas9 variants with modified PAM specificities.},
}
@article {pmid41323291,
year = {2025},
author = {Li, Y and Hall-Ponselè, AM},
title = {Plant Cell Strain Improvement Through Engineering Biology for Industrial Plant Cell Culture.},
journal = {Engineering biology},
volume = {9},
number = {1},
pages = {e70002},
pmid = {41323291},
issn = {2398-6182},
abstract = {Plant cell culture (PCC) presents a promising and sustainable alternative to traditional agricultural methods for producing specialty bioactive compounds. However, its widespread industrial application has been hindered by challenges such as low yields, cell line instability and inconsistent product quality. engineering biology (EB) offers a powerful toolkit to overcome these limitations by systematically improving plant cell lines. This review focuses on the application of EB principles to enhance PCC for the production of high-value bioactives from an industry-oriented perspective. We explore three core pillars of the EB toolkit: (1) Multiomics and in silico design, which leverage comprehensive data integration and predictive modelling for rational target identification; (2) gene manipulation and pathway bioengineering, encompassing precise genome editing (e.g., CRISPR/Cas), synthetic gene circuits and directed evolution for targeted metabolic reprogramming and (3) biosensors for high-throughput screening and real-time monitoring, enabling rapid testing and optimisation of engineered cell lines. The synergistic integration of these tools within the iterative design-build-test-learn (DBTL) cycle is highlighted as a key strategy for accelerating strain improvement. Ultimately, the convergence of these EB approaches is transforming PCC into a robust platform for producing pharmaceuticals, functional foods and green chemicals, contributing to a biobased economy with a minimal ecological footprint.},
}
@article {pmid41321825,
year = {2025},
author = {Senthilraja, G and Sandhya, M and Priyadharshini, E and Anand, T and Kavitha, M and Tharmalingam, N},
title = {Targeting effector proteins of plant pathogens as a strategy for durable plant disease resistance.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1681047},
pmid = {41321825},
issn = {1664-302X},
}
@article {pmid41317788,
year = {2025},
author = {Brogan, DJ and Lin, CP and Benetta, ED and Wang, T and Chen, F and Li, H and Lin, C and Komives, EA and Akbari, OS},
title = {Synthetic type III-E CRISPR-Cas effectors for programmable RNA-targeting.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {169566},
doi = {10.1016/j.jmb.2025.169566},
pmid = {41317788},
issn = {1089-8638},
abstract = {The recent discovery of the type III-E class of CRISPR-Cas effectors has reshaped our fundamental understanding of CRISPR-Cas evolution and classification. Type III-E effectors are composed of several Cas7-like domains and a single Cas11-like domain naturally fused together to create a single polypeptide capable of targeting and degrading RNA. Here we identified a novel type III-E-like effector composed of three Cas7 domains and a Cas1 domain which was not active but could be engineered into an active chimeric RNA-targeting Cas effector by domain additions and swaps from other type III-E effectors. The results reveal that various domains in type III-E effectors can be swapped for the equivalent domain from a different type III-E effector. Remarkably, the Cas1 domain located at the C-terminus of Cas7-1 could be swapped in place of the Cas11 domain located between the Cas7.1 and the Cas7.2 domains of DiCas7-11. The results reveal a new modality for engineering type III-E effectors from the blueprints found in nature.},
}
@article {pmid41315365,
year = {2025},
author = {Wang, Y and Liao, Y and Sun, Y and Mitra, B and Guo, R and Piedras, BI and White, S and Tang, HY and Asara, JM and Tempera, I and Lieberman, PM and Gewurz, BE},
title = {The CTLH ubiquitin ligase substrates ZMYND19 and MKLN1 negatively regulate mTORC1 at the lysosomal membrane.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10731},
pmid = {41315365},
issn = {2041-1723},
support = {R01 AI164709/AI/NIAID NIH HHS/United States ; PF-24-1250090-01-IBCD//American Cancer Society (American Cancer Society, Inc.)/ ; P01CA269043//U.S. Department of Health &amp; Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; P01 CA269043/CA/NCI NIH HHS/United States ; R01AI164709//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; PF-23-1144614-01-IBCD//American Cancer Society (American Cancer Society, Inc.)/ ; PF-24-1194768-01-TBE//American Cancer Society (American Cancer Society, Inc.)/ ; PF-24-1308318-01-TBE//American Cancer Society (American Cancer Society, Inc.)/ ; R01 DE033907/DE/NIDCR NIH HHS/United States ; R00 DE031016/DE/NIDCR NIH HHS/United States ; },
mesh = {Humans ; *Lysosomes/metabolism ; *Mechanistic Target of Rapamycin Complex 1/metabolism/genetics ; *Ubiquitin-Protein Ligases/metabolism/genetics ; HEK293 Cells ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Intracellular Membranes/metabolism ; Signal Transduction ; Phosphatidylinositol 3-Kinases/metabolism ; Cell Proliferation ; },
abstract = {Most Epstein-Barr virus-associated gastric carcinoma (EBVaGC) harbor non-silent mutations that activate phosphoinositide 3 kinase (PI3K) to drive downstream metabolic signaling. To gain insights into PI3K/mTOR pathway dysregulation in this context, we perform a human genome-wide CRISPR/Cas9 screen for hits that synergistically blocked EBVaGC proliferation together with the PI3K antagonist alpelisib. Multiple subunits of carboxy terminal to LisH (CTLH) E3 ligase, including the catalytic MAEA subunit, are among top screen hits. CTLH negatively regulates gluconeogenesis in yeast, but not in higher organisms. The CTLH substrates MKLN1 and ZMYND19, which highly accumulated upon MAEA knockout, associate with one another and with lysosome outer membranes to inhibit mTORC1. Rather than perturbing mTORC1 lysosomal recruitment, ZMYND19 and MKLN1 block the interaction between mTORC1 and Rheb and also with mTORC1 substrates S6 and 4E-BP1. Thus, CTLH enables cells to rapidly tune mTORC1 activity at the lysosomal membrane via the ubiquitin/proteasome pathway.},
}
@article {pmid41261109,
year = {2025},
author = {Zhang, T and Zheng, Y and Sheng, M and Wang, Q and Jin, Z and Li, Z and Chen, W and Huang, J and Yang, X},
title = {Electrochemical Biosensing Platform Based on MOF Carrier Signal Probes and CRISPR/Cas12a Cleavage for Sensitive Detection of MicroRNAs.},
journal = {Analytical chemistry},
volume = {97},
number = {47},
pages = {26265-26273},
doi = {10.1021/acs.analchem.5c05821},
pmid = {41261109},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis ; *Metal-Organic Frameworks/chemistry ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; DNA, Single-Stranded/chemistry ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; Methylene Blue/chemistry ; Limit of Detection ; Bacterial Proteins ; },
abstract = {Dysregulation of microRNA (miRNA) expression is associated with a variety of human diseases, including cancers, and the development of highly sensitive miRNA detection is important for the early diagnosis of cancer. In this study, we developed an electrochemical biosensing platform for miRNA detection through the integration of a DNA-gated metal-organic framework (MOF) signal probe, duplex-specific nuclease (DSN)-assisted signal amplification, and a CRISPR/Cas12a system. The zirconium-based MOF UiO-66-NH2 was engineered as a nanocarrier for methylene blue (MB) entrapment, and single-stranded DNA (ssDNA) was capped on the MOF surface as a "gatekeeper" that allowed the controlled release of MB molecules, resulting in a DNA-gated MOF electrochemical signal probe. In the presence of target miRNA, target-initiated DSN cyclic amplification and digestion of the CRISPR/Cas12a initiator DNA sequence blocked the collateral cleavage activity of Cas12a toward ssDNA, thus preventing the release of MB and generating a high electrochemical signal. Conversely, the CRISPR/Cas12a system would be activated to cleave ssDNA in the absence of the target, leading to the release of a signal molecule and a low response. With the usage of the proposed biosensing strategy, sensitive detection of miRNA let-7a, a biomarker associated with nonsmall cell lung cancer, has been achieved. Therefore, this work expands the application scope of a MOF as a nanocarrier to prepare electrochemical signal probes and provides a valuable biosensing method for clinical diagnosis.},
}
@article {pmid41254269,
year = {2025},
author = {Hernández-Huertas, L and Moreno-Sánchez, I and Crespo-Cuadrado, J and Vargas-Baco, A and da Silva Pescador, G and Zhang, Y and Wen, Z and Florens, L and Santos-Pereira, JM and Bazzini, AA and Moreno-Mateos, MA},
title = {CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of maternal-to-zygotic transition in teleosts.},
journal = {The EMBO journal},
volume = {44},
number = {23},
pages = {7021-7059},
pmid = {41254269},
issn = {1460-2075},
support = {RyC-2017-23041//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; PGC2018-097260-B-I00//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; PID2021-127535NB-I00//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; MDM-2016-0687//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; CEX2020-001088-M//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; CNS2022-135564//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; PRE2019-087721//Ministerio de Ciencia e Innovaci&#xf3;n (MCIN)/ ; PREDOC_01569//Junta de Andaluc&#xed;a (Andalusian Board)/ ; EMC21_00188//Junta de Andaluc&#xed;a (Andalusian Board)/ ; P20_00866//Junta de Andaluc&#xed;a (Andalusian Board)/ ; UPO-1380590//Junta de Andaluc&#xed;a (Andalusian Board)/ ; Margarita Salas Postdoctoral contract//EC | NextGenerationEU (NGEU)/ ; PRTR-C17.I1//EC | NextGenerationEU (NGEU)/ ; NIHR01GM136849//HHS | National Institutes of Health (NIH)/ ; NIH R21OD034161//HHS | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; *Zygote/metabolism ; *Zebrafish Proteins/genetics/metabolism ; *Protein Processing, Post-Translational ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; Female ; Phosphorylation ; },
abstract = {The maternal-to-zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screen, in which we targeted mRNAs encoding kinases and phosphatases or proteins regulating them in zebrafish. This screen identified branched-chain ketoacid dehydrogenase kinase, Bckdk, as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA, and expression of a phospho-mimetic mutant of Phf10 rescued the developmental defects observed after bckdk mRNA depletion, as well as restored H3K27ac levels. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation.},
}
@article {pmid41219509,
year = {2025},
author = {Johnson, KA and Goswami, HN and Catchpole, RJ and Ahmadizadeh, F and Zhao, P and Wells, L and Li, H and Terns, MP},
title = {A phage-encoded anti-CRISPR protein co-opts host enolase to prevent type III CRISPR immunity.},
journal = {Nature microbiology},
volume = {10},
number = {12},
pages = {3162-3175},
pmid = {41219509},
issn = {2058-5276},
support = {R35GM118160//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; GR000347//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; 2400220//National Science Foundation (NSF)/ ; },
mesh = {*Phosphopyruvate Hydratase/metabolism/genetics ; *Streptococcus thermophilus/virology/immunology/genetics ; *Viral Proteins/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; *Streptococcus Phages/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems provide powerful adaptive immunity against phage infection. In response, phages use anti-CRISPR (Acr) proteins to evade CRISPR immunity. The few type III Acrs identified so far show conditional effectiveness in countering type III immunity or rely on unknown or poorly understood inhibitory mechanisms. Here we report the discovery of AcrIIIA2, a type III-A Acr encoded by Streptococcus thermophilus phages. Biochemical and structural analyses reveal that phage AcrIIIA2 co-opts host enolase, a highly abundant glycolysis enzyme, to form a ternary complex with the S. thermophilus type III-A (Csm) CRISPR ribonucleoprotein complex, obstructing its immune responses. The enolase-chaperoned AcrIIIA2 blocks the initial step of phage RNA binding, thereby preventing downstream type III anti-phage immune responses. Enolase participates in the anti-immune response by serving as an essential structural scaffold, stabilizing Acr-CRISPR interactions. These findings uncover a new anti-defence strategy that exploits a well-conserved host factor to block CRISPR immunity.},
}
@article {pmid41205968,
year = {2025},
author = {Zhang, Z and Chen, H and Tang, K and Xu, J and Jiang, L and Ning, Y},
title = {Triple-helix molecular switch-based aptasensor integrating exonuclease I-assisted target recycling and CRISPR-Cas13a-mediated signal amplification for fluorescence detection of adenosine triphosphate.},
journal = {International journal of biological macromolecules},
volume = {333},
number = {Pt 1},
pages = {148753},
doi = {10.1016/j.ijbiomac.2025.148753},
pmid = {41205968},
issn = {1879-0003},
mesh = {*Adenosine Triphosphate/analysis ; *Exodeoxyribonucleases/metabolism ; *Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Methicillin-Resistant Staphylococcus aureus ; Graphite/chemistry ; Fluorescence ; Limit of Detection ; Spectrometry, Fluorescence ; },
abstract = {It is essential to develop new methods for detecting adenosine triphosphate (ATP), as it plays crucial roles in the regulating numerous biological processes and its levels are significantly correlated with various diseases. This study developed a fluorescent aptasensor for ATP detection utilizing a triple-helix molecular switch (THMS) based on exonuclease I (Exo I)-assisted target recycling and CRISPR-Cas13a-mediated signal amplification. Without the target, all carboxyfluorescein (fluorescence-active molecule; FAM)-labeled probes remain close to the graphene oxide (GO) substrate, resulting in the fluorescence quenching of FAM. In contrast, the target initiates THMS opening, which triggers the simultaneous target recycling and signal amplification. The iterative reactions lead to FAM accumulation, and the fluorescence can be assayed at excitation/emission wavelengths of 480/514 nm, respectively. The target was detected at concentrations from 1 fM to 1 nM with a detection limit of 0.54 fM. The aptasensor showed satisfactory performance for evaluating ATP in methicillin-resistant Staphylococcus aureus and studying drug action mechanisms, highlighting its potential for clinical diagnostics and screening antibacterial agents.},
}
@article {pmid41174223,
year = {2025},
author = {Liao, Y and Yan, J and Kong, IY and Li, Z and Ding, W and Clark, S and Maestri, D and Yoshida, T and Giulino-Roth, L and Gewurz, BE},
title = {Lysine-specific histone demethylase complex restricts Epstein-Barr virus lytic reactivation.},
journal = {Nature microbiology},
volume = {10},
number = {12},
pages = {3290-3304},
pmid = {41174223},
issn = {2058-5276},
support = {R01 AI164709/AI/NIAID NIH HHS/United States ; U01CA275301//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01DE033907//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 DE033907/DE/NIDCR NIH HHS/United States ; U01 CA275301/CA/NCI NIH HHS/United States ; P01CA269043//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01CA228700//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 CA228700/CA/NCI NIH HHS/United States ; R01AI164709//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; P01 CA269043/CA/NCI NIH HHS/United States ; },
mesh = {*Herpesvirus 4, Human/physiology/genetics ; Humans ; *Histone Demethylases/metabolism/genetics ; *Virus Activation ; Animals ; Virus Latency ; Mice ; Epstein-Barr Virus Infections/virology ; Co-Repressor Proteins/metabolism/genetics ; Histones/metabolism ; Lysine/metabolism ; Trans-Activators/metabolism/genetics ; Cell Line, Tumor ; Methylation ; Burkitt Lymphoma/virology ; CRISPR-Cas Systems ; Host-Pathogen Interactions ; Nerve Tissue Proteins ; },
abstract = {Epstein-Barr virus (EBV) infects >95% of adults and contributes to several human cancers. EBV can remain latent where viral lytic genes are silenced, precluding the use of antiviral agents such as ganciclovir. Little is known about the host factors involved in EBV latency. Here we performed a human genome-wide CRISPR-Cas9 screen in Burkitt lymphoma B cells, which identified lysine-specific histone demethylase 1 (LSD1) and its corepressors REST corepressor 1 (CoREST) and zinc finger protein 217 (ZNF217) as critical for EBV latency. Gene knockout or LSD1 inhibition triggered EBV reactivation, and the latter sensitized cells to ganciclovir cytotoxicity, including in murine tumour xenografts. Mechanistically, ZNF217 recruits LSD1 and CoREST to form a complex that binds a specific DNA motif associated with regions implicated in EBV reactivation. It removes histone 3 lysine 4 (H3K4) methylation marks and restricts host DNA looping. Alternatively, the H3K4 lysine methyltransferase 2D supports EBV lytic reactivation. Our results highlight H3K4 methylation as a major EBV lytic switch regulator and therapeutic target.},
}
@article {pmid40992203,
year = {2025},
author = {Xu, Y and Wang, L and Jiang, J and Zhao, G and Wang, Z},
title = {Knockdown of argininosuccinate lyase influences the growth of Mycolicibacterium smegmatis in vitro and in vivo.},
journal = {Tuberculosis (Edinburgh, Scotland)},
volume = {155},
number = {},
pages = {102693},
doi = {10.1016/j.tube.2025.102693},
pmid = {40992203},
issn = {1873-281X},
mesh = {Animals ; *Mycobacterium smegmatis/growth &amp; development/drug effects/enzymology/genetics ; *Argininosuccinate Lyase/genetics/metabolism ; Gene Knockdown Techniques ; *Mycobacterium Infections, Nontuberculous/microbiology/genetics/drug therapy/enzymology ; Disease Models, Animal ; *Bacterial Proteins/genetics/metabolism ; Arginine/pharmacology/metabolism ; Liver/microbiology ; Antitubercular Agents/pharmacology ; CRISPR-Cas Systems ; Kidney/microbiology ; },
abstract = {The rising prevalence of drug-resistant tuberculosis (DR-TB), coupled with stagnation in the development of novel therapeutics, underscores the urgent need for new drug targets and innovative anti-tuberculosis agents. In this study, we demonstrate that CRISPR interference-mediated knockdown of argH, a nitrogen metabolism-associated gene encoding argininosuccinate lyase, significantly impairs the growth of Mycolicibacterium smegmatis (formerly Mycobacterium smegmatis). This growth defect was alleviated in a concentration-dependent manner by arginine supplementation. In a goldfish infection model, argH knockdown led to a marked reduction in bacterial burden within both liver and kidney tissues. Notably, bacitracin and 5-fluorouracil exhibited synergistic effects when combined with argH knockdown. Metabolomic profiling revealed significant perturbations in multiple amino acids, as well as in succinyl-CoA and lactate levels, suggesting that suppression of argH impairs M. smegmatis proliferation by disrupting amino acid homeostasis and interfering with aerobic respiration.},
}
@article {pmid41315225,
year = {2025},
author = {Wen, HP and Yu, C and Bi, S and Jiang, LH and Wang, ZG and Yao, Z and Pang, DW and Liu, SL},
title = {Programmable targeted RNA degradation via dCas13d-directed chaperone-mediated autophagy (dCasCMA).},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10738},
pmid = {41315225},
issn = {2041-1723},
support = {22293032//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22374138//National Natural Science Foundation of China (National Science Foundation of China)/ ; 21977054//National Natural Science Foundation of China (National Science Foundation of China)/ ; 24JCZDJC01240//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; 23JCYBJC01880//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; },
mesh = {Humans ; Animals ; *Chaperone-Mediated Autophagy/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA Stability/genetics ; *CRISPR-Cas Systems/genetics ; Mice ; HEK293 Cells ; Autophagy ; },
abstract = {RNA-targeted degradation technologies offer significant promise for treating diseases by selectively disrupting gene expression. However, a robust method to specifically, efficiently, and programmability degrade targeted RNAs in mammalian cells is still in demand. Here, we present a versatile platform, dCas13d-directed chaperone-mediated autophagy (dCasCMA), which integrates the precise targeting capabilities of dCas13/CRISPR with the degradation efficiency of chaperone-mediated autophagy (CMA) to achieve efficient degradation of specific RNAs. By combining dCas13d with a CMA-targeting motif and customizable guide RNA (gRNA), the platform allows for accurate targeting of both exogenous and endogenous RNAs in cells. Moreover, the incorporation of multiplexed gRNA expression arrays enables the simultaneous degradation of multiple RNA targets during viral pathogenesis in live cells and in vivo. Our findings emphasize the platform's modular design, which enables flexible combinations of dCCTM components with user-defined gRNA sequences. This versatility positions it as a promising tool for developing innovative therapies for various diseases.},
}
@article {pmid41315077,
year = {2025},
author = {Loedige, KW and White, AL and McMurrough, TA and Stead, BE and Edgell, DR},
title = {A buffer-tuning strategy to profile domain-specific activity of chimeric I-TevI/CRISPR gene editors in vitro.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {42742},
pmid = {41315077},
issn = {2045-2322},
support = {RGPIN-2022-05459//Natural Sciences and Engineering Research Council of Canada/ ; RGPIN-2022-05459//Natural Sciences and Engineering Research Council of Canada/ ; ALLRP 571374 - 21//Mitacs/ ; ALLRP 571374 - 21//Mitacs/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Buffers ; RNA, Guide, CRISPR-Cas Systems/genetics ; Protein Domains ; DNA Cleavage ; *Endodeoxyribonucleases/metabolism/genetics ; Humans ; DNA/metabolism ; },
abstract = {Protein-DNA interactions can be manipulated in vitro by changing buffer conditions. Here, we develop a methodology to map the cleavage preferences of chimeric gene editors that are fusions of the I-TevI nuclease domain to CRIPSR nucleases by manipulating in vitro salt concentrations. We found that DNA cleavage by the I-TevI (Tev) nuclease domain at CNNNG sites was de-coupled from the gRNA-targeted site in low salt buffers. For TevCas12a, this non-targeted cleavage activity was enriched at Tev CNNNG cleavage motifs optimally positioned within a 30-bp window upstream of a Cas12a TTTV PAM site. Non-targeted cleavage did not require Cas12a nuclease activity or specific Cas12a gRNA targeting. Similar non-targeted products were observed in low salt buffer conditions for TevSaCas9, Tev-meganuclease and Tev-zinc finger editors. Cas12a and SaCas9 activity at gRNA-directed sites and sites with multiple mismatches were also sensitive to buffer salt concentration. Oxford Nanopore sequencing revealed a remarkably similar Tev CNNNG cleavage preference at different salt concentrations and in different fusion contexts, emphasizing the robustness and specificity of Tev activity. More generally, our work highlights the sensitivity of gene editors to in vitro reaction conditions and how these conditions can be leveraged to functionally dissect the activity of individual domains of chimeric gene editors.},
}
@article {pmid41314912,
year = {2025},
author = {Chen, L and Ouyang, W and Hu, Y and Peng, L and Chen, P and Guo, W and Yang, H and Xu, J and Pan, M and Xu, D and Wang, X and Zhang, C and Chen, S and Hao, Q and Yuan, S and Huang, Y and Shan, Z and Yang, Z and Xia, R and Hewezi, T and Chen, H and Tran, LP and Zhou, X and Cao, D},
title = {Creating artificial miR2118a/b to boost yield and broad-spectrum resistance in soybean via CRISPR/Cas9-targeted mutation.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.10.022},
pmid = {41314912},
issn = {1879-3096},
abstract = {While regulatory functions of mature miRNAs are well established, the functions of miRNAs* and their potential for genetic engineering in crop improvement remain underexplored. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) to generate artificial miR2118a/b (amiR2118a/b) by editing miR2118a/b-5p and obtained several amir2118a/b mutants in soybean (Glycine max). miR2118a/b-5p modifications altered the secondary structure of precursor amiR2118a/b (pre-amiR2118a/b) and reduced mature miR2118a/b levels. These amir2118a/b mutants retained the ability to initiate biogenesis of phased small interfering RNAs (phasiRNAs), albeit with a reduced abundance compared with wild-type (WT) plants. Furthermore, these mutants upregulated the expression of genes related to growth and defense under normal and Pseudomonas syringae pv. glycinea (Psg)-infected conditions, respectively. Notably, two transgene-free amir2118 mutants exhibited enhanced resistance to Psg, soybean cyst nematode (SCN), and root-knot nematode (RKN), and achieved increased yield under pathogen-free field conditions. This study provides a strategy to generate artificial miRNAs (amiRNAs) for crop improvement through the CRISPR/Cas system by mutating miRNAs* in crops.},
}
@article {pmid41314751,
year = {2025},
author = {Fatima, M and Tariq, I and Tariq, A and Talib, S and Fatima, M and Shehzadi, M and Aqib, AI},
title = {Pharmacogenomics and CRISPR-based therapies.},
journal = {Progress in brain research},
volume = {297},
number = {},
pages = {319-343},
doi = {10.1016/bs.pbr.2025.08.009},
pmid = {41314751},
issn = {1875-7855},
mesh = {Humans ; *Pharmacogenetics/methods ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Precision Medicine/methods ; *Genetic Therapy/methods ; Animals ; },
abstract = {Pharmacogenomics and CRISPR-based treatments are two areas of precision medicine that are advancing together. Pharmacogenomics involves studying how differences in someone's genes can change the effect of medications on them. Pharmacogenomics helps reduce adverse reactions to drugs and improve healing by choosing and measuring drugs according to a patient's genetic information. Additionally, CRISPR-Cas systems now serve as leading genome editing tools that allow precise alterations at given points of the genome. CRISPR technology's use in pharmacogenomics creates new opportunities for modifying gene expression, fixing harmful mutations, and creating innovative treatment approaches. A more proactive approach to illness treatment is supported by this synergy, in which genetic factors serve as both direct targets for intervention and a basis for medication selection. This chapter examines the theoretical and practical frameworks that link CRISPR-based treatments with pharmacogenomics, emphasizing recent uses in pharmacoresistance, cancer, and monogenic diseases. To guarantee safe and fair deployment, it also covers the ethical, legal, and technical issues that need to be resolved. When combined, these technologies hold the potential to revolutionize medicine by facilitating individualized and curative drugs.},
}
@article {pmid41310509,
year = {2025},
author = {Ekrami, A and Taheri, B and Daneshfar, S and Moradi, M and Ghorbani, A and Akhash, N and Jafarzadeh, Z and Farshadzadeh, Z and Saki, M},
title = {Occurrence of CRISPR-Cas genes and lack of association with antibiotic resistance in Shigella isolates collected from patients with diarrhea in Ahvaz, southwest Iran.},
journal = {BMC infectious diseases},
volume = {25},
number = {1},
pages = {1666},
pmid = {41310509},
issn = {1471-2334},
abstract = {BACKGROUND: So far, few studies have examined the association between CRISPR-Cas and antibiotic resistance in Shigella isolates. Hence, this study sought to address this issue in Shigella species isolated from stool samples of patients with diarrhea in Ahvaz, southwest Iran.<br><br>METHODS: In this cross-sectional study, stool samples were collected from 103 children (3&#x2013;14 years) with diarrhea admitted to Abuzar Hospital affiliated to the Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. All samples were cultured on the xylose lysine desoxycholate agar and hektoen enteric agar and incubated at 37&#xa0;&#xb0;C for 24&#xa0;h. Primary identification of the Shigella species was performed by biochemical tests and polymerase chain reaction (PCR). Antibiotic resistance rates were evaluated by the Kirby-Bauer disc diffusion. The prevalence of 12 CRISPR-Cas genes was investigated by PCR.<br><br>RESULTS: Overall, 83 Shigella isolates were identified by standard biochemical tests. Finally, 72 Shigella isolates including 45 S. flexneri (46.8%) and 27 (53.2%) S. sonnei were confirmed by PCR. The most effective antibiotics were trimethoprim/sulfamethoxazole (n&#x2009;=&#x2009;48, 66.7%), imipenem (n&#x2009;=&#x2009;46, 63.9%), and ceftazidime (n&#x2009;=&#x2009;43, 59.7%), respectively. All isolates harbored at least one of the CRISPR-Cas genes. Occurrence of CRISPR-Cas genes was as follows: CRa (100.0%), CRb (100.0%), CRc (100.0%), CSe2 (100.0%), CRf (95.8%), CSe1_Cas3 (87.5%), Cas2_Cas (84.7%), Cas (81.9%), CRd (80.6%), Cas7 (76.4%), Cas6e_Cas5 (72.2%), and CRe (48.6%). There was no significant association between the occurrence of CRISPR-Cas elements and resistance to any antibiotic in Shigella isolates (P-value&#x2009;&#x2265;&#x2009;0.9999), except for Cas gene with ceftriaxone and cefepime.<br><br>CONCLUSION: This study revealed high resistance rates of various antibiotics in Shigella isolates. However, there was no significant association between the existences of CRISPR-Cas genes with antibiotic resistance. Further investigation with higher sample size is needed to confirm this observation.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12879-025-12100-0.},
}
@article {pmid41310261,
year = {2025},
author = {Spaans, GW and van der Berg, JP and Bouwman, LMS and Kleter, GA},
title = {Advancements in genomic crop techniques and considerations for regulation and food safety.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {49},
pmid = {41310261},
issn = {1573-9368},
mesh = {*Crops, Agricultural/genetics/growth &amp; development ; *Plants, Genetically Modified/genetics/growth &amp; development ; *Food Safety ; Gene Editing ; Plant Breeding ; *Genomics/methods ; Food, Genetically Modified ; Mutagenesis ; Humans ; Genome, Plant ; },
abstract = {Advancements in genomic crop techniques have led to the development of new genetic technologies, such as base- and prime editing, but improvements have been made to existing conventional techniques as well. Fields in which these advancements occur include targeted mutagenesis, conventional random mutagenesis, and developments with null segregants, e.g., crops from which transgenic elements have been crossed out. In this review, we describe the developments in these three fields and provide considerations concerning regulatory and safety aspects. Because of differences in legislation of modern biotechnology between countries or regions, regulatory challenges are to be expected given the ongoing developments in genomic crop techniques. Moreover, the nature of the mutations induced with these newly developed techniques is not different from those induced with conventional techniques, making the modified crop plants indistinguishable from non-modified counterparts of the same crop species. Thus, enforcement of regulations cannot solely rely on technical analytical methods. Also, potential off-target or unintended effects in the primary mutants remain underexplored. Yet, these do not raise safety concerns owing to the experience with the crop breeding practice of iterative cycles for desirable traits selection, as well as the segregation and discard of unwanted phenotypes. Given that regulation will always change after innovation and developments within the sector advance rapidly, we advocate that both authorities and the breeding sector pro-actively implement a food safety culture. Such a safety culture will help developers of genomic technologies in crops to identify potential food safety issues at an early stage of development of future products.},
}
@article {pmid41309901,
year = {2025},
author = {Moroi, K and Yamamoto, T and Kurita, T},
title = {Double-strand break-free and transgene-free genome editing in the microalga Nannochloropsis oceanica using removable vectors containing the CRISPR base editing system.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {42431},
pmid = {41309901},
issn = {2045-2322},
mesh = {*Gene Editing/methods ; *Microalgae/genetics ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; Plasmids/genetics ; DNA Breaks, Double-Stranded ; Transgenes ; *Stramenopiles/genetics ; },
abstract = {The accumulation of lipids by algae makes them attractive for carbon-neutral fuel production; however, the industrial-scale production of algal lipids has yet to be achieved. Currently, researchers are trying to improve the lipid productivity of algal strains using genome editing for molecular breeding with CRISPR-Cas9, which allows the efficient alteration of genomic information. However, CRISPR-based gene modification via double-strand breaks sometimes induces unintended large deletions that are toxic to host cells. Here, we applied the cytidine base editor combined with an episomal vector backbone containing a centromere and autonomous replication sequence to the microalga Nannochloropsis oceanica. The cytosine base editor introduces cytidine-to-thymidine base substitutions using deaminase without double-strand breaks, and an episomal vector enables plasmid removal after base substitution. We succeeded in inducing cytidine-to-thymidine substitution at the six target sites of five endogenous genes. The base substitution activity ranged from 29.2% to 47.6% on cytidine bases at the 16th to 19th positions from the protospacer adjacent motifs. The removal of base editor plasmids was also detected, which is essential for constructing transgene-free strains. Our results provide insights into the applicability of further technologies in the genetic modification of microalgae.},
}
@article {pmid41309688,
year = {2025},
author = {Zhang, F and Peng, Y and Fan, D and Song, G and Gao, X and Tian, Y},
title = {Engineering a CRISPR-associated IscB system for developing miniature genome-editing tools in human cells and mouse embryos.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10693},
pmid = {41309688},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; Humans ; Mice ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Embryo, Mammalian/metabolism ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {IscB, as the putative ancestor of Cas9, possesses a compact size, making it suitable for in vivo delivery. OgeuIscB is the first IscB protein known to function in eukaryotic cells but requires a complex TAM (NWRRNA). Here, we characterize a CRISPR-associated IscB system, named DelIscB, which recognizes a flexible TAM (NAC). Through systematically engineering its protein and sgRNA, we obtain enDelIscB with an average 48.9-fold increase in activity. By fusing enDelIscB with T5 exonuclease (T5E), we find that enDelIscB-T5E displays robust efficiency comparable to that of enIscB-T5E in human cells. Moreover, by fusing cytosine or adenosine deaminase with enDelIscB nickase, we establish efficient miniature base editors (ICBE and IABE). Finally, we efficiently generate mouse models by microinjecting mRNA/sgRNA of enDelIscB and enDelIscB-T5E into mouse embryos. Collectively, our work presents a set of enDelIscB-based miniature genome-editing tools with great potential for diverse applications in vivo.},
}
@article {pmid41242303,
year = {2025},
author = {Dong, M and Zhang, J and Long, L and Wang, H and Zhang, A and Zeng, W and Li, J},
title = {Establishment of a liver-specific Albumin-Cre recombinase transgenic golden hamster model using gRosa26-Targeted dual-fluorescent reporter system for hepatocyte-specific genetic manipulation.},
journal = {Biochemical and biophysical research communications},
volume = {792},
number = {},
pages = {152912},
doi = {10.1016/j.bbrc.2025.152912},
pmid = {41242303},
issn = {1090-2104},
mesh = {Animals ; *Integrases/genetics/metabolism ; *Mesocricetus/genetics ; *Hepatocytes/metabolism ; Cricetinae ; *Genes, Reporter ; Animals, Genetically Modified ; *Liver/metabolism ; *Albumins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; *RNA, Untranslated/genetics ; Male ; Mice ; Organ Specificity ; },
abstract = {BACKGROUND AND AIMS: Despite its superior physiological relevance to human liver metabolism and diseases, the absence of reliable tissue-specific gene manipulation systems has considerably limited genetic research in the Syrian golden hamster (Mesocricetus auratus). The Cre/loxP-based Rosa26-targeted dual-fluorescent reporter system (mT/mG) is widely utilized in murine models for accurate lineage tracing and conditional gene editing. However, such powerful genetic tools are not available for golden hamsters. Herein, we established a functional Rosa26-targeted mT/mG reporter system and a hepatocyte-specific albumin-Cre Recombinase (Alb-Cre) driver line in golden hamsters, providing an important platform for hepatocyte-specific gene manipulation.<br><br>METHODS: The Rosa26 homolog (gRosa26) was identified in the golden hamster genome. Then, a CRISPR/Cas9-based two-cell microinjection system was developed to precisely knock-in gRosa26. The dual-fluorescent Cre reporter (mT/mG) was inserted into gRosa26. Thereafter, a liver-specific Alb-Cre transgenic line was developed using murine Alb enhancer/promoter elements. Finally, Cre-mediated recombination efficiency was evaluated in double transgenic hamsters.<br><br>RESULTS: The gRosa26 locus exhibited >70&#xa0;% sequence conservation with mice. Golden hamsters with the mT/mG reporter exhibited ubiquitous tdTomato expression, with efficient EGFP activation after Cre-mediated recombination. Furthermore, Alb-Cre transgenic golden hamsters exhibited hepatocyte-specific Cre expression. In double transgenic hamsters, robust hepatocyte-specific tdTomato-to-EGFP recombination was observed by postnatal days 21 and 42. This validated the functionality of the gRosa26-targeted mT/mG system and Alb-driven Cre expression.<br><br>CONCLUSIONS: We reported the development of a functional Rosa26-targeted mT/mG dual-reporter system and a liver-specific Alb-Cre driver in Syrian golden hamsters for the first time. This platform facilitates precise spatiotemporal gene editing in hepatocytes and serves as an essential genetic tool for liver research in this emerging model organism.},
}
@article {pmid41237878,
year = {2025},
author = {Ashok, K and Ellango, R and Venkatesh, R and Bhargava, CN and Pavithran, S and Dhawane, Y and Manamohan, M and Jha, GK and Asokan, R},
title = {Differential expression of genes in pre-blastoderm embryos of oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) microinjected with white locus CRISPR/Cas9 ribo nucleo protein (RNP) complex.},
journal = {International journal of biological macromolecules},
volume = {333},
number = {Pt 2},
pages = {148735},
doi = {10.1016/j.ijbiomac.2025.148735},
pmid = {41237878},
issn = {1879-0003},
mesh = {Animals ; *Tephritidae/genetics/embryology ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics ; *Gene Expression Regulation, Developmental ; Microinjections ; Gene Editing ; Gene Expression Profiling ; Insect Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics ; Transcriptome ; },
abstract = {CRISPR/Cas9-based genome editing is currently revolutionizing applications in agriculture, human health, and other domains. Targeted gene editing employing CRISPR/Cas9 is achieved by delivering the various components viz. guide RNA, traCrRNA, and the Cas9 protein in DNA or RNA or RNP format. RNP enables fast, DNA-free editing without genomic integration. While innate immunity to guide RNA is studied in humans and plants, similar research in insects remains unexplored. In the present study, the RNP complex (sgRNA+Cas9) was administered via microinjection of embryos (eggs) in the pre-blastoderm (G0) stage. Transcriptome sequencing was performed using the Illumina Novoseq 6000 platform to understand the prevalence of innate immunity against the delivered guide RNA for the white locus of Bactrocera dorsalis. 33 up- and 67 down-regulated genes were found in the differential gene expression analysis from the RNA-Seq data. The correlation between RT-qPCR and RNA-Seq gene expression levels showed a strong coefficient of determination (R2 = 0.984). The genes related to stress, intron removal, and effector recognition were overexpressed. However, the genes responsible for growth and development were significantly downregulated. Thus, the present study identified the possible reasons that could play a vital role in influencing the editing outcomes employed through the RNP complex.},
}
@article {pmid41205942,
year = {2025},
author = {Feng, Z and He, X and Lin, M and Zhao, H and Chen, Y and Chen, J and Li, Z and Shen, Y and Chen, J and Yang, X and Chen, Q},
title = {Genome-wide CRISPR screen identifies ALG5, ALG6, NF2, and FUT8 as key host proteins involved in transmissible gastroenteritis virus infection.},
journal = {International journal of biological macromolecules},
volume = {333},
number = {Pt 2},
pages = {148770},
doi = {10.1016/j.ijbiomac.2025.148770},
pmid = {41205942},
issn = {1879-0003},
mesh = {Animals ; *Transmissible gastroenteritis virus/physiology ; Swine ; *Fucosyltransferases/genetics/metabolism ; *Gastroenteritis, Transmissible, of Swine/virology/genetics/metabolism ; *CRISPR-Cas Systems ; Cell Line ; Virus Internalization ; Glycosylation ; *Host-Pathogen Interactions/genetics ; Virus Replication ; Galactoside 2-alpha-L-fucosyltransferase ; },
abstract = {Transmissible gastroenteritis virus (TGEV) represents a significant threat to global swine production. In the absence of effective antiviral therapies, control relies primarily on vaccination. To identify potential therapeutic targets, we performed a genome-wide CRISPR/Cas9 screen in porcine IPEC-J2 cells, which revealed asparagine-linked glycosylation 5 (ALG5), asparagine-linked glycosylation 6 (ALG6), neurofibromin 2 (NF2), and fucosyltransferase 8 (FUT8) as essential host factors for TGEV infection. Functional characterization demonstrated that ALG5, ALG6, and NF2 knockout impaired viral adsorption and internalization through disruption of aminopeptidase N (pAPN) transcription or N-glycosylation. Consistently, tunicamycin-mediated inhibition of N-glycosylation suppressed TGEV infection. In contrast, FUT8 knockout specifically affects viral internalization and early replication by preventing the formation of double-membrane vesicles (DMVs) but does not affect pAPN expression. This role was independent of FUT8's fucosyltransferase activity, as the enzymatic inhibitor FDW028 had no effect. Mechanistically, we found that FUT8 interacts with the TGEV nonstructural proteins NSP3 and NSP4 to facilitate DMV biogenesis. Our findings delineate distinct mechanisms by which host factors support TGEV infection and provide novel insights for the development of targeted antiviral strategies.},
}
@article {pmid41135303,
year = {2026},
author = {Lin, Y and Zhou, Y and Yuan, X and Wang, X and Xu, H and Lei, F and Wang, L and Lan, Q and Zhu, B},
title = {RPA-CRISPR-Cas12a/RPA-CRISPR-Cas12a-LFD: Two detection platforms for the rapid identification of five forensic body fluids.},
journal = {Forensic science international. Genetics},
volume = {81},
number = {},
pages = {103376},
doi = {10.1016/j.fsigen.2025.103376},
pmid = {41135303},
issn = {1878-0326},
mesh = {Humans ; Female ; Semen/chemistry ; Menstruation ; RNA, Messenger/genetics/metabolism ; Saliva/chemistry ; *CRISPR-Cas Systems ; *Forensic Genetics/methods ; Male ; Vagina/metabolism ; Genetic Markers ; Cervix Mucus/chemistry ; beta-Globins/genetics ; Real-Time Polymerase Chain Reaction ; },
abstract = {Identifying tissue source types of human peripheral blood, menstrual blood, vaginal secretion, semen, and saliva left at a crime scene can offer crucial clues for case investigation. Although messenger RNA (mRNA) profiling has become a robust tool for body fluid identification, conventional methods remain constrained by time-consuming workflows and dependence on laboratory infrastructure, hindering rapid on-site forensic analysis. To address these limitations, this study designed the RPA-CRISPR-Cas12 experiment and independently developed an RPA-CRISPR-Cas12a-LFD detection system, which achieved simple, rapid (<40 min), and accurate forensic identification of five kinds of critical body fluids. Initially, we mined the Genotype-Tissue Expression database and related literatures to screen candidate mRNA markers, subsequently employed RT-qPCR to evaluate their expression levels, and applied the Boruta algorithm to optimize the number of mRNA markers, ultimately identifying five mRNA markers: HBB (peripheral blood), MMP10 (menstrual blood), DKK4 (vaginal secretion), AKAP4 (semen), and HTN3 (saliva). The developed system exhibited exceptional specificity. Furthermore, the sensitivity values of HBB, MMP10, DKK4, AKAP4, and HTN3 markers in RPA-CRISPR-Cas12 and RPA-CRISPR-Cas12a-LFD detection systems were 0.1, 0.1, 1, 0.1, 0.1 ng; and 0.1, 0.1, 1, 1 and 1 ng, respectively. Among them, the sensitivity of HBB and DKK4 markers was superior to that of previous studies. Notably, the platform successfully resolved mixed samples (excluding AKAP4 marker), and MMP10 and DKK4 markers retained detectability in aged menstrual blood and vaginal secretion samples. The proposed method provides the groundwork for further development of tissue origin identification tools of forensic body fluids, which may eventually be suitable for on-site rapid detection environments.},
}
@article {pmid41074741,
year = {2025},
author = {Karmakar, A and Hota, A and Tanga, S and Kumar, V and Das, P and S, AE and Thapa, M and Maji, B},
title = {Engineered Thermostable Chemically Responsive GlowCas9 System for Real-Time Therapeutic Monitoring Applications.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {64},
number = {49},
pages = {e202511707},
doi = {10.1002/anie.202511707},
pmid = {41074741},
issn = {1521-3773},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; Animals ; Temperature ; *Protein Engineering ; Genetic Therapy ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has revolutionized gene therapy applications due to its ease of design and efficiency, albeit accompanied by off-target effects. Spatiotemporally regulated Cas9 offers safer gene editing methods due to its lower off-target mediated genotoxicities; however, probing the Cas9 in real time for assessing the delivery efficiency, gene editing timeframe, and spatial target information remains elusive. Here, we report an engineered Cas9 system, GlowCas9, that can be probed chemically in real-time across all assay systems, including cell lysate, native gel, ex vivo, and in vivo. Importantly, we rationally engineered the Cas9 system to attain remarkable thermostability with enhanced gene editing activities. Besides its sensitive reporter and enhanced gene editing activity, the GlowCas9 system exhibits precision HDR-based gene knock-in ability in cells and significantly outperformed the WTCas9. Overall, we report a new thermostable engineered SpCas9 system for real-time theratracking applications compatible with diverse assay formats, including live ex vivo and in vivo. This GlowCas9 system will add a new dimension of non-invasive real-time tracking in gene therapy development, both ex vivo and in vivo.},
}
@article {pmid40957713,
year = {2025},
author = {Tani, M and Hatano, K and Ishizuya, Y and Oka, T and Kanaki, T and Inoguchi, S and Yoshimura, A and Horibe, Y and Liu, Y and Nesrine, S and Okuda, Y and Yamamoto, A and Uemura, T and Yamamichi, G and Hayashi, T and Yamamoto, Y and Kato, T and Kawashima, A and Yamaguchi, T and Obika, S and Yusa, K and Nonomura, N and Nimura, K},
title = {Development of a Synthetic Lethality-Based Combination Therapy Using LIG1 and PARP Inhibitors for Prostate Cancer.},
journal = {Cancer science},
volume = {116},
number = {12},
pages = {3403-3416},
doi = {10.1111/cas.70194},
pmid = {40957713},
issn = {1349-7006},
support = {19K18610//Japan Society for the Promotion of Science/ ; 21K09345//Japan Society for the Promotion of Science/ ; 24K12482//Japan Society for the Promotion of Science/ ; },
mesh = {Humans ; Male ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; Animals ; Cell Line, Tumor ; Mice ; Phthalazines/pharmacology ; Piperazines/pharmacology ; *Synthetic Lethal Mutations/drug effects ; Xenograft Model Antitumor Assays ; Apoptosis/drug effects ; *DNA Ligase ATP/antagonists &amp; inhibitors/genetics/metabolism ; *Prostatic Neoplasms, Castration-Resistant/drug therapy/genetics/pathology ; *Prostatic Neoplasms/drug therapy/genetics ; DNA Damage/drug effects ; Drug Synergism ; *Antineoplastic Combined Chemotherapy Protocols/pharmacology ; Drug Resistance, Neoplasm/genetics ; CRISPR-Cas Systems ; Mice, Nude ; Tumor Suppressor Protein p53/genetics/metabolism ; },
abstract = {Despite advances in androgen receptor signaling inhibitors (ARSIs) and poly (ADP-ribose) polymerase inhibitors (PARPIs), metastatic castration-resistant prostate cancer (mCRPC) remains lethal. PARPIs clinical efficacy is limited in patients with homologous recombination repair deficiencies, such as BRCA1/2 mutations, due to resistance. Thus, identifying novel synthetic lethal interactions with PARP may expand treatment options and improve therapeutic efficacy. Here, to identify genes that influence sensitivity to the PARPI olaparib, we conducted a genome-wide CRISPR-Cas9 knockout screening of 18,010 genes in DU145, 22Rv1, and LNCaP prostate cancer cell lines. Our screening identified PARP and LIG1 as synthetic lethality-inducing factors, whereas TP53 conferred resistance to PARPIs. Simultaneous inhibition of LIG1 and PARP increased DNA damage and apoptosis. Additionally, the combination of the LIG1 inhibitor L82-G17 with olaparib exhibited synergistic effects. To the best of our knowledge, we validated this combination therapy in vivo for the first time, suppressing tumor growth in a DU145 xenograft model while minimizing toxicity in normal tissues. Immunohistochemical analysis revealed that LIG1 was overexpressed in CRPC tissues, suggesting its potential as a therapeutic target. This study established LIG1 as a novel synthetic lethality-inducing factor in prostate cancer, showing that L82-G17 enhances the efficacy of olaparib, regardless of the BRCA mutation status. These findings suggest that the combination of PARP and LIG1 inhibitors could be a novel therapeutic strategy for mCRPC.},
}
@article {pmid40745506,
year = {2025},
author = {Minh, NNT and Lee, E and Kim, SA},
title = {The cochaperone BAG3 promotes the stabilization of p53 under heat stress conditions.},
journal = {FEBS open bio},
volume = {15},
number = {12},
pages = {1945-1954},
doi = {10.1002/2211-5463.70096},
pmid = {40745506},
issn = {2211-5463},
support = {RS-2024-00358563//National Research Foundation of Korea/ ; },
mesh = {Humans ; *Tumor Suppressor Protein p53/metabolism/genetics ; *Heat-Shock Response/physiology/genetics ; *Adaptor Proteins, Signal Transducing/metabolism/genetics ; *Apoptosis Regulatory Proteins/metabolism/genetics ; HeLa Cells ; HSP70 Heat-Shock Proteins/metabolism ; *Molecular Chaperones/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; },
abstract = {Bcl-2-associated athanogene 3 (BAG3) is the only member of the BAG cochaperone family that is induced by stressful stimuli such as heat shock and heavy metals. In the present study, bag3 knockout (KO) HeLa cells were generated via the CRISPR-Cas9 system, and the role of BAG3 in relation to p53 under heat stress conditions was investigated. Normally, the levels of p53 were low in both wild-type (WT) and KO cells, while heat shock increased the levels of nuclear p53 in both cell lines. However, the increased level of p53 was much greater in WT cells than in KO cells, which suggested that BAG3 played a role in controlling the level of p53 under heat stress conditions. The mRNA level of p53 did not increase in either WT or KO cells during the heat stress period, which suggested that the differences in the levels of p53 were not due to transcriptional regulation. Both treatment with the proteasome inhibitor MG132 and heat shock drastically increased p53 levels to a similar extent in WT cells. Interestingly, both BAG3 and Hsp70 rapidly translocated to the nucleus and formed a complex with p53 upon heat stress. During a 1-h recovery period from heat stress, the transcriptional activity of p53 increased up to 4-fold in WT cells, but only 1.69-fold in KO cells. These results demonstrate that Hsp70 and BAG3 are involved in the quality control of p53 under heat stress conditions and suggest a role for BAG3 as a cochaperone protein.},
}
@article {pmid40643191,
year = {2025},
author = {Zhou, R and Seth, SR and Reeves, J and Burns, AH and Hsieh, C and Horn, TW and Xue, LJ and Tsai, CJ},
title = {Populus VariantDB v3.2 facilitates CRISPR and functional genomics research.},
journal = {Tree physiology},
volume = {45},
number = {13},
pages = {143-148},
doi = {10.1093/treephys/tpaf081},
pmid = {40643191},
issn = {1758-4469},
support = {ERKP886//Office of Biological and Environmental Research of the US Department of Energy, Office of Science/ ; DE-SC0023166//Office of Biological and Environmental Research of the US Department of Energy, Office of Science/ ; DE-SC0023338//Office of Biological and Environmental Research of the US Department of Energy, Office of Science/ ; //Georgia Research Alliance Hank Haynes Forest Biotechnology Endowment/ ; },
mesh = {*Populus/genetics ; *Genomics/methods ; *Genome, Plant ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Databases, Genetic ; Gene Editing ; },
abstract = {The success of CRISPR genome editing studies depends critically on the precision of guide RNA (gRNA) design. Sequence polymorphisms in outcrossing tree species pose design hazards that can render CRISPR genome editing ineffective. Despite recent advances in tree genome sequencing with haplotype resolution, sequence polymorphism information remains largely inaccessible to various functional genomics research efforts. The Populus VariantDB v3.2 addresses these challenges by providing a user-friendly search engine to query sequence polymorphisms of heterozygous genomes. The database accepts short sequences, such as gRNAs and primers, as input for searching against multiple poplar genomes, including hybrids, with customizable parameters. We provide examples to showcase the utilities of VariantDB in improving the precision of gRNA or primer design. The platform-agnostic nature of the probe search design makes Populus VariantDB v3.2 a versatile tool for the rapidly evolving CRISPR field and other sequence-sensitive functional genomics applications. The database schema is expandable and can accommodate additional tree genomes to broaden its user base.},
}
@article {pmid41309578,
year = {2025},
author = {Arana, S and Du, PP and Vaughan-Jackson, A and Enright, N and Spees, K and Valbuena, R and Garcia, CA and Nguyen, T and Venida, A and Seczynska, M and Bintu, L and Lehner, PJ and Prolo, LM and Bassik, MC},
title = {Reduced Cas9 transgene silencing by incorporation of intron sequences.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10656},
pmid = {41309578},
issn = {2041-1723},
support = {R01HG011866//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*Introns/genetics ; *Transgenes/genetics ; *Gene Silencing ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; HEK293 Cells ; Chromatin/metabolism/genetics ; Animals ; Cell Line ; },
abstract = {Silencing remains a significant challenge for exogenous gene expression, limiting both the penetrance and expressivity of transgenes. In particular, silencing of Cas9 expression is a major technical limitation for many gene editing and CRISPR screening applications. Here, we demonstrate that including introns in Cas9 expression cassettes significantly reduces silencing across multiple cell lines. Notably, the incorporation of an intron into a CRISPRa construct results in reduced silencing, increased expression levels, and markedly enhanced activation of target genes. We investigate diverse intron sequences and discover that T-rich introns over 2 kb confer the greatest protection against silencing. In addition, we find that introns can work synergistically with chromatin opening elements to further mitigate silencing, suggesting regulatory mechanisms are acting at both the DNA and RNA level to silence exogenous genes. Our work highlights the potential of introns to optimize genetic constructs for enhanced expression and improved cellular engineering requiring constitutive expression of large transgenes.},
}
@article {pmid41260510,
year = {2025},
author = {Jiang, W and Yang, A and Ma, J and Lv, D and Liu, M and Wang, C and Chen, S and Fang, H and Chu, Y and He, Z and Li, W and Liu, Y and Zhao, Y and Zhou, Z and Long, G and Jiang, H},
title = {Genetic determinants of efficacy of antiviral drugs revealed by genome-wide CRISPR screens.},
journal = {Antiviral research},
volume = {244},
number = {},
pages = {106309},
doi = {10.1016/j.antiviral.2025.106309},
pmid = {41260510},
issn = {1872-9096},
mesh = {*Antiviral Agents/pharmacology ; Animals ; Humans ; *CRISPR-Cas Systems ; Mice ; Virus Replication/drug effects ; Cell Line, Tumor ; Ribavirin/pharmacology ; Adenosine Monophosphate/analogs &amp; derivatives/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Nucleoside and nucleobase analog antiviral drugs are pivotal in antiviral therapy, but comprehensive methods to understand their cellular response mechanisms and genetic regulators are still lacking. Here, we show that Eμ-Myc; Arf[-/-] mouse lymphoma cells, which are highly apoptosis-prone, enabled genome-wide CRISPR-Cas9 screening on such drugs to identify genes that modulate their efficacy. Using retroviral sgRNA libraries and MAGeCK analysis, we uncovered key regulators of drug transport, activation, and inactivation for these drugs. For ribavirin, adenosine kinase (ADK) and adenylsuccinate synthase (ADSS) were critical for nucleotide metabolism and bioactivation. Remdesivir uptake and activation depended on the transporter SLC29A3 and phosphoamidase HINT1, whereas favipiravir resistance was linked to NT5C2-mediated dephosphorylation. Viral replication assays in Huh7 cells validated that knockout of SLC29A3, HINT1, or NT5C2 significantly altered antiviral efficacy. This study delineates the genetic network governing nucleotide analog response, providing mechanistic insights and potential biomarkers for personalized antiviral therapy.},
}
@article {pmid40905205,
year = {2025},
author = {Bataa, D and Kajiura, H and Sawada-Choi, RLS and Yamashita, Y and Ishimizu, T and Misaki, R and Takeda, A and Fujiyama, K},
title = {CRISPR/Cas9-Mediated Knockouts of the ALG3 and GNTI in N. benthamiana and Their Application to Pharmaceutical Production.},
journal = {Plant biotechnology journal},
volume = {23},
number = {12},
pages = {5894-5916},
doi = {10.1111/pbi.70326},
pmid = {40905205},
issn = {1467-7652},
support = {22K06143//Japan Society for the Promotion of Science/ ; 22K19186//Japan Society for the Promotion of Science/ ; },
mesh = {*Nicotiana/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Glycosylation ; Polysaccharides/metabolism ; Gene Knockout Techniques ; Plants, Genetically Modified/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {N-Glycosylation critically influences the efficacy, safety and pharmacokinetic properties of biopharmaceuticals. Plant expression platforms offer multiple advantages for the production of N-glycosylated proteins, but their use is impeded by the presence of plant-specific N-glycan epitopes, which raise concerns of possible immunogenicity to humans. In this study, N-glycoengineered Nicotiana benthamiana plants that produce more homogeneous N-glycans without plant-specific epitopes were generated using multiplex CRISPR/Cas9 genome editing. To achieve this N-glycosylation modification, ALG3 and GNTI, which function in N-glycosylation processes in the ER and Golgi, respectively, were characterised, and single- and double-knockout mutant plants were generated. Comprehensive N-glycan profiling revealed that while the ALG3-knockout plant line, alg3, maintained predominantly plant-specific N-glycans with altered mannose content, the GNTI-knockout line, gntI, produced exclusively high-mannose-type N-glycans. Notably, the alg3gntI double-knockout mutants yielded highly uniform trimannosidic N-glycans. To validate our N-glycoengineering approach, we expressed two model biopharmaceuticals, Varlilumab (anti-CD27 antibody) and β-glucocerebrosidase (GCase), in wild-type and mutant plants. While the antibodies expressed in alg3 and alg3gntI showed a certain level of glucosylated endoplasmic reticulum-type N-glycan, with increased non-N-glycosylated heavy chains, GCase exhibited a more consistent N-glycosylation profile, reflecting the engineered N-glycosylation pathway. Our findings provide valuable insights into N-glycan biosynthesis in N. benthamiana and demonstrate the potential of targeted N-glycoengineering for producing biopharmaceuticals with more homogeneous mannose-type N-glycan profiles.},
}
@article {pmid40905066,
year = {2025},
author = {Lin, XX and Gong, BQ and Wang, FZ and Wan, JB and Xiong, X and Li, JF},
title = {Versatile Applications of CRISPR-Based Programmable T-DNA Integration in Plants.},
journal = {Plant biotechnology journal},
volume = {23},
number = {12},
pages = {5950-5964},
doi = {10.1111/pbi.70353},
pmid = {40905066},
issn = {1467-7652},
support = {32125004//National Natural Science Foundation of China/ ; 2023B0303000022//Guangdong Major Project of Basic and Applied Basic Research grant/ ; },
mesh = {*Arabidopsis/genetics ; *DNA, Bacterial/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Gene Editing/methods ; Agrobacterium/genetics ; DNA Breaks, Double-Stranded ; },
abstract = {Agrobacterium-mediated T-DNA integration into plant genomes represents a cornerstone for transgenic expression in plant basic research and synthetic biology. However, random T-DNA integration can disrupt essential endogenous genes or compromise transgene expression, stressing the need for targeted integration strategies. Here we explored CRISPR-aided targeted T-DNA integration (CRISTTIN) in Arabidopsis, leveraging CRISPR-induced double-strand breaks (DSBs) to facilitate precise T-DNA insertion. Contrary to our initial hypothesis, conventional Cas9 outperformed a designed Cas9-adaptor fusion nuclease that may recruit Agrobacterium VirD2/T-DNA complexes to DSB sites via the adaptor-VirD2 interaction. Using Cas9-based CRISTTIN, we streamlined the parallel generation of FERONIA null alleles and in-locus complementation alleles expressing a wild-type or mutated gene. This enabled phenotypic comparisons under identical genomic contexts and significantly accelerated gene characterisation and critical residue identification. Additionally, CRISTTIN was employed to simultaneously knockout AGAMOUS and in-locus integrate a RUBY reporter, yielding plants with pink double-petaled flowers. CRISTTIN also enabled site-specific insertion of 35S enhancers for transcriptional upregulation of adjacent genes or reporter constructs for promoter activity monitoring. CRISTTIN's effectiveness was further validated in rice. These results demonstrated CRISTTIN as a versatile tool for gene functional studies and precise control of transgene expression in plants.},
}
@article {pmid40836480,
year = {2025},
author = {Qin, B and Hou, X and Fan, S and Li, C and Wang, X and Chen, Q and Yang, Y and Lin, P and Lu, LR and Fu, C and Yang, N and Gao, Q and Zheng, S and Liu, S},
title = {Simultaneous Knockout of Tk1-SST and Tk1-FFT via CRISPR/Cas9 Enhances the Natural Rubber Accumulation in Taraxacum kok-saghyz.},
journal = {Plant biotechnology journal},
volume = {23},
number = {12},
pages = {5654-5676},
doi = {10.1111/pbi.70329},
pmid = {40836480},
issn = {1467-7652},
support = {2023YFA0914801//National Key Research and Development Program of China/ ; 31970364//National Natural Science Foundation of China/ ; ZDYF2024XDNY213//Hainan Provincial Science and Technology Special Fund/ ; 324RC533//Hainan Provincial Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Taraxacum/genetics/metabolism ; *Rubber/metabolism ; Gene Knockout Techniques ; Inulin/metabolism/biosynthesis ; Sucrose/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Plant Leaves/metabolism ; },
abstract = {Taraxacum kok-saghyz (TKS) synthesises natural rubber (NR) and inulin using sucrose as a carbon source. However, molecular mechanisms regulating inulin and NR accumulation remain largely unclear. Here, we report the generation of double-gene homozygous mutants, 1-sst1-fft, by simultaneously knocking out two key genes responsible for inulin biosynthesis (Tk1-SST and Tk1-FFT) using CRISPR/Cas9 technology. The 1-sst1-fft mutants exhibited significant increases in rosette leaf number, flower number, leaf area, whole-plant biomass and seed set. Moreover, inulin biosynthesis was abolished in 1-sst1-fft, leading to significant changes in sugar composition, particularly a marked increase in sucrose levels. Notably, NR accumulation more than doubled, with no significant change in molecular weight and most terpenoid accumulation also increased in 1-sst1-fft, both being positively correlated with sucrose levels. For the first time, this study reports the generation of an inulin synthesis-deficient mutant in plants, emphasising the essential roles of 1-SST and 1-FFT in regulating carbon partitioning and, consequently, modulating important traits and metabolite accumulation. Transcriptomic analysis revealed fundamental genes involved in sucrose metabolism, sugar signalling and transport and NR elongation were significantly upregulated in 1-sst1-fft; accompanied by enhanced enzymatic activities of sucrose-phosphate synthase and invertase. These findings demonstrate that blocking inulin biosynthesis in 1-sst1-fft redirects sucrose towards NR biosynthesis, highlighting the dual role of sucrose as both a carbon source and signalling molecule in modulating plant growth and development and metabolite synthesis. Our study provides a successful approach to enhancing NR accumulation by modifying carbon allocation in TKS, offering novel insights into high-yield breeding strategies.},
}
@article {pmid41309382,
year = {2025},
author = {Dao, TO and Park, HE and Lee, JH and Kim, KM and Trinh, MP and Kang, HL and Yoo, HS and Shin, MK},
title = {Advances and Challenges in Mycobacterial Genetic Engineering: Techniques for Knockout, Knockdown and Overexpression.},
journal = {Journal of microbiology and biotechnology},
volume = {35},
number = {},
pages = {e2507051},
doi = {10.4014/jmb.2507.07051},
pmid = {41309382},
issn = {1738-8872},
mesh = {*Genetic Engineering/methods ; *Gene Knockout Techniques/methods ; *Mycobacterium/genetics ; CRISPR-Cas Systems ; *Gene Knockdown Techniques/methods ; DNA Transposable Elements ; Homologous Recombination ; Gene Transfer Techniques ; },
abstract = {Genetic engineering of mycobacteria is challenging due to their hydrophobic cell wall structure and slow growth rates. Despite these obstacles, significant progress has been made to develop genetic engineering tools to study gene function and pathogenesis in these organisms. This review comprehensively explores the current methodologies employed in the genetic modification of mycobacteria, focusing on gene knockout, knockdown, and overexpression systems. Techniques covered include homologous recombination, recombineering, transposon mutagenesis, CRISPR-Cas systems, conditional expression strategies, and phage-mediated gene delivery. The mechanism, advantages, and limitations of those methods are critically analyzed, with particular emphasis on the adaptability of these tools to various mycobacterial species. By providing a detailed comparative analysis of available genetic tools, this review is a practical guide for researchers aiming to develop targeted and efficient genetic modifications in Mycobacterium species, accelerating discoveries in pathogenesis, drug resistance, and vaccine development.},
}
@article {pmid41167015,
year = {2025},
author = {Song, X and Peng, Q and Zhang, X and Zhao, Y and Zhang, G and Guo, R and Li, Y and Shen, Y and Yang, S and Hu, M and Zhang, X and Li, J and Fan, B and Li, B},
title = {Enhanced immunogenicity of swine coronavirus PEDV by engineering transcriptional regulatory sequences.},
journal = {Vaccine},
volume = {68},
number = {},
pages = {127914},
doi = {10.1016/j.vaccine.2025.127914},
pmid = {41167015},
issn = {1873-2518},
mesh = {Animals ; *Porcine epidemic diarrhea virus/immunology/genetics ; Swine ; Antibodies, Viral/blood/immunology ; *Coronavirus Infections/prevention &amp; control/veterinary/immunology ; *Viral Vaccines/immunology/genetics/administration &amp; dosage ; *Swine Diseases/prevention &amp; control/immunology/virology ; Antibodies, Neutralizing/blood/immunology ; Spike Glycoprotein, Coronavirus/immunology/genetics ; Female ; *Immunogenicity, Vaccine ; Vaccines, Inactivated/immunology/administration &amp; dosage ; Immunoglobulin G/blood ; CRISPR-Cas Systems ; Pregnancy ; Immunoglobulin A/blood ; Gene Editing ; },
abstract = {Porcine epidemic diarrhea virus (PEDV) is a porcine enteropathogenic coronavirus that can cause severe diarrhea and high mortality in neonatal piglets. Therefore, the development of safe and effective vaccines remains a top priority for controlling PEDV infection. Previous studies have found that the transcription regulatory sequence (TRS) of the viral M gene has the strongest regulatory ability for the expression of exogenous gene. In order to investigate whether the transcription level of the S gene is increased after swapping body TRS of the S gene (TRS-S) with the body-TRS of M gene (TRS-M), CRISPR/Cas9 gene editing technology were used to replace the TRS-S of PEDV with TRS-M, and found that replacing TRS-S by TRS-M, as well as the downstream bases of TRS-M, can effectively promote the expression of S protein. Subsequently, the recombinant strain with high expression of S protein was prepared into an inactivated vaccine, which induced higher levels of S-specific immunoglobulin G (IgG), IgA, and neutralizing antibodies in immunized pregnant sows. The newborn spiking pigs also obtained high levels of antibodies through breastfeeding, and after being challenged, it was found that clinical symptoms and microscopic lesions were significantly reduced. In conclusion, these data suggest that replacing TRS-S with the transcriptionally efficient TRS-M effectively promotes PEDV S protein expression, and this modified strain exhibits improved immunogenicity and better protection against viral challenge, which provides new method for the research and development of PEDV vaccines.},
}
@article {pmid41067593,
year = {2026},
author = {Wijerathna, HMSM and Jung, S and Lee, J},
title = {Enhancing Edwardsiella piscicida resistance through CRISPR/Cas9-mediated deletion of toll-like receptor 5a (tlr5a) in zebrafish.},
journal = {Fish &amp; shellfish immunology},
volume = {168},
number = {},
pages = {110915},
doi = {10.1016/j.fsi.2025.110915},
pmid = {41067593},
issn = {1095-9947},
mesh = {Animals ; *Toll-Like Receptor 5/genetics/immunology ; Edwardsiella/physiology ; *Fish Diseases/immunology/microbiology/genetics ; *Zebrafish/immunology/genetics ; *Enterobacteriaceae Infections/immunology/veterinary/genetics ; CRISPR-Cas Systems ; *Immunity, Innate/genetics ; *Disease Resistance/genetics ; *Zebrafish Proteins/genetics/immunology ; },
abstract = {Toll-like receptor 5 (TLR5), a pattern recognition receptor that detects bacterial flagellin, plays a critical role in innate immune responses. Teleost fish possess two paralogs, tlr5a and tlr5b, of which the functional role of tlr5a during flagellated bacterial infections remains unclear. In this study, we investigated the involvement of tlr5a in the immune response against Edwardsiella piscicida infection in zebrafish (Danio rerio). To this end, we generated tlr5a-deficient (tlr5a[-/-]) zebrafish using CRISPR/Cas9 technology. Survival analysis following E. piscicida challenge revealed significantly higher survival rates and reduced bacterial loads in tlr5a[-/-] larvae and adults compared to wild-type (WT) controls. tlr5a[-/-] fish exhibited significantly lower expression of pro-inflammatory cytokines (tnfa, il6, and il-1β), chemokine (il8), and pathway genes (nfκb, myd88, and mapk14a) at multiple time points post-infection (0, 6, 12, 24, 48, and 72 hpi). DCFH-DA staining and Sudan Black/Neutral Red staining revealed elevated reactive oxygen species (ROS) levels and immune cell recruitment, respectively, indicating reduced ROS production and diminished neutrophil and macrophage infiltration in tlr5a[-/-] larvae. Antioxidant gene expression analysis revealed reduced levels of cat and nrf2 in tlr5a[-/-] larvae compared to WT. The results of this study indicate that tlr5a knockout attenuates excessive inflammation and improves resistance against E. piscicida infection, likely by reducing bacterial adhesion and suppressing NF-κB-mediated pro-inflammatory pathways. This study highlights tlr5a as a potential immunomodulatory target to enhance disease resistance in teleost aquaculture.},
}
@article {pmid40834600,
year = {2025},
author = {Chang, Y and Huang, H and Zhao, R and Diao, L and Xiong, W and Wan, P},
title = {Development of the CRISPR/Cas9 system for genome editing in Riemerella anatipestifer.},
journal = {Poultry science},
volume = {104},
number = {11},
pages = {105696},
pmid = {40834600},
issn = {1525-3171},
mesh = {*Riemerella/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods/veterinary ; Animals ; Poultry Diseases/microbiology ; Flavobacteriaceae Infections/veterinary/microbiology ; },
abstract = {Riemerella anatipestifer (R. anatipestifer) is an important pathogen responsible for high mortality rates and severe economic losses in the poultry industry. Research on R. anatipestifer is constrained by limited genetic manipulation tools, highlighting the need for an effective genome editing toolkit. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 system from Streptococcus pyogenes has been widely used as a genome editing tool for various bacteria, but has not been used in R. anatipestifer. In this study, we developed a CRISPR/Cas9-based genome editing system, pCasRA-SacB, specifically for R. anatipestifer. This shuttle vector contains the replication origin from R. anatipestifer plasmid pRA0726 and p15A origin for replication in both R. anatipestifer and Escherichia coli (E. coli). It also includes chloramphenicol and cefoxitin resistance genes, which allow for selection in E. coli and R. anatipestifer. The vector features BsaI and SalI sites, enabling the cloning of single guide RNA (sgRNA) and homologous arms. In addition, it contains the high-expression promoter of B739_0921 gene and the promoter of rpsL gene, facilitating the expression of sgRNA and Cas9 protein. Furthermore, the vector features the sucrose-sensitive gene sacB and the oriT region, which enable plasmid curing and the transfer of plasmid via conjugation. Notably, the pCasRA-SacB system enables rapid, efficient, and scarless genome editing in R. anatipestifer, including gene deletion, insertion, and point mutation in the dprA gene, with editing efficiencies of 54.2 %, 100.0 %, and 50.0 %, respectively. In summary, the pCasRA-SacB system not only expands the genome editing toolbox of R. anatipestifer but also is helpful in fundamental research in R. anatipestifer.},
}
@article {pmid40659814,
year = {2025},
author = {Levesque, S and Bauer, DE},
title = {CRISPR-based therapeutic genome editing for inherited blood disorders.},
journal = {Nature reviews. Drug discovery},
volume = {24},
number = {12},
pages = {907-925},
pmid = {40659814},
issn = {1474-1784},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Hematologic Diseases/therapy/genetics ; Anemia, Sickle Cell/therapy/genetics ; },
abstract = {Therapeutic genome editing promises to transform medicine. Pivotal discoveries have provided a diverse and versatile set of tools to correct pathogenic mutations or produce protective alleles using CRISPR-based technologies. These innovative therapies are especially adaptable for blood and immune disorders, where clinical methods allow haematopoietic stem cells (HSCs) to be mobilized, harvested, engineered ex vivo and transplanted back into a patient to permanently replace their blood system. This paradigm has been exemplified with the first US Food and Drug Administration (FDA)-approved CRISPR-Cas9 therapy for sickle cell disease and β-thalassaemia, exa-cel (Casgevy). Although promising, efficient delivery of gene edits involves complicated ex vivo manipulation and toxic myeloablative conditioning. The quiescent and elusive nature of HSCs also brings associated challenges. In this Review, we explore the state-of-the-art genome editing technologies of nucleases, base editors and prime editors, which hold promise to address unmet clinical needs for patients with inherited haematological disorders. We highlight the progress made for several disorders and discuss the challenges that remain for ex vivo and in vivo targeting of HSCs for next-generation gene therapies.},
}
@article {pmid41309229,
year = {2025},
author = {Song, BS and Baek, YH and Kim, EH and Kwon, HI and Kim, AH and Lee, SH and Son, YB and Kim, SH and Song, MS and Choi, YK and Park, SJ},
title = {Development of an RT-LAMP-CRISPR/Cas12a assay for rapid and specific detection of Bandavirus dabieense.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {11},
pages = {e2506013},
doi = {10.71150/jm.2506013},
pmid = {41309229},
issn = {1976-3794},
support = {2022R1C1C1004704//National Research Foundation of Korea/ ; RS-2023-00301974//Ministry of Education/ ; //Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry/ ; RS-2022-IP322088//Ministry of Agriculture, Food and Rural Affairs/ ; },
mesh = {*Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Humans ; *CRISPR-Cas Systems ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology ; RNA, Viral/genetics ; *RNA Viruses/genetics/isolation &amp; purification ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Bandavirus dabieense, a single-stranded RNA virus, is the causative agent of severe fever with thrombocytopenia syndrome (SFTS), a disease associated with high fatality rates. Early and accurate diagnosis is essential for improving clinical outcomes, particularly given the limited therapeutic options and high mortality rates associated with SFTS. However, while highly sensitive, conventional diagnostic methods such as PCR and qRT-PCR require specialized laboratory facilities and trained personnel, making them impractical for rapid detection in resource-limited settings. To address these challenges, we developed a rapid and highly sensitive assay for Bandavirus dabieense detection by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) with CRISPR/Cas12a technology. LAMP primers and guide RNA sequences were designed to target the L gene, ensuring broad detection across viral genotypes. The optimized assay demonstrated a detection limit of 5 RNA copies per reaction, showing more sensitivity than qRT-PCR, and exhibited 100% concordance with qRT-PCR results in clinical samples. Given its speed, accuracy, and field applicability, this LAMP-CRISPR/Cas12a-based assay represents a promising diagnostic tool for early SFTSV detection, particularly in resource-constrained environments where conventional molecular diagnostics are not readily available.},
}
@article {pmid41307588,
year = {2025},
author = {Kumar, V and Verma, P},
title = {Advances in microbial biotechnology for sustainable wastewater reclamation: recent trends and future prospects.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {12},
pages = {478},
pmid = {41307588},
issn = {1573-0972},
mesh = {*Wastewater/microbiology/chemistry ; *Biotechnology/methods/trends ; *Water Purification/methods ; Biodegradation, Environmental ; Bacteria/metabolism/genetics ; Microalgae/metabolism ; Bioelectric Energy Sources ; Waste Disposal, Fluid/methods ; Microbial Consortia ; },
abstract = {The growing demand for freshwater, coupled with the increasing volume of industrial and municipal wastewater, has intensified the need for sustainable and eco-friendly reclamation strategies. Recent advancements in microbial biotechnology have emerged as promising tools for developing cost-effective, efficient, and environmentally sustainable wastewater treatment (WWT) strategies for reuse and safe disposal. This mini-review explores current innovations, such as microbial consortia, bioaugmentation, and the microalgae-bacteria nexus, which have shown promising results in nutrient removal, enhanced degradation of complex pollutants (including emerging contaminants), and biomass valorization. Moreover, bioelectrochemical systems, such as microbial fuel cells (MFC) and microbial electrolysis cells (MEC), have revolutionized WWT by facilitating pollutant degradation while simultaneously generating bioelectricity or biohydrogen. This article also critically examines the role of CRISPR-based tools and 'omics' approaches, which have enabled the development of novel microbial strains and degradative pathways, enhancing wastewater reclamation in challenging environments. Furthermore, advancements through the integration of multi-omics and artificial intelligence, digital twins, and Internet of Things (IoT) for microbial optimization and real-time process control are discussed. The review highlights the role of microbial systems in resource recovery, supporting a circular economy by transforming wastewater into valuable bioresources. Additionally, this review addresses the major challenges and proposes future research directions for effective wastewater treatment. The novelty of this manuscript is that no single review explores the cutting-edge microbial biotechnologies for wastewater reclamation, uniquely integrating CRISPR-Cas genome editing, multi-omics analyses, and artificial intelligence-driven optimization to advance pollutant degradation and real-time process control in one place. This study concludes that by implementing multi-omics and artificial intelligence (AI)-driven optimization process for wastewater treatment can be effective towards wastewater treatment while simultaneously minimizing the environmental pollution.},
}
@article {pmid41307501,
year = {2025},
author = {Sahu, S and Boukherroub, R and Ritzenthaler, C and Szunerits, S},
title = {Emerging technologies for in-field plant virus detection: innovations and future directions.},
journal = {The Journal of general virology},
volume = {106},
number = {11},
pages = {},
pmid = {41307501},
issn = {1465-2099},
mesh = {*Plant Viruses/isolation &amp; purification/genetics ; *Plant Diseases/virology ; Biosensing Techniques/methods ; High-Throughput Nucleotide Sequencing ; CRISPR-Cas Systems ; Nanotechnology/methods ; },
abstract = {Plant virus infections pose a substantial threat to crop quality and productivity, contributing to considerable economic losses in global agriculture annually. Traditionally, laboratories have widely adopted serological techniques, such as ELISA, and molecular methods, including quantitative PCR, for virus diagnostics. More recently, sophisticated next-generation sequencing approaches have been introduced to improve the efficiency and reliability of virus detection and identification. However, the development of sensitive, rapid and low-cost methods for the on-site detection, quantification and identification of plant viruses remains an ongoing challenge and is still in its early days. Point-of-care technologies have not fully realized their potential in agriculture due to numerous challenges, such as the elevated cost of development, lack of standardized validation and insufficient field testing. Therefore, future success depends on addressing these technical, economic and regulatory hurdles, as well as considering the specific user needs within the agricultural context. In this mini-review, recent advancements in biosensing for on-site plant virus monitoring, involving nanotechnology-based sensors, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems, electrochemical and modern field-effect transistor-based sensors offering high sensitivity, speed and portability, are discussed. These technologies, when integrated with smartphone applications and/or machine learning modules, could enable real-time, field-deployable diagnostics for early disease management and sustainable agriculture. The aim is to raise awareness among plant virologists about this panel of emerging diagnostic concepts that could help improve current methods, ultimately facilitating the management of plant viral diseases.},
}
@article {pmid41306592,
year = {2025},
author = {Shi, L and Chen, H and Zhang, Z and Wang, Y and Ren, W and Huang, J},
title = {Evolving HPV diagnostics: current practice and future frontiers.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1681779},
pmid = {41306592},
issn = {2235-2988},
mesh = {Humans ; *Papillomavirus Infections/diagnosis/virology ; Female ; *Molecular Diagnostic Techniques/methods/trends ; *Papillomaviridae/genetics/isolation &amp; purification ; Uterine Cervical Neoplasms/virology/diagnosis ; Early Detection of Cancer/methods ; High-Throughput Nucleotide Sequencing ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Polymerase Chain Reaction/methods ; Mass Screening/methods ; },
abstract = {Human papillomavirus (HPV) infection serves as a primary causative agent of cervical cancer, highlighting the importance of early screening and detection in mitigating the incidence and mortality rates of HPV-related diseases. Over the past decades, HPV detection technologies have evolved considerably, transitioning from traditional methods to more advanced, patient-centered approaches. This review provides a comprehensive overview of both established and emerging HPV detection strategies, with a particular focus on their clinical applicability, technical advantages, and limitations. Conventional methods such as hybrid capture and PCR-based assays remain the backbone of clinical screening, offering robust sensitivity and specificity. However, their reliance on invasive sampling and centralized laboratory infrastructure limits accessibility and patient compliance, particularly in low-resource settings. To address these limitations, emerging technologies-including CRISPR/Cas systems, droplet digital PCR (ddPCR), next-generation sequencing (NGS), isothermal amplification techniques (IAT) and artificial intelligence (AI) combined with hpv screening offer enhanced accuracy, rapid turnaround, and the potential for point-of-care deployment. In parallel, innovations in sampling such as self-collected vaginal swabs and liquid biopsy using urine, blood, or extracellular vesicles are improving test acceptability and broadening screening coverage. By summarizing current progress and highlighting ongoing challenges, this review aims to guide the development of more precise, non-invasive, and scalable HPV detection strategies to reduce the global burden of HPV-related disease, support global prevention efforts, and guide public health policies.},
}
@article {pmid41305866,
year = {2025},
author = {Naderi, S and Williamson, J and Sun, H and Joshi, S and Spera, RJ and Zaib, S and Sharma, S and Sun, C and Brodovskiy, A and Zawar, I and Kapur, J},
title = {Hydroxycarboxylic Acid Receptor 2 Mediates β-hydroxybutyrate's Antiseizure Effect in Mice.},
journal = {Annals of neurology},
volume = {},
number = {},
pages = {},
doi = {10.1002/ana.78098},
pmid = {41305866},
issn = {1531-8249},
support = {R01NS120945//United States National Institute of Health (NINDS)/ ; R37N119012//United States National Institute of Health (NINDS)/ ; },
abstract = {OBJECTIVE: The ketogenic diet, a high-fat, low-carbohydrate regimen, is often used to treat drug-resistant seizures and is being studied for Alzheimer's disease and other neuropsychiatric disorders. However, its mechanism of action remains unclear. β-hydroxybutyrate, a primary circulating ketone body produced by the ketogenic diet, may mediate its effects on seizures by binding to a recently identified Gi-coupled receptor: hydrocarboxylic acid receptor 2 (HCAR2).<br><br>METHODS: RNAscope in situ hybridization assay and real-time quantitative polymerase chain reaction were used to assess HCAR2 expression in the mouse brain. We generated HCAR2[-]/[-] using the CRISPR-Cas technique on an S129 mouse background. Whole-cell current-clamp was performed to measure the passive and active membrane properties of hippocampal dentate granule cells. The voltage-clamp was performed to record synaptic currents. Two complementary in vivo mouse models-continuous hippocampal stimulation to induce status epilepticus (SE) and kindling-were used to induce seizures.<br><br>RESULTS: HCAR2 was localized in dentate granule cells and microglia. In mice with HCAR2, &#x3b2;-hydroxybutyrate reduced neuronal excitability by hyperpolarizing the resting membrane potential, raising the action potential threshold, and reducing the firing frequency of dentate granule cells. &#x3b2;-hydroxybutyrate suppressed excitatory synaptic transmission. These effects were nullified in HCAR2[-]/[-] mice. HCAR2[-]/[-] mice showed no cognitive impairment. Moreover, &#x3b2;-hydroxybutyrate did not affect seizures in HCAR2[-]/[-] mice. However, it diminished both the duration and severity of seizures in HCAR2[+]/[+] mice.<br><br>INTERPRETATION: These findings demonstrate that HCAR2 mediates &#x3b2;-hydroxybutyrate's antiseizure effects by regulating neuronal excitability and synaptic transmission. These studies propose a new mechanism for the antiseizure action of the ketogenic diet. ANN NEUROL 2025.},
}
@article {pmid41305525,
year = {2025},
author = {Verma, N and O'Mahony, A and Mohammad, R and Keiser, D and Mosman, CW and Holden, D and Starr, K and Bauer, J and Bauer, B and Suntisukwattana, R and Atthaapa, W and Tantituvanont, A and Nilubol, D and Gladue, DP},
title = {The First CRISPR-Based Therapeutic (SL_1.52) for African Swine Fever Is Effective in Swine.},
journal = {Viruses},
volume = {17},
number = {11},
pages = {},
pmid = {41305525},
issn = {1999-4915},
mesh = {Animals ; *African Swine Fever/therapy/virology ; Swine ; *African Swine Fever Virus/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Viral ; },
abstract = {African swine fever virus (ASFV) is a high-consequence pathogen that causes African swine fever (ASF), for which mortality rates can reach 90-100%, with death typically occurring within 14 days. ASF is currently a highly contagious pandemic disease responsible for extensive losses in pig production in multiple affected countries suffering from extended outbreaks. While a limited number of vaccines to prevent ASF are in use in south-east Asia, vaccines are not widely available, are only effective against highly homologous strains of ASFV, and must be used prior to an outbreak on a farm. Currently, there is no treatment for ASF and culling affected farms is the only response to outbreaks on farms to try and prevent spreading. CRISPR/Cas systems evolved as an adaptive immune response in bacteria and archaea that function by cleaving and disrupting the genomes of invading bacteriophage pathogens. CRISPR technology has since been leveraged into an array of endonuclease-based systems used for nucleic acid detection, targeting, genomic cleavage, and gene editing, making them particularly well-suited for development as sequence-specific therapeutic modalities. The programmability of CRISPR-based therapeutics offers a compelling new way to rapidly and specifically target pathogenic viral genomes simply by using different targeting guide RNAs (gRNA) as an adaptable antiviral modality. Here, we demonstrate for the first time a specific CRISPR/Cas9 multiplexed gRNA system that targets the African swine fever viral genome, resulting in sequence-specific cleavage, leading to the reduction in the viral load in infected animals, and subsequent recovery from an otherwise lethal dose of ASFV. Moreover, animals that recovered had protective immunity to subsequent homologous ASFV infection.},
}
@article {pmid41062067,
year = {2025},
author = {Izumikawa, T and Moriya, A and Nakato, E and Yamamoto, K and Sano, R and Akiyama, T and Kinoshita-Toyoda, A and Toyoda, H and Nakato, H},
title = {In vivo analysis of Drosophila chondroitin sulfate biosynthetic genes.},
journal = {The Journal of biological chemistry},
volume = {301},
number = {11},
pages = {110783},
pmid = {41062067},
issn = {1083-351X},
mesh = {Animals ; *Chondroitin Sulfates/biosynthesis/genetics ; *Sulfotransferases/genetics/metabolism ; *N-Acetylgalactosaminyltransferases/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Drosophila melanogaster/genetics/metabolism ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Chondroitin sulfate (CS) is an evolutionarily conserved class of glycosaminoglycans and is found in most animal species. Previous studies of CS-deficient Drosophila models, Chondroitin sulfate synthase (Chsy), and Chondroitin polymerizing factor (Chpf) mutants demonstrated the importance of CS in the structural integrity of the basement membrane and organ shape maintenance. However, biosynthetic mechanisms of Drosophila CS remain to be elucidated. To investigate the CS biosynthesis in Drosophila, we generated mutants for two additional biosynthetic enzyme genes, CS N-acetylgalactosaminyltransferase (Csgalnact) and CS 4-O sulfotransferase (C4st), using CRISPR-Cas9 mutagenesis. Csgalnact-null mutants show moderate changes in CS biosynthesis, including reduced CS in the larval brain and altered CS chain length. We found that this gene is dispensable for normal viability and morphogenesis. On the other hand, C4st mutants show more severe defects, including a high level of lethality and a folded wing phenotype. The C4st mutation not only eliminates CS sulfation but increases production of unsulfated chondroitin, suggesting the existence of a compensatory feedback mechanism. Both Csgalnact and C4st mutants show impaired adult negative geotaxis behavior, consistent with the role of CS proteoglycan in the neuromuscular systems. Our study revealed unique and poorly understood features of invertebrate CS biosynthesis and provides novel in vivo toolsets to investigate CSPG functions in development.},
}
@article {pmid41005478,
year = {2025},
author = {Veit, MC and Stauder, R and Bai, Y and Gabhrani, R and Schmidt, M and Klähn, S and Lai, B},
title = {The necessity of multi-parameter normalization in cyanobacterial research: A case study of the PsbU in Synechocystis sp. PCC 6803 using CRISPRi.},
journal = {The Journal of biological chemistry},
volume = {301},
number = {11},
pages = {110763},
pmid = {41005478},
issn = {1083-351X},
mesh = {*Synechocystis/genetics/metabolism/growth &amp; development ; *Photosystem II Protein Complex/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Oxygen/metabolism ; Photosynthesis ; *CRISPR-Cas Systems ; Chlorophyll A/metabolism ; },
abstract = {Photosystem II (PSII) is a multiprotein complex and plays a central role in oxygenic photosynthesis. PsbU, a 12 kDa subunit of PSII, is associated with thermotolerance and structural stabilization of the oxygen-evolving complex in cyanobacteria. Corresponding knockout strains showed decreased oxygen evolution rates, although the growth was not impaired. In this study, we provide further insights into the consequences of PsbU perturbations and propose to revisit the impact of PsbU on cell physiology. We made use of CRISPRi to knock down the psbU gene in Synechocystis sp. PCC 6803, and assessed previously described effects referred to different biomass parameters including optical density, chlorophyll a content and cell number. After knocking down psbU, the growth rate was decreased by 15% based on counting the cell number, while this effect was not observed when monitoring optical density. Furthermore, the oxygen evolution rate per cell in the psbU knockdown strain did not show a significant difference compared to the control groups, which was probably due to its larger cell size and higher chlorophyll a content per cell. The decreased quantum efficiency of pigments was compensated by the increased pigment content on the single-cell level in the knockdown strain. Our results complement previous analyses and highlight the importance of evaluating cyanobacterial physiology based on different biomass quantitative units to avoid misinterpretation of the results.},
}
@article {pmid39938795,
year = {2025},
author = {Fu, ZH and Cheng, S and Li, JW and Zhang, N and Wu, Y and Zhao, GR},
title = {Synthetic tunable promoters for flexible control of multi-gene expression in mammalian cells.},
journal = {Journal of advanced research},
volume = {78},
number = {},
pages = {351-361},
doi = {10.1016/j.jare.2025.02.008},
pmid = {39938795},
issn = {2090-1224},
mesh = {*Promoter Regions, Genetic/genetics ; Humans ; *Synthetic Biology/methods ; Genes, Reporter ; CRISPR-Cas Systems/genetics ; Animals ; HEK293 Cells ; *Gene Expression Regulation/genetics ; },
abstract = {INTRODUCTION: Synthetic biology revolutionizes our ability to decode and recode genetic systems. The capability to reconstruct and flexibly manipulate multi-gene systems is critical for understanding cellular behaviors and has significant applications in therapeutics.<br><br>OBJECTIVES: This study aims to construct a diverse library of synthetic tunable promoters (STPs) to enable flexible control of multi-gene expression in mammalian cells.<br><br>METHODS: We designed and constructed synthetic tunable promoters (STPs) that incorporate both a universal activation site (UAS) and a specific activation site (SAS), enabling multi-level expression control via the CRISPR activation (CRISPRa) system. To evaluate promoter activity, we utilized Massively Parallel Reporter Assays (MPRA) to assess the basal strengths of the STPs and their activation responses. Next, we constructed a three-gene reporter system to assess the capacity of the synthetic promoters for achieving multilevel control of single-gene expression within multi-gene systems.<br><br>RESULTS: The promoter library contains 24,960 unique non-redundant promoters with distinct sequence characteristics. MPRA revealed a wide range of promoter activities, showing different basal strengths and distinct activation levels when activated by the CRISPRa system. When regulated by targeting the SAS, the STPs exhibited orthogonality, allowing multilevel control of single-gene expression within multi-gene systems without cross-interference. Furthermore, the combinatorial activation of STPs in a multi-gene system enlarged the scope of expression levels achievable, providing fine-tuned control over gene expression.<br><br>CONCLUSION: We provide a diverse collection of synthetic tunable promoters, offering a valuable toolkit for the construction and manipulation of multi-gene systems in mammalian cells, with applications in gene therapy and biotechnology.},
}
@article {pmid39914488,
year = {2025},
author = {Yu, H and Zhang, G and Liu, J and Liu, P and Peng, H and Teng, Z and Li, Y and Ren, X and Fu, C and Tang, J and Li, M and Wang, Y and Wang, L and Peng, L},
title = {A functional cascading of lignin modification via repression of caffeic acid O-methyltransferase for bioproduction and anti-oxidation in rice.},
journal = {Journal of advanced research},
volume = {78},
number = {},
pages = {1-9},
doi = {10.1016/j.jare.2025.01.048},
pmid = {39914488},
issn = {2090-1224},
mesh = {*Lignin/metabolism ; *Oryza/metabolism/genetics ; *Methyltransferases/metabolism/genetics ; Oxidation-Reduction ; Antioxidants/metabolism ; Cadmium/metabolism ; Biomass ; Coumaric Acids/metabolism ; Plant Proteins/metabolism/genetics ; Mutation ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: Crop straws provide substantial biomass resources that are transformable for sustainable biofuels and valuable bioproducts. However, the natural lignocellulose recalcitrance results in an expensive biomass process and secondary waste liberation. As lignin is a major recalcitrant factor, genetic engineering of lignin biosynthesis is increasingly being implemented in bioenergy crops, but much remains unclear about the desired lignocellulose alteration and resulting function.<br><br>OBJECTIVES: This study attempted to explore the mechanisms of lignin modification responsible for efficient lignocellulose conversion in vitro and an effective plant anti-oxidation response in vivo.<br><br>METHODS: We initially selected specific rice mutants by performing modern CRISPR/cas9 editing with caffeic acid O-methyltransferase involved in the synthetic pathways of monolignols (G, S) and ferulic acid (FA), and then explored lignocellulose conversion and plant cadmium (Cd) accumulation using advanced chemical, biochemical and thermal-chemical analyses.<br><br>RESULTS: Notable lignin modification was achieved from the predominately synergistic down-regulation of S-monomer synthesis in three mutants. This consequently upgraded lignocellulose porosity by up to 1.8 folds to account for significantly enhanced biomass saccharification and bioethanol production by 20&#xa0;%-26&#xa0;% relative to the wild-type. The modified lignin also favors the dissection of diverse lignin nanoparticles with dimensions reduced by 1.5-1.9 folds, applicable for thermal-chemical conversion into the carbon quantum dots with increased yields by 15&#xa0;% and 31&#xa0;%. The proportions of G-monomers and FA were significantly increased in the mutants, and the lignin extractions were further assayed with higher activities for two standard antioxidants (DPPH and ABTS) in vitro compared to the wild-type, revealing a distinctively enhanced plant antioxidative capacity in the mutants. Water culture showed that young mutant seedlings accumulated more Cd than wild-type did (p&#xa0;<&#xa0;0.01, n&#xa0;=&#xa0;3), suggesting effective heavy metal phytoremediation in the mutants.<br><br>CONCLUSION: A hypothetical model of characteristic lignin modification for specific S-monomer reduction, accountable for improved lignocellulose recalcitrance, was proposed. It provides a powerful strategy for achieving high-yield biofuels and value-added bioproducts or enhancing plant antioxidative capacity for heavy metal phytoremediation.},
}
@article {pmid41305485,
year = {2025},
author = {Iftehimul, M and Hasan, NA and Bass, D and Bashar, A and Haque, MM and Santi, M},
title = {Combating White Spot Syndrome Virus (WSSV) in Global Shrimp Farming: Unraveling Its Biology, Pathology, and Control Strategies.},
journal = {Viruses},
volume = {17},
number = {11},
pages = {},
doi = {10.3390/v17111463},
pmid = {41305485},
issn = {1999-4915},
support = {2022/21/Other//Ocean Country Partnership Programme (OCPP), Blue Planet Fund/ ; },
mesh = {*White spot syndrome virus 1/pathogenicity/immunology/physiology/genetics ; Animals ; *Aquaculture/methods ; *Penaeidae/virology/immunology ; Immunity, Innate ; Viral Vaccines/immunology ; },
abstract = {White Spot Syndrome Virus (WSSV) is one of the most devastating viral pathogens affecting shrimp, causing severe economic losses to the global farmed shrimp trade. The globalization of live shrimp trade and waterborne transmission have facilitated the rapid spread of WSSV across major shrimp-producing countries since its initial emergence. The present review gives an updated account of WSSV biology, pathology, transmission dynamics, and recent developments in control measures. The virus, a double-stranded DNA virus of the Nimaviridae family, utilizes advanced immune evasion strategies, resulting in severe mortality. Shrimp lack adaptive immunity and hence rely predominantly on innate immunity, which is insufficient to mount an effective response against severe infections. Traditional disease control measures such as augmented biosecurity, selective breeding, and immunostimulants have, despite extensive research, achieved only limited success. New biotechnological tools such as RNA interference, CRISPR-Cas gene editing, and nanotechnology offer tremendous potential for disease mitigation. In parallel, the development of DNA and RNA vaccines targeting WSSV structural proteins, such as VP28, holds significant promise for stimulating the shrimp immune system. This review highlights the urgent need for a convergent approach to sustainable disease management in global shrimp aquaculture, with interdisciplinarity playing a pivotal role in shaping the future of WSSV control.},
}
@article {pmid41304933,
year = {2025},
author = {Yue, Y and Xu, Z and Soteyome, T and Premarathna, M and Yin, X and Liu, J},
title = {Phage Encapsulation and Delivery Technology: A Strategy for Treating Drug-Resistant Pathogenic Microorganisms.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {18},
number = {11},
pages = {},
doi = {10.3390/ph18111688},
pmid = {41304933},
issn = {1424-8247},
abstract = {Antimicrobial resistance (AMR) is one of the most critical challenges to global public health in the 21st century, posing a significant threat to healthcare systems and human health due to treatment failure and high mortality. The World Health Organization (WHO) estimates that, without effective interventions, AMR-associated infections could cause 10 million deaths annually and economic losses of up to 100 trillion US dollars by 2050. The rapid spread of drug-resistant strains, especially in hospital and community settings, has significantly reduced the efficacy of traditional antibiotics. With the continuous advancements in relevant research, bacteriophage (Phage) therapy is constantly innovating in the antimicrobial field. The application of frontier technologies, such as phage cocktails and engineered phages, has significantly enhanced the broad spectrum and high efficiency of phage therapy, which is gradually becoming a new generation of tools to replace antibiotics and effectively combat pathogenic bacteria. However, phage therapy is facing several challenges, including phage inactivation by gastric acid, enzymes, ultraviolet light, and mechanical stress, as well as the potential risk of bacterial phage resistance. Advanced encapsulation technologies such as electrospun fibers, liposomes, chitosan nanoparticles, and electrospray provide solutions to these problems by protecting phage activity and enabling controlled release and targeted delivery. This review addresses phage therapeutic studies of Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes, summarizes the recent advances in phage research, and details the current development and applications of encapsulated phage technologies across various delivery modes.},
}
@article {pmid41304797,
year = {2025},
author = {Sharma, A and Sharma, V and Sharma, S and Sharma, S and Sharma, M and Sivanesan, I},
title = {Advanced Nanosystems and Emerging Therapies: Innovations in Tuberculosis Treatment and Drug Resistance.},
journal = {Pharmaceutics},
volume = {17},
number = {11},
pages = {},
doi = {10.3390/pharmaceutics17111459},
pmid = {41304797},
issn = {1999-4923},
abstract = {Tuberculosis (TB) remains a significant worldwide health challenge due to the limitations of conventional treatments and the rising incidence of drug-resistant Mycobacterium tuberculosis strains. This review consolidates the advancements in nanotechnology-based therapeutics, inhalable formulations, CRISPR-Cas tools, host-directed therapies (HDTs), and nanoparticle-based vaccine development aimed at enhancing TB management. Novel nanocarriers such as liposomes, solid-lipid nanoparticles (SLNs), dendrimers, and polymeric nanoparticles (NPs) offer enhanced bioavailability of drugs, sustained release, as well as targeted delivery to infected macrophages, thereby reducing systemic toxicity and dosing frequency. Inhalable nanomedicines provide localized delivery to the pulmonary site, enhancing the concentration of the drug at the primary site of infection. CRISPR-Cas technology is emerging as a transformative approach to disabling drug-resistant genes and enhancing diagnostic precision. HDTs, including agents like vitamin D and metformin, show potential in modulating host immune responses and enhancing pathogen clearance. Nanoparticle-based vaccines, including mRNA and antigen-conjugated platforms, aim to overcome the limitations of the BCG vaccine by enhancing antigen presentation and eliciting stronger, longer-lasting immunity. Collectively, these modalities mark a shift toward more personalized, effective, and less toxic TB therapies. However, challenges such as regulatory approval, safety, scalability, and accessibility remain. This review highlights the integrated potential of nanomedicine, gene editing, and immunomodulation to transform TB care and combat drug resistance, paving the way for more robust and durable treatment strategies.},
}
@article {pmid41304764,
year = {2025},
author = {Alidriss, OM and AlSudais, H and Alhumaidan, OS and Altwaijry, HD and Bakhsh, A and Almuhanna, Y and Alkudmani, ZS and Alqarni, IA and Alenazi, D and Aljasham, AT and Jamous, YF},
title = {Targeted Drug Delivery Strategies in Overcoming Antimicrobial Resistance: Advances and Future Directions.},
journal = {Pharmaceutics},
volume = {17},
number = {11},
pages = {},
doi = {10.3390/pharmaceutics17111426},
pmid = {41304764},
issn = {1999-4923},
abstract = {Antimicrobial resistance (AMR) is a present, pressing global public health crisis associated with rising morbidity and mortality rates due to previously curable infectious disease. Targeted drug delivery is an important approach to address AMR due to its ability to improve the therapeutic performance of antibiotics without leading to any adverse effects or organ toxicities. In this review we explore molecular mechanisms of AMR and drawbacks of conventional antibiotic therapies and discuss unique drug delivery approaches to compensate these. Nanoparticulate carrier systems, stimuli-responsive systems, antibody-drug conjugates, and CRISPR-Cas systems are some of the carrier method designs that are promising for tackling hard to treat infections related to pathogenic strains and biofilms due to their features. Many of these are among the most significant advances in the field. However, there are many challenges to be overcome, with biological limitations, scaling and regulatory challenges, etc., before they can be employed in commercial applications. Materials are being developed, and an approach standardized and applicable to future work is in development to improve the efficiency of targeted delivery systems. Controlled drug delivery, which could be the answer to an increasing AMR problem, will not only help in alerting awareness among individuals but will also help in prolonging the activity of antibiotics by providing synergistic interdisciplinary solutions. This review emphasizes the complementary role of targeted drug delivery in transitioning from laboratory investigations to clinical therapy. It addresses underrepresented aspects, including new materials, scalability, regulatory considerations, and ethical implications, while offering a roadmap for translating innovations into next-generation antimicrobials.},
}
@article {pmid41304743,
year = {2025},
author = {Zhang, H and Li, Y and Li, J and Li, X and Li, T},
title = {Base and Prime Editing for Inherited Retinal Diseases: Delivery Platforms, Safety, Efficacy, and Translational Perspectives.},
journal = {Pharmaceutics},
volume = {17},
number = {11},
pages = {},
doi = {10.3390/pharmaceutics17111405},
pmid = {41304743},
issn = {1999-4923},
support = {U22A20311//he National Natural Science Foundation of China/ ; 82571246//the National Natural Science Foundation of China/ ; 82388101//the National Natural Science Foundation of China/ ; 23J41900200//Science and Technology Commission of Shanghai Municipality/ ; SHWSRS(2025)_071//Shanghai "Rising Stars of Medical Talents" Youth Development Program/ ; },
abstract = {Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous spectrum of disorders that lead to progressive and irreversible vision loss. Gene therapy is the most promising emerging treatment for IRDs. While gene augmentation strategies have demonstrated clinical benefit and results within the first approved ocular gene therapy, their application is restricted by adeno-associated virus (AAV) packaging capacity and limited efficacy for dominant mutations. Recent breakthroughs in precision genome editing, particularly base editing (BE) and prime editing (PE), have provided alternatives capable of directly correcting pathogenic variants. BE enables targeted single-nucleotide conversions, whereas PE further allows for precise insertions and deletions, both circumventing the double-strand DNA cleavage or repair processes typically induced by conventional CRISPR-Cas editing systems, thereby offering advantages in post-mitotic retinal cells. Preclinical investigations across murine and non-human primate models have demonstrated the feasibility, molecular accuracy, and preliminary safety profiles of these platforms in targeting IRD-associated mutations. However, critical challenges remain before clinical application can be realized, including limited editing efficiency in photoreceptors, interspecies variability in therapeutic response, potential risks of off-target effects, and barriers in large-scale vector manufacturing. Moreover, the delivery of genome editors to the outer retina remains suboptimal, prompting intensive efforts in capsid engineering and the development of non-viral delivery systems. This review synthesizes the current progress in BE and PE optimization, highlights innovations in delivery platforms that encompass viral and emerging non-viral systems and summarizes the major barriers to clinical translation. We further discuss AI-driven strategies for the rational design of BE/PE systems, thereby outlining their future potential and perspectives in the treatment of IRDs.},
}
@article {pmid41303344,
year = {2025},
author = {Zhang, X and Che, J and Li, Z and Bao, B and Fan, C},
title = {The Mutation of piezo1 Weakens the Intermuscular Bones in Zebrafish and Crucian Carp.},
journal = {International journal of molecular sciences},
volume = {26},
number = {22},
pages = {},
doi = {10.3390/ijms262210851},
pmid = {41303344},
issn = {1422-0067},
support = {2023YFD2400300//National Key Research and Development Program of China/ ; 32170514//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Carps/genetics ; *Ion Channels/genetics/metabolism ; *Mutation ; *Zebrafish Proteins/genetics/metabolism ; *Bone and Bones/metabolism ; CRISPR-Cas Systems ; *Fish Proteins/genetics/metabolism ; },
abstract = {Intermuscular bones (IBs), unique skeletal features found only in teleost fishes, pose significant challenges to food processing and consumption. While recent studies have identified several key genetic regulators of IB development, the role of mechanosensory mechanisms remains largely unexplored. This study investigated the role of Piezo1, a critical mechanosensitive ion channel, in IB formation using zebrafish and crucian carp models. Our findings demonstrated that piezo1 was expressed in the myoseptum of zebrafish, and CRISPR/Cas9-mediated knockout of this gene resulted in shorter and smaller IBs. Similar knockout experiments in crucian carp confirmed the conserved role of Piezo1 across cyprinid species. These results established Piezo1 as a key regulator of IB development, providing new insights into the molecular mechanisms underlying this process and suggesting potential strategies for breeding IB-free fish strains through modulation of mechanosensory pathways.},
}
@article {pmid41302913,
year = {2025},
author = {Ayaz, S and Kong, WW and Wang, J and Liu, SH and Xu, JP},
title = {Host Immunity Mechanisms Against Bacterial and Viral Infections in Bombyx mori.},
journal = {Insects},
volume = {16},
number = {11},
pages = {},
doi = {10.3390/insects16111167},
pmid = {41302913},
issn = {2075-4450},
abstract = {The domesticated silkworm, Bombyx mori, is a highly valued biodiversity and economic asset, acclaimed for its silk production, besides making important contributions to various scientific disciplines. However, the sericulture industry faces ongoing threats from bacterial and viral infections, which severely impact silkworm health and silk yield. This review provides a comprehensive overview of the innate immune response of B. mori against bacterial and viral pathogens, emphasizing the fundamental molecular and cellular defense mechanisms. We explore the humoral and cellular immune response using antimicrobial peptides (AMPs), pattern recognition receptors (PRRs) like peptidoglycan recognition protein (PGRP), and glucan recognition protein (GRP), which activate canonical signaling pathways. The review further highlights the molecular mechanisms underlying the silkworm's defense against viruses, incorporating RNA interference (RNAi), apoptosis, and distinct signaling pathways such as Toll and Imd, JAK/STAT, and STING. We also discussed the viral suppression strategies and modulation of host metabolism during infection. Furthermore, the review explores the recent use of CRISPR-Cas gene editing to enhance disease resistance, presenting a promising avenue for mitigating pathogen-induced losses in sericulture. By elucidating these mechanisms, the work provides a synthesis that is critical in terms of developing particular interventions and developing more resistant silkworm strains to ensure that the industry of sericulture becomes viable and productive.},
}
@article {pmid41301838,
year = {2025},
author = {Dziedzic, A and Kubina, R and Skonieczna, M and Madej, M and Fiegler-Rudol, J and Abid, M and Nadhim, D and Tanasiewicz, M},
title = {CRISPR Genome Editing in Personalized Therapy for Oral and Maxillofacial Diseases: A Scoping Review.},
journal = {Biomedicines},
volume = {13},
number = {11},
pages = {},
doi = {10.3390/biomedicines13112745},
pmid = {41301838},
issn = {2227-9059},
abstract = {Background: CRISPR/Cas genome editing is emerging as a powerful tool in oral and maxillofacial medicine, with potential applications in personalized therapies for conditions that currently lack durable treatments. Objectives: This scoping review aimed to map existing evidence on CRISPR-based applications in oral and maxillofacial fields, rather than to assess treatment effectiveness. Methods: A systematic search of PubMed, Scopus, Web of Science, and ClinicalTrials.gov (2012-2024) identified studies and registered trials involving CRISPR with oral health relevance. Eligible articles included peer-reviewed experimental reports and clinical trials. Results: From 1437 records, 121 studies met inclusion criteria: 106 preclinical reports and 15 clinical or translational studies. Investigated domains included oral cancer therapy, hereditary craniofacial syndromes, regenerative strategies, infectious disease models, and pathogen detection. Early clinical efforts focus mainly on CRISPR-edited T-cell immunotherapies in oncology. Major barriers include off-target effects, delivery challenges, regulatory complexity, and ethical concerns. Conclusions: CRISPR-based bioengineering shows strong promise for precision care in oral and maxillofacial medicine. However, current evidence remains largely preclinical and heterogeneous. No clinical recommendations can yet be made, and translation will depend on rigorous late-phase trials, ethical oversight, and health-economic evaluation.},
}
@article {pmid41301629,
year = {2025},
author = {Wang, Y and Li, L and Liang, Y and Xu, K and Ye, Y and He, M},
title = {Phage Therapy for Acinetobacter baumannii Infections: A Review on Advances in Classification, Applications, and Translational Roadblocks.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {14},
number = {11},
pages = {},
doi = {10.3390/antibiotics14111134},
pmid = {41301629},
issn = {2079-6382},
support = {82302568//National Natural Science Foundation of China/ ; 2308085QH283//Anhui Provincial National Science Foundation/ ; 2022xkjT012//Basic and Clinical Collaboration Enhancement Program Foundation of Anhui Medical University/ ; },
abstract = {The global spread of carbapenem-resistant Acinetobacter baumannii (CRAB) poses a severe public health threat, driving growing interest in phage-based precision antibacterial strategies. This systematic review synthesizes recent advances in the field of A. baumannii phage. Modern taxonomy, based on whole-genome phylogeny, has reclassified the majority of A. baumannii phages into the class Caudoviricetes, revealing distinct evolutionary clades that correlate with host tropism and biological properties, superseding the traditional morphological families (Myoviridae, Siphoviridae, Podoviridae). To overcome limitations of natural phage therapy, such as narrow host range, cocktail therapies (ex vivo resistance mutation rates < 5%) and phage-antibiotic synergism (enabling antibiotic efficacy at 1/4 minimum inhibitory concentration) have significantly enhanced antibacterial efficacy. Preclinical models demonstrate that phage therapy efficiently clears pathogens in pneumonia models and promotes the healing of burn wounds and diabetic ulcers via immunomodulatory mechanisms. Technical optimizations include nebulized inhalation delivery achieving 42% alveolar deposition, and thermosensitive hydrogels enabling sustained release over 72 h. Genetic engineering approaches, such as host range expansion through tail fiber recombination and CRISPR/Cas-mediated elimination of lysogeny, show promise. However, the genetic stability of engineered phages requires further validation. Current challenges remain, including limited host spectrum, the absence of clinical translation standards, and lagging regulatory frameworks. Future efforts must integrate metagenomic mining and synthetic biology strategies to establish a precision medicine framework encompassing resistance monitoring and personalized phage formulation, offering innovative solutions against CRAB infections.},
}
@article {pmid41301547,
year = {2025},
author = {Mohammed, A and Ibrahim, NA and Basher, NS},
title = {Protein Engineering and Drug Discovery: Importance, Methodologies, Challenges, and Prospects.},
journal = {Biomolecules},
volume = {15},
number = {11},
pages = {},
doi = {10.3390/biom15111628},
pmid = {41301547},
issn = {2218-273X},
support = {IMSIU-DDRSP2501//Imam Mohammad ibn Saud Islamic University/ ; },
mesh = {*Drug Discovery/methods ; *Protein Engineering/methods ; Humans ; Animals ; Recombinant Proteins/therapeutic use/chemistry/genetics ; },
abstract = {Protein engineering is a rapidly evolving field that plays a critical role in transforming drug discovery and development. This innovative field harnesses the unique structural and functional properties of engineered proteins, such as monoclonal antibodies, nanobodies, therapeutic enzymes, and cytokines, to address complex diseases more effectively than traditional small-molecule drugs. These biologics not only enhance therapeutic specificity but also minimize adverse effects, marking a significant advancement in patient care. However, the journey of protein engineering is not without challenges. Issues related to protein folding, stability, and potential immunogenicity pose significant complications. Additionally, navigating the complex regulatory landscape can delay the transition from laboratory to clinical application. Addressing these hurdles requires the integration of cutting-edge technologies, including phage and yeast display technology, CRISPR, and advanced computational modeling, which enhance the predictability and efficiency of protein design. In this review, we explore the multifaceted impact of protein engineering on modern medicine, highlighting its potential to transform treatment paradigms, methodologies, challenges, and the successful development and approval of recombinant protein-based therapies. By navigating the complexities and leveraging technological advancements, the field is poised to unlock new therapeutic possibilities, ultimately improving patient outcomes and transforming healthcare.},
}
@article {pmid41301473,
year = {2025},
author = {Gibril, BAA and Chai, X and Xu, J},
title = {From Correlation to Causation: Defining Gene and RNA Function in Poultry Muscle Biology Using In Vivo Genetic Tools.},
journal = {Biomolecules},
volume = {15},
number = {11},
pages = {},
doi = {10.3390/biom15111554},
pmid = {41301473},
issn = {2218-273X},
support = {20242BCE50051//Science and Technology Research Project of the Education Department of Jiangxi Province/ ; },
mesh = {Animals ; *Muscle, Skeletal/metabolism ; Muscle Development/genetics ; *Poultry/genetics ; Muscular Diseases/genetics ; Transcriptome ; CRISPR-Cas Systems ; *RNA/genetics/metabolism ; },
abstract = {A central challenge in functional genomics is understanding the difference between correlative transcriptomic observations and definitive causal understanding of gene function in vivo. Poultry skeletal muscle, a system of significant agricultural and biological importance, demonstrates this challenge. While transcriptomic studies have cataloged extensive RNA expression dynamics during muscle development and in growth-related myopathies like wooden breast, establishing causative roles for these molecules is lacking. This review synthesizes how advanced genetic tools are now enabling a shift from correlation to causation in avian muscle biology. We detail how viral vectors (e.g., adenovirus, lentivirus, and RCAS) and CRISPR/Cas9 systems have provided direct in vivo validation of the functional roles of specific mRNAs, miRNAs, lncRNAs, and circRNAs in regulating myogenesis, hypertrophy, and atrophy. We contrast this success in fundamental biology with the study of myopathies, which remains largely descriptive. Here, a wealth of transcriptomic data has identified dysregulated pathways, including ECM remodeling, metabolism, and inflammation, but functional validation for most candidates is absent. We argue that the critical next step is to apply this established functional genomics toolkit to disease models. By defining causal mechanisms, this research will not only address a major agricultural issue but also provide a model for using genetic tools to dissect complex traits in a post-genomic era.},
}
@article {pmid41300819,
year = {2025},
author = {Kansal, R},
title = {Curing Sickle Cell Disease by Allogeneic Hematopoietic Stem Cell (HSC) Transplantation Toward In Vivo HSC Gene Therapy.},
journal = {Genes},
volume = {16},
number = {11},
pages = {},
doi = {10.3390/genes16111367},
pmid = {41300819},
issn = {2073-4425},
mesh = {*Anemia, Sickle Cell/therapy/genetics ; Humans ; *Hematopoietic Stem Cell Transplantation/methods ; *Genetic Therapy/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; Transplantation, Homologous ; Animals ; Hematopoietic Stem Cells/metabolism ; },
abstract = {Sickle cell disease comprises a group of prevalent inherited disorders defined by an underlying sickle cell allele that forms sickle hemoglobin. The incidence of this disease is rising, with more than 500,000 children born with it globally. The disease carries significant morbidity and mortality. Its only curative treatment was an allogeneic hematopoietic stem cell (HSC) transplant (HSCT) until late 2023, when two one-time gene therapies were approved for treating patients aged 12 years or older with severe sickle cell disease. This work aims to inform readers about these two gene therapies: one lentiviral-based and the other nonviral. The latter is based on the Nobel Prize-winning discovery of clustered, regularly interspaced, short, palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 proteins and single-guide RNA (sgRNA)-based genome editing. Both approved gene therapies require an autologous HSCT with ex vivo genetically edited autologous hematopoietic stem and progenitor cells. Therefore, access to these gene therapies is limited to specialized centers with expertise in HSCTs. This review is meant for students, researchers, and clinical practitioners. It explains the basis for both approved gene therapies, their mechanisms of action, differences, risks, and other lentiviral-based and CRISPR-Cas9-based ex vivo gene therapies for sickle cell disease in clinical development. Additionally, it discusses the current state of preclinical studies for in vivo HSC gene therapy for sickle cell disease, which utilize advanced genome editing technologies developed after CRISPR-Cas9-sgRNA-based genome editing. In vivo HSC gene therapy, after it is clinically developed, would eliminate the need for an HSCT in receiving gene therapy and vastly increase access for numerous patients worldwide, even in low-income countries with the most significant disease burden.},
}
@article {pmid41300715,
year = {2025},
author = {Sapakhova, Z and Kanat, R and Daurov, D and Daurova, A and Shamekova, M and Zhambakin, K},
title = {The Enhancement of Fungal Disease Resistance in Major Staple Crops Using CRISPR-Cas Technology.},
journal = {Genes},
volume = {16},
number = {11},
pages = {},
doi = {10.3390/genes16111263},
pmid = {41300715},
issn = {2073-4425},
support = {BR21882269//Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; *Crops, Agricultural/genetics/microbiology ; Gene Editing/methods ; Fungi/pathogenicity/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Fungal pathogens represent a major constraint to global agricultural productivity, causing a wide range of plant diseases that severely affect staple crops such as cereals, legumes, and vegetables. These infections result in substantial yield losses, deterioration of grain and produce quality, and significant economic impacts across the entire agri-food sector. Among phytopathogens, fungi are considered the most destructive, causing a wide range of diseases such as powdery mildew, rusts, fusarium head blight, smut, leaf spot, rots, late blight, and other fungal pathogens. Traditional plant protection methods do not always provide long-term effectiveness and environmental safety, which requires the introduction of innovative approaches to creating sustainable varieties. CRISPR-Cas technology opens up new opportunities for targeted genome editing, allowing the modification or silencing of susceptibility genes and thus increasing plant resistance to fungal infections. This review presents current achievements and prospects for the application of CRISPR-Cas technology to increase the resistance of major agricultural crops to fungal diseases. The implementation of these approaches contributes to the creation of highly productive and resistant varieties, which is crucial for ensuring food security in the context of climate change.},
}
@article {pmid41300710,
year = {2025},
author = {Iksat, N and Madirov, A and Zhanassova, K and Masalimov, Z},
title = {Artificial Intelligence-Assisted CRISPR/Cas Systems for Targeting Plant Viruses.},
journal = {Genes},
volume = {16},
number = {11},
pages = {},
doi = {10.3390/genes16111258},
pmid = {41300710},
issn = {2073-4425},
support = {grant No. BR21882269//the Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Artificial Intelligence ; *Plant Viruses/genetics ; *Gene Editing/methods ; *Plant Diseases/virology/genetics ; Crops, Agricultural/genetics/virology ; },
abstract = {Plant viral infections continue to pose a significant and ongoing threat to global food security, especially in the context of climatic instability and intensive agricultural practices. The CRISPR/Cas system has emerged as a powerful tool for developing virus-resistant crops by enabling precise modifications to viral genomes or plant susceptibility factors. Nonetheless, the efficacy and dependability of CRISPR-based antiviral approaches are limited by challenges in guide RNA design, off-target effects, insufficiently annotated datasets, and the intricate biological dynamics of plant-virus interactions. This paper summarizes the latest advancements in the incorporation of artificial intelligence (AI) methodologies, including machine learning and deep learning algorithms, into the CRISPR design and optimization framework. It examines how convolutional and recurrent neural networks, transformer architectures, and generative models like AlphaFold2, RoseTTAFold, and ESMFold can be used to predict protein structures, score sgRNAs, and model host-virus interactions. AI-enhanced methods have been proven to improve target specificity, Cas protein performance, and in silico validation. This paper aims to establish a foundation for next-generation genome editing strategies against plant viruses and promote the adoption of AI-powered CRISPR technologies in sustainable agriculture.},
}
@article {pmid41299842,
year = {2025},
author = {Shi, C and Yu, Z and Tan, H and Li, W and Wang, Y and Wang, Y and Zhang, Q and Man, Y},
title = {One-Pot CRISPR-Based Isothermal Amplification for Nucleic Acid Detection: A Comparative Review of Different Strategies.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c00806},
pmid = {41299842},
issn = {2379-3694},
abstract = {Nucleic acid detection plays an important role in pathogen monitoring and disease diagnosis. CRISPR one-pot assays combined with isothermal amplification are emerging as promising point-of-care technologies that simplify workflows while increasing sensitivity and specificity. However, the incompatibility inherent in the one-pot reaction of isothermal amplification and CRISPR detection limits their practical application. This review comprehensively analyzes diverse advanced one-pot CRISPR-based isothermal amplification strategies developed to overcome this fundamental challenge. These strategies primarily encompass physical separation strategies (utilizing lid-bottom, internal ledge, nested tube, and membrane approaches), phase separation strategies (employing glycerol, sucrose, and gel matrices), reaction system optimization strategies (fine-tuning reaction parameters and incorporating specialized additives), non-PAM and suboptimal PAM strategies, improved Cas enzyme strategies (enhanced Cas12 and Cas13 variants), light-controlled approaches (PC-oligonucleotides, NPOM-dt modification, and acylation modification), and microfluidic chip integration strategies (centrifugal microfluidic chips, droplet microfluidic chips, and microarray chips). These methodological approaches have achieved important advances in simplifying operational processes, enhancing sensitivity, shortening detection cycles, and minimizing cross-contamination risks. The review further synthesizes critical insights regarding current opportunities, technical challenges, and future directions for one-pot CRISPR-based isothermal amplification technologies in nucleic acid detection, providing valuable guidance for researchers and practitioners in this evolving field.},
}
@article {pmid41298934,
year = {2025},
author = {Lee, SY and Birkholz, N and Lee, JH and Fineran, PC and Park, HH},
title = {Regulation of anti-CRISPR operons by structurally distinct families of Aca proteins.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1698},
pmid = {41298934},
issn = {2399-3642},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; PE25150//Korea Polar Research Institute (KOPRI)/ ; },
mesh = {*Operon ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Bacterial ; *Viral Proteins/genetics/metabolism/chemistry ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Protein Binding ; Bacteriophages/genetics ; Crystallography, X-Ray ; Models, Molecular ; },
abstract = {CRISPR-Cas systems provide bacteria with adaptive immunity against bacteriophages and mobile genetic elements, driving an evolutionary arms race in which phages deploy anti-CRISPR (Acr) proteins. Acr proteins are often co-encoded in operons with anti-CRISPR-associated (Aca) proteins, which coordinate the regulation of acr gene expression. Here, we reveal the molecular basis of DNA binding that mediates transcriptional repression by two distinct Aca family members: Aca7 and Aca11. Crystal structures of Aca7 and Aca11 highlight conserved helix-turn-helix (HTH) motifs within α-helix bundles, providing a universal DNA-binding platform. Aca7 forms a symmetrical dimer to recognize a 19-bp inverted repeat (IR) within the acrIF11-aca7 operon. Strikingly, Aca11 binds 22-bp IRs in two distinct promoters, suggesting that Aca proteins can control multiple target operons. Mutagenesis and electrophoretic mobility shift assays (EMSAs) confirm that dimerization and sequence-specific IR recognition are essential for DNA binding. Despite mechanistic similarities, these and other Aca proteins exhibit notable differences. Structural comparisons across Aca families reveal that while monomer structures are generally similar with conserved HTH motifs, the structures of their dimeric functional units vary significantly. These structural differences might be essential for Aca proteins to bind to various promoters and regulate the expression of different Acr proteins.},
}
@article {pmid41298570,
year = {2025},
author = {Kiseleva, AA and Timonova, EM and Berezhnaya, AA and Kolozhvari, AE and Kochetov, AV and Salina, EA},
title = {Fine tuning wheat heading time through genome editing of transcription factor binding sites in Ppd-1 gene promoter.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {42034},
pmid = {41298570},
issn = {2045-2322},
support = {FWNR&#x2011;2024&#x2011;0009//The Ministry of Science and Higher Education of the Russian Federation/ ; 075&#x2011;15&#x2011;2025&#x2011;51//The Kurchatov Genomic Center of ICG SB RAS/ ; },
mesh = {*Triticum/genetics/growth &amp; development ; *Promoter Regions, Genetic ; *Gene Editing/methods ; *Transcription Factors/metabolism/genetics ; Binding Sites ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; Photoperiod ; },
abstract = {Increasing the productivity and adaptability of agricultural plants often depends on optimizing heading time. To develop common wheat lines with accelerated heading and investigate its regulation, we targeted the PPD-1 genes, which control photoperiod sensitivity. Large deletions in the promoter regions of these genes are known to disrupt their expression, resulting in early heading. Using CRISPR/Cas9 genome editing, we generated wheat plants with mutations in the promoter regions of the Ppd-D1 and Ppd-B1 genes. These mutations included nucleotide substitutions, deletions, and insertions ranging from several to hundreds of base pairs, occurring within probable transcription factor binding sites, that may influence gene expression. Under short-day conditions, we assessed PPD-1 gene expression in T0 plants and T2 lines with different mutations. Our analysis revealed that deletions spanning the CHE transcription repressor binding sites altered gene expression patterns, supporting the hypothesis regarding the role of these cis-elements in regulating PPD-1 expression. Furthermore, plants with different mutations displayed distinct diurnal expression patterns, suggesting the involvement of additional transcription factors in the regulation of this gene. Evaluation of heading time in T1 and T2 families with different mutations demonstrated that plants with mutations affecting the "core region", including the CHE binding sites, initiated heading significantly earlier than those without mutations.},
}
@article {pmid41296365,
year = {2025},
author = {Chen, W and Fan, L and Dong, M and Gao, L and Du, X and Wang, L and Su, S and Liu, LE and Wu, Y and Ding, L},
title = {Emerging CRISPR/Cas-Based Strategies for Extracellular Vesicle Detection: A Comprehensive Review.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c02441},
pmid = {41296365},
issn = {2379-3694},
abstract = {Extracellular vesicles (EVs), which carry a variety of molecules such as proteins and nucleic acids, have great potential for broad application in liquid biopsy. However, achieving highly sensitive detection of biomarkers within EVs remains a significant challenge. The emergence of CRISPR/Cas systems─adaptive immune mechanisms found in bacteria and archaea that defend against foreign genetic elements─offers new opportunities to address this issue through powerful nucleic acid recognition and cleavage capabilities. Compared to other EV detection techniques, CRISPR/Cas-based biosensors exhibit superior sensitivity, specificity, and operational efficiency, making them a compelling platform for clinical translation. Thus, to promote the application of EVs in disease diagnosis, disease monitoring, and therapeutic evaluation, this review focuses on the state-of-the-art CRISPR/Cas systems (specifically CRISPR/Cas9, CRISPR/Cas12, CRISPR/Cas13, and CRISPR/Cas14) as well as the latest applications of CRISPR/Cas-based EV detection techniques.},
}
@article {pmid41294842,
year = {2025},
author = {Nagaoka, K and Kobayashi, Y and Kakimi, K},
title = {NeoPAIR-T: Functional Mapping of Neoantigen-TCR Pairs Using a CRISPR-Engineered Jurkat Reporter System.},
journal = {Cells},
volume = {14},
number = {22},
pages = {},
pmid = {41294842},
issn = {2073-4409},
support = {24ama221321h0002//Japan Agency for Medical Research and Development (AMED)/ ; 23H02762//JSPS KAKENHI/ ; },
mesh = {Humans ; Jurkat Cells ; *Receptors, Antigen, T-Cell/metabolism/genetics ; *Antigens, Neoplasm/immunology/genetics/metabolism ; Genes, Reporter ; *CRISPR-Cas Systems/genetics ; Coculture Techniques ; Antigen-Presenting Cells/immunology/metabolism ; },
abstract = {Targeting mutation-derived neoantigens is a promising strategy for personalized immunotherapies. However, identifying true neoantigens and cognate T cell receptors (TCRs) remains challenging because computational prediction of neoantigen peptides is uncertain and most tumor-infiltrating lymphocytes are bystanders rather than tumor-reactive, necessitating functional validation. Here, we developed NeoPAIR-T (Neoantigen-TCR Pairing Assay using reporter T cells), a functional assay based on co-culture of TCR-T reporter cells and autologous antigen-presenting cells (APCs) to screen neoantigen-TCR pairs. Reporter T cells are Jurkat-derived cells engineered to express a luciferase/eGFP dual reporter, providing quantitative readouts of TCR activation, while APCs are immortalized autologous cells transfected with tandem minigenes (TMGs) encoding predicted neoantigens, bypassing peptide synthesis. NeoPAIR-T also includes TCRα-knockout with targeted knock-in of candidate TCRs at the TCRβ locus to prevent mispairing and enables parallel testing of multiple reporter T cell clones co-cultured with the same APCs for efficient identification of functional pairs. Using lung cancer samples, whole-exome and RNA sequencing predicted 63 candidate peptides assembled into three TMGs. Single-cell RNA/TCR sequencing identified eight TCR clonotypes, introduced into reporter T cells and tested in parallel. Co-culture with TMG-expressing APCs revealed two functional neoantigen-TCR pairs validated by peptide assays (EC50: 10[-9.2]-10[-6.7] M). Collectively, NeoPAIR-T streamlines neoantigen-TCR identification for vaccine and TCR-T applications.},
}
@article {pmid41294822,
year = {2025},
author = {Bucheeri, S and Alcibahy, Y and Bucheeri, Y and Bucheeri, S and Alhermi, A and Butler, AE},
title = {CRISPR as a Tool to Uncover Gene Function in Polycystic Ovary Syndrome: A Literature Review of Experimental Models Targeting Ovarian and Metabolic Genes.},
journal = {Cells},
volume = {14},
number = {22},
pages = {},
pmid = {41294822},
issn = {2073-4409},
mesh = {*Polycystic Ovary Syndrome/genetics/metabolism ; Animals ; Female ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Disease Models, Animal ; *Ovary/metabolism/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Polycystic ovary syndrome (PCOS) is a complex disorder characterized by reproductive abnormalities such as hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology, and is frequently accompanied by metabolic disturbances such as insulin resistance, obesity and dyslipidemia. Genome-wide association studies (GWASs) have identified several susceptibility loci, yet little is known about their functional implications. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has emerged as a powerful gene editing tool in bridging this gap by allowing researchers to directly target candidate genes in ovarian and metabolic pathways. For instance, experimental models have highlighted the role of CYP17A1 and DENND1A.V2 in androgen excess, anti-Müllerian hormone (AMH) in follicular arrest, and insulin receptor substrate 1 (IRS1) and PPARγ in insulin signaling and adipogenesis. To highlight the multifactorial nature of PCOS, animal models, including zebrafish and rodents, have been used to reveal interactions between reproductive and metabolic phenotypes. Nevertheless, most studies remain restricted to single-gene models, and dual-gene models or combined gene editing and hormonal induction models remain underexplored. Future research integrating precision editing, multi-omic platforms, and patient-derived organoids may provide more accurate disease models and novel therapeutic strategies.},
}
@article {pmid41294729,
year = {2025},
author = {Guo, W and Jiang, M and Xie, Y and Xu, H and Sun, Z},
title = {Recognition Element-Based Strategies for Rapid Detection of Foodborne Pathogens: Recent Progress and Perspectives.},
journal = {Biosensors},
volume = {15},
number = {11},
pages = {},
pmid = {41294729},
issn = {2079-6374},
support = {BK20241924//Natural Science Foundation of Jiangsu Province/ ; SH2024010//Key Research and Development Program of Zhenjiang City/ ; SH2024112//Key Research and Development Program of Zhenjiang City/ ; NY2023002//Key Research and Development Program of Zhenjiang City/ ; },
mesh = {*Biosensing Techniques/methods ; *Food Microbiology ; Humans ; *Foodborne Diseases/microbiology/diagnosis ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; Molecular Imprinting ; },
abstract = {The detection of foodborne pathogens is of great significance for safeguarding food safety and public health. In recent years, rapid detection technologies based on diverse recognition elements have advanced considerably, driven by progress in molecular biology, materials science, and information technology. This review takes recognition elements as the central theme and systematically outlines the mechanisms and research progress of antibodies, nucleic acid aptamers, nucleic acid amplification techniques, CRISPR/Cas systems, molecular imprinting technology, peptides, and small-molecule receptors in foodborne pathogen detection, while comparing their performance in terms of specificity, sensitivity, stability, and applicability. In addition, this review further elaborates on the developmental trends of detection platforms, including multi-target and multimodal integration, microfluidics combined with portable point-of-care testing (POCT) systems, and intelligent terminals empowered by artificial intelligence algorithms. These trends provide new perspectives for improving detection systems in terms of throughput, portability, and intelligence. Overall, this review aims to serve as a comprehensive reference for the development of rapid, accurate, and intelligent detection systems for foodborne pathogens.},
}
@article {pmid41293956,
year = {2025},
author = {Zang, J and Niklaus, S and Neuhauss, SCF},
title = {An EAAT2b/SLC1A2b-mediated chloride leak current enables rapid cone photoreceptor signalling.},
journal = {Open biology},
volume = {15},
number = {11},
pages = {250347},
pmid = {41293956},
issn = {2046-2441},
support = {/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {Animals ; *Retinal Cone Photoreceptor Cells/metabolism ; Zebrafish/genetics/metabolism ; *Excitatory Amino Acid Transporter 2/metabolism/genetics ; *Signal Transduction ; Electroretinography ; *Chlorides/metabolism ; *Zebrafish Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Gene Editing ; },
abstract = {Excitatory amino acid transporters not only mediate high-affinity glutamate uptake but also conduct an uncoupled chloride current. In zebrafish, a whole-genome duplication gave rise to two eaat2 paralogues with distinct roles. Excitatory amino acid transporter 2a (SLC1A2b, GLT-1) functions primarily in Müller glia as a glutamate transporter, whereas excitatory amino acid transporter 2b is expressed in cone photoreceptors and exhibits a prominent glutamate-independent chloride current. We hypothesized that this leak current stabilizes the cone resting membrane potential, thereby supporting rapid visual signalling. In order to test this hypothesis, we generated eaat2b knockout zebrafish using CRISPR-Cas9-mediated genome editing. While eaat2b mutants showed no gross morphological abnormalities, they exhibited reduced electroretinogram b-wave amplitudes. Consistent with our hypothesis, eaat2b-deficient larvae displayed a significant reduction in flicker fusion electroretinogram power at each stimulus frequency, indicating impaired temporal processing likely due to delayed repolarization of cone photoreceptors. Our findings reveal a critical role for an excitatory amino acid transporter 2b-mediated chloride anion leak current in regulating the kinetics of photoreceptor responses. This functional innovation, enabled by a whole-genome duplication in the teleost lineage, highlights how gene duplications can lead to the acquisition of physiologically relevant new functions.},
}
@article {pmid41292787,
year = {2025},
author = {Majumder, P and Cahir, CW and Roberts, CG and Patel, DJ and Marraffini, LA},
title = {Cap1 forms a cyclic tetra-adenylate-induced membrane pore during the type III-A CRISPR-Cas immune response.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.11.13.688252},
pmid = {41292787},
issn = {2692-8205},
abstract = {During type III CRISPR-Cas immunity in prokaryotes, RNA-guided recognition of viral (phage) transcripts stimulates the Cas10 complex to convert ATP into cyclic oligoadenylates. These act as signaling molecules that bind to CARF proteins and activate their effector domains. Here, we report the structure and function of the Cap1 effector, composed of a pair of transmembrane helices (TM1/2), a CARF-like (CARFL) domain and a domain of unknown function (DUF4579). Cryo-EM studies on apo- and ligand-bound states of Cap1 in glyco-diosgenin detergent revealed the formation of tetrameric complexes in both states, with one cyclic tetra-adenylate molecule bound in a pocket composed by the four CARFL domains. Binding of cA 4 triggers conformational changes that widen an otherwise narrow pore formed by the four TM1/2 domains. In vivo , Cap1 activation results in membrane depolarization, a growth arrest of the bacterial host and the abrogation of the viral lytic cycle. Our findings reveal the mechanistic basis of membrane depolarizarion mediated by cyclic nucleotide signaling during the type III CRISPR-Cas response.},
}
@article {pmid41291345,
year = {2025},
author = {Wang, Z and Zhang, Y and Xu, N and Liu, L and Zhang, M and Huang, S and Su, C and Liu, T and Duan, K},
title = {Improving soybean fatty acid profiles by CRISPR/Cas12a-mediated gene editing of GmFAD2 and GmFAD3.},
journal = {Plant cell reports},
volume = {44},
number = {12},
pages = {282},
pmid = {41291345},
issn = {1432-203X},
support = {32172499//National Natural Science Foundation of China/ ; JSSCTD202342//Shuangchuang Program of Jiangsu Province/ ; },
mesh = {*Glycine max/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Fatty Acids/metabolism ; Soybean Oil/metabolism ; Plants, Genetically Modified ; Linoleic Acid/metabolism ; *Plant Proteins/genetics/metabolism ; Oleic Acid/metabolism ; Fatty Acid Desaturases/genetics/metabolism ; },
abstract = {Using CRISPR/Cas12a, we engineered novel soybean germplasms by knocking out GmFAD2 (GmFAD2-1A, GmFAD2-1B) and GmFAD3 (GmFAD3A, GmFAD3B) genes, yielding elevated oleic or linoleic acid content. Soybean oil contains high levels of polyunsaturated fatty acids (PUFAs), which are known to reduce cholesterol levels and help prevent hypertension, thereby contributing significantly to human health. However, the chemical instability of PUFAs makes them susceptible to oxidation, a process that generates harmful trans-fatty acids. To address this issue, precise modulation of fatty acid composition in soybeans becomes critically important for health applications. In this study, we employed CRISPR/Cas12a gene editing technology to selectively knock out the GmFAD2 (GmFAD2-1A, GmFAD2-1B) and GmFAD3 (GmFAD3A, GmFAD3B) genes in soybean. This approach successfully created novel soybean germplasms with distinct fatty acid profiles: one with elevated oleic acid content and another with increased linoleic acid levels. These engineered variants provide valuable options for utilizing soybean oil with optimized fatty acid compositions tailored for specific health and nutritional purposes.},
}
@article {pmid41291135,
year = {2025},
author = {Hann, E and Majumdar, D and Layton, D and Fareh, M and Cahill, DM and Ziemann, M and Ujvari, B and Schat, KA and Challagulla, A},
title = {Systematic evaluation of CrRNA design parameters for optimized Cas13d-mediated RNA targeting in chicken cells.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {256},
pmid = {41291135},
issn = {1438-7948},
mesh = {Animals ; Chickens/genetics ; *CRISPR-Cas Systems ; Cell Line ; RNA, Messenger/genetics ; },
abstract = {The CRISPR-Cas13 system has emerged as a powerful platform for programmable RNA targeting, offering efficient and sequence-specific silencing of coding and non-coding transcripts. The RNA-targeting capabilities of CRISPR-Cas13 have been harnessed to silence transcripts harbouring pathogenic mutations and combat infectious diseases. However, the molecular basis of on-target and collateral activity are not completely understood, limiting the utility of Cas13 systems. In this study, we delineate the principles for the development of effective crRNAs by targeting DsRed fluorescence reporter and synthetic influenza mRNA in chicken fibroblast DF1 cells. To systematically determine the optimal design for RfxCas13d crRNA, we investigated the minimum length of the crRNA, importance of protospacer flanking sequence, degree of mismatch tolerance, and off target effects. Our data reveal variable knockdown levels between crRNAs, in which several crRNAs achieved over 95% target knockdown. We show that crRNAs exhibit a high degree of tolerance to single-nucleotide mismatches, regardless of their position in the spacer sequence. However, 4-nt mismatches between the spacer and the target significantly reduces targeting efficacy, whereas eight nucleotide mismatches completely abolish the activity of RfxCas13d. Finally, we compared targeting efficiency and collateral activity of two widely used RfxCas13d and HfCas13d variants. Our data extend current understanding of Cas13d-mediated RNA targeting and offer a framework for rational crRNA design to enhance effectiveness in diverse applications, including antiviral strategies.},
}
@article {pmid41290964,
year = {2025},
author = {El Menofy, NG and Payoumi, AN and Eissa, MA and El-Sharif, A},
title = {Association of the existence of CRISPR-Cas system and antimicrobial resistance in multi-drug resistant Klebsiella pneumoniae in Egypt.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {41814},
pmid = {41290964},
issn = {2045-2322},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification ; Egypt ; *Drug Resistance, Multiple, Bacterial/genetics ; *CRISPR-Cas Systems/genetics ; *Anti-Bacterial Agents/pharmacology ; Humans ; Microbial Sensitivity Tests ; *Klebsiella Infections/microbiology/drug therapy/epidemiology ; beta-Lactamases/genetics ; Colistin/pharmacology ; Bacterial Proteins/genetics ; },
abstract = {The CRISPR-Cas systems are supposed to be associated with antibiotic susceptibility. Klebsiella pneumoniae (K. pneumoniae) is a major multidrug-resistant (MDR) pathogen that may cause severe infections. This study aimed to detect the antimicrobial resistance (AMR) of K. pneumoniae isolates in addition to determine the association between the existence of CRISPR-Cas systems and the presence of AMR in Egypt. The antibiotic susceptibility patterns of 100 K. pneumoniae isolates were determined using the Kirby Bauer disc diffusion and broth microdilution methods. The frequency of carbapenem resistance encoding genes (blaKPC, blaOXA, blaIMP, blaNDM, and blaVIM), ESBLs encoding genes (blaTEM), aminoglycoside resistance encoding genes (aac(3)-Ia, aac(3)-IIa, colistin resistance encoding genes (mcr-1, mcr-2) and tetracycline resistance encoding genes (tetB) were determined using polymerase chain reaction (PCR). The presence or absence of CRISPR-Cas systems was determined by detection of Cas genes (Cas1 or Cas3) in conjunction with one of CRISPR arrays 1, 2 or 3. Kirby Bauer disc diffusion revealed that 95% of isolates were MDR. The resistance rates of K. pneumoniae isolates to amikacin, meropenem, and colistin were 76%, 67%, and 41% respectively by broth microdilution assay. Among selected 41 K. pneumoniae, the frequency of ESBLs; blaTEM was 92.7%, while the frequency of blaNDM blaOXA blaVIM blaIMP and blaKPC was 95.1%, 95.1%, 39%, 19.5% and 14.6% respectively. The frequency of mcr-1 and mcr-2 was 70.7% and 65.9%. Additionally, the frequency of aac(3)-Ia was 12.2%, and aac(3)-IIa was 87.8%, while the frequency of tetB was 100%. Our isolates exhibited varied profiles for CRISPR-Cas systems, where 65.9% were positive for CRISPR-Cas system. No general significant positive correlation between AMR and the presence ofCRISPR-Cas system was detected; however, a significant difference is present for imipenem, colistin and chloramphenicol phenotypic resistance andfor aac(3)-IIa and mcr-1 genes (P value > 0.1). A significant positive correlation was detected between AMR for imipenem, colistin and chloramphenicol and for aac(3)-IIa and mcr-1 genes and thepresence of CRISPR-Cas system.},
}
@article {pmid41230612,
year = {2025},
author = {Hao, J and Gong, X and Duan, X and Qin, W and Ren, H and ShenTu, X and Ye, Z and Yu, X},
title = {Ultrasensitive electrochemical detection of parvovirus B19 DNA by combining CRISPR-Cas12a and multivalent framework nucleic acids.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {46},
pages = {9357-9364},
doi = {10.1039/d5ay01285f},
pmid = {41230612},
issn = {1759-9679},
mesh = {*Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Parvovirus B19, Human/genetics/isolation &amp; purification ; *DNA, Viral/analysis/genetics ; *Biosensing Techniques/methods ; Humans ; Limit of Detection ; CRISPR-Associated Proteins ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; },
abstract = {The rapid and ultrasensitive detection of parvovirus B19 (B19V) DNA is critical for preventing severe complications in high-risk populations, such as fetal hydrops in pregnant women and aplastic crisis in immunocompromised patients. The absence of clinically approved vaccines or antivirals against B19V thus mandates the urgent development of accessible in vitro diagnostics to enable time-critical interventions and contain community transmission. Herein, we developed an electrochemical biosensor as a proof-of-concept for B19V by integrating CRISPR-Cas12a with multivalent framework nucleic acids (FNAs), namely, 12 nm tetrahedral DNA nanostructures (TDNs). Target B19V DNA activates Cas12a to indiscriminately cleave the biotin-modified ssDNA protruding from the four vertices of the TDNs, while the TDNs precisely orient ssDNA probes on electrodes, minimizing nonspecific adsorption. This method takes advantage of the target-specific cleavage ability of CRISPR-Cas12a (Cas12a-crRNA complex, 10-12 nm) and the unique structural and functional features of 12 nm TDNs. The comparable dimensions of the Cas12a-crRNA complex and the TDN suggest a potential synergistic effect, which contributes to the observed signal amplification and high detection sensitivity. The developed platform is user-friendly, has a low detection limit (2.19 fM), and shows high selectivity. This work establishes a foundational biosensing platform, demonstrating potential for ultrasensitive nucleic acid detection. By combining the accuracy of CRISPR-Cas12a and the benefits of FNAs, this method provides a more efficient, amplification-free, and reliable approach that holds promise for future development in point-of-care diagnostics and other applications.},
}
@article {pmid41038164,
year = {2025},
author = {Cheng, Y and Dang, S and Zhang, Y and Chen, Y and Yu, R and Liu, M and Jin, S and Han, A and Katz, S and Wang, S},
title = {Sequencing-free whole-genome spatial transcriptomics at single-molecule resolution.},
journal = {Cell},
volume = {188},
number = {24},
pages = {6953-6970.e12},
doi = {10.1016/j.cell.2025.09.006},
pmid = {41038164},
issn = {1097-4172},
mesh = {Animals ; Humans ; Mice ; In Situ Hybridization, Fluorescence/methods ; *Transcriptome/genetics ; *Gene Expression Profiling/methods ; Single-Cell Analysis/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Single Molecule Imaging/methods ; Genome ; CRISPR-Cas Systems ; },
abstract = {Recent breakthroughs in spatial transcriptomics technologies have enhanced our understanding of diverse cellular identities, spatial organizations, and functions. Yet existing spatial transcriptomics tools are still limited in either transcriptomic coverage or spatial resolution, hindering unbiased, hypothesis-free transcriptomic analyses at high spatial resolution. Here, we develop reverse-padlock amplicon-encoding fluorescence in situ hybridization (RAEFISH), an image-based spatial transcriptomics method with whole-genome coverage and single-molecule resolution in intact tissues. We demonstrate the spatial profiling of transcripts from 23,000 human or 22,000 mouse genes in single cells and tissue sections. Our analyses reveal transcript-specific subcellular localization, cell-type-specific and cell-type-invariant zonation-dependent transcriptomes, and gene programs underlying preferential cell-cell interactions. Finally, we further develop our technology for the direct spatial readout of guide RNAs (gRNAs) in an image-based, high-content CRISPR screen. Overall, these developments offer a broadly applicable technology that enables high-coverage, high-resolution spatial profiling of both long and short, native and engineered RNAs in many biomedical contexts.},
}
@article {pmid41034579,
year = {2025},
author = {Sullivan, AE and Nabhani, A and Izrailevsky, DS and Schinkel, K and Hoffman, CRK and Robbins, LK and Nagy, TA and Duncan, ML and Ledvina, HE and Erbse, AH and Kibby, EM and Tak, U and Dinh, DM and Ednacot, EMQ and Nguyen, CM and Burroughs, AM and Aravind, L and Whiteley, AT and Morehouse, BR},
title = {The Panoptes system uses decoy cyclic nucleotides to defend against phage.},
journal = {Nature},
volume = {647},
number = {8091},
pages = {988-996},
pmid = {41034579},
issn = {1476-4687},
mesh = {*Nucleotides, Cyclic/metabolism ; Models, Molecular ; Dinucleoside Phosphates/metabolism ; Crystallography, X-Ray ; *Bacteriophages/physiology/immunology ; *Second Messenger Systems ; *Operon/genetics ; CRISPR-Cas Systems/genetics ; Protein Domains ; Viral Proteins/metabolism/chemistry ; },
abstract = {Bacteria combat phage infection using antiphage systems and many systems generate nucleotide-derived second messengers upon infection that activate effector proteins to mediate immunity[1]. Phages respond with counter-defences that deplete these second messengers, leading to an escalating arms race with the host. Here we outline an antiphage system we call Panoptes that indirectly detects phage infection when phage proteins antagonize the nucleotide-derived second-messenger pool. Panoptes is a two-gene operon, optSE, wherein OptS is predicted to synthesize a nucleotide-derived second messenger and OptE is predicted to bind that signal and drive effector-mediated defence. Crystal structures show that OptS is a minimal CRISPR polymerase (mCpol) domain, a version of the polymerase domain found in type III CRISPR systems (Cas10). OptS orthologues from two distinct Panoptes systems generated cyclic dinucleotide products, including 2',3'-cyclic diadenosine monophosphate (2',3'-c-di-AMP), which we showed were able to bind the soluble domain of the OptE transmembrane effector. Panoptes potently restricted phage replication, but phages that had loss-of-function mutations in anti-cyclic oligonucleotide-based antiphage signalling system (CBASS) protein 2 (Acb2) escaped defence. These findings were unexpected because Acb2 is a nucleotide 'sponge' that antagonizes second-messenger signalling. Our data support the idea that cyclic nucleotide sequestration by Acb2 releases OptE toxicity, thereby initiating inner membrane disruption, leading to phage defence. These data demonstrate a sophisticated immune strategy that bacteria use to guard their second-messenger pool and turn immune evasion against the virus.},
}
@article {pmid41034576,
year = {2025},
author = {Doherty, EE and Adler, BA and Yoon, PH and Hsieh, K and Loi, K and Armbruster, EG and Lahiri, A and Bolling, CS and Wilcox, XE and Akkati, A and Iavarone, AT and Pogliano, J and Doudna, JA},
title = {A miniature CRISPR-Cas10 enzyme confers immunity by inhibitory signalling.},
journal = {Nature},
volume = {647},
number = {8091},
pages = {997-1004},
pmid = {41034576},
issn = {1476-4687},
mesh = {Humans ; *CRISPR-Cas Systems/genetics/immunology ; *Signal Transduction/immunology ; Oligonucleotides/biosynthesis/immunology ; Dinucleoside Phosphates/metabolism ; Membrane Proteins/metabolism/chemistry ; Immune Evasion ; Cell Death ; Adenylyl Cyclases/metabolism/chemistry ; HEK293 Cells ; Models, Molecular ; },
abstract = {Microbial and viral co-evolution has created immunity mechanisms involving oligonucleotide signalling that share mechanistic features with human antiviral systems[1]. In these pathways, including cyclic oligonucleotide-based antiphage signalling systems (CBASSs) and type III CRISPR systems in bacteria and cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) in humans, oligonucleotide synthesis occurs upon detection of virus or foreign genetic material in the cell, triggering the antiviral response[2-4]. Here, in an unexpected inversion of this process, we show that the CRISPR-related enzyme mCpol synthesizes cyclic oligonucleotides constitutively as part of an active mechanism that represses a toxic effector. Cell-based experiments demonstrated that the absence or loss of mCpol-produced cyclic oligonucleotides triggers cell death, preventing the spread of viruses that attempt immune evasion by depleting host cyclic nucleotides. Structural and mechanistic investigation revealed mCpol to be a di-adenylate cyclase whose product, c-di-AMP, prevents toxic oligomerization of the effector protein 2TMβ. Analysis of cells by fluorescence microscopy showed that lack of mCpol allows 2TMβ-mediated cell death due to inner membrane collapse. These findings unveil a powerful defence strategy against virus-mediated immune suppression, expanding our understanding of the role of oligonucleotides in immunity.},
}
@article {pmid41005744,
year = {2025},
author = {Li, E and Wen, L and Yin, C and Wang, N and Yang, S and Feng, W and Chen, M},
title = {Copper ionophore-autophagy interference nanoregulators for tumor self-defense reprograming to amplify cuproptotic stress and antitumor immunity.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {388},
number = {Pt 1},
pages = {114262},
doi = {10.1016/j.jconrel.2025.114262},
pmid = {41005744},
issn = {1873-4995},
mesh = {Humans ; *Copper/administration &amp; dosage/chemistry ; Autophagy/drug effects ; Animals ; Autophagy-Related Protein 5/genetics ; *Neoplasms/immunology/drug therapy ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Naphthoquinones/administration &amp; dosage/chemistry ; *Antineoplastic Agents/administration &amp; dosage ; Hyaluronan Receptors/metabolism ; Polyethyleneimine/chemistry/administration &amp; dosage ; Female ; Mice ; },
abstract = {Cuproptosis as a copper-dependent cell death modality driven by pathological aggregation of lipoylated proteins and proteotoxic stress resulting from destabilization of iron-sulfur (FeS) cluster proteins, represents a promising therapeutic strategy for cancer. However, the therapeutic efficacy of cuproptosis can be compromised by the intrinsic compensatory mechanisms within cancers, particularly low intracellular copper ion concentration and protective autophagy, which facilitates cellular adaptation and survival under stress. To overcome this limitation, a self-amplifying cuproptosis nanoregulator (SHK-Cu/[TK]PF/pATG5@HA, abbreviated as SC/TpA@HA) for CD44-targeted delivery is developed that integrate shikonin‑copper (SHK-Cu) coordination complexes with CRISPR/Cas9 plasmids targeting ATG5, condensed by fluorinated polyethyleneimine ([TK]PF)-condensed to enhance cancer therapy. Briefly, Cu[+] is released from the dissociated SHK-Cu complex upon intracellular GSH activation induces dihydrolipoamide S-acetyltransferase (DLAT) oligomerization and reduces FeS cluster proteins, triggering tricarboxylic acid (TCA) cycle collapse and irreversible mitochondrial damage. Concurrently, CRISPR/Cas9-mediated ATG5 knockout prevents autophagosome formation, creating an autophagic flux trap that accumulates copper-damaged mitochondria. Notably, such mitochondrial dysfunction as induced by copper overload combined with impaired cellular damage clearance from autophagy blockade elevates cuproptosis. In addition, the immunogenic cell death through cuproptosis in cancer cells, as validated by the exposure of calreticulin and the extracellular release of HMGB1, triggers a potent anti-tumor immune response. This response is enhanced through autophagy inhibition, as ATG5 deletion blocks the downstream signaling of copper-activated Unc-51-like autophagy activating kinase 1/2 (ULK1/2), ultimately amplifying cytotoxic T lymphocyte infiltration. Therefore, this dual intervention through copper overload and autophagy blockade potentiates both cuproptosis and anti-tumor immune effect, representing an innovative strategy of cuproptosis treatment.},
}
@article {pmid40848222,
year = {2025},
author = {Liu, L and Song, L and Qi, C and Cao, X and Huang, S and Zhang, G and Chen, G and Men, X and Zhang, H},
title = {Enhanced production of sabinene by engineered Saccharomyces cerevisiae from corn hydrolysates.},
journal = {Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]},
volume = {56},
number = {4},
pages = {2327-2337},
pmid = {40848222},
issn = {1678-4405},
support = {2022YFC2106200//National Key R&amp;D Program of China/ ; ZR2023MC163//Natural Science Foundation of Shandong Province/ ; QNESL 0P202308//Qingdao New Energy Shandong Laboratory/ ; TSQN201909159//Young Taishan Scholars/ ; Y2021063//Youth Innovation Promotion Association CAS/ ; },
mesh = {*Saccharomyces cerevisiae/metabolism/genetics ; *Zea mays/metabolism/chemistry ; *Metabolic Engineering ; Fermentation ; CRISPR-Cas Systems ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; *Monoterpenes/metabolism ; },
abstract = {Sabinene is a type of monoterpene that is widely used in flavors, fragrances and pharmaceuticals. Though sabinene biosynthesis has been investigated in a variety of microorganisms, application of sabinene is still limited due to its high production cost and lesser yielding strains. The baker's yeast Saccharomyces cerevisiae, which is generally recognized as safe (GRAS), is a suitable cell factory for the food and beverage industries. In this study, we aimed to enhance the production of sabinene from corn hydrolysates by employing genetic engineering techniques on S. cerevisiae. Here, we engineered S. cerevisiae for the production of sabinene by overexpressing sabinene synthase (SabS) and geranyl diphosphate synthase (GPPS) via CRISPR-Cas9, which is a simple and efficient tool for targeted and marker-free genome engineering. Subsequently, the culture medium and process conditions were optimized to enhance sabinene production and achieve ~ 23.6 mg/L under flask fermentation conditions. Based on the optimized culture conditions, we further investigated the production of sabinene from corn hydrolysates, which is a major source of dietary nutrients worldwide and an inexpensive source of sugars, and a high-level production of 60.0 mg/L was achieved in shake-flask fermentation. Our results implied that corn hydrolysates was a suitable medium for sabinene production and that CRISPR-Cas9 could boost the marker-free engineered yeast strain, which was more suitable for the food and beverage industry. Altogether, our work represents the progress in the bioproduction of food-grade sabinene from an inexpensive raw material.},
}
@article {pmid41288772,
year = {2025},
author = {Sun, W and Ren, X and Huang, J and Wang, Y and Liu, S},
title = {B-PER tandem assembly CRISPR/Cas12a cascade amplification strategy based fluorescence/colorimetric dual-mode for detection of MC-LR.},
journal = {Mikrochimica acta},
volume = {192},
number = {12},
pages = {853},
pmid = {41288772},
issn = {1436-5073},
support = {32072330//National Natural Science Foundation of China/ ; ZR2022MB066//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Microcystins/analysis ; Quantum Dots/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Colorimetry/methods ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; Marine Toxins ; Fluorescence ; Spectrometry, Fluorescence/methods ; Cadmium Compounds/chemistry ; },
abstract = {Microcystins are the most common, most powerful and most toxic types of cyanobacteria, which seriously threaten the public health and ecological environment. Biosensor has been widely used in Microcystin-Leucine-Arginine (MC-LR) detection. In this study, we report an efficient single-hairpin with double primers (the trigger chain T hybridizes with the left end a, and the amplified product ab hybridizes with the right end a) for bidirectional PER (B-PER) amplification strategy for uniform visual and fluorescence detection of trace amounts of MC-LR in the environment using quantum dots (QDs) as signal reporters. The biosensor is triggered by a single stranded DNA of MC-LR specificity to initiate the amplification by B-PER. From the long product of the B-PER, the trans-cleavage activity of CRISPR/Cas12a is activated, and the Ag[+] is released from the C-Ag[+]-C. The released Ag[+] undergoes cation exchange reaction (CER) with CdTe QDs (QDs), quenching QDs fluorescence and generating visual and fluorescent dual signals. The biosensor can simultaneously complete the display of dual signals of naked eyes and fluorescence, and can successfully used for the detection of the actual environment sample. In addition, the biosensor has low detection limits, high sensitivity, good accuracy and high selectivity. The detection range under the best conditions was 0.05-500 nM, and the detection limit was 0.705 pM. In summary, this strategy provides a general detection platform for detecting trace pollutants in the environment by using biosensors.},
}
@article {pmid41288291,
year = {2025},
author = {Liu, H and Liu, Y and Feng, R and Qian, M and Li, Y and Zhai, S and Song, J and Qiu, X},
title = {Homogeneous Femtomolar Detection of P-tau181 via Proximity Extension and CRISPR/Cas Technique.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c04856},
pmid = {41288291},
issn = {1520-6882},
abstract = {Accurate quantification of site-specific tau phosphorylation in plasma holds great promise for the noninvasive early diagnosis of Alzheimer's disease (AD). Here, we integrated the proximity extension assay (PEA) with nucleic acid amplification techniques-polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA)-and coupled them with CRISPR/Cas12a-mediated fluorescence detection to enable quantitative and homogeneous measurement of threonine-181-phosphorylated tau (p-tau181), a key biomarker of AD. Binding of two PEA probes to a single p-tau181 molecule induces proximity-mediated probe hybridization and extension, thereby converting the protein signal into an amplifiable nucleic acid signal. The resulting double-stranded DNA is subsequently amplified by PCR or RPA and detected through Cas12a trans-cleavage activity. The limits of detection (LODs) for the PEA-PCR-CRISPR/Cas and PEA-RPA-CRISPR/Cas assays were 149.0 fM (6.8 pg·mL[-1]) and 45.4 fM (2.1 pg·mL[-1]), respectively. In fetal bovine serum, LODs of 231.4 fM (10.6 pg·mL[-1]) and 139.2 fM (6.3 pg·mL[-1]) were achieved, demonstrating excellent antimatrix performance. The accuracy of the PEA-RPA-CRISPR/Cas assay in human serum was further validated using a commercial enzyme-linked immunosorbent assay (ELISA) kit. This homogeneous, wash-free approach combines operational simplicity with ultrahigh sensitivity, showing great potential for routine clinical detection and early stage monitoring of AD biomarkers.},
}
@article {pmid41288175,
year = {2025},
author = {Prokhorova, PV and Vlasova, NN and Yuzhakov, AG and Gulyukin, AM},
title = {Modern approaches to the construction and use of recombinant viruses.},
journal = {Voprosy virusologii},
volume = {70},
number = {5},
pages = {417-430},
doi = {10.36233/0507-4088-323},
pmid = {41288175},
issn = {2411-2097},
mesh = {Humans ; *Genetic Vectors/genetics ; *Oncolytic Viruses/genetics ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; *Vaccines, Synthetic/genetics/immunology ; Animals ; Oncolytic Virotherapy/methods ; Homologous Recombination ; Plasmids/genetics ; },
abstract = {The review describes certain viral vectors and considers various methods for constructing recombinant viruses with special attention paid to the homologous recombination and CRISPR/Cas9 system, and also describes the capabilities of using various cloning vectors (different plasmids, BAC etc.). The review also presents a comparative analysis of the effectiveness and safety of using various viral vectors, both for creating recombinant vaccines and for obtaining oncolytic viruses, as well as medicines for gene therapy.},
}
@article {pmid41285890,
year = {2025},
author = {Kim, S and Won, H and Bae, J and Kim, J and Choi, J and Richar, H and Kim, YG and Choi, HJ},
title = {Structural and functional insights into internal domain replacement in SpCas9 for protein engineering.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {41528},
pmid = {41285890},
issn = {2045-2322},
support = {SRFC-MA1801-09//Samsung Science and Technology Foundation/ ; },
mesh = {*Protein Engineering/methods ; *CRISPR-Associated Protein 9/genetics/chemistry/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; *Streptococcus pyogenes/enzymology/genetics ; Escherichia coli/genetics/enzymology ; Protein Domains ; },
abstract = {The CRISPR-Cas9 system has emerged as a powerful tool for precise genome editing, with ongoing research focused on enhancing its reliability and expanding its versatility. One effective strategy involves the integration of foreign functional domains into Cas9 to confer new capabilities. However, successful integration requires identification of insertion sites that preserve the protein's structural integrity and function. In this study, we identified a C-terminal region of Streptococcus pyogenes Cas9 (SpCas9), spanning residues 1242-1263, as a viable site for domain replacement. Structural and biochemical analyses of a SpCas9 variant lacking this region confirmed its dispensability for SpCas9 activity. As a proof of concept, we substituted this segment with the evolved E. coli tRNA adenosine deaminase (TadA), a key component of adenine base editors. Functional evaluation of this engineered SpCas9-TadA variant demonstrated deamination efficiency comparable to that of the ABE8e, with the potential to modulate the editing window through linker design. These results highlight the potential of targeted engineering of this region to develop more precise and versatile genome editing tools.},
}
@article {pmid41285767,
year = {2025},
author = {Rahimov, F and Ghosh, S and Petiwala, S and Schmidt, M and Nyamugenda, E and Shi, M and Tam, J and Verduzco, D and Singh, S and Avram, V and Modi, A and Espinoza, CA and Lu, C and Wang, J and Keller, A and Macoritto, M and Mahi, NA and Anton, T and Chung, N and Flister, MJ and Katlinski, KV and Biswas, A and den Hollander, AI and Waring, JF and Stender, JD},
title = {A genome-wide CRISPR screen identifies the TNRC18 gene locus as a regulator of inflammatory signaling.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10346},
pmid = {41285767},
issn = {2041-1723},
mesh = {Humans ; *Signal Transduction/genetics ; *Interleukin-1beta/metabolism/genetics ; *Inflammation/genetics/metabolism ; Lipopolysaccharides/pharmacology ; CRISPR-Cas Systems ; U937 Cells ; Finland ; Gene Knockout Techniques ; Genome-Wide Association Study ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation ; },
abstract = {Interleukin-1β (IL-1β) is dysregulated in chronic inflammatory diseases, yet the genetic factors influencing IL-1β production remain largely unknown. Myeloid-derived cells are the primary producers of IL-1β, which prompted a genome-wide CRISPR knockout screen in the human myeloid-derived U937 cells treated with lipopolysaccharide (LPS) to mimic inflammatory conditions and sorted for high and low intracellular IL-1β levels. A total of 295 genes are identified as regulators of IL-1β production, with 57 overlapping loci associated with inflammatory diseases, including the TNRC18 gene locus associated with multiple diseases in the Finnish population. U937 cells engineered with the Finnish-enriched rs748670681 risk allele demonstrate decreased expression of TNRC18 and an adjacent gene WIPI2, reduction in LPS-dependent gene activation and cytokine production, but elevation of interferon-responsive gene programs. Transcriptomic profiles for individual knockouts of TNRC18 and WIPI2 attribute the loss of LPS-dependent signaling primarily to TNRC18, which occurs through the modulation of H3K27 acetylation around inflammatory regulatory regions via TNRC18 and its protein interaction network. In contrast, the loss of WIPI2 is characterized by an exacerbation of interferon signaling. These findings delineate the global regulatory mechanisms of IL-1β production and provide molecular insights to the role of the rs748670681 variant in inflammatory diseases.},
}
@article {pmid41285753,
year = {2025},
author = {Mameli, E and Samantsidis, GR and Viswanatha, R and Kwon, H and Hall, DR and Butnaru, M and Hu, Y and Mohr, SE and Perrimon, N and Smith, RC},
title = {A genome-wide CRISPR screen in Anopheles mosquito cells identifies fitness and immune cell function-related genes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10323},
pmid = {41285753},
issn = {2041-1723},
support = {AI166857//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 2336877//National Science Foundation (NSF)/ ; GM132087//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; GM132087//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {Animals ; *Anopheles/genetics/immunology/drug effects ; *Mosquito Vectors/genetics/immunology/drug effects ; Liposomes ; Clodronic Acid/pharmacology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Malaria/transmission/prevention &amp; control ; Genome, Insect ; *Genetic Fitness ; Female ; },
abstract = {Anopheles mosquitoes are the sole vector of malaria, the most burdensome vector-borne disease worldwide. At present, strategies for reducing mosquito populations or limiting their ability to transmit disease show the most promise for disease control. Therefore, improving our understanding of mosquito biology and immune function may aid new approaches to limit malaria transmission. Here, we perform genome-wide CRISPR screens in Anopheles mosquito cells to identify genes required for fitness and that confer resistance to clodronate liposomes, which are used to ablate immune cells. The cellular fitness screen identifies 1280 fitness-related genes (393 at highest confidence) that are highly enriched for roles in fundamental cell processes. The clodronate screen identifies resistance factors that impair clodronate liposome function. For the latter, we confirm roles in liposome uptake and processing through in vivo validation in Anopheles gambiae that provide new mechanistic detail of phagolysosome formation and clodronate liposome processing. Altogether, we present a genome-wide CRISPR knockout platform in a major malaria vector and identify genes important for fitness and immune-related processes.},
}
@article {pmid41285544,
year = {2026},
author = {Gong, C and Wang, Z and Gao, X and He, S},
title = {A review of CRISPR-Cas as a "molecular catcher" for tracking circulating tumor cells and extracellular vesicles.},
journal = {Analytica chimica acta},
volume = {1381},
number = {},
pages = {344675},
doi = {10.1016/j.aca.2025.344675},
pmid = {41285544},
issn = {1873-4324},
mesh = {*Neoplastic Cells, Circulating/pathology/metabolism ; Humans ; *Extracellular Vesicles/metabolism/chemistry ; *CRISPR-Cas Systems ; Biomarkers, Tumor/genetics ; },
abstract = {BACKGROUND: Liquid biopsy is reshaping the landscape of cancer diagnostics by turning a simple blood sample into a rich source of real-time molecular insights. Among its most promising targets are circulating tumor cells (CTCs) and extracellular vesicles (EVs), which carry valuable clues about tumor progression, metastasis, and treatment response. However, traditional workflows for analyzing CTCs and EVs typically rely on immunoaffinity-based enrichment followed by molecular assays such as quantitative Polymerase Chain Reaction (qPCR) or Enzyme-Linked Immunosorbent Assay (ELISA). These approaches are often limited by low sensitivity, high costs, and complex procedures, hindering their widespread clinical use.<br><br>RESULTS: Recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technologies offer a multifaceted approach to biomarker analysis. Cas9 is primarily used for functional gene interrogation to identify and validate targets, while Cas12a and Cas13a serve as direct diagnostic tools, enabling ultrasensitive signal amplification for DNA, RNA, and protein markers. By integrating these distinct Cas effectors with aptamer-based recognition, nanomaterial-assisted enrichment, and hybrid amplification techniques like Hybridization Chain Reaction (HCR), Rolling Circle Amplification (RCA), researchers have developed highly sensitive and programmable platforms for analyzing CTCs and EVs.<br><br>SIGNIFICANCE: Despite ongoing challenges such as off-target effects, Protospacer Adjacent Motif (PAM) sequence limitations, and clinical variability, the field is rapidly evolving. The convergence of CRISPR diagnostics with artificial intelligence, device miniaturization, and multiplexed sensing is accelerating clinical translation. Collectively, these innovations are paving the way for a new era of precision oncology that is fast, flexible, and achievable with only a drop of blood.},
}
@article {pmid41284662,
year = {2025},
author = {Abad Santos, JC and Garimella, SS and Khanchandani, AN and Shah, PS},
title = {Rapid Optimization of a Light-Inducible System to Control Mammalian Gene Expression.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {225},
pages = {},
doi = {10.3791/68779},
pmid = {41284662},
issn = {1940-087X},
mesh = {Humans ; HEK293 Cells ; *Optogenetics/methods ; Light ; *CRISPR-Cas Systems ; Flow Cytometry/methods ; },
abstract = {Inducible gene expression tools can open novel applications in human health and biotechnology, but current options are often expensive, difficult to reverse, and have undesirable off-target effects. Optogenetic systems use light-responsive proteins to control the activity of regulators such that expression is controlled with the "flip of a switch". This study optimizes a simplified light activated CRISPR effector (2pLACE) system, which provides tunable, reversible, and precise control of mammalian gene expression. The OptoPlate-96 enables high-throughput screening via flow cytometry for single-cell analysis and rapid optimization of 2pLACE. This study demonstrates how to use the 2pLACE system with the OptoPlate-96 in HEK293T cells to identify the optimal component ratios for maximizing dynamic range and to find the blue light intensity response curve. Similar workflows can be developed for other mammalian cells and for other optogenetic systems and wavelengths of light. These advancements enhance the precision, scalability, and adaptability of optogenetic tools for biomanufacturing applications.},
}
@article {pmid41281758,
year = {2025},
author = {Jia, Z and Wu, J and Zhang, J and Zheng, P and Zhang, H and Lin, Y and Pan, T and Wu, M and Song, Y},
title = {Precision Reprogramming in CAR-T Cell Therapy: Innovations, Challenges, and Future Directions of Advanced Gene Editing.},
journal = {International journal of biological sciences},
volume = {21},
number = {15},
pages = {6884-6906},
pmid = {41281758},
issn = {1449-2288},
mesh = {Humans ; *Gene Editing/methods ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics ; CRISPR-Cas Systems/genetics ; Neoplasms/therapy ; T-Lymphocytes ; Animals ; },
abstract = {Chimeric antigen receptor (CAR)-T cell therapy represents a breakthrough in cancer immunotherapy, demonstrating impressive clinical outcomes, particularly for hematologic malignancies. However, its broader therapeutic application, especially against solid tumors, remains limited. Key challenges include T cell exhaustion, limited persistence, cytokine-mediated toxicities, and logistical hurdles associated with manufacturing autologous products. Emerging gene editing technologies, such as CRISPR/Cas systems, base editing, and prime editing, offer novel approaches to optimize CAR-T cells, aiming to enhance efficacy while managing toxicity and improving accessibility. This review comprehensively examines the current landscape of these gene editing tools in CAR-T cell therapy, highlighting the latest advancements, persisting challenges, and future directions. Leveraging gene editing holds the potential to transform CAR-T therapy into a more potent, safer, and broadly applicable modality for cancer and beyond.},
}
@article {pmid41281724,
year = {2025},
author = {Barraclough, A and Bär, I and van Duijl, T and Fijnvandraat, K and Eikenboom, JCJ and Leebeek, FWG and Bierings, R and Voorberg, J and Trasanidou, D},
title = {Correction: Rewriting the script: gene therapy and genome editing for von Willebrand Disease.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1719330},
doi = {10.3389/fgeed.2025.1719330},
pmid = {41281724},
issn = {2673-3439},
abstract = {[This corrects the article DOI: 10.3389/fgeed.2025.1620438.].},
}
@article {pmid41232934,
year = {2025},
author = {Li, Z and Li, F and Hua, L and Chai, F and Xie, L and Wang, D and Zhang, S and Zheng, C and Wang, Z and Jiang, X},
title = {Unlocking Zeptomolar Single-Molecule Detection by Synergizing Digital Microfluidics and Digital CRISPR.},
journal = {Journal of the American Chemical Society},
volume = {147},
number = {47},
pages = {43870-43883},
doi = {10.1021/jacs.5c15767},
pmid = {41232934},
issn = {1520-5126},
mesh = {Humans ; Immunoassay/methods ; Limit of Detection ; *CRISPR-Cas Systems ; Natriuretic Peptide, Brain/blood ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Tumor Necrosis Factor-alpha/blood ; *Single Molecule Imaging ; Interleukin-6/blood ; *Microfluidics ; Biomarkers/blood ; Peptide Fragments/blood ; },
abstract = {Accurate diagnosis relies on the highly sensitive and quantitative detection of multiple immune-related biomarkers. However, current detection methods still face significant limitations in sensitivity, specificity, and background signal control. Here, we introduce DDA (Dual-Digital immunoAssay), a fully automated, universal immunoassay platform that synergizes digital microfluidics with digital Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based amplification. This "dual-digital" strategy pushes the detection limit into the zeptomolar (zM) regime, enabling unprecedented sensitivity for single-molecule analysis. The DDA platform is built upon a digital microfluidic microwell array chip, integrating magnetic bead-based immunocapture with RNA-guided CRISPR/Cas13a signal amplification. This system enables a fully automated, "sample-in, answer-out" workflow. By systematically optimizing the entire process, DDA significantly reduces background noise and enhances detection sensitivity, achieving a limit of detection (LOD) down to 100 zM for key protein biomarkers. This represents a >100-fold improvement over leading commercial ultrasensitive assays. With single-molecule resolution and full automation, DDA provides a robust solution for the precise quantification of low-abundance immune biomarkers. As a proof-of-concept, we demonstrate its ability to accurately quantify key heart-failure-associated biomarkers, including NT-proBNP (LOD: 1 aM), IL-6 (LOD: 1.5 aM), and TNF-α (LOD: 2.5 aM), directly in complex serum samples. This platform holds great promise for automated multibiomarker screening and risk assessment, showcasing its powerful potential for the early diagnosis of major diseases such as cardiovascular diseases, cancers, neurodegenerative disorders, and infectious diseases.},
}
@article {pmid41232706,
year = {2026},
author = {Puri, B and Majumder, S and Gaikwad, AB},
title = {Targeting LncRNA MEG3 to modulate ER stress and autophagy: A CRISPR/Cas9-based strategy in AKI-to-CKD transition.},
journal = {Experimental cell research},
volume = {454},
number = {1},
pages = {114826},
doi = {10.1016/j.yexcr.2025.114826},
pmid = {41232706},
issn = {1090-2422},
mesh = {*Endoplasmic Reticulum Stress/genetics ; *Autophagy/genetics ; *RNA, Long Noncoding/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Animals ; *Acute Kidney Injury/genetics/pathology/metabolism ; Rats ; *Renal Insufficiency, Chronic/genetics/pathology/metabolism ; Fibrosis ; Cell Line ; Apoptosis/genetics ; },
abstract = {Acute kidney injury (AKI) to chronic kidney disease (CKD) transition is a progressive, long-term kidney dysfunction driven by complex pathophysiological processes, including persistent endoplasmic reticulum (ER) stress and impaired autophagy, contributing to fibrosis. Long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3) has been implicated in endoplasmic reticulum (ER) stress and autophagy in several diseases, but its role in kidney injury and fibrosis during AKI-to-CKD transition remains unclear. Our previous transcriptomic analysis revealed that MEG3 is dysregulated during this transition, prompting us to explore its functional role. In this study, we investigated the function of MEG3 in ER stress-autophagy crosstalk during the AKI-to-CKD transition. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9)-mediated MEG3 knockout in NRK-52E cells was confirmed by T7 endonuclease assay, quantitative real-time polymerase chain reaction (qRT-PCR), and fluorescence in-situ hybridization (FISH) assay. Functionally, MEG3 knockout markedly attenuated ER stress and apoptosis, as shown by reduced expression of BiP, CHOP, ATF6, ATF4, p-PERK, p-eIF2α, along with restoration of the Bax/Bcl-2 balance. Autophagy activity was restored, with increased Beclin-1 and LC3B expression and decreased p62 accumulation. Furthermore, fibrotic progression was reduced, as indicated by lower levels of fibronectin and collagen I. Notably, tauroursodeoxycholic acid (TUDCA, 400 μM) acted synergistically with MEG3 knockout, further suppressing ER stress and fibrosis markers compared to either treatment alone. These findings demonstrate that MEG3 promotes maladaptive ER stress and impaired autophagy in tubular epithelial cells, driving AKI-to-CKD transition. Targeting MEG3 through CRISPR-based strategies or in combination with TUDCA may represent a promising therapeutic strategy to mitigate fibrosis and slow disease progression.},
}
@article {pmid41211945,
year = {2025},
author = {Laffin, LJ and Nicholls, SJ and Scott, RS and Clifton, PM and Baker, J and Sarraju, A and Singh, S and Wang, Q and Wolski, K and Xu, H and Nielsen, J and Patel, N and Duran, JM and Nissen, SE},
title = {Phase 1 Trial of CRISPR-Cas9 Gene Editing Targeting ANGPTL3.},
journal = {The New England journal of medicine},
volume = {393},
number = {21},
pages = {2119-2130},
doi = {10.1056/NEJMoa2511778},
pmid = {41211945},
issn = {1533-4406},
mesh = {Humans ; Angiopoietin-Like Protein 3 ; Male ; Middle Aged ; Female ; *Angiopoietin-like Proteins/genetics ; Adult ; *Gene Editing ; *CRISPR-Cas Systems ; Aged ; Loss of Function Mutation ; *Dyslipidemias/genetics/drug therapy ; Hypercholesterolemia/drug therapy/genetics ; Hypertriglyceridemia/drug therapy/genetics ; },
abstract = {BACKGROUND: Angiopoietin-like protein 3 (ANGPTL3) inhibits lipoprotein and endothelial lipases. ANGPTL3 loss-of-function genetic variants are associated with decreased levels of low-density lipoprotein cholesterol and triglycerides and a decreased lifetime risk of atherosclerotic cardiovascular disease.<br><br>METHODS: We conducted an ascending-dose phase 1 trial to assess the safety and efficacy of CTX310, a lipid-nanoparticle-encapsulated clustered regularly interspaced short palindromic repeats-Cas9 endonuclease (CRISPR-Cas9) messenger RNA (mRNA) and guide RNA targeting hepatic ANGPTL3 to induce a loss-of-function mutation. Adults who had uncontrolled hypercholesterolemia, hypertriglyceridemia, or mixed dyslipidemia and were receiving maximally tolerated lipid-lowering therapy received a single intravenous dose of CTX310 (0.1, 0.3, 0.6, 0.7, or 0.8 mg per kilogram of body weight). The primary end point was adverse events, including dose-limiting toxic effects.<br><br>RESULTS: A total of 15 participants received CTX310 and had at least 60 days of follow-up. No dose-limiting toxic effects related to CTX310 occurred. Serious adverse events occurred in two participants (13%): one participant had a spinal disk herniation, and the other died suddenly 179 days after treatment with the 0.1-mg-per-kilogram dose. Infusion-related reactions were reported in three participants (20%), and one participant (7%) who had elevated levels of aminotransferases at baseline had a transient elevation in aminotransferases to between three times and five times as high as those at baseline, peaking on day 4 and returning to baseline by day 14. The mean percent change in ANGPTL3 level was 9.6% (range, -21.8 to 71.2) with the dose of 0.1 mg per kilogram, 9.4% (range, -25.0 to 63.9) with 0.3 mg per kilogram, -32.7% (range, -51.4 to -19.4) with 0.6 mg per kilogram, -79.7% (range, -86.8 to -72.5) with 0.7 mg per kilogram, and -73.2% (range, -89.0 to -66.9) with 0.8 mg per kilogram.<br><br>CONCLUSIONS: Editing of ANGPTL3 was associated with few adverse events and resulted in reductions from baseline in ANGPTL3 levels. (Funded by CRISPR Therapeutics; Australia New Zealand Clinical Trials Registry number, ACTRN12623000809639.).},
}
@article {pmid41064856,
year = {2025},
author = {Jiang, Z and Su, L and Chen, C and He, R and Jiang, L and Shu, Y and Dai, D and Wu, M and Guo, A and Liu, J and Liu, S and Liu, Z},
title = {Atf3 Deficiency Promotes Mesodermal Commitment and Enhances Endothelial Differentiation in Embryonic Stem Cells.},
journal = {Arteriosclerosis, thrombosis, and vascular biology},
volume = {45},
number = {12},
pages = {2226-2242},
pmid = {41064856},
issn = {1524-4636},
mesh = {*Activating Transcription Factor 3/genetics/deficiency/metabolism ; Animals ; *Cell Differentiation ; Mice, Knockout ; Mice ; *Endothelial Cells/metabolism ; *Mouse Embryonic Stem Cells/metabolism ; *Mesoderm/metabolism/cytology ; Cells, Cultured ; CRISPR-Cas Systems ; Cell Lineage ; Signal Transduction ; },
abstract = {BACKGROUND: Ischemic diseases have become a major threat to global health, with endothelial cell (EC) damage closely associated with their pathogenesis and progression. Cell therapies targeting endothelial repair have thus become a treatment approach of great interest, yet the procurement of clinically approved ECs for these applications has not been fully established. Modulating the expression of Atf3 (activating transcription factor 3) represents a potential strategy for deriving ECs from stem cells; however, its precise function in the development and differentiation of ECs from stem cells remains elusive. In the present study, we sought to elucidate the potential role of Atf3 in the differentiation of embryonic stem cells into ECs.<br><br>METHODS: CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) system was used to knockout Atf3 (Atf3KO [Atf3 knockout]) in mouse embryonic stem cells. EC differentiation was initially induced using the hanging drop method to promote embryoid bodies formation, followed by embryoid bodies attachment onto culture slides. The expression changes of EC markers during differentiation were assessed by RNA sequencing, Western blotting, immunofluorescence staining, flow cytometry, and reverse transcription quantitative polymerase chain reaction. Functional comparisons of differentiated ECs were performed by assessing LDL (low-density lipoprotein) uptake and NO production. Potential molecular mechanisms were further explored via bioinformatic analysis of RNA sequencing data.<br><br>RESULTS: Atf3KO led to a significant upregulation in the expression levels of progenitor and mesoderm cell markers on days 3 and 6 of differentiation. By day 9, the expression of mature EC markers also exhibited a notable increase. Moreover, Atf3KO enhanced the functional properties of differentiated Atf3KO ECs. In addition, our findings revealed that the activation of the Rap1 (Ras-related protein 1) signaling pathway, triggered by Atf3KO, contributed to ECs development and maturation.<br><br>CONCLUSIONS: Atf3KO directs embryonic stem cells toward the mesodermal lineage and activates the Rap1 signaling pathway, thereby promoting ECs development. These findings highlight a key role of Atf3 in regulating early stage of vascular endothelial development.},
}
@article {pmid40842147,
year = {2025},
author = {Ye, S and Kim, JS and Kim, M and Kim, KY and Won, YH and Park, T and An, S and Jeong, H and Chung, HJ and Lee, IS and Kang, MH and Kang, CY and Kim, MY and Chung, JH and Gim, JA and Hwang, W and Kim, Y and Kim, SC and Lee, S and Hur, JK and Hur, JW},
title = {MUTE-Seq: An Ultrasensitive Method for Detecting Low-Frequency Mutations in cfDNA With Engineered Advanced-Fidelity FnCas9.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {37},
number = {47},
pages = {e05208},
doi = {10.1002/adma.202505208},
pmid = {40842147},
issn = {1521-4095},
support = {2021R1A6A1A03040260//National Institute for International Education/ ; RS-2023-00260529//National Institute for International Education/ ; RS-2024-00435385//Korea National Institute of Health/ ; RS-2021-KD000007//Korea Medical Device Development Fund/ ; RS-2021-NR056589//National Research Foundation of Korea/ ; RS-2023-00260529//National Research Foundation of Korea/ ; RS-2023-00262309//National Research Foundation of Korea/ ; RS-2025-02218918//National Research Foundation of Korea/ ; RS-2022-KH129266//Korea Ministry of Health &amp; Welfare/ ; K2125811//Korea University/ ; },
mesh = {Humans ; *Mutation ; CRISPR-Cas Systems ; Carcinoma, Non-Small-Cell Lung/genetics ; *Cell-Free Nucleic Acids/genetics ; Lung Neoplasms/genetics ; Leukemia, Myeloid, Acute/genetics ; Pancreatic Neoplasms/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {In this study, we present the development of the Mutation tagging by CRISPR-based Ultra-precise Targeted Elimination in Sequencing (MUTE-Seq) method. We engineered a highly precise advanced-fidelity FnCas9 variant, named FnCas9-AF2, to effectively discriminate single-base mismatches at all positions of the single guide RNA (sgRNA) target sequences. FnCas9-AF2 exhibited significantly lower off-target effects compared to existing high-fidelity CRISPR-Cas9 variants. MUTE-Seq leverages FnCas9-AF2 for the enrichment of mutant DNA through the exclusive cleavage of perfectly matched wild-type DNA, allowing for sensitive detection of low-frequency cancer-associated mutant alleles. MUTE-Seq enabled sensitive monitoring of minimal residual disease (MRD) from the bone marrow of patients with Acute Myeloid Leukemia (AML). Furthermore, MUTE-Seq was applied in a multiplexed manner on cell-free DNA (cfDNA) from patients diagnosed with non-small cell lung cancer (NSCLC) and pancreatic cancer. This approach demonstrated a significant improvement in the sensitivity of simultaneous mutant detection and highlighted its clinical utility for early-stage cancer patients with extremely low levels of circulating tumor DNA (ctDNA). We anticipate that the FnCas9-AF2-based MUTE-Seq could offer a valuable clinical tool to facilitate improved molecular diagnosis, prognosis evaluation, and treatment planning for cancers in various stages.},
}
@article {pmid41280798,
year = {2025},
author = {Fronza, F and Verardo, R and Schneider, C},
title = {RepFluo, a Fast Fluorescent In Vitro Assay of Cas9 Activity Exploiting Melting Curve Analysis.},
journal = {ACS omega},
volume = {10},
number = {45},
pages = {53816-53826},
pmid = {41280798},
issn = {2470-1343},
abstract = {Demand for less labor-intensive in vitro assays of the activity of CRISPR/Cas proteins is rising to extend the potential applications of CRISPR in the field of diagnostics. RNA guided DNA endonucleases of the Cas family generate double-strand breaks in the target DNA, which results in two shorter DNA fragments. We hypothesized that this cleavage event could be studied using melting curve analysis, and using SpyCas9, we demonstrate that it is possible to evaluate the activity of Cas proteins by measuring the melting curves of their products. We present here a novel assay for the in vitro activity of Cas9 that exploits melting curve analysis (MCA) to be fast, inexpensive, and widely accessible. The assay can, in fact, be performed with readily available components(?)in its simplest form a real-time thermal cycler and an intercalating dye (SYBR Green I)(?)and produces reliable results with a run-time of 15 min. It does not require external intervention to stop the reaction, which is done by thermal denaturation of the protein directly in the thermal cycler machine. The described advantages, combined with the provided data analysis package, make the assay robust and amenable to high-throughput applications. To increase the accessibility of our assay, we provided an R package that simplifies the analytical process.},
}
@article {pmid41280279,
year = {2026},
author = {Li, X and Ye, C and Liu, T and Li, S and Zhang, M and Zhao, Y and Jin, Y and Cheng, J and Yang, G and Li, P},
title = {Engineering genetic elements for microbial protein expression systems: Advances, challenges, applications, and prospects.},
journal = {Synthetic and systems biotechnology},
volume = {11},
number = {},
pages = {370-384},
pmid = {41280279},
issn = {2405-805X},
abstract = {The rising global demand for sustainable protein sources poses critical challenges across food, pharmaceutical, and industrial biotechnology sectors. Microbial expression systems provide scalable and versatile platforms for producing recombinant proteins, including enzymes, therapeutic molecules, and functional food ingredients. These platforms enable efficient biosynthesis of high-value proteins from renewable substrates often via precision fermentation, surpassing conventional methods in yield, cost-efficiency, and environmental sustainability. This review summarizes the genetic regulatory elements that govern gene expression in microbial hosts, with comparative coverage of prokaryotic and eukaryotic systems at transcriptional and translational levels. Key regulatory components, such as promoters, ribosome binding sites (RBS), untranslated regions (UTRs), signal peptides, and terminators, are discussed in the context of host-specific engineering strategies. We highlight advanced engineering approaches, including artificial intelligence (AI) assisted sequence design, CRISPR-Cas-based genome editing, and modular combinatorial optimization of genetic elements. Particular attention is given to the integration of high-throughput screening and predictive modeling tools that accelerate the rational design and optimization of microbial production systems. The review also discusses practical applications in food, pharmaceutical, and industrial enzyme production, emphasizing how genetic element engineering bridges fundamental research and biomanufacturing. Finally, key challenges and future prospects are analyzed to guide the development of next-generation microbial cell factories for sustainable protein production and industrial innovation.},
}
@article {pmid41279041,
year = {2025},
author = {Vera, AO and Avilés-Vázquez, FJ and Ha, T and Choudhary, A and Raines, RT},
title = {Nuclear Localization Signals Enable the Cellular Delivery of an Anti-CRISPR Protein to Control Genome Editing.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.10.28.685205},
pmid = {41279041},
issn = {2692-8205},
abstract = {Precise regulation of Cas9 activity is essential to minimize off-target effects, mosaicism, chromosomal alterations, immunogenicity, and genotoxicity in genome editing. Although type II anti-CRISPR proteins (Acrs) can inhibit and regulate Cas9, their size and anionic charge generally prevent them from crossing the cell membrane. Existing Acr delivery methods employing vectors or electroporation are either slow and persistent or require external equipment, limiting their therapeutic utility. To address these challenges, we developed a cell-permeable Acr (6×NLS-Acr), which uses nuclear localization signals (NLSs) to cross the cell membrane. We conjugated 6×NLS-Acr to a fluorescent dye to elucidate its cellular entry mechanism and directly visualized its binding to a fluorescent Cas9·gRNA complex to study its inhibitory mechanism. 6×NLS-Acr (IC 50 = 0.47 µM) directly transduces human cells, including immortalized cell lines, embryonic stem cells, and 3D cell cultures, within 5 min, inhibiting up to 99% of Cas9 activity and increasing genome-editing specificity by nearly 100%. We further compared 6×NLS-Acr with our anthrax-derived Acr delivery platform. Our results demonstrate that 6×NLS-Acr is the most efficacious cell-permeable CRISPR-Cas inhibitor, significantly enhancing the precision and therapeutic potential of CRISPR-based genome editing.},
}
@article {pmid41277735,
year = {2025},
author = {Zhang, L and He, X and Hu, J and Bai, H and Yao, Y and Hu, WW},
title = {Recent advances in nanozyme-CRISPR/Cas biosensors.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc05585g},
pmid = {41277735},
issn = {1364-548X},
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system, renowned for precise DNA recognition and potent trans-cleavage activity, has become a promising tool for biosensing. Nanozymes, a new class of artificial enzymes that combine the physicochemical properties of nanomaterials with the catalytic functions of natural enzymes, offer high surface area and versatile functionalization, enabling efficient target binding and catalysis to markedly boost biosensing performance. With advances across disciplines, the integration of nanozymes and CRISPR/Cas has become prominent in biosensing. Nanozyme-enhanced CRISPR/Cas biosensors can substantially improve detection sensitivity and expand sensing modes for bioanalysis, with potential for direct target detection without pre-amplification. In this review, we comprehensively examine recent strategies in nanozyme-enhanced CRISPR/Cas biosensing, with particular emphasis on advances in bioanalytical applications. We further critically assess the challenges and prospects of using nanozymes to improve CRISPR/Cas performance for biosensing and offer insights for the design of next-generation biosensors for precise, rapid, on-site detection of nucleic acids, proteins, and small molecules in clinical samples.},
}
@article {pmid41277692,
year = {2025},
author = {Jang, H and Kang, JE and Kim, H and Kim, JR and Park, J and Go, SR and Lee, YH and Kang, H and Park, Y and Kim, S and Jung, Y and Kim, SJ and Lim, EK and Jung, J and Woo, EJ and Park, KH and Kang, T},
title = {CRISPR/Cas12a2 enables ultra-sensitive amplification-free RNA detection.},
journal = {Nucleic acids research},
volume = {53},
number = {21},
pages = {},
pmid = {41277692},
issn = {1362-4962},
support = {//National Research Foundation of Korea/ ; 2021M3H4A1A02051048//Ministry of Science and ICT/ ; 2023R1A2C2005185//Ministry of Science and ICT/ ; RS-2024-00438316//Ministry of Science and ICT/ ; RS-2024-00348576//Ministry of Science and ICT/ ; RS-2024-00459749//Ministry of Science and ICT/ ; RS-2025-00554718//Ministry of Science and ICT/ ; 2021M3A9G802559922//Ministry of Science and ICT/ ; RS-2022-NR071772//Ministry of Science and ICT/ ; RS-2021-NR059435//Ministry of Science and ICT/ ; CRC22024-500//Ministry of Science and ICT/ ; 2020R1A5A1018052//Ministry of Science and ICT/ ; GTL25061-000//Ministry of Science and ICT/ ; //Korea Evaluation Institute of Industrial Technology/ ; RS-2022-00154853//Ministry of Trade, Industry and Energy/ ; RS-2024-00432382//Ministry of Trade, Industry and Energy/ ; RS-2024-00403563//Ministry of Trade, Industry and Energy/ ; //Korea Environmental Industry and Technology Institute/ ; 2021003370003//Ministry of Environment/ ; //Korea Health Industry Development Institute/ ; RS-2025-02213315//Ministry of Health and Welfare/ ; RS-2024-00401639//Korea Institute of Planning and Evaluation for Technology in Food, Agriculture/ ; KGM1322511//Korea Research Institute of Bioscience and Biotechnology Research Initiative Program/ ; KGM1032511//Korea Research Institute of Bioscience and Biotechnology Research Initiative Program/ ; KGM1062511//Korea Research Institute of Bioscience and Biotechnology Research Initiative Program/ ; KGM5382531//Korea Research Institute of Bioscience and Biotechnology Research Initiative Program/ ; //Research Institute of Bioscience and Biotechnology/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Viral/genetics/analysis ; *SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; *COVID-19/diagnosis/virology ; Limit of Detection ; Endodeoxyribonucleases ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; },
abstract = {Advances in clustered regularly interspaced short palindromic repeat (CRISPR) technologies have led to the use of diverse CRISPR-associated (Cas) proteins in diagnostic applications. Herein, we present a CRISPR/Cas12a2-based amplification-free RNA detection method that exhibits sub-attomolar sensitivity and substrate versatility. Cas12a2, a recently characterized RNA-guided nuclease, uniquely integrates bimolecular recognition through CRISPR RNA (crRNA)-target complementarity and protospacer flanking sequence identification, enabling highly specific trans-cleavage of single-stranded DNA, double-stranded DNA, and RNA. We have optimized key biochemical parameters, including pH, ionic strength, and temperature, to enhance the catalytic efficiency of Cas12a2. Based on the optimal activity conditions of Cas12a2, we have achieved ultra-sensitive viral RNA detection with a limit of detection of 46.7 aM through the strategic design and cooperative activation of crRNAs targeting conserved regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome. The diagnostic accuracy of the Cas12a2-based assay has been demonstrated for 26 SARS-CoV-2 variants, and it has further resulted in the definitive diagnosis of 317 clinical samples. This work establishes Cas12a2 as a promising molecular diagnostic tool that provides an amplification-free, rapid, and versatile solution for RNA detection. The adaptability and simplicity of the platform render it particularly well suited for point-of-care applications, paving the way for next-generation CRISPR diagnostics.},
}
@article {pmid41222440,
year = {2025},
author = {Ming, W and Zhu, Y and Li, L and Wang, T and Pan, A and Xu, Q and Ji, H and Qin, Y and Wu, L},
title = {Allele-Specific CRISPR-Cas9-Based Ratiometric Fluorescence Platform for Portable EGFR L858R Mutation Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {46},
pages = {25832-25839},
doi = {10.1021/acs.analchem.5c05548},
pmid = {41222440},
issn = {1520-6882},
mesh = {Humans ; ErbB Receptors/genetics ; *CRISPR-Cas Systems/genetics ; Alleles ; Mutation ; *Lung Neoplasms/genetics/diagnosis ; *Carcinoma, Non-Small-Cell Lung/genetics/diagnosis ; Fluorescence ; Point-of-Care Testing ; },
abstract = {Effective detection of low-abundance EGFR L858R mutation from circulating tumor DNA (ctDNA) is critical for early stage NSCLC diagnosis. Here, a portable CRISPR-Cas9-based ratiometric fluorescence sensor was proposed. Typically, allele-specific activation of Cas9 and the trans-cleavage of Cy5/BHQ2-labeled blocker DNAs were achieved by engineering sgRNA to position the L858R mutation within the PAM region of Cas9, resulting in increased Cy5 fluorescence. Simultaneously, the attenuated fluorescence of HBC-530 was observed because the released input RNA bound to the Pepper aptamer, which resulted from the decreased melting temperature (Tm) of the blocker DNA-RNA hybrid. Thus, a dual-channel ratiometric readout can be readily attained. Ultimately, visual point-of-care testing (POCT) of L858R at 0.01% allele frequency with single-nucleotide specificity was realized using a compact 3D-printed device. The validation result of 22 collected plasma samples demonstrated 100% concordance with the clinical diagnoses. This platform provides a cost-effective and accessible solution for NSCLC screening, making it particularly suitable for resource-limited healthcare settings.},
}
@article {pmid40854986,
year = {2025},
author = {Lu, Z and Chen, C and Zhang, H and Li, B and Liu, Y and Guo, J and Xu, R and Shi, K and Ma, Q and Zhang, M and Cai, Y and Huang, J and Geng, H and Fan, L and Ning, C and Li, Y and Chen, S and Tian, W and Hu, K and Li, H and Yang, X and Huang, C and Wei, Y and Zhu, X and Li, X and Xiong, Z and Cai, M and Wang, X and Zhang, S and Chen, H and Dai, M and Chen, K and Jin, M and Jin, M and Zhu, Y and Tian, J and Miao, X},
title = {Characterization of cis-regulatory elements and functional variants in colorectal cancer using epigenomics and CRISPRi screenings.},
journal = {Nature cancer},
volume = {6},
number = {11},
pages = {1777-1799},
pmid = {40854986},
issn = {2662-1347},
support = {NSFC-82130098//National Science Foundation of China | Key Programme/ ; },
mesh = {Humans ; *Colorectal Neoplasms/genetics/pathology ; *Epigenomics/methods ; Epigenesis, Genetic ; Gene Expression Regulation, Neoplastic ; *Regulatory Sequences, Nucleic Acid/genetics ; Transcription Factor 7-Like 2 Protein/genetics ; Repressor Proteins/genetics ; CRISPR-Cas Systems ; Polymorphism, Single Nucleotide ; Cell Proliferation/genetics ; Cell Line, Tumor ; Genetic Predisposition to Disease ; },
abstract = {Genetic variants associated with colorectal cancer (CRC) are primarily noncoding and reside in cis-regulatory elements (CREs), yet their underlying mechanisms remain elusive. Here we established a dynamic epigenetic atlas using multiomics data from 533 colorectal tissues spanning normal to advanced adenoma to cancer, identifying 7,492 differential CREs linked to 5,490 target genes. High-throughput CRISPR interference screening revealed 265 functional CREs involved in CRC cell proliferation. A polygenic risk score (PRS) based on functional CRE variants effectively predicted CRC and precancerous lesions among 476,770 individuals. Notably, the functional variant rs10871066 was significantly associated with increased risk of precancerous lesions and CRC (odds ratio = 1.27, P = 1.03 × 10[-13]). Mechanistically, rs10871066 triggers silencer-to-enhancer switching mediated by FOXP1 and TCF7L2, distally upregulating KLF5 to activate oncogenic pathways and PIBF1 to suppress natural killer cell cytotoxicity. Our study provides a comprehensive resource of dynamic epigenomic atlas, a functionally informed PRS for risk prediction and insights into epigenetic mechanisms underlying CRC development.},
}
@article {pmid40669499,
year = {2025},
author = {Tadokoro, T and Olson, EN and Liu, N},
title = {Gene Editing Applications as Future Cardiovascular Therapies.},
journal = {Annual review of genetics},
volume = {59},
number = {1},
pages = {119-145},
doi = {10.1146/annurev-genet-011725-094039},
pmid = {40669499},
issn = {1545-2948},
mesh = {*Gene Editing/methods/trends ; Humans ; CRISPR-Cas Systems/genetics ; *Cardiovascular Diseases/therapy/genetics ; *Genetic Therapy/methods/trends ; Animals ; },
abstract = {Cardiovascular disease is the leading cause of global morbidity and mortality, despite advances in pharmacological and surgical interventions. The emergence of CRISPR-Cas9 genome editing technology offers promising approaches for correcting genetic causes of hereditary cardiovascular disorders and modulating pathogenic signaling pathways implicated in various heart diseases. However, several challenges with respect to in vivo delivery of gene editing components, as well as important safety considerations, remain to be addressed in the path toward possible clinical application. We review current gene editing strategies, their potential therapeutic applications in the context of a variety of cardiovascular disorders, and their respective merits, limitations, and regulatory considerations. The rapid advances in this field combined with the many opportunities for deploying gene editing therapies for cardiovascular disorders augur well for the future of this transformative technology.},
}
@article {pmid41277686,
year = {2025},
author = {Stohr, AM and Hansen, H and Richards, B and Park, H and Goncalves, AG and Agrawal, A and Blenner, M and Chen, W},
title = {Metabolite-responsive scaffold RNAs for dynamic CRISPR transcriptional regulation.},
journal = {Nucleic acids research},
volume = {53},
number = {21},
pages = {},
doi = {10.1093/nar/gkaf1290},
pmid = {41277686},
issn = {1362-4962},
support = {MCB2317398//National Science Foundation/ ; GM133803/NH/NIH HHS/United States ; //U.S. Department of Defense/ ; P200A210065//GAANN Fellowship Program/ ; },
mesh = {*CRISPR-Cas Systems ; Theophylline/metabolism/pharmacology ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Tryptophan/metabolism/pharmacology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcriptional Activation ; Transcription, Genetic ; *Gene Expression Regulation ; },
abstract = {CRISPR activation is a powerful tool to upregulate a vast array of genes in many different contexts. However, there are few dynamic CRISPR transcriptional programs, which limit its usage in the creation of living biosensors, self-regulating microbial factories, or conditional therapeutics. Here, we address this limitation by embedding a molecular switch directly into a guide RNA to create a combined sensor-actuator called a metabolite-responsive scaffold RNA (MR-scRNA). We demonstrate the regulatory potential for MR-scRNAs by conditionally activating genes in three different kingdoms of life. We create MR-scRNAs responsive to two distinct metabolites, theophylline and tryptophan, by swapping the molecular switch used. MR-scRNAs respond quickly in a dose-dependent manner specifically to their target metabolite and enhance biochemical production when used as a dynamic regulator of pathway enzyme expression. The broad functionality and ease of design of the MR-scRNAs offer a promising tool for dynamic cellular regulation.},
}
@article {pmid41277071,
year = {2025},
author = {Hotta, M and Inoue, YU and Asami, J and Hoshino, M and Inoue, T},
title = {Generation of a Triple Tag Knock-In Mouse to Visualize Precise Protein Localization Patterns for Type II Classic Cadherins During Brain Development.},
journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms},
volume = {30},
number = {6},
pages = {e70070},
doi = {10.1111/gtc.70070},
pmid = {41277071},
issn = {1365-2443},
support = {NCNP 3-9//Intramural Research Grant for Neurological and Psychiatric Disorders of National Center of Neurology and Psychiatry/ ; NCNP 6-9//Intramural Research Grant for Neurological and Psychiatric Disorders of National Center of Neurology and Psychiatry/ ; },
mesh = {Animals ; *Cadherins/metabolism/genetics ; Mice ; *Brain/metabolism/embryology ; Gene Knock-In Techniques ; Gene Expression Regulation, Developmental ; Mice, Transgenic ; CRISPR-Cas Systems ; },
abstract = {Classic cadherin cell-cell adhesion molecules with self-organizing activities play roles in segregating distinct populations of cells at developing brain regions and/or boundaries. However, the protein dynamics of each cadherin subclass in the mouse embryonic brain is poorly described due to the low antigenicity. Here, we generate Cdh6-HA and Cdh8-PA tag knock-in (KI) mice by CRISPR/Cas9-mediated genome editing and establish Cdh6[HA/HA]; Cdh8[PA/PA]; Cdh11[EGFP/EGFP] triple tag KI homo mice with normal viability and fertility. Immunostaining with specific antibodies for these tags reveals differential protein expression profiles almost comparable with mRNA in situ hybridization (ISH) results during embryonic brain development. We can additionally detect considerable levels of immunostaining signals outside the mRNA ISH-positive areas, specifically along the nerve tracts, suggesting physiological accumulation of these type II cadherin proteins along axons. By using super-resolution imaging, we further evaluate cadherin subcellular localization dynamics around the zona limitans intrathalamica to confirm that the prosomere 2/3 compartment boundary at E12.5 is maintained by the distinctive integration of Cdh6 or Cdh11 at apical attachment sites of the ventricular cells. These results highlight the value of the genetic tag KI strategy for proteins with low antigenicity and the functional relevance of type II classic cadherins in brain development.},
}
@article {pmid41276731,
year = {2025},
author = {Eren Eroglu, AE and Toklu, K and Yasa, &#x130;},
title = {Functional genomics of a food-related thermotolerant Acetobacter oryzifermentans strain AAB5: genetic determinants of stress response, CAZyme repertoire, and CRISPR-Cas system.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {253},
pmid = {41276731},
issn = {1438-7948},
}
@article {pmid41276041,
year = {2025},
author = {Khan, MF and Javed, M and Kaur, J and Badwal, AK and Singh, S},
title = {CRISPR-Cas mediated targeting of resistance genes for combating ESKAPE pathogen infections: A Review.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {149180},
doi = {10.1016/j.ijbiomac.2025.149180},
pmid = {41276041},
issn = {1879-0003},
abstract = {Advancements in the treatment of antimicrobial infections have highlighted the importance of the CRISPR-Cas system in targeting resistance genes in bacterial pathogens resistant to conventional drugs. Various CRISPR-Cas techniques, such as CRISPR-Cas9, Cas3, dCas9 and the mini-CRISPR system, have been utilized for this purpose in ESKAPE pathogens. Novel strategies like Associates Toxin Antitoxin and CRISPR-Cas to kill multidrug resistant pathogens-CRISPR-regulated toxin antitoxin module (ATTACK-CreTA) and CRISPR interference refine CRISPR-Cas efficacy. This review explores the mechanism of action of resistance genes (e.g., tetM, ermB, VanA, aph-3, aac3, oxa23, blaNDM etc.) prevalent within these pathogens and highlights the notable achievements of CRISPR-Cas technology in targeting these genes, thereby offering a pathway to sensitize resistant bacteria. This article also discusses various delivery approaches for CRISPR components in pathogens, mainly focusing on engineered bacteriophages, including phagemids, temperate phages and virulent phages. Additionally nanoparticles, bacterial conjugation and natural phages hold promise for administering the CRISPR system inside bacteria. Specific targeting of resistance genes in resistant pathogens via CRISPR-Cas based methods would pave a way for combating ESKAPE pathogen infections by reversing the resistance phenotype.},
}
@article {pmid41273791,
year = {2025},
author = {Espinoza-Erazo, VP and Vela-Chauvin, MG and Collantes-Vela, JC and Zapata-Mena, S and Machado, A},
title = {Biofilms of Salmonella: Implications for Food Safety and Public Health.},
journal = {Foodborne pathogens and disease},
volume = {},
number = {},
pages = {},
doi = {10.1177/15353141251389597},
pmid = {41273791},
issn = {1556-7125},
abstract = {Salmonella enterica is a leading cause of foodborne illness worldwide, responsible for an estimated 93.8 million cases and approximately 155,000 deaths annually, according to the World Health Organization. This foodborne pathogen imposes a significant burden on public health and the global economy. A key factor contributing to the persistence and widespread impact of S. enterica is its potential to form biofilms, which may enhance its survival in clinical, industrial, and agricultural environments, making it a major and ongoing public health concern. Biofilms are structured microbial communities encapsulated in a self-produced extracellular matrix that protects against environmental stressors, disinfectants, and antimicrobial agents. This complex phenotype enables Salmonella to colonize food-contact surfaces, medical devices, and host tissues, hampering efforts to eliminate contamination and control transmission. The poultry industry, a key component of the global food supply, is particularly vulnerable to emerging Salmonella strains with increased virulence, stress tolerance, and disinfectant resistance, making biofilm control a top priority. This review aims to provide an updated and comprehensive overview of the mechanisms involved in Salmonella biofilm formation, its implications for food safety, and recent advances in detection and control strategies. Emerging technologies such as CRISPR-Cas systems are receiving particular attention due to their potential as precise molecular tools for investigating genes implicated in biofilm formation. By integrating current findings, this review underscores the urgent need for novel and effective strategies for biofilm control. It highlights the importance of a One Health approach that links human, animal, and environmental health to address the risks posed by Salmonella biofilms in the food production and public health sectors.},
}
@article {pmid41273435,
year = {2025},
author = {Yu, SM and Li, TT and Fu, BQ and Zhang, NZ},
title = {Molecular diagnosis of Trichinella spp.: current status and future prospects.},
journal = {Parasitology research},
volume = {124},
number = {11},
pages = {136},
pmid = {41273435},
issn = {1432-1955},
support = {2023YFD1801000//National Key Research and Development Program of China/ ; QYXTZX-RKZ2024-03-3//Science and Technology Project of Tibet Autonomous Region/ ; },
mesh = {Animals ; *Trichinellosis/diagnosis/parasitology/veterinary ; *Trichinella/genetics/isolation &amp; purification ; *Molecular Diagnostic Techniques/methods/trends ; CRISPR-Cas Systems ; Humans ; },
abstract = {Trichinellosis, a significant parasitic zoonotic disease, poses a risk to public health as well as economic implications for the safety of animal feed. Consumption of raw or undercooked meat containing Trichinella larvae can lead to trichinellosis infection. Many molecular diagnostic methods have been developed to identify Trichinella spp. at muscle or intestine stages. However, no molecular diagnostic technique is currently advised for routine testing on Trichinella infection in food animals, particularly at the early stage of infection. Here, the authors review the development of molecular diagnostic techniques of Trichinella spp., such as PCR, RT-PCR, LAMP, RPA and other methods to detect Trichinella DNA. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR-Cas) technology holds great promise for diagnostic testing by providing rapid, sensitive and specific methods for detection. Diagnosis of Trichinella spp. based on CRISPR-Cas system may be a promising method meeting the needs of individual testing.},
}
@article {pmid41273433,
year = {2025},
author = {Schreiber, D and Yang, R and Guan, X and Schalper, KT and Hou, C and Li, Z and Hegde, P and Liu, C},
title = {3D-Printed CRISPR-based detection system powered by a reusable handwarmer.},
journal = {Biomedical microdevices},
volume = {27},
number = {4},
pages = {53},
pmid = {41273433},
issn = {1572-8781},
support = {U01CA269147/NH/NIH HHS/United States ; UConn Research Excellence Program (REP) award//University of Connecticut/ ; },
mesh = {*Printing, Three-Dimensional ; Humans ; Human papillomavirus 16/genetics ; *CRISPR-Cas Systems/genetics ; *Lab-On-A-Chip Devices ; DNA, Viral/analysis/genetics ; Equipment Reuse ; },
abstract = {Nucleic acid-based molecular diagnostics are essential for the prevention, early detection, and treatment of cancer and infectious diseases. In this study, we developed a 3D-printed, electricity-free detection system for CRISPR-based nucleic acid detection. To eliminate the need for costly electrical heaters, we developed a reusable heating platform powered by a sodium acetate-based handwarmer. To maintain optimal temperatures for the CRISPR reaction, we designed and fabricated a 3D-printed heatsink filled with docosane wax to regulate the temperature. The fully 3D-printed microfluidic chip integrates finger-activated fluid transport via a 3D-printed flexible blister, a CRISPR reaction chamber, and a lateral flow strip for visual readout. We demonstrated the system's analytical performance by detecting HPV-16 DNA with a sensitivity as low as 1 femtomolar. Additionally, we validated its clinical pilot feasibility using clinical cervical samples, achieving results consistent with standard PCR assays. Overall, this low-cost, reusable, and electricity-free detection system offers a practical solution for point-of-care molecular testing, particularly in resource-limited settings.},
}
@article {pmid41273185,
year = {2025},
author = {Verma, A and Kaur, L and Kandoth, PK},
title = {Agrobacterium rhizogenes-Mediated Hairy Root Transformation for Genome Editing in Recalcitrant Legume Lathyrus sativus.},
journal = {Current protocols},
volume = {5},
number = {11},
pages = {e70256},
doi = {10.1002/cpz1.70256},
pmid = {41273185},
issn = {2691-1299},
mesh = {*Lathyrus/genetics ; *Agrobacterium/genetics ; *Plant Roots/genetics ; *Gene Editing/methods ; *Transformation, Genetic ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; },
abstract = {Lathyrus sativus, commonly known as the grass pea, is a nutritious legume that is resilient to climate change, allowing it to grow in drought, waterlogged, and saline soils. However, developing effective functional genomic tools for this crop has been challenging, primarily due to the absence of reliable and stable transformation protocols. Agrobacterium rhizogenes-mediated hairy root transformation provides a practical and rapid method for validating gene functions using the CRISPR/Cas system. This method has not been applied to grass pea despite its potential. In this article, we present the first protocol for A. rhizogenes-mediated hairy root transformation and CRISPR/Cas genome editing aimed at the functional characterization of candidate genes in L. sativus. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Designing CRISPR/Cas9 construct for targeted gene editing in L. sativus Support Protocol 1: Escherichia coli competent cell preparation and transformation Support Protocol 2: A. rhizogenes competent cell preparation and transformation Basic Protocol 2: A. rhizogenes-mediated hairy root transformation in L. sativus Basic Protocol 3: Screening of transgenic hairy root lines Support protocol 3: DNA isolation from L. sativus hairy roots.},
}
@article {pmid41272318,
year = {2025},
author = {Duan, B and Jin, X and An, X and Xiao, Y and Yang, Q and Zhao, H and Huang, Y and Wang, J and Wang, Q and Du, F and Lu, L and Sun, L and Chen, Z and Zhao, B},
title = {Molecular basis of SAM-AMP synthesis and degradation in the type III-B CRISPR-Cas system.},
journal = {Nature chemical biology},
volume = {},
number = {},
pages = {},
pmid = {41272318},
issn = {1552-4469},
support = {2021hwyq36//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {Upon sensing nonself target RNA, the CorA-associated type III-B CRISPR-Cas system catalyzes S-adenosyl methionine (SAM) and ATP to synthesize SAM-AMP, which activates the effector CorA and triggers immune responses. SAM-AMP can be degraded by NrN and SAM lyase, potentially deactivating the system. Here we find that the type III-B effector complex from Bacteroides fragilis uses a specific mechanism to recognize nonself target RNA and synthesize SAM-AMP. The 3' anti-tag of nonself target RNA induces conformational changes in the Cmr2 subunit, triggering SAM-AMP synthesis independently of the stalk loop of Cmr3 subunit. SAM-AMP binding induces NrN to transit from an open to a closed conformation, enabling hydrolysis of the 3'-5' phosphodiester bond. SAM lyase forms a triangular trimer that specifically degrades SAM-AMP into 5'-methylthioadenosine-AMP and homoserine lactone. These findings unveil unique mechanisms for SAM-AMP synthesis and degradation and provide deeper insights into the molecular basis of type III CRISPR-Cas signaling.},
}
@article {pmid41271814,
year = {2025},
author = {Ueno, R and Ito, S and Oyama, T},
title = {A CRISPR/Cas9-induced restoration of bioluminescence reporter system for single-cell gene expression analysis in plants.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {41271},
pmid = {41271814},
issn = {2045-2322},
support = {JPMJSP2110//Japan Science and Technology Agency/ ; JPMJAL1108//Japan Science and Technology Agency/ ; JP20K06342//Japan Society for the Promotion of Science/ ; 17KT0022//Japan Society for the Promotion of Science/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Arabidopsis/genetics/metabolism ; *Single-Cell Analysis/methods ; *Genes, Reporter ; Plants, Genetically Modified/genetics ; *Luminescent Measurements/methods ; *Gene Expression Regulation, Plant ; Luciferases/genetics/metabolism ; Single-Cell Gene Expression Analysis ; },
abstract = {Bioluminescence monitoring techniques have greatly contributed to revealing a variety of biological regulatory systems in living organisms, including circadian clocks. In plant science, these techniques are applied to long-term quantitative analyses of gene expression behavior. Transient transfection with a luciferase reporter using the particle bombardment method has been used for bioluminescence observations at the single-cell level. This allows for capturing heterogeneity and temporal fluctuations in cellular gene expression, although bioluminescence could fluctuate according to variation in physiological factors associated with the luciferase reaction. We developed a novel CRISPR/Cas9-induced restoration of bioluminescence reporter system, CiRBS, to monitor cellular bioluminescence from a reporter gene in the genome of transgenic Arabidopsis. In this method, the enzymatic activity of an inactive luciferase mutant, LUC40Ins26bp, which has a 26-bp insertion at the 40th codon, was restored by introducing an indel at the insertion site using CRISPR/Cas9. We succeeded in long-term monitoring of the cellular bioluminescence of Arabidopsis plants expressing LUC40Ins26bp, which was restored by transient transfection with CRISPR/Cas9-inducible constructs using particle bombardment. Recombination events via indels were mostly complete within 24 h of CRISPR/Cas9 induction, and 7.2% of CRISPR/Cas9-transfected cells restored bioluminescence. It was estimated that 94% of the bioluminescence-restored cells carried only one chromosome having the optimal recombination construction. Thus, CiRBS allows for reliable single-cell gene expression analysis of cell-to-cell heterogeneity and temporal fluctuations from a single locus.},
}
@article {pmid41271724,
year = {2025},
author = {Elsharkasy, OM and Hegeman, CV and Driedonks, TAP and Liang, X and Lansweers, I and Cotugno, OL and de Groot, IY and de Wit, ZEMNJ and Garcia-Guerra, A and Moorman, NJA and Boonstra, SH and Bosman, EDC and Lefferts, JW and de Voogt, WS and François, JJ and van Wesel, ACW and El Andaloussi, S and Schiffelers, RM and Kooijmans, SAA and Mastrobattista, E and Vader, P and de Jong, OG},
title = {A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10309},
pmid = {41271724},
issn = {2041-1723},
support = {VI.Veni.192.174//Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organisation for Scientific Research)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Extracellular Vesicles/metabolism ; *Aptamers, Nucleotide/metabolism/genetics ; Humans ; *Gene Editing/methods ; Ultraviolet Rays ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; },
abstract = {CRISPR-Cas9 gene editing technology offers the potential to permanently repair genes containing pathological mutations. However, efficient intracellular delivery of the Cas9 ribonucleoprotein complex remains a major hurdle in its therapeutic application. Extracellular vesicles (EVs) are biological nanosized membrane vesicles that play an important role in intercellular communication, and have an innate capability of intercellular transfer of biological cargos, including proteins and RNA. Here, we present a versatile, modular strategy for EV-mediated loading and delivery of Cas9. We leverage the high affinity binding of MS2 coat proteins fused to EV-enriched proteins to MS2 aptamers incorporated into guide RNAs, in combination with a UV-activated photocleavable linker domain, PhoCl. Moreover, we demonstrate that Cas9 can readily be exchanged for other variants, including transcriptional activator dCas9-VPR and adenine base editor ABE8e. Taken together, we describe a robust, modular strategy for successful Cas9 delivery, which can be applied for CRISPR-Cas9-based genetic engineering and transcriptional regulation.},
}
@article {pmid41271690,
year = {2025},
author = {Huang, Z and Dong, Y and Yang, Y and Han, X and Wang, F and Lyon, CJ and Ding, S and Peng, Y and Zhang, G and Hu, C and Huang, H and Yang, L and Zhao, G and Fan, XY and Lu, S and Hu, T and Wang, J},
title = {Thermally programmed one-pot CRISPR assay for on-site pandemic surveillance.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10286},
pmid = {41271690},
issn = {2041-1723},
support = {31922046//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Pandemics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Point-of-Care Systems ; Sensitivity and Specificity ; Temperature ; },
abstract = {The ongoing monkeypox virus outbreak highlights the need for rapid and accurate diagnostics to enhance epidemic control. CRISPR-based assays hold promise, but clinical translation is hindered by high complexity and low throughput. Here, we describe a thermally regulated asynchronous CRISPR-enhanced (TRACE) assay that rapidly and sensitively detects multiple DNA targets in a streamlined, one-pot format. TRACE exhibits a 2.5 copies/test limit of detection - 40 times lower than a canonical one-pot CRISPR. When applied to clinical samples, it achieves 99.5% accuracy across diverse sample types, and can detect MPXV within 11 minutes. Point-of-care TRACE assays meet ASSURED criteria and deliver comparable performance to qPCR, with a fivefold reduced report time, in outpatient settings. Moreover, TRACE enables simultaneous detection of pathogen and host genes at comparable sensitivity to address a critical limitation of current CRISPR assays, which lack internal controls. TRACE thus enables rapid, on-site surveillance to facilitate bench-to-bedside translation of CRISPR diagnostics.},
}
@article {pmid41271362,
year = {2025},
author = {Rahman, MA and Akter, S and Ashrafudoulla, M and Jung, SJ and Rapak, MT and Ha, SD},
title = {CRISPR-Cas systems as emerging tools for precision biofilm control for food safety: Mechanisms and applications.},
journal = {Food research international (Ottawa, Ont.)},
volume = {222},
number = {Pt 2},
pages = {117803},
doi = {10.1016/j.foodres.2025.117803},
pmid = {41271362},
issn = {1873-7145},
mesh = {*Biofilms/growth &amp; development ; *CRISPR-Cas Systems ; *Food Safety/methods ; Gene Editing/methods ; *Food Microbiology/methods ; },
abstract = {Biofilms on food-contact surfaces pose persistent challenges to sanitation, safety, and product quality within food processing. Traditional cleaning methods and broad-spectrum antimicrobials often fail to disrupt the resilient matrix and multispecies communities characteristic of these biofilms. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) systems offer a transformative approach to enhancing food safety, enabling precise modulation of microbial gene networks with applications in diagnostics, programmable sanitation, and targeted microbial control. This review synthesizes recent advances in CRISPR-Cas technology, encompassing Cas9/Cas12-based gene editing, Cas13-mediated RNA targeting, and dead Cas9 (dCas9)-based transcriptional regulation (CRISPR interference/activation, CRISPRi/a), and evaluates their relevance to biofilm prevention and eradication in food environments. We critically assess delivery platforms, including plasmids, nanocarriers, phagemids, and conjugative systems, for their efficiency in complex biofilm settings. The review highlights innovations such as multiplexed repression of redundant pathways, activation of latent antibiofilm functions. These genetic strategies are increasingly being integrated with omics-based analytics (e.g., transcriptomics, proteomics, metabolomics) to reveal systems-level cellular responses and regulatory shifts triggered by biofilm-targeted interventions. We also address the practical limitations, such as delivery barriers, off-target effects, regulatory hurdles, and ethical considerations specific to food applications. Ultimately, we propose a framework for translating CRISPR-Cas technology into scalable, safety-compliant tools for precision control of biofilms in food processing environments. This review aims to guide future research and inform stakeholders on leveraging CRISPR-Cas technology for safe, sustainable, and targeted management of food-associated biofilms.},
}
@article {pmid41202983,
year = {2026},
author = {Hocq, R and Chartier, G and Lopes Ferreira, N and Wasels, F},
title = {CRISPR/anti-CRISPR genome editing in Clostridium beijerinckii.},
journal = {Journal of biotechnology},
volume = {409},
number = {},
pages = {165-169},
doi = {10.1016/j.jbiotec.2025.11.002},
pmid = {41202983},
issn = {1873-4863},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Clostridium beijerinckii/genetics/drug effects ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Listeria monocytogenes/genetics ; Genome, Bacterial ; CRISPR-Associated Protein 9/genetics ; },
abstract = {The development of CRISPR technologies has revolutionized genome editing. However, in bacteria, CRISPR-based methods can be difficult to implement due to the cytotoxicity of CRISPR-associated proteins, which often impair or entirely prevent transformation. In this work, we combine inducible expression of classical CRISPR-Cas9 components with the anti-CRISPR protein AcrIIA4 from Listeria monocytogenes to tightly regulate Cas9 activity. Using this approach, we demonstrate efficient and iterative genome editing in the genetically recalcitrant Clostridium beijerinckii DSM 6423. While deletion of upp alone was not sufficient to render the strain sensitive to 5-fluorouracil, the additional deletion of a second gene involved in the uracil salvage pathway conferred resistance to the drug and validated our gene editing strategy. Collectively, our results show that CRISPR/anti-CRISPR systems can overcome a key limitation of CRISPR-based genome editing and may offer a broadly applicable strategy for engineering otherwise intractable bacterial species.},
}
@article {pmid41202696,
year = {2025},
author = {Ito, S and Nakamura, K and Murata, K and Nakajima, R and Kanou, M and Koketsu, M and Yamana, K and Yamanouchi, K and Ueda, H},
title = {Generation of Cre/LoxP-mediated extracellular TurboID knock-in rats with CRISPR/Cas9 system.},
journal = {Biochemical and biophysical research communications},
volume = {791},
number = {},
pages = {152898},
doi = {10.1016/j.bbrc.2025.152898},
pmid = {41202696},
issn = {1090-2104},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; Rats ; *Integrases/metabolism/genetics ; Biotinylation ; Cell Membrane/metabolism ; },
abstract = {The plasma membrane plays a central role in regulating signal transduction from the extracellular environment to the intracellular space and represents a major site of drug targeting. Proximity-dependent biotinylation with biotin ligases such as BioID and its derivatives, including TurboID and AirID, enables identification of novel protein‒protein interactions by fusion to a target protein. In vivo application of biotin ligases for cell membrane analysis has been attempted; however, methods for expressing biotin ligases on the target cell membrane remain largely limited to viral vector delivery. In this study, we inserted a Cre-dependent TurboID expression cassette into the rat genome to express TurboID on the cell surface via the CRISPR/Cas9 system. We generated TurboID knock-in (KI) rats that express TurboID on the cell surface, enabling biotinylation of extracellular proteins. The TurboID KI rats thus provide a valuable model for in vivo analysis of cell-surface molecules and may facilitate identification of novel drug targets or antigens.},
}
@article {pmid41130365,
year = {2026},
author = {Zou, M and Tao, Y and Shi, B and Xu, R and Zhu, D and Li, Y and Han, R and Wang, R},
title = {CRISPR/Cas9-based gene deletion and targeted metabolomics reveal ectoine flux reprogramming in Halomonas campaniensis.},
journal = {Journal of biotechnology},
volume = {409},
number = {},
pages = {67-76},
doi = {10.1016/j.jbiotec.2025.10.006},
pmid = {41130365},
issn = {1873-4863},
mesh = {*Halomonas/genetics/metabolism ; *Amino Acids, Diamino/metabolism/biosynthesis/genetics ; Metabolomics/methods ; Gene Deletion ; *CRISPR-Cas Systems/genetics ; Betaine/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Ectoine and betaine are widely used compatible solutes. In Halomonas campaniensis XH26, the hom gene is involved in betaine biosynthesis, and the doeA gene participates in ectoine degradation. Deletion of hom and doeA may lead to poorly understood changes in metabolic flux within the ectoine biosynthesis pathway. The metabolically deficient XH26/Δhom and XH26/Δhom/ΔdoeA strains were constructed using a CRISPR/Cas9 approach. Comparative analyses of colony morphology, growth characteristics, and intracellular ectoine yield were conducted to evaluate the regulatory roles of the hom and doeA genes. RT-qPCR and targeted metabolomics were used to assess changes in gene expression related to ectoine biosynthesis and shifts in central carbon metabolic flux. The metabolically deficient strains XH26/Δhom and XH26/Δhom/ΔdoeA were constructed. Compared to the strain XH26, both mutant strains exhibited smaller colony diameters and shorter, broader cells. Intracellular ectoine yield increased by 13.3 % and 33.3 %, respectively, while betaine yield significantly decreased by 73.08 % and 76.92 %. RT-qPCR analysis revealed the significant upregulation of asd, lysC, ectA, ectB, and ectC, suggesting an enhanced metabolic flux toward ectoine biosynthesis. Targeted metabolomics indicated that the differentially abundant metabolites were mainly involved in four key energy metabolism pathways. These results indicate that knocking out the key genes hom and doeA in the ectoine biosynthesis pathway led to the restructuring of carbon metabolic flux in H. campaniensis. More carbon entered the ectoine biosynthesis pathway, resulting in the enhanced production of ectoine and a concomitant reduction in its degradation. These findings offer theoretical support for engineering high-yield ectoine-producing strains.},
}
@article {pmid41048015,
year = {2025},
author = {Li, Y and Zhao, W and Wu, Y and Li, R and Zhang, J and Xie, H and Zhang, K and Li, J},
title = {In vivo CRISPR biosensing.},
journal = {Chemical Society reviews},
volume = {54},
number = {23},
pages = {10977-11016},
doi = {10.1039/d5cs00921a},
pmid = {41048015},
issn = {1460-4744},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA/genetics/analysis ; Gene Editing ; },
abstract = {In vivo biosensing is essential for real-time monitoring of biological processes and disease progression within living organisms. Leveraging the programmable specificity and multifunctionality of CRISPR effectors, in vivo CRISPR-based biosensing has emerged as a powerful tool for highly sensitive and target-specific detection in complex physiological environments. This review presents the fundamental principles, design strategies, and bioanalytical applications of these advanced sensors, focusing on three key approaches: CRISPR-mediated highly efficient in vivo sequence recognition, CRISPR-driven trans-cleavage activity for signal amplification, and the use of base editors and prime editors for sensing-coupled genetic modulation. Critical design parameters-including delivery strategies, intracellular dynamics, and signal amplification mechanisms-are discussed in detail. We further highlight a broad range of applications, including in vivo DNA/RNA imaging, quantification of proteins and small molecules, gene-controlled drug release, dynamic signal recording, environmental response sensing, and lineage tracing in embryogenesis and tumor progression. The current challenges and outlining future directions are also discussed, underscoring the transformative potential of in vivo CRISPR biosensing in both fundamental biology and clinical translation.},
}
@article {pmid40555270,
year = {2025},
author = {Liang, L and Yang, Y and Jacqueline Elise, F and Yu, J and Yin, X and Lu, G and Chen, B and Xing, J},
title = {Potential Applications of the CRISPR-Cas9 System for Research and Treatment of Osteoarthritis.},
journal = {Zeitschrift fur Orthopadie und Unfallchirurgie},
volume = {163},
number = {6},
pages = {510-517},
doi = {10.1055/a-2616-0819},
pmid = {40555270},
issn = {1864-6743},
support = {2308085QH292//Anhui Natural Science Foundation/ ; 82305280//This research did receive grant from National Natural Science Foundation of China/ ; },
mesh = {*Osteoarthritis/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Genetic Therapy/methods ; *Gene Editing/methods ; Disease Models, Animal ; },
abstract = {Osteoarthritis is a common degenerative disease of joint cartilage that affects millions of people in the world, especially the elderly. Progression of osteoarthritis is associated with a plethora of genetic and non-genetic factors. The CRISPR/Cas9 system is emerging as a powerful tool for genome engineering and has remarkable potential for guiding further research into osteoarthritis and may be a viable means for treating the disease. This review discusses existing and potential applications of the CRISPR/Cas9 system in osteoarthritis studies and treatments. Firstly, we briefly summarize the current status and mechanism of this technology. Next, we focus on the latest advances in the application of CRISPR/Cas9 system in elucidating the contributions of various factors to the pathogenesis of osteoarthritis as demonstrated through in vitro studies and animal models. Finally, we provide our perspective on the direction and challenges of studying and treating osteoarthritis with CRISPR/Cas9.},
}
@article {pmid41270733,
year = {2025},
author = {Wei, Z and Lan, Y and Meng, L and Wang, H and Li, L and Li, Y and Zhang, N and Lu, R and Cui, Z and Song, Y and Wang, Y and Li, Y and Yue, Z and Fan, G and Li, Q and Gu, Y and Liu, S and Qian, PY and Meng, L and Shao, C},
title = {Hologenomic insights into the molecular adaptation of deep-sea coral Bathypathes pseudoalternata.},
journal = {Cell host &amp; microbe},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.chom.2025.10.020},
pmid = {41270733},
issn = {1934-6069},
abstract = {Deep-sea coral ecosystems support biodiversity and nutrient cycling through interactions with symbionts. However, their molecular mechanisms remain unexplored. Here, hologenomic analyses of Bathypathes pseudoalternata are applied to uncover molecular adaptations underpinning host-symbiont interactions. Genomic evidence reveals that B. pseudoalternata exhibits adaptations in nutrient transport, immune response, and lysosomal digestion, reflecting its genomic adjustments for a stable symbiosis. Candidatus Nitrosopumilus bathypathes (78.43% ± 3.65%) is inferred to oxidize host-derived ammonia to synthesize amino acids and vitamins to provision the host. The presence of CRISPR-Cas and restriction-modification (R-M) systems suggests that Ca. Bathyplasma bathypathes and Ca. Thalassoplasma bathypathes (10.68% ± 2.99%) may protect the host from viral infections. Ca. Bathybacter bathypathes (8.39% ± 1.53%) is hypothesized to synthesize heme, lipoic acid, and glutathione, which serve dual functions as antioxidants and nutrients. These findings collectively provide insights into how the hologenome contributes to the survival of B. pseudoalternata in the extreme environment.},
}
@article {pmid41270608,
year = {2025},
author = {Zhou, Y and Zhai, J and Chen, H and Qu, Y and Fang, Z and Chen, B and Bao, Z and Chen, D},
title = {Dz-SiG CRISPR: A DNAzyme-Switched G-quadruplex-lock CRISPR system for isothermal and rapid detection of lead ions.},
journal = {Talanta},
volume = {299},
number = {},
pages = {129139},
doi = {10.1016/j.talanta.2025.129139},
pmid = {41270608},
issn = {1873-3573},
abstract = {Lead (Pb[2+]) poses serious risks to health and ecosystems, necessitating rapid, ultrasensitive detection. CRISPR/Cas12a systems offers exceptional specificity and intrinsic signal amplification. Nevertheless, their adaptation to Pb[2+] is hindered by the lack of programmable interfaces to convert small-molecule binding into Cas12a activation. Herein, we report a DNAzyme-Switched G-quadruplex-locked CRISPR (Dz-SiG CRISPR) strategy, enabling ultrasensitive, femtomolar-level detection of Pb[2+]. A RNA G-quadruplex (RG4) structure is conjugated to the 5' end of the crRNA, serving as a conformational lock that suppresses Cas12a's trans-cleavage activity. Upon Pb[2+] binding, the GR-5 DNAzyme catalyzes a hydrolytic cleavage that acts as a molecular switch, releasing the RG4 domain and unleashing active crRNA to trigger Cas12a-mediated cleavage of a fluorogenic reporter, yielding a sharp "off-to-on" fluorescent signal. The Dz-SiG CRISPR system achieves an ultralow limit of detection of 18.91 fM for Pb[2+] and demonstrates outstanding performance in real water and soil samples, with recovery rates ranging from 94.44 % to 99.03 %. The assay can be completed within 30 min, making it highly suitable for rapid on-site lead ion detection. Importantly, the modular Dz-SiG CRISPR framework can be readily reprogrammed for other small molecules by simply substituting the DNAzyme module, offering a generalizable strategy for rapid, ultrasensitive environmental monitoring.},
}
@article {pmid41202468,
year = {2025},
author = {Zingarelli, F and Nanni, J and Cristiano, G and Zannoni, L and Curti, A},
title = {CRISPR-Cas9 in acute myeloid leukaemia: Current state-of-art and future perspectives.},
journal = {Current opinion in pharmacology},
volume = {85},
number = {},
pages = {102582},
doi = {10.1016/j.coph.2025.102582},
pmid = {41202468},
issn = {1471-4973},
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/therapy/diagnosis ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *Genetic Therapy/methods ; },
abstract = {CRISPR-Cas9 gene editing technology has gained attention as a new, reliable and manageable tool for the treatment of previously incurable monogenic diseases. Besides exciting results in this setting, ethical, safety and crucial technical issues have not been fully clarified. More importantly, the role of this potent editing tool in the context of a genetically complex and heterogeneous hematologic malignancy such as acute myeloid leukemia (AML) has not yet been defined to date. In this review we aim to summarize and exploring the ultimate CRISPR-cas9 based strategies for diagnosis, risk stratification and treatment in the context of AML.},
}
@article {pmid41177337,
year = {2025},
author = {Liu, B and Li, Y and Yang, Z and Wu, J and Jiang, Y and Zhao, L and Ge, J},
title = {A rapid and visual detection for canine Adenovirus-2 using CRISPR-Cas13a-based SHERLOCK technology.},
journal = {Journal of microbiological methods},
volume = {239},
number = {},
pages = {107314},
doi = {10.1016/j.mimet.2025.107314},
pmid = {41177337},
issn = {1872-8359},
mesh = {Animals ; Dogs ; *CRISPR-Cas Systems/genetics ; *Adenoviridae Infections/diagnosis/veterinary/virology ; *Adenoviruses, Canine/isolation &amp; purification/genetics ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *Dog Diseases/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Canine adenovirus type 2 (CAdV-2) is an important pathogen causing infectious tracheobronchitis (ITB) and viral enteritis in puppies, often exacerbating clinical symptoms through co-infection with other viruses. However, existing diagnostic methods for CAdV-2 exhibit notable limitations. Specifically, they are time-consuming, require additional nucleic acid purification steps, depend on expensive detection equipment, and necessitate operation by professional personnel. Collectively, these limitations prevent the achievement of rapid and accurate CAdV-2 detection in resource-limited settings. In this study, we established a novel CAdV-2 detection method by integrating CRISPR/Cas13a collateral cleavage activity with HUDSON rapid nucleic acid extraction, recombinase-aided amplification (RAA), and a lateral flow strip. This isothermal assay allows for visual, naked-eye result interpretation and achieves a sensitivity of 10[2] copies/μL as read by lateral flow strips (corresponding to approximately 750 copies per reaction). It showed excellent specificity with no cross-reactivity observed against five other major canine viruses. When tested on 20 clinical samples, the assay demonstrated a 95 % concordance rate with the conventional simplex PCR results. The entire detection process is simple to perform, requires only basic equipment, and delivers results within 90 min. The developed CRISPR/Cas13a-based detection method exhibits significant application potential for CAdV-2 detection. This study develops a CRISPR/Cas13a-based point-of-care diagnostic tool for CAdV-2, delivering rapid, sensitive, and visual detection that significantly facilitates field-based pathogen surveillance and control efforts, while advancing the application of CRISPR diagnostics in veterinary infectious diseases.},
}
@article {pmid41167358,
year = {2025},
author = {Rahangdale, S and Vishwakarma, A and Chauhan, R and Singh, S and Singh, PK},
title = {Engineered sgRNA captures single-stranded donor template and delivers at the DSB site to enhance HDR.},
journal = {International journal of biological macromolecules},
volume = {332},
number = {Pt 2},
pages = {148614},
doi = {10.1016/j.ijbiomac.2025.148614},
pmid = {41167358},
issn = {1879-0003},
mesh = {Saccharomyces cerevisiae/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; *DNA Breaks, Double-Stranded ; *Recombinational DNA Repair ; *DNA, Single-Stranded/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {The CRISPR-Cas9 system from Streptococcus pyogenes has revolutionized genome modification through precise editing across a wide range of organisms. Yeast supports efficient genome editing via plasmid-based Cas9-gRNA expression, while higher eukaryotes often require genome-integrated cassettes or RNP delivery. In this study, we engineered CRISPR components to enhance nuclear targeting and editing efficiency. We demonstrated the proof of concept in Saccharomyces cerevisiae using its CAN1 locus. We developed a dual-host compatible vector, encoding Cas9 nuclease fused with three nuclear localization signals (Cas9-3xNLS). The recombinant protein, expressed in E. coli and purified on a Ni-NTA column, showed DNA cleavage in an in vitro assay. Genome editing efficacy of Cas9-3xNLS was demonstrated in S. cerevisiae AH109 strain. Further, we engineered sgRNAs by extending their ends to facilitate the annealing to ssODN. We synthesized ssODNs having a complementary sequence either at 3' or 5' to anchor with sgRNAs. sgRNAs (unmodified and end extended) and ssODNs were introduced into yeast in various combinations. sgRNA with a 3' ssDNA-anchoring motif and ssODN representing the antisense strand of the target gene with sgRNA complementary motif at the 5' end (free homology arm at 3') improved HDR efficiency significantly. This combination yielded about a 1.64-fold increase in canavanine-resistant colonies as compared to the control via precise insertion of a stop codon. In contrast, extension of sgRNA at the 5' end did not show any advantage. This approach is flexible and easy to use and has the potential to enhance homology-directed repair in diverse organisms.},
}
@article {pmid41093192,
year = {2025},
author = {Chou, SJ and Wang, CH and Chang, YL and Fang, WC and Hwang, DK and Hsiao, YJ and Luo, YH and Lo, WL and Viet, NQ and Tang, KY and Lan, YT and Hsu, CC and Chen, SJ and Lin, TC and Yang, YP and Chiou, SH},
title = {Dual delivery of supramolecular nanoparticle-carried minicircle donor DNA with Cas9/gRNA improved HITI knock-in efficiency in X-linked juvenile retinoschisis.},
journal = {International journal of biological macromolecules},
volume = {332},
number = {Pt 2},
pages = {148300},
doi = {10.1016/j.ijbiomac.2025.148300},
pmid = {41093192},
issn = {1879-0003},
mesh = {Humans ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; *Retinoschisis/genetics/therapy ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *DNA, Circular/genetics ; Gene Editing/methods ; *Gene Knock-In Techniques/methods ; Gene Transfer Techniques ; CRISPR-Associated Protein 9/genetics ; Genetic Therapy/methods ; HEK293 Cells ; Eye Proteins/genetics ; Plasmids/genetics ; },
abstract = {X-linked retinoschisis (XLRS) is a hereditary mutation of the RS1 gene and is characterized by early-onset maculopathy with severe visual impairment. Current gene therapy utilizing CRISPR-associated protein 9 (Cas9) is ongoing; however, the optimization of nonviral/nanoparticle CRISPR/Cas9-based therapeutics for gene delivery into nondivided retinal neurons remains undetermined. Minicircles DNA, a circular DNA molecule lacking bacterial backbone sequences, has gained recognition for improving transfection efficiency and biosafety. Here, we developed a supramolecular nanoparticle (SMNP)-editing platform in which SMNPs carrying CRISPR/Cas9 integrated minicircle donor DNA (mc dDNA) to achieve highly efficient and precise gene knock-in. To increase the efficiency of RS1 gene knock-in, we replaced the conventional mc dDNA with a single flanking-Cas9/cut site. Furthermore, using homology-independent targeted integration (HITI) as an editing-nondivided cell strategy, SMNP-carried CRISPR/Cas9 could effectively facilitate the dual delivery of mc-RS1/GFP dDNA and Cas9/gRNA plasmids. Compared with the delivery of the Cas9/gRNA plasmid alone (~20 %), the delivery of mc-RS1/GFP dDNA via SMNPs had significantly higher transfection efficiency (90 %). Further flow cytometry analysis revealed that 5.99 % of the FACS-positive cells were detected in the mc-RS1/GFP dDNA group, markedly exceeding the 2.21 % with long-term expression in the conventional dDNA group. Moreover, when XLRS/iPSC-derived retinal neuron organoids were used as a patient-based disease model, compared with conventional plasmid-based delivery, robust RS1 expression with integration sustained transgene expression in XLRS/iPSC-derived retinal organoids. Collectively, these findings indicated that SMNP-mediated dual delivery of the Cas9/gRNA plasmid and mc-RS1/GFP dDNA substantially enhanced RS1-targeted integration with long-term transgene expression, providing safer and effective gene therapy for the treatment of XLRS.},
}
@article {pmid41270157,
year = {2025},
author = {Li, Y and Wu, Y and Zheng, Z and Wu, Y and Zhang, Y and Zhang, J and Quan, F and Zhao, W and Xu, R and Li, Y and Gao, H and Zhang, K},
title = {Renal clearable CRISPR nanosensor targeting mitochondrial DNA mutation for noninvasive monitoring of tumor progression and metastasis.},
journal = {Science advances},
volume = {11},
number = {47},
pages = {eadz4594},
pmid = {41270157},
issn = {2375-2548},
mesh = {*DNA, Mitochondrial/genetics ; *Mutation ; Humans ; *CRISPR-Cas Systems ; Animals ; Disease Progression ; Neoplasm Metastasis ; Mice ; Biomarkers, Tumor/genetics/urine ; Cell Line, Tumor ; *Biosensing Techniques/methods ; Mitochondria/genetics ; Lung Neoplasms/genetics ; },
abstract = {Mitochondrial DNA (mtDNA) mutations are emerging as important molecular features of tumorigenesis. Liquid biopsies, involving analysis of cell-free mtDNA, enable early cancer detection but suffer from low sensitivity due to scarce analytes. Here, we developed a CRISPR/Cas12a-mediated urinary biomarker, termed CasUber, for in vivo monitoring of tumor progression and metastasis. Our results demonstrate that CasUber can deliver a CRISPR detection system into tumor cell mitochondria, leverage the single-nucleotide variant recognition ability and trans-cleavage activity of Cas12a to convert tumor-specific mtDNA mutations into renal-clearable fluorescent biomarkers, and exocytosed along with the natural efflux pathway of damaged mtDNA. As a result, CasUber enables discrimination of ultrasmall tumor lesions (~1 cubic millimeter) and detection of lung tumor nodules earlier than bioluminescence imaging in a blood-lung metastasis model. This renal clearable nanosensor allows in situ recognition of specific gene mutation to generate amplified signals, overcoming the limitation of low mtDNA abundance and enabling noninvasive and ultrasensitive monitoring of tumor progression and metastasis via a simple urine test.},
}
@article {pmid41266743,
year = {2025},
author = {Zhang, W and Shi, J and Wang, B and Qu, H and Wu, X and Wang, X},
title = {An integrated Aptamer-CRISPR-Cas12a method for rapid and sensitive detection of carbendazim.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {41119},
pmid = {41266743},
issn = {2045-2322},
support = {22A310016//Henan Province Higher Education Key Scientific Research Project/ ; },
mesh = {*Carbamates/analysis ; *Aptamers, Nucleotide/genetics/chemistry ; *CRISPR-Cas Systems ; *Benzimidazoles/analysis ; Limit of Detection ; *Biosensing Techniques/methods ; *Pesticide Residues/analysis ; Reproducibility of Results ; },
abstract = {Concerns over carbendazim (CBZ) pesticide residues in agricultural products and medicinal herbs have intensified due to their potential health and environmental risks. While existing detection techniques offer distinct advantages, they are often limited by complex procedures, specialized equipment, and high costs. To address these challenges, we developed a novel Aptamer-CRISPR/Cas12a assay, which combines the specificity of aptamers with the high sensitivity and precision of the CRISPR/Cas12a system. This assay achieves a linear detection range of 10-5000 ng/mL (R[2] = 0.9639 at 15 min and R[2] = 0.9774 at 30 min) and a limit of detection (LOD) of 10 ng/mL. In real samples, the average recovery rate of CBZ ranges from 92.10% to 102.86%, demonstrating robust accuracy and reliability. Notably, the method is user-friendly, requires minimal equipment, and delivers results in about 40 min, making it suitable for field applications. Furthermore, the crRNA serves as a universal sequence, enabling the detection of different targets by simply replacing the aptamer and complementary strand, while keeping the CRISPR/Cas12a system intact. This streamlined approach enhances flexibility and broad applicability. In conclusion, the Aptamer-CRISPR/Cas12a assay offers a practical solution for monitoring agricultural products, Chinese herbal medicine, and environmental safety.},
}
@article {pmid41266199,
year = {2025},
author = {Wu, X and Wang, M and Luo, S and Zhou, Z and Wang, Y and Du, G and Chen, J and Liu, X},
title = {Dual enhancement of mycoprotein nutrition and sustainability via CRISPR-mediated metabolic engineering of Fusarium venenatum.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.09.016},
pmid = {41266199},
issn = {1879-3096},
abstract = {Mycoprotein (MP) production represents a promising environmentally sustainable strategy to address global protein deficit. To enhance the nutritional profile and production efficiency of MP, we employed CRISPR/Cas9-mediated scarless gene knockout and obtained a Fusarium venenatum strain (designated FCPD), which exhibited a 32.9% increase in essential amino acid index (EAAI) through targeted truncation of competitive metabolic pathways and regulation of amino acid metabolism or biosynthesis. FCPD achieved a 44.3% reduction in substrate consumption while improving MP production rate by 88.4% compared with the wild type (WT) strain. The cradle-to-gate life cycle assessment (LCA) shows that FCPD could reduce environmental impacts such as global warming potential (GWP) by 4-61.3% under production scenarios in six representative countries. Comparative environmental performance demonstrated the superiority of FCPD-MP over cell-cultured meat and chicken meat. These findings establish CRISPR/Cas technology and metabolic engineering as the dual-purpose tool for both nutritional enhancement and environmental impact mitigation in alternative protein production.},
}
@article {pmid41265250,
year = {2025},
author = {Ramachandran, H and Becker, A and Dobner, J and Hildebrandt, B and Distelmaier, F and Rossi, A and Anand, R},
title = {CRISPR/Cas9-mediated editing of MIC13 in human induced pluripotent stem cells: A model for mitochondrial hepato-encephalopathy.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103870},
doi = {10.1016/j.scr.2025.103870},
pmid = {41265250},
issn = {1876-7753},
abstract = {MIC13 is essential for cristae formation and functions as a key component of the large mitochondrial multi subunit MICOS complex. Mutations in MIC13 causes severe mitochondrial disease called mitochondrial hepato-encephalopathy. In this study, we describe the generation of a human induced pluripotent stem cell (iPSC) line carrying a patient-specific MIC13 mutation, introduced using a CRISPR/Cas knock-in approach. The resulting iPSC line will provide a valuable model to study the pediatric severe mitochondrial disease and to determine the pathological mechanisms as well as to facilitate the identification of potential therapeutic targets in the future.},
}
@article {pmid41264989,
year = {2025},
author = {Liu, X and Yang, M and Sun, D and Lu, C and Ma, Y and Jiang, Y and Ouyang, R and Miao, Y},
title = {Integrating amplification strategies and functional nanomaterials for advanced electrochemical biosensing of MicroRNA.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {168},
number = {},
pages = {109170},
doi = {10.1016/j.bioelechem.2025.109170},
pmid = {41264989},
issn = {1878-562X},
abstract = {MicroRNAs (miRNAs) are crucial disease biomarkers, yet their short length, low abundance, and high sequence homology pose significant challenges for sensitive detection. Electrochemical biosensing presents a promising alternative, though effective signal amplification remains essential. This review summarizes recent advances in amplification strategies for electrochemical miRNA detection, covering nucleic acid-based techniques-such as hybridization chain reaction (HCR), rolling circle amplification (RCA), and catalytic hairpin assembly (CHA)-as well as nanomaterial-assisted approaches using metal-organic frameworks and transition metal dichalcogenides. Key mechanisms, advantages, and limitations of each method are discussed, along with performance metrics (e.g., detection limit and linear range) and emerging hybrid systems like RCA-CRISPR/Cas. Current challenges, including probe complexity and nanomaterial aggregation, are also addressed. Finally, the review highlights future directions involving multi-mechanism integration and clinical translation, offering insights for the development of highly sensitive and reliable electrochemical biosensors to advance precision medicine.},
}
@article {pmid41233602,
year = {2025},
author = {Strefeler, A and Baker, ZN and Chollet, S and Foged, MM and Guerra, RM and Ivanisevic, J and Gallart-Ayala, H and Pagliarini, DJ and Jourdain, AA},
title = {Uridine-sensitized screening identifies demethoxy-coenzyme Q and NUDT5 as regulators of nucleotide synthesis.},
journal = {Nature metabolism},
volume = {7},
number = {11},
pages = {2221-2235},
pmid = {41233602},
issn = {2522-5812},
support = {310030_200796//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Pyrophosphatases/metabolism/genetics ; Humans ; *Nucleotides/biosynthesis ; *Uridine/metabolism/pharmacology ; Phosphoribosyl Pyrophosphate/metabolism ; CRISPR-Cas Systems ; Pyrimidines/biosynthesis ; },
abstract = {Rapidly proliferating cells require large amounts of nucleotides, making nucleotide metabolism a widely exploited therapeutic target against cancer, autoinflammatory disorders and viral infections. However, regulation of nucleotide metabolism remains incompletely understood. Here, we reveal regulators of de novo pyrimidine synthesis. Using uridine-sensitized CRISPR-Cas9 screening, we show that coenzyme Q (CoQ) is dispensable for pyrimidine synthesis, in the presence of the demethoxy-CoQ intermediate as alternative electron acceptor. We further report that the ADP-ribose pyrophosphatase NUDT5 directly binds PPAT, the rate-limiting enzyme in purine synthesis, which inhibits its activity and preserves the phosphoribosyl pyrophosphate (PRPP) pool. In the absence of NUDT5, hyperactive purine synthesis exhausts the PRPP pool at the expense of pyrimidine synthesis, which promotes resistance to purine and pyrimidine nucleobase analogues. Of note, the interaction between NUDT5 and PPAT is disrupted by PRPP, highlighting an intricate allosteric regulation. Overall, our findings reveal a fundamental mechanism of nucleotide balance and position NUDT5 as a regulator of nucleobase analogue metabolism.},
}
@article {pmid40992601,
year = {2026},
author = {Demirayak, PS and Akay Sazaklioglu, S},
title = {CRISPR for detection of drug resistance genes.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {579},
number = {},
pages = {120626},
doi = {10.1016/j.cca.2025.120626},
pmid = {40992601},
issn = {1873-3492},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Drug Resistance/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Resistance to antibiotics, anticancer, antiviral, and antiparasitic drugs has become one of the greatest threats to modern medicine, seriously straining global health systems. Antimicrobial resistance threatens the integrity of the health system by reducing the effectiveness of treatment protocols such as chemotherapy, organ transplantation, and major surgical interventions. In this case, not only the development of new drugs but also the rapid, sensitive, and specific detection of resistant microorganisms and genetic markers is of vital importance. Therefore, the need for more innovative diagnostic approaches suitable for field applications is increasing. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based molecular diagnostic systems developed in recent years stand out as strong candidates that can fill the gap in this area. Thanks to their ability to recognize and target specific DNA or RNA sequences with high specificity, CRISPR systems enable rapid and sensitive detection of drug resistance genes. Various CRISPR effector proteins, such as Cas9, Cas12, and Cas13, have the potential to revolutionize diagnostic technologies due to their ability to both target-specifically cut and generate signals. This review will focus on the application of CRISPR technology for detecting drug resistance genes. In addition, the sensitivity, specificity, application areas, and technical challenges of the systems will be discussed through literature examples of current applications. The review aims to synthesize scientific developments in this field by examining how CRISPR-based diagnostic approaches can play a role in the global fight against drug resistance and to provide a guiding resource for future research.},
}
@article {pmid40992599,
year = {2026},
author = {Jiang, T and Zhang, C and Wang, D and Guo, Z and Guo, Y and Liu, H and Wang, Z},
title = {Rapid molecular diagnostic method for Gardnerella vaginalis based on CRISPR-Cas12a and recombinase-aided amplification (RAA).},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {579},
number = {},
pages = {120625},
doi = {10.1016/j.cca.2025.120625},
pmid = {40992599},
issn = {1873-3492},
mesh = {*Gardnerella vaginalis/genetics/isolation &amp; purification ; Humans ; Female ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; *Molecular Diagnostic Techniques/methods ; *Vaginosis, Bacterial/diagnosis/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Imbalance of the vaginal microbiota, particularly the overgrowth of Gardnerella vaginalis, is the primary cause of bacterial vaginosis (BV), which poses a significant threat to women's reproductive health. Therefore, early and rapid diagnosis of BV is crucial. Current laboratory diagnostic methods for BV mainly rely on Amsel's clinical criteria, bacterial culture, and PCR techniques. However, these methods have notable limitations: Amsel's criteria are subject to operator subjectivity, culture methods are time-consuming and require specialized expertise, while PCR necessitates expensive instrumentation. These constraints hinder their widespread clinical application. To address this issue, developing a highly accurate and low-cost molecular diagnostic method holds significant clinical value for BV detection. In recent years, recombinase-aided amplification (RAA) and CRISPR-Cas12a gene-editing technologies have achieved groundbreaking progress in nucleic acid detection. This study innovatively integrates RAA isothermal amplification with CRISPR-Cas12a detection to successfully establish a rapid nucleic acid detection platform for Gardnerella vaginalis. Experimental results demonstrate that this platform achieves a detection sensitivity of 10 copies/mL for Gardnerella vaginalis genomic DNA, with no cross-reactivity against other common reproductive tract pathogens. In validation tests using 44 clinical vaginal swab samples, the platform showed a 100.00 % positive agreement rate compared to qPCR. These findings confirm that the CRISPR-Cas12a-based detection platform exhibits excellent specificity, sensitivity, and reliability, serving as an effective tool for monitoring Gardnerella vaginalis colonization levels. This approach provides a novel molecular diagnostic solution for early BV screening and prevention.},
}
@article {pmid41264695,
year = {2025},
author = {Rönspies, M and Khosravi, S and Helia, O and Valisi, A and Fajkus, J and Fojtová, M and Houben, A and Puchta, H},
title = {CRISPR-Cas-mediated heritable chromosome fusions in Arabidopsis.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6775},
pages = {843-848},
doi = {10.1126/science.adz8505},
pmid = {41264695},
issn = {1095-9203},
mesh = {*Arabidopsis/genetics ; *Chromosomes, Plant/genetics ; *CRISPR-Cas Systems ; Gene Editing ; *Genome, Plant ; Karyotype ; Meiosis ; Recombination, Genetic ; Gene Fusion ; },
abstract = {The genome of Arabidopsis thaliana consists of 10 chromosomes. By inducing CRISPR-Cas-mediated breaks at subcentromeric and subtelomeric sequences, we fused entire chromosome arms, obtaining two eight-chromosome lines. In one line, both arms of chromosome 3 were fused to chromosome 1. In another line, the arms were transferred to chromosomes 1 and 5. Both chromosome number-reduced lines were fertile. Phenotypic and transcriptional analyses revealed no differences compared with wild-type plants. After crossing with the wild type, the progeny showed reduced fertility. The meiotic recombination patterns of the transferred chromosome arms were substantially changed. Directed chromosome number changes in plants may enable new breeding strategies, redefining linkage groups and establishing genetic barriers. Moreover, our data indicate that plants are highly robust to engineered karyotype changes.},
}
@article {pmid41263111,
year = {2025},
author = {Feng, X and Li, Y and Zheng, J and Chen, X and Yang, S and Chen, Y and Li, SC},
title = {MicrobialScope: an integrated genomic resource with rich annotations across bacteria, archaea, fungi, and viruses.},
journal = {Nucleic acids research},
volume = {},
number = {},
pages = {},
doi = {10.1093/nar/gkaf1234},
pmid = {41263111},
issn = {1362-4962},
support = {C2004-23Y//Young Collaborative Research/ ; JCYJ20220818101201004//Shenzhen Science and Technology Program/ ; 32300527//National Natural Science Foundation of China/ ; 32470695//National Natural Science Foundation of China/ ; 2022A1515110784//Guangdong Basic and Applied Basic Research Foundation/ ; 2023B0303040004//Key-Area Research and Development Program of Guangdong Province/ ; TC2024JC43//Basic Research Programs of Taicang, 2024/ ; //Shenzhen-Hong Kong Institute of Brain Science/ ; //SIAT-HKUST Joint Laboratory of Brain Science/ ; },
abstract = {Microorganisms, including bacteria, archaea, fungi, and viruses, are the most taxonomically diverse and ecologically dominant life forms on Earth, playing critical roles in ecosystems, human health, and industrial applications. While existing microbial databases such as BV-BRC and IMG archive both monoisolate and metagenome-assembled genomes (MAGs) across domains, challenges remain in standardized, multi-level annotations and interactive tools for all microbial groups. Here, we present MicrobialScope (https://microbial.deepomics.org/), a comprehensive microbial genomic platform that integrates large-scale genome collections, multilevel annotations, and interactive visualizations. MicrobialScope harbors 2 411 503 bacterial, 24 472 archaeal, 20 203 fungal, and 188 267 viral genomes derived from both monoisolate assemblies and MAGs. Integrating 15 state-of-the-art bioinformatics tools and 10 specialized databases, MicrobialScope provides extensive annotations encompassing basic genomic features, genomic element prediction (e.g., genes, tRNAs, tmRNAs, CRISPR-Cas and anti-CRISPR elements, secondary metabolite biosynthetic clusters, signal peptides, and transmembrane proteins), and functional and structural annotations. This includes 1 072 114 935 proteins with diverse annotations, 24 640 186 tRNAs and tmRNAs, 140 888 CRISPR-Cas systems, 173 256 anti-CRISPR elements, 105 121 secondary metabolite biosynthetic clusters, 13 235 096 signal peptides, and 50 811 729 transmembrane proteins. In addition, MicrobialScope offers unrestricted access to all data resources, interactive visualization tools, and built-in online analytical modules for intuitive exploration and comparative analysis. With its extensive genome collection, comprehensive annotations, and user-friendly interface, MicrobialScope serves as a scalable platform to advance genome research across diverse microbial domains.},
}
@article {pmid41262458,
year = {2025},
author = {Gao, Z and Gao, Y and Wang, S and Li, X and Cao, W and Deng, W and Yao, L and Wei, X and Zhang, Z and Wang, S and Zhang, Y and Li, M and Xie, Y},
title = {Application progress and biosafety challenges of gene editing and synthetic biotechnology in diagnosis, treatment and prevention of infectious diseases.},
journal = {Biosafety and health},
volume = {7},
number = {5},
pages = {312-322},
pmid = {41262458},
issn = {2590-0536},
abstract = {Global infectious disease prevention faces escalating challenges due to the continual emergence of novel pathogens and rapid viral mutations. Synthetic biology has revolutionized this field by enabling precise diagnostics, innovative vaccine platforms, and targeted therapeutics, yet it simultaneously raises concerns regarding dual-use potential, biosafety, and ethical governance. This systematic review (2015-2025, PubMed, Web of Science, Scopus) focuses on CRISPR-based diagnostics, synthetic vaccines, and engineered probiotics. CRISPR/Cas systems such as DETECTR (Cas12a) and SHERLOCK (Cas13a) demonstrate high sensitivity and rapid pathogen detection (e.g., SARS-CoV-2, Ebola), but their misuse could enhance pathogen virulence or enable bioweapon development. mRNA and viral vector vaccines offer flexible and rapid responses to emerging infections but encounter limitations in molecular stability, delivery system toxicity, and ecological safety. Engineered probiotics, designed as "living therapeutics," can detect pathogens and modulate immune responses, yet pose potential risks of horizontal gene transfer and host-specific variability. Overall, while synthetic biology provides transformative tools for infectious disease control, it necessitates robust global regulatory frameworks, standardized biosafety practices, and ethical oversight to ensure responsible and sustainable application.},
}
@article {pmid41262327,
year = {2025},
author = {Jia, Y and Horvath, K and Rananaware, SR and Jain, PK and Sampath, J},
title = {Exploring the temperature stability of CRISPR-Cas12b using molecular dynamics simulations.},
journal = {Molecular systems design &amp; engineering},
volume = {},
number = {},
pages = {},
pmid = {41262327},
issn = {2058-9689},
abstract = {The thermal stability of CRISPR-Cas nucleases is a critical factor for their successful application in 'one-pot' diagnostic assays that utilize high-temperature isothermal amplification. To understand the atomistic mechanism of stabilization in a previously engineered variant of the thermostable BrCas12b protein, we performed all-atom molecular dynamics (MD) simulations on the wild-type and mutant forms of apo BrCas12b. High-temperature simulations reveal a small structural change along with greater flexibility in the PAM-interacting domain of the mutant BrCas12b, with marginal structural and flexibility changes in the other mutated domains. Comparative essential dynamics analysis between the wild-type and mutant BrCas12b at both ambient and elevated temperatures provides insights into the stabilizing effects of the mutations. Our findings offer comprehensive insights into the important protein motions induced by these mutations. These results provide insights into thermal stability mechanisms in BrCas12b that may inform the future design of CRISPR-based tools.},
}
@article {pmid41262251,
year = {2025},
author = {Stelcer, E and Wozniak, A and Magner, D and Zeyland, J},
title = {Genetically modified pigs with α1,3-galactosyltransferase knockout and beyond: a comprehensive review of xenotransplantation strategies.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1663246},
pmid = {41262251},
issn = {1664-3224},
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Galactosyltransferases/genetics/deficiency ; *Animals, Genetically Modified ; Swine ; *Graft Rejection/immunology/genetics/prevention &amp; control ; Gene Knockout Techniques ; Humans ; Heterografts/immunology ; },
abstract = {Xenotransplantation holds promise to eliminate the shortage of organs intended for humans in need. Pigs constitute the most suitable organ xenograft donor due to the fact that their organ anatomy physiological metabolism and immune system resemble those of humans. However, swine organs rapidly cause hyperacute rejection (HAR) and acute humoral xenograft rejection (AHXR) after transplantation. HAR and AHXR are caused by the presence of xenoreactive natural immunoglobulins directed toward a galactose alpha1-3-galactose (alpha-Gal) epitope on porcine vascular endothelium. In order to suppress both types of rejection, pigs with alpha1,3-galactosyltransferase gene knockout (GT-KO) and other genetic modifications (like simultaneous expression of the human complementary regulatory proteins) are intensively investigated. This review highlights the usefulness of GT-KO pig - derived organs such as kidney, heart, corneal, and lung in xenotransplantation. To obtain transgenic pigs researchers can use several techniques based on pronuclear and cytoplasmic microinjection, somatic cell nuclear transfer (SCNT), viral transduction of DNA and DNA transposable element -based technology, site specific nucleases and modifications of the CRISPR/Cas bacterial immune system. Some additional strategies like targeted immunosuppression or tolerance induction of B and T cells will be essential for sustained survival of xenografts. Although xenotransplantation with the use of pigs is a very rapidly evolving field, more research is needed to create perfectly compatible with the human immune system organs.},
}
@article {pmid41261856,
year = {2025},
author = {Xiao, Y and Zhao, R and Bao, Y and Lu, B and Jiang, Y and Tang, Y and Li, B},
title = {Cas12a-assisted split crRNA complex for analysis and detection of diverse entities.},
journal = {Nucleic acids research},
volume = {53},
number = {21},
pages = {},
pmid = {41261856},
issn = {1362-4962},
support = {SKL202402017//The Science and Technology Development Plan Project of Jilin Province/ ; SKL202302030//The Science and Technology Development Plan Project of Jilin Province/ ; 20240101005JJ//Jilin Province Science Fund for Distinguished Young Scholars/ ; 23GZZ03//Major Project of Changchun State Key Laboratory/ ; 20230203193SF//Key R&amp;D Program of Jilin Province/ ; 22525405//National Nature Science Foundation of China/ ; 22504138//National Nature Science Foundation of China/ ; 22474135//National Nature Science Foundation of China/ ; 22374142//National Nature Science Foundation of China/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Cas Systems ; MicroRNAs/genetics/analysis ; DNA, Single-Stranded/genetics ; DNA/genetics ; *Bacterial Proteins/genetics/metabolism ; *Biosensing Techniques/methods ; },
abstract = {The Cas12a-crRNA system possesses inherent sequence-specific recognition of double-stranded DNA/single-stranded DNA (dsDNA/ssDNA) coupled with trans-cleavage activity toward ssDNA, making it a powerful tool for nucleic acid diagnostics. However, its application beyond nucleic acid targets remains challenging, limiting its potential as a universal detection platform. In this study, we systematically explore the key parameters governing the activation of a Cas12a-split crRNA system and established a comprehensive set of design guidelines. Building on these findings, we developed CASCADE (Cas12a-Assisted Split crRNA Complex for Analysis and Detection of Diverse Entities), an adaptable detection platform that extends Cas12a's application beyond nucleic acids. Using microRNA as a model, we validated the system's sensitivity, specificity, and mismatch discrimination capability. Additionally, we successfully demonstrated its capability for non-nucleic acid target detection by detecting tobramycin, kanamycin, biotin, and tetracycline repressor protein, confirming its sensitivity and specificity. Finally, by integrating a lateral flow assay (LFA), we enhanced the portability of CASCADE, enabling user-friendly, on-site detection. This work expands the application scope of the Cas12a system and offers a promising strategy for point-of-care diagnostics or environmental monitoring.},
}
@article {pmid41261174,
year = {2025},
author = {Seker-Polat, F and Rogozinska, M and Ban, Y and Abdula, F and Buyukcelebi, K and Xie, P and Fan, J and Abbaszadeh, N and Kingham, Y and Paylakhi, SZ and Zhang, B and Adli, M},
title = {Druggable genome CRISPR screening identifies the KEAP1/NRF2 axis as a mediator of PD-L1 expression.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1610},
pmid = {41261174},
issn = {2399-3642},
support = {R01CA267544//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; U54CA268084//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*B7-H1 Antigen/genetics/metabolism ; Humans ; *Kelch-Like ECH-Associated Protein 1/metabolism/genetics ; *NF-E2-Related Factor 2/metabolism/genetics ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Animals ; Female ; Mice ; CRISPR-Cas Systems ; Pancreatic Neoplasms/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Signal Transduction ; Ovarian Neoplasms/genetics ; },
abstract = {Cancer cells rapidly induce PD-L1 expression in response to inflammatory cytokines such as IFNγ from cytotoxic T cells. Increased surface PD-L1 is a primary mechanism of cancer cells evading cytotoxic T-cell-mediated immune clearance. Identifying how cancer cells increase PD-L1 expression may yield clinically relevant immune checkpoint regulators. However, the key regulators and molecular mechanisms mediating rapid PD-L1 induction are yet to be understood entirely. To identify targetable mechanisms controlling cytokine-induced PD-L1 expression, we performed functional CRISPR gene KO screening with a custom-designed sgRNA library that targets "druggable" genes. We performed the screening in 6 different cancer lines: 3 ovarian (OVCAR4, CaOV3, and SKOV3) and three pancreatic cancer (MiaPaca2, ASPC1 and KP4) cell lines. The screening recovered the known regulators of PD-L1 expression and uncovered several novel regulators of PD-L1 that control its expression in all cell lines or in a cancer-type-specific fashion. For example, while genetic or pharmacological depletion of CSNK1A1 results in reduced PD-L1 expression in ovarian cancer cells, CDK1 depletion modulates PD-L1 in pancreatic cancer cell lines. Significantly, we discovered that KEAP1 depletion or pharmacological inhibition diminishes PD-L1 in all cell lines tested (n = 6). Mechanistically, KEAP1 depletion-mediated reduced PD-L1 is due to transcriptional repression of the PD-L1 gene by NRF2 activation. As such, depletion of NRF2 restores PD-L1 expression, while its overexpression leads to diminished PD-L1 expression. Supporting this, pharmacological NRF2 activation resulted in significant antitumor immunity with increased cytotoxic effector T cell infiltration and reduced exhausted T cells, resulting in smaller xenografted tumors. These findings establish the KEAP1/NRF2 axis as a novel and potentially druggable mechanism of IFNγ-meditated PD-L1 expression in cancer cells.},
}
@article {pmid41260544,
year = {2025},
author = {Gupta, A and Beg, MA and Badhwar, S and Srivastava, S and Srivastava, R and Puri, N and Saxena, A and Abdin, MZ and Selvapandiyan, A},
title = {Nucleoside diphosphate kinase (LdNDK2): A metacyclogenesis-regulating kinase essential for Leishmania parasite survival within eukaryotic host cells.},
journal = {Microbial pathogenesis},
volume = {210},
number = {},
pages = {108192},
doi = {10.1016/j.micpath.2025.108192},
pmid = {41260544},
issn = {1096-1208},
abstract = {Nucleoside diphosphate kinase (NDK) transfers phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphates (NDPs) via a ping-pong mechanism, benefiting both prokaryotes and eukaryotes. In Leishmania donovani, we identified a putative NDK2 (LdNDK2), hypothesized to play a crucial role in nucleotide metabolism and cellular energy regulation. To investigate its function and enzymatic properties, we cloned, expressed, and purified recombinant LdNDK2, confirming enzymatic activity via the ADP-Glo assay. The secondary structure and thermal stability were analyzed using circular dichroism spectroscopy, while intrinsic tryptophan fluorescence assays revealed that ATP's gamma phosphate is first transferred to rLdNDK2, forming an intermediate phospho-enzyme complex. We further evaluated the role of rLdNDK2 in ATP-mediated cytolysis of LPS-activated THP-1 cells by measuring lactate dehydrogenase release. Using the CRISPR-Cas9 method, we fluorescently tagged LdNDK2, localizing it to distinct regions of the endomembrane system. In vitro growth studies of LdNDK2 deleted (LdNDK2[-/-]) procyclic promastigotes, through CRISPR-Cas9, revealed that these mutants exhibit a longer flagellum, a reduced cell body, an altered cell division cycle, and increased growth compared to wild-type parasites. LdNDK2[-/-] parasites were arrested at the metacyclic stage, contrasting with continuous differentiation in parental counterparts. Morphological alterations and growth defects were confirmed by re-expressing LdNDK2 in knockouts. Infection with LdNDK2[-/-] parasites significantly reduced the viability and recovery of mast cells and THP-1 macrophages and flow cytometry indicated increased apoptosis in host cells, likely due to parasites inability to utilize NDK enzyme for preventing cytolysis. Our findings highlight LdNDK2's crucial role in metacyclogenesis and immune evasion, underscoring its therapeutic potential for leishmaniasis.},
}
@article {pmid41205603,
year = {2025},
author = {Regan, SB and Medhi, D and Xu, Y and White, TB and Jiang, YZ and Kim, JE and Wang, SC and Deng, Q and Jia, S and Baasan, D and Connelly, JP and Chang, TC and Pruett-Miller, SM and Jasin, M},
title = {Megabase-scale loss of heterozygosity provoked by CRISPR-Cas9 DNA double-strand breaks.},
journal = {Molecular cell},
volume = {85},
number = {22},
pages = {4119-4137.e10},
doi = {10.1016/j.molcel.2025.10.015},
pmid = {41205603},
issn = {1097-4164},
mesh = {*DNA Breaks, Double-Stranded ; Humans ; *CRISPR-Cas Systems ; Animals ; *Loss of Heterozygosity ; Mice ; DNA End-Joining Repair ; Gene Editing/methods ; Flow Cytometry ; CRISPR-Associated Protein 9/genetics/metabolism ; Epithelial Cells/metabolism ; },
abstract = {Harnessing DNA double-strand breaks (DSBs) is a powerful approach for gene editing, but it may provoke loss of heterozygosity (LOH), a common feature of tumor genomes. To interrogate this risk, we developed a flow cytometry-based system (Flo-LOH), detecting LOH in ∼5% of mouse embryonic and human epithelial cells following a DSB. Inhibition of both non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) massively increases LOH, although the dependence on individual pathways differs in the two cell types. Multiple mechanisms lead to LOH, including chromosome truncations with de novo telomere addition and whole chromosome loss. LOH spans megabases distal from the DSB but also frequently tens of megabases centromere-proximal, which can arise from breakage-fusion-bridge events. Unlike DSBs, Cas9 nicks and adenine base editing did not noticeably impact LOH. The capacity for large-scale LOH must therefore be considered when using DSB-based gene editing, especially in conjunction with end-joining inhibition.},
}
@article {pmid41194386,
year = {2025},
author = {Sanchez-Quirante, T and Kužmová, E and Riopedre-Fernandez, M and Golojuch, S and Vopálenský, P and Raindlová, V and El-Sagheer, AH and Brown, T and Hocek, M},
title = {Enzymatic Synthesis of Modified RNA Containing 5-Methyl- or 5-Ethylpyrimidines or Substituted 7-Deazapurines and Influence of the Modifications on Stability, Translation, and CRISPR-Cas9 Cleavage.},
journal = {ACS chemical biology},
volume = {20},
number = {11},
pages = {2755-2767},
doi = {10.1021/acschembio.5c00692},
pmid = {41194386},
issn = {1554-8937},
mesh = {*CRISPR-Cas Systems ; *Pyrimidines/chemistry/metabolism ; *RNA/chemistry/metabolism ; Humans ; *Purines/chemistry/metabolism ; Protein Biosynthesis ; DNA-Directed RNA Polymerases/metabolism ; Viral Proteins ; },
abstract = {A set of modified 5-methyl- and 5-ethylpyrimidine (uracil and cytosine) and 7-methyl-, 7-ethyl-, and 7-unsubstituted 7-deazapurine (deazaadenine and deazaguanine) ribonucleoside triphosphates was synthesized and used for enzymatic synthesis of base-modified RNA using in vitro transcription (IVT). They all were good substrates for T7 RNA polymerase in the IVT synthesis of model 70-mer RNA, mRNA encoding Renilla luciferase, and 99-mer single-guide RNA (sgRNA). The effect of modifications in the particular RNA on the stability and efficiency in in vitro and in cellulo translation as well as in CRISPR-Cas9 gene cleavage was quantified. In the in vitro translation assay, we observed moderately enhanced luciferase production with 5-methyluracil and -cytosine, while any 7-deazaadenines completely inhibited the translation. Surprisingly, in cellulo experiments showed a significant enhancement of translation with mRNA containing 7-deazaguanine and moderate enhancement with 5-methyl- or 5-ethylcytosine. Most of the modifications had a minimal effect on the efficiency of the gene cleavage in CRISPR-Cas9 except for 7-alkyl-7-deazaadenines that completely inhibited the cleavage. The results are important for further design of potential base-modified RNA therapeutics.},
}
@article {pmid41131871,
year = {2025},
author = {Campbell, KB and Ouye, RB and Wong, BL and Jiang, A and Okada, K and McKenney, RJ and Fisher, AJ and Beal, PA},
title = {Control of ADAR2 Dimerization and RNA Editing Efficiency by Site-Specific 2'-Fluoro Modification of Guide RNAs.},
journal = {ACS chemical biology},
volume = {20},
number = {11},
pages = {2637-2648},
doi = {10.1021/acschembio.5c00493},
pmid = {41131871},
issn = {1554-8937},
mesh = {*Adenosine Deaminase/metabolism/chemistry/genetics ; *RNA Editing ; *RNA-Binding Proteins/metabolism/chemistry/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; RNA, Double-Stranded/metabolism/chemistry ; Protein Multimerization ; Binding Sites ; },
abstract = {Adenosine Deaminases Acting on RNA (ADARs) are an important class of RNA editing enzymes that catalyze the deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). Since inosine is typically read as guanosine (G) during translation, ADARs can produce A to G transitions in dsRNA. Site-directed RNA editing (SDRE) is a promising therapeutic tool wherein guide RNAs can be used to direct endogenous human ADARs to reverse disease-causing mutations in specific RNA transcripts. Guide RNA (gRNA) modifications at locations that contact the ADAR active site are often used to improve editing efficiency. However, little is known about rate-enhancing chemical modifications in the gRNA at the dsRNA binding domain (dsRBD)-RNA interface. Analysis of published crystal structures of ADAR2 bound to dsRNA suggested positions at this interface would be sensitive to gRNA modification. In this work, gRNAs bearing 2'-modifications in the dsRBD binding site were synthesized and subsequently tested to determine their effects on the editing rate of therapeutically relevant ADAR targets. We found that replacing a single 2'-OH at specific positions on the gRNA with a 2'-F substantially increased the rate of in vitro ADAR2-catalyzed adenosine deamination for two different sequences, whereas 2'-OMe at these positions was inhibitory. This effect was also validated in cellulo. The rate of ADAR1-catalyzed deamination is not stimulated by these 2'-F modifications. A crystal structure of an ADAR2 fragment bound to duplex RNA bearing a single 2'-F at guide position +13 suggested a favorable interaction between the side chain of N241 of the auxiliary ADAR2 monomer and the 2'-F modification. Furthermore, electrophoretic mobility shift assays and mass photometry indicate 2'-F at position +13 facilitates ADAR2 dimerization on the RNA substrate. This work advances our understanding of the RNA features that define superior ADAR substrates and inform the design of gRNAs for therapeutic RNA editing.},
}
@article {pmid41099664,
year = {2025},
author = {Spavieri, JM and Inacio, TG and Seguchi, G and de Souza, BC and Pereira, GAG and de Mello, F},
title = {Impact of CRISPRi-Mediated Titration of GPD Genes on the Fermentative Performance of S. cerevisiae.},
journal = {ACS synthetic biology},
volume = {14},
number = {11},
pages = {4412-4423},
doi = {10.1021/acssynbio.5c00316},
pmid = {41099664},
issn = {2161-5063},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; Fermentation/genetics ; Ethanol/metabolism ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; Glycerol/metabolism ; *CRISPR-Cas Systems/genetics ; *Glycerol-3-Phosphate Dehydrogenase (NAD+)/genetics/metabolism ; Gene Editing/methods ; },
abstract = {Glycerol is one of the main byproducts in ethanol fermentation due to its importance in redox balance and response to osmotic stress in Saccharomyces cerevisiae. Since its production diverts carbon from alcohol production, traditional gene-editing methods have been applied to the glycerol synthesis pathway. However, such approaches generate undesirable phenotypes for industrial applications. In the present study, we employed the CRISPR-dCas9 system to moderately downregulate the expression of GPD1 and GPD2, the two main genes involved in this metabolism. GPD2 gene expression downregulation and a graded reduction in glycerol production after repression of four different target sites in each paralogue were achieved. Employment of the CRISPRi approach for GPD gene modulation resulted in higher specific ethanol productivity (SEP) than that of single knockout cells. Targeted modulation in a region -140 basepairs upstream of the transcription start site (TSS) of GPD1 resulted in a 3% increase in ethanol production compared to the wild type and gpd Δ strains. Such regulation, combined with GPD2 deletion, revealed the higher SEP among all tested strains. Furthermore, a GPD1-modulated strain maintained tolerance to high osmolarity in very high-gravity (VHG) fermentation while maintaining its ethanol production levels above those observed in the control strain.},
}
@article {pmid41072420,
year = {2025},
author = {Zhao, G and Liu, Y and Zhang, G and Wang, J and Zou, Z and Wang, Y and Xu, S and Han, D and Xu, Z and Chen, Z and Yang, M and Zeng, Z and Wu, Y and Zhou, X and Huang, J},
title = {Engineered hypercompact Fanzor-ωRNA system with enhanced genome editing activity.},
journal = {Molecular cell},
volume = {85},
number = {22},
pages = {4138-4151.e4},
doi = {10.1016/j.molcel.2025.09.031},
pmid = {41072420},
issn = {1097-4164},
mesh = {Humans ; *Gene Editing/methods ; HEK293 Cells ; Animals ; Dependovirus/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Genome, Human ; CRISPR-Cas Systems ; Mice ; },
abstract = {As programmable RNA-guided DNA nucleases found in eukaryotes, Fanzors have shown promise in genome editing with their compact size. A wide variety of RuvC-containing Fanzor nucleases have been identified in various eukaryotes and their viruses. However, low editing efficiency limits the application of Fanzor in mammalian genome editing. In this study, we introduce SpuFz1 V4, an engineered RNA-guided DNA endonuclease with vigorous editing activity in the human genome. Furthermore, we demonstrate the substantial potential of the Fanzor system as a base editor. SpuFz1 V4 is currently the most active RNA-guided DNA nuclease of eukaryotic origin, belonging to the Fanzor1 family. Due to its compact size, SpuFz1 V4 can be efficiently delivered via a single adeno-associated virus (AAV) into the retina, achieving robust in vivo genome editing, which has the potential to be applied in both basic research and disease treatment applications.},
}
@article {pmid41017740,
year = {2025},
author = {Cao, X and Zeng, Z and Cao, X and He, Y and Wang, L and Li, D and Zhang, X},
title = {Generation and Phenotypic Analysis of the IL-10RAR104W/R104W Mouse Model.},
journal = {Inflammatory bowel diseases},
volume = {31},
number = {11},
pages = {3149-3159},
doi = {10.1093/ibd/izaf099},
pmid = {41017740},
issn = {1536-4844},
support = {32025023//National Natural Science Foundation of China/ ; 22YF1437700//Shanghai Science and Technology Program/ ; },
mesh = {Animals ; Mice ; *Disease Models, Animal ; Phenotype ; *Inflammatory Bowel Diseases/genetics/pathology ; *Interleukin-10 Receptor alpha Subunit/genetics ; *Colitis/pathology/genetics ; *Point Mutation ; Humans ; Macrophages ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; },
abstract = {BACKGROUND: Very-early-onset inflammatory bowel disease (VEO-IBD) is a form of IBD that manifests in infants and young children, with a significant proportion of them carrying interleukin 10 receptor alpha (IL-10RA) mutations. Despite the increasing incidence rate, the pathogenesis of VEO-IBD remains elusive, and treatment options are limited. The utilization of a humanized mouse model holds promise for further investigation into VEO-IBD. Previous study has revealed that VEO-IBD patients had a homozygous C > T mutation at IL-10RA position 301, which can be pathogenic.<br><br>METHODS: We generated the corresponding point mutation mouse model via CRISPR/Cas9 technology. Subsequently, we performed various experiments to assess the colitis phenotype in mice and conducted a preliminary exploration of the model's utility.<br><br>RESULTS: The mouse model progressively developed spontaneous colitis between 6 and 12 weeks. Hematoxylin and eosin (H&amp;E) staining revealed abnormal colonic structure and massive local immune cell infiltration. The mouse model has abnormal levels of inflammatory cytokines in the colonic tissue, with an expansion of F4/80+ macrophages, CD4+ T cells, and B220+ B cells. Among the macrophages, the level of tissue-resident macrophages associated with anti-inflammation was reduced in IL-10RAR104W/R104W mice, while the level of immature macrophages associated with pro-inflammation was increased. Furthermore, we found that bone marrow transplantation can alter the composition of intestinal macrophage populations and treat intestinal inflammation in mutant mice. Finally, the result of subcutaneous tumor-bearing experiments indicated a faster tumor growth rate in the mutant mice.<br><br>CONCLUSIONS: In summary, we have successfully constructed a humanized mouse model with a stable spontaneous colitis phenotype, which is a valuable model for the therapeutic exploration of VEO-IBD.},
}
@article {pmid40954215,
year = {2025},
author = {Romero-Moya, D and Torralba-Sales, E and Calvo, C and Marin-Bejar, O and Magallon-Mosella, M and Distefano, M and Pera, J and Castaño, J and De Giorgio, F and Gonzalez, J and Iglesias, A and Berenguer-Balaguer, C and Schilling, M and Plass, M and Pasquali, L and Català, A and Molina, O and Wlodarski, MW and Bigas, A and Giorgetti, A},
title = {CRISPR-engineered human GATA2 deficiency model uncovers mitotic dysfunction and premature aging in HSPCs, impairing hematopoietic fitness.},
journal = {Leukemia},
volume = {39},
number = {12},
pages = {3015-3025},
pmid = {40954215},
issn = {1476-5551},
support = {PID2020-15591RB-100//Ministry of Economy and Competitiveness | Agencia Estatal de Investigaci&#xf3;n (Spanish Agencia Estatal de Investigaci&#xf3;n)/ ; FORT23/00032//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; AC23_2/00040//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; AC23_2/00014//Ministry of Economy and Competitiveness | Instituto de Salud Carlos III (Institute of Health Carlos III)/ ; KOG-202109-01162//European Hematology Association (EHA)/ ; 101029927//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Sk&#x142;odowska-Curie Actions (H2020 Excellent Science - Marie Sk&#x142;odowska-Curie Actions)/ ; LCF-PR-HR24-00150//"la Caixa" Foundation (Caixa Foundation)/ ; PID2023-151556OB-I00//"la Caixa" Foundation (Caixa Foundation)/ ; PID2022-142966OB-I00//Ministerio de Econom&#xed;a y Competitividad (Ministry of Economy and Competitiveness)/ ; },
mesh = {Humans ; *Hematopoietic Stem Cells/metabolism/pathology ; *GATA2 Transcription Factor/genetics/deficiency ; *CRISPR-Cas Systems ; *Mitosis/genetics ; *GATA2 Deficiency/genetics/pathology ; Animals ; Mutation ; Mice ; Cell Proliferation ; *Cellular Senescence/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {GATA2 deficiency is a monogenic transcriptopathy disorder characterized by bone marrow failure (BMF), immunodeficiency, and a high risk of developing myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML). Although informative mouse models have been developed, the mechanisms by which GATA2 haploinsufficiency drives disease initiation in humans remain incompletely understood. To address this, we developed a novel humanized model using CRISPR/Cas9 technology to knock-in GATA2-R398W variant in primary cord blood CD34[+] cells. Additionally, we introduced specific mutations in SETBP1 and ASXL1 to model distinct premalignant stages of GATA2 deficiency. Through clonal competition and serial transplantation assays, we demonstrated that human CD34[+] cells harboring the GATA2 mutation exhibit significantly reduced fitness in vivo when compete with wild-type cells. Notably, this fitness disadvantage persists even when GATA2 mutations are combined with oncogenic SETBP1 and ASXL1 drivers, underscoring the dominant, deleterious effect of GATA2 deficiency on hematopoietic stem cell function. Functional in vitro analyses revealed that GATA2-R398W mutation impairs cell proliferation, disrupts cell cycle progression, and induces mitotic defects, which may contribute to hematopoietic stem/progenitor cell loss and impaired self-renewal. Transcriptomic profiles of GATA2-mutant cells revealed that these functional defects are associated with reduced HSC self-renewal capacity and upregulation of the pre-aging phenotype. Our work highlights the feasibility of generating a human GATA2 deficiency model suitable for studying the biological consequences of various GATA2 variants and the generation of a platform to test potential phenotype-rescuing therapeutics.},
}
@article {pmid40753279,
year = {2025},
author = {Tan, GZH and Maurya, KS and Krishnamoorthi, S and Boonyaves, K and Urano, D},
title = {Regulatory helix deletion in glutamate decarboxylase reduces GABA and enhances Agrobacterium-mediated transient expression in lettuce.},
journal = {Journal of plant research},
volume = {138},
number = {6},
pages = {1033-1044},
pmid = {40753279},
issn = {1618-0860},
support = {A19E4a0101//Agency for Science, Technology and Research/ ; },
mesh = {*gamma-Aminobutyric Acid/metabolism ; *Glutamate Decarboxylase/genetics/metabolism ; *Lactuca/genetics/metabolism/enzymology ; *Agrobacterium/genetics ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; *Plant Proteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Gamma-aminobutyric acid (GABA) is a metabolite involved in plant growth and stress responses, with its synthesis regulated by glutamate decarboxylase (GAD). Plant GAD enzymes have an autoinhibitory α-helix at the C-terminus, which calmodulin (CaM) binding typically relieves. Eliminating this C-terminal motif usually increases GABA levels in crops. In this case study, we generated a CRISPR/Cas9-edited lettuce line with a 14-amino acid deletion in the C-terminal helix of LsGAD2, the isozyme primarily expressed in most tissues. This targeted truncation removes CaM-binding residues while retaining the key Lys cluster (Lys489, Lys490, Lys491) responsible for autoinhibition, resulting in a significant reduction in GABA content without affecting growth. The LsGAD1/2-ΔC line showed a transcriptomic profile resembling stress responses in the wildtype under unstressed conditions. Reduced GABA levels appeared to upregulate genes involved in stress perception, signalling, and defense-related metabolic and hormonal changes, potentially mediated by WRKY-family transcription factors. Likely due to lower GABA levels and altered defense responses, LsGAD1/2-ΔC plants showed increased Agrobacterium-mediated transient expression of β-glucuronidase. Overall, our study suggests that targeted genetic manipulation of the C-terminal helix of GAD enzymes can reduce GABA levels while enhancing transformation efficiency in lettuce, thus presenting a means for engineering for such purposes.},
}
@article {pmid41259334,
year = {2025},
author = {Siroosi, M and Ghasemi, F and Jabalameli, F and Emaneini, M and Salehi, M and Beigverdi, R and Amoozegar, MA},
title = {Investigating the impact of type I-E CRISPR-Cas systems and acrEI10 on multidrug-resistance in clinical isolates of Klebsiella pneumoniae.},
journal = {PloS one},
volume = {20},
number = {11},
pages = {e0335756},
doi = {10.1371/journal.pone.0335756},
pmid = {41259334},
issn = {1932-6203},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Multiple, Bacterial/genetics ; Humans ; *Bacterial Proteins/genetics ; Anti-Bacterial Agents/pharmacology ; Klebsiella Infections/microbiology/drug therapy ; Microbial Sensitivity Tests ; beta-Lactamases/genetics ; },
abstract = {Klebsiella pneumoniae is a pathogen related to nosocomial infections with a high rate of antibiotic resistance. The aim of this study was to understand the impact of the presence of CRISPR-Cas systems and an anti-CRISPR gene on multidrug-resistance in K. pneumoniae isolates. The study analyzed 100 clinical K. pneumoniae isolates obtained from a hospital setting. The investigation involved determining antibiotic resistance profiles, including ESBL production, identifying specific carbapenemase and aminoglycoside resistance genes, detecting the presence of CRISPR-Cas systems, identifying the anti-CRISPR gene acrEI10, and sequencing CRISPR arrays. Correlation analysis between resistance genes and CRISPR-Cas systems was also performed. All isolates in this study were determined to be multidrug-resistant (MDR), with resistance rates exceeding 70% for the majority of antibiotics tested. The most prevalent carbapenemase genes were blaOXA-48 and blaNDM, while aminoglycoside resistance was primarily mediated by aac(6´)-Ia and ant(2")-Ia. Only 7% of the isolates harbored CRISPR-Cas systems and the gene acrEI10, which encodes an anti-CRISPR protein, was detected in one of the CRISPR-Cas positive isolates. Sequencing of the CRISPR array from this isolate showed similarities between the spacers and sequences found in plasmids and K. pneumoniae chromosome. No strong correlation was identified between the antibiotic resistance genes and CRISPR-Cas systems. Findings from this study suggest a complex interplay between these factors in MDR isolates of K. pneumoniae and show that further investigations are needed to have a better understanding of the mechanisms related to the coexistence of these elements and their impact on dissemination of antibiotic resistance genes.},
}
@article {pmid41257956,
year = {2025},
author = {Feng, C and Wang, Y and Liu, C and Dong, F and Guo, M and Liu, F and Li, Y and Zhang, L},
title = {Programmable no-nonspecific genetic analytical system via dual-circle-based rolling circle amplification with an efficient CRISPR/Cas12a biosensing strategy.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {40723},
pmid = {41257956},
issn = {2045-2322},
support = {CSTB2024NSCQ-MSX0785//Natural Science Foundation Project of Chongqing, Chongqing Science and Technology Commission/ ; 2023YQB061//Young Ph.D. Talent Incubation Program of the Second Affiliated Hospital of Army Medical University/ ; CQYC20220303658//Chongqing Science and Technology Innovation Leading Talent Support Program/ ; 2024ZDXM012//Chongqing Municipal Science and Health Joint Medical Research Project Key Project/ ; 82472384//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Humans ; DNA, Single-Stranded/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; DNA, Circular/genetics ; Limit of Detection ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Ultrasensitive and specific detection of DNA is highly important for early cancer screening and diagnosis. Nucleic acid amplification technology is the most commonly used method for oncogene detection, but nonspecific amplification may occur. We designed a nicking endonuclease (NEase)-mediated exponential rolling circle amplification (RCA) that avoids nonspecific amplification for the CRISPR/Cas12a preamplification process. The purpose was to construct a NEase-assisted target recycling (NATR)-triggered no-nonspecific exponential RCA (NER) reaction integrated with a CRISPR/Cas12a (NATR-NER/Cas12a) system, enabling ultrasensitive and high-fidelity target detection. Innovatively, two circular single-stranded DNAs (ssDNAs) with NEase recognition sites were designed as the preprimer and template for RCA. In the presence of the target, the endonuclease Nt.BstNBI cleaves the circular preprimers into linear fragments, triggering the NER reaction. This generates many short ssDNA fragments, which are recognized by CRISPR/Cas12a and generates a fluorescence signal. The proposed strategy exhibited a wide linear range (10 fM-1 nM), a low detection limit (0.77 fM), and specifically recognized single mismatched DNA. In serum samples, this method exhibited good agreement with real-time quantitative polymerase chain reaction (qPCR) results at lower cost. The developed NATR-NER/Cas12a system provides a promising tool for the early screening and clinical diagnosis of cancer in resource-limited areas.},
}
@article {pmid41256794,
year = {2025},
author = {Zhou, X and Ye, C and Xie, M and Wei, Y and Zhao, Y and Liu, X and Ma, J and Qing, J and Chen, Z},
title = {Advances in the application of CRISPR technology in pathogen detection: amplification-based and amplification-free strategies.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1645699},
pmid = {41256794},
issn = {2235-2988},
mesh = {Humans ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics/isolation &amp; purification ; *Communicable Diseases/diagnosis ; },
abstract = {CRISPR technology, with its high specificity and programmability, has become an important tool for the detection of human pathogens. The timely and accurate detection of pathogens is crucial for public health. In recent years, significant progress has been made in the application of CRISPR technology for pathogen detection. However, several challenges remain, including detection sensitivity, specificity, and operational convenience. This review summarizes the latest advances in CRISPR technology for pathogen detection, with a focus on the principles and performance comparisons of amplification-based CRISPR (such as those combined with isothermal amplification techniques like RPA and LAMP) and amplification-free CRISPR (such as cascade CRISPR, sensor technologies, and digital droplet CRISPR). It also discusses their applications in pathogen detection. In addition, the article analyzes the advantages and limitations of CRISPR detection technology and looks forward to future development trends, providing a theoretical basis for the optimization of rapid diagnostic techniques for pathogens.},
}
@article {pmid41255389,
year = {2025},
author = {Pizzoccheri, R and Falchi, FA and Alloni, A and Caldarulo, M and Camboni, T and Zambelli, F and Pavesi, G and Visentin, C and Camilloni, C and Sertic, S and Briani, F},
title = {Pathological PNPase variants with altered RNA binding and degradation activity affect the phenotype of bacterial and human cell models.},
journal = {NAR molecular medicine},
volume = {2},
number = {1},
pages = {ugae028},
pmid = {41255389},
issn = {2976-856X},
abstract = {Human PNPase (hPNPase) is an essential RNA exonuclease located in mitochondria, where it contributes to RNA import from the cytoplasm, degradation of mitochondrial RNA and R-loop homeostasis. Biallelic mutations in the hPNPase PNPT1 gene cause different genetic diseases, ranging from hereditary hearing loss to Leigh syndrome. In this work, we used an Escherichia coli model to test the effects of four pathological PNPT1 mutations associated with diseases of different severity. Moreover, we generated a new human cell model by introducing PNPT1 mutations into 293T cells via CRISPR-Cas editing. Notably, the bacterial cells expressing the different mutant alleles exhibited similar phenotypes consistent with hPNPase loss of function. In contrast, the human cell model responded differently to the two mutations tested, with responses correlating with the severity of the respective pathologies. We interpreted the data derived from both models in the light of the in vitro RNA binding and degradation activity of the wild-type and mutated hPNPase variants. We found that all pathogenic mutations tested caused defects in protein assembly and affected the degradation and RNA binding efficiency to varying degrees. However, the severity of the conditions caused by different mutations did not correlate with the catalytic activity of the mutant proteins.},
}
@article {pmid41254867,
year = {2025},
author = {Podkowik, M and Tillman, A and Takats, C and Carion, H and Putzel, G and McWilliams, J and See, B and Wang, G and Munoz-Gomez, S and Otto, C and Drlica, K and Marraffini, L and Pironti, A and Hochman, S and Kerantzas, C and Shopsin, B},
title = {CRISPR-Cas-associated SCCmec variants in methicillin-resistant Staphylococcus aureus evade rapid diagnostic detection.},
journal = {The Journal of infectious diseases},
volume = {},
number = {},
pages = {},
doi = {10.1093/infdis/jiaf575},
pmid = {41254867},
issn = {1537-6613},
abstract = {Rapid molecular assays guiding treatment of methicillin-resistant Staphylococcus aureus (MRSA) detect SCCmec (Xpert) or the SCCmec-orfX junction (BCID2). Sequence variation in this region can disrupt primer binding, yielding false-negative results. Investigation of a missed bloodstream infection linked escape to a CRISPR-Cas-associated SCCmec variant, leading to identification of 64 variants from 45 patients-2% of 2,432 screened. Misdiagnosis was restricted to clonal complex 5, a hospital-associated lineage; 11 of 40 SCCmec/junctions evaded detection by BCID2 or Xpert. Variants had mecA instability and circulated in healthcare settings. Our findings reveal a unique escape mechanism and underscore a threat to diagnostic accuracy.},
}
@article {pmid41254256,
year = {2025},
author = {Wang, Q and Wang, Z and Liu, H and Lv, Y and Zhou, C and Li, C and Fan, H and Ouyang, X and Tao, L and Pei, X and Xie, T},
title = {PAM-readID is a rapid, simple, and accurate PAM determination method for CRISPR-Cas enzymes in mammalian cells.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1601},
pmid = {41254256},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; HEK293 Cells ; *CRISPR-Associated Protein 9/metabolism/genetics ; Animals ; DNA Breaks, Double-Stranded ; },
abstract = {One CRISPR-Cas enzyme's recognized protospacer adjacent motif (PAM) profile always shows intrinsic differences between assays with different working environments, such as in vitro, in bacterial cells, or in mammalian cells. The developed methods in mammalian cells are technically complex and not readily amenable to be broadly adopted, highlighting the urgent need for a well-established PAM-determining method in mammalian cells. In this study, we construct a rapid, simple, and accurate method for determining the PAM recognition profile of CRISPR-Cas nucleases in mammalian cells. The developed method is termed PAM-readID, PAM REcognition-profile-determining Achieved by Double-stranded oligodeoxynucleotides Integration in DNA double-stranded breaks. Using PAM-readID, the PAM recognition profiles of SaCas9, SaHyCas9, Nme1Cas9, SpCas9, SpG, SpRY, and AsCas12a in mammalian cells are well produced. An accurate PAM preference for SpCas9 can be identified by analysis with extremely low sequence depth (500 reads). PAM-readID can also define a PAM recognition profile of Cas9 based on Sanger sequencing with a significantly lower cost of time and price than that of high-throughput sequencing. We present an easy-to-use method for comprehensively revealing functional PAM of CRISPR-Cas nucleases in mammalian cells, which can contribute towards accelerating the advancement of exploiting novel genome editing nucleases.},
}
@article {pmid41253931,
year = {2025},
author = {Ni, J and Gong, J and Ran, Y and Bai, R and Huang, P and Zheng, Z and Zhou, M and Lan, F and Gu, W and Liu, X},
title = {Optimization of gene knockout approaches and sgRNA selection in hPSCs with inducible Cas9 expression.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {40461},
pmid = {41253931},
issn = {2045-2322},
support = {JCYJ20220531091615034//Shenzhen Fundamental Research Program/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Associated Protein 9/genetics/metabolism ; Cell Line ; INDEL Mutation ; },
abstract = {CRISPR/Cas9 has been extensively used for gene knockout, enabling functional studies of genetic loss-of-function in human pluripotent stem cells (hPSCs). However, commonly used Cas9 systems usually exhibit limited and variable efficiencies, and identifying single-guide RNAs (sgRNAs) with high cleavage activity-while avoiding ineffective ones-remains a major challenge. To address these issues, we generated a doxycycline-inducible spCas9-expressing hPSCs (hPSCs-iCas9) line and developed it into an optimized gene knockout system through systematically refining critical parameters. Through this optimization, the system achieved stable INDELs (Insertions and Deletions) efficiencies of 82-93% for single-gene knockouts, over 80% for double-genes knockouts, and up to 37.5% homozygous knockout efficiency for large DNA fragment deletions. Moreover, using this optimized system, we precisely evaluated three widely used gRNA scoring algorithms and integrated Western blotting to rapidly identify the ineffective sgRNA. As a result, among the tested algorithms, Benchling provided the most accurate predictions. Notably, we identified an ineffective sgRNA targeting exon 2 of ACE2, where the edited cell pool exhibited 80% INDELs but retained ACE2 protein expression. Together, these findings provide a robust framework for improving gene knockout efficiency in hPSCs and offer practical guidance for reliable sgRNA selection in gene editing experiments.},
}
@article {pmid41252463,
year = {2025},
author = {Fehrenbach, A and Mitrofanov, A and Alkhnbashi, OS and Backofen, R and Baumdicker, F},
title = {An evolutionary approach to predict the orientation of CRISPR arrays.},
journal = {PLoS computational biology},
volume = {21},
number = {11},
pages = {e1013706},
doi = {10.1371/journal.pcbi.1013706},
pmid = {41252463},
issn = {1553-7358},
abstract = {CRISPR-Cas is a defense system of bacteria and archaea against phages. Parts of the foreign DNA, called spacers, are incorporated into the CRISPR array which constitutes the immune memory. The orientation of CRISPR arrays is crucial for analyzing and understanding the functionality of CRISPR systems and their targets. Several methods have been developed to identify the orientation of a CRISPR array. To predict the orientation, different methods use different features such as the repeat sequences between the spacers, the location of the leader sequence, the Cas genes, or PAMs. However, those features are often not sufficient to predict the orientation with certainty, or different methods disagree. Remarkably, almost all CRISPR systems have been found to insert spacers in a polarized manner at the leader end of the array. We introduce CRISPR-evOr, a method that leverages the resulting patterns to predict the acquisition orientation for (a group of) CRISPR arrays by reconstructing and comparing the likelihood of their evolutionary history with respect to both possible acquisition orientations. The new method is independent of Cas type, leader existence and location, and transcription orientation. CRISPR-evOr is thus particularly useful for arrays that other CRISPR orientation tools cannot predict confidently and to verify or resolve conflicting predictions from existing tools. CRISPR-evOr currently confidently predicts the orientation of 28.3% of the arrays in the considered subset of CRISPRCasdb, which other tools like CRISPRDirection and CRISPRstrand cannot reliably orient. As our tool leverages evolutionary information we expect this percentage to grow in the future when more closely related arrays will be available. Additionally, CRISPR-evOr provides confident decisions for rare subtypes of CRISPR arrays, where knowledge about repeats and leaders and their orientation is limited.},
}
@article {pmid41252186,
year = {2025},
author = {Calhoun, CCS and Capps, MES and Muya, K and Gannaway, WC and Martina, V and Conklin, CL and Klein, MC and Webster, JM and Torija-Olson, EG and Thyme, SB},
title = {Removal of developmentally regulated microexons has a minimal impact on larval zebrafish brain morphology and function.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {41252186},
issn = {2050-084X},
mesh = {Animals ; *Zebrafish/genetics/growth &amp; development ; *Brain/anatomy &amp; histology/growth &amp; development/physiology ; Larva/growth &amp; development/genetics ; *Exons/genetics ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Gene Expression Regulation, Developmental ; *Alternative Splicing ; Phenotype ; },
abstract = {Microexon splicing is a vertebrate-conserved process through which small, often in-frame, exons are differentially included during brain development and across neuron types. Although the protein sequences encoded by these exons are highly conserved and can mediate interactions, the neurobiological functions of only a small number have been characterized. To establish a more generalized understanding of their roles in brain development, we used CRISPR/Cas9 to remove 45 microexons in zebrafish and assessed larval brain activity, morphology, and behavior. Most mutants had minimal or no phenotypes at this developmental stage. Among previously studied microexons, we uncovered baseline and stimulus-driven phenotypes for two microexons (meA and meB) in ptprd and reduced activity in the telencephalon in the tenm3 B0 isoform. Although mild neural phenotypes were discovered for several microexons that have not been previously characterized, including in ppp6r3, sptan1, dop1a, rapgef2, dctn4, vti1a, and meaf6. This study establishes a general approach for investigating conserved alternative splicing events and prioritizes microexons for downstream analysis.},
}
@article {pmid41232532,
year = {2025},
author = {Yang, Y},
title = {What we can learn from the first personalized CRISPR-treated baby to tackle genetic brain disorders.},
journal = {Neuron},
volume = {113},
number = {22},
pages = {3697-3702},
doi = {10.1016/j.neuron.2025.10.023},
pmid = {41232532},
issn = {1097-4199},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; *Brain Diseases/genetics/therapy ; *Precision Medicine/methods ; Infant ; },
abstract = {The landmark report on personalized CRISPR genome editing to treat an infant (baby KJ) with a life-threatening liver disease sparked widespread attention,[1] ushering in a new era of precision genetic intervention. This piece discusses the key challenges and opportunities in translating this milestone into treatments for genetic brain disorders.},
}
@article {pmid41222482,
year = {2025},
author = {Chen, W and He, Y and Yuan, J and Yang, F},
title = {Catalytic hairpin assembly cascade-initiated proximity with self-priming amplification for CRISPR-enhanced ultrasensitive detection of coronary heart disease-associated microRNAs.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {45},
pages = {9254-9261},
doi = {10.1039/d5ay01456e},
pmid = {41222482},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis/blood ; Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *Coronary Disease/genetics/diagnosis ; *Biosensing Techniques/methods ; },
abstract = {Accurate detection of specific microRNAs (miRNAs) is essential for the early diagnosis of coronary heart disease. Emerging technologies, including functional nuclease-mediated target amplification and DNA nanotechnology, offer substantial potential for precise miRNA identification in clinical diagnostics. This study presents a highly sensitive and specific biosensing platform that integrates catalytic hairpin assembly (CHA) cascade-initiated proximity based self-priming amplification and CRISPR/Cas12a-mediated signal generation for miRNA quantification. Target miRNA initiates the CHA cascade, yielding a toehold-bearing CHA product. This toehold subsequently enables "Variable primer" extension, transcribing double-stranded DNA (dsDNA). The resultant dsDNA activates CRISPR/Cas12a, triggering collateral cleavage and signal amplification. Leveraging this dual-amplification strategy (CHA and CRISPR/Cas12a), the assay achieves a sub-femtomolar detection limit (0.36 fM). Dual-sequence verification, including CHA and CRISPR/Cas12a recognition, ensures exceptional specificity. Validation using spiked serum samples confirmed precise miRNA quantification. Collectively, this biosensor demonstrates significant promise for clinical molecular diagnostics.},
}
@article {pmid41217062,
year = {2025},
author = {Vieyra, F and Pindi, C and Lisi, GP and Morzan, UN and Palermo, G},
title = {Design Rules for Expanding PAM Compatibility in CRISPR-Cas9 from the VQR, VRER and EQR variants.},
journal = {The journal of physical chemistry. B},
volume = {129},
number = {46},
pages = {11949-11958},
doi = {10.1021/acs.jpcb.5c06153},
pmid = {41217062},
issn = {1520-5207},
mesh = {Molecular Dynamics Simulation ; *CRISPR-Cas Systems ; DNA/chemistry/metabolism/genetics ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; Gene Editing ; },
abstract = {Expanding the range of Protospacer Adjacent Motifs (PAMs) recognized by CRISPR-Cas9 is essential for broadening genome-editing applications. Here, we combine molecular dynamics simulations with graph-theory and centrality analyses to dissect the principles of PAM recognition in three Cas9 variants - VQR, VRER, and EQR - that target noncanonical PAMs. We show that efficient recognition is not dictated solely by direct contacts between PAM-interacting residues and DNA but also by a distal network that stabilizes the PAM-binding domain and preserves long-range communication with REC3, a hub that relays signals to the HNH nuclease. A key role emerges for the D1135 V/E substitution, which enables stable DNA binding by K1107 and preserves key DNA phosphate locking interactions via S1109, securing stable PAM engagement. In contrast, variants carrying only R-to-Q substitutions at PAM-contacting residues, though predicted to enhance adenine recognition, destabilize the PAM-binding cleft, perturb REC3 dynamics, and disrupt allosteric coupling to HNH. Together, these findings establish that PAM recognition requires local stabilization, distal coupling, and entropic tuning, rather than a simple consequence of base-specific contacts. This framework provides guiding principles for engineering Cas9 variants with expanded PAM compatibility and improved editing efficiency.},
}
@article {pmid41183414,
year = {2026},
author = {Luo, Q and Zheng, C and Huang, Y and Liang, L and Gong, Y and Zhang, J and Tang, Q and Zhang, K and Liao, X},
title = {Polydopamine-stabilized CsPbBr3 enables toxicity-reduced ECL detection of MMP-2 via CRISPR/Cas12a trans-cleavage.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118193},
doi = {10.1016/j.bios.2025.118193},
pmid = {41183414},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Indoles/chemistry ; *Polymers/chemistry ; *Matrix Metalloproteinase 2/isolation &amp; purification/chemistry/blood ; Gold/chemistry ; Electrochemical Techniques/methods ; CRISPR-Cas Systems ; Humans ; Limit of Detection ; Luminescent Measurements/methods ; Lead/chemistry ; Titanium/chemistry ; },
abstract = {Lead-halide perovskites offer bright electrochemiluminescence (ECL) but suffer from aqueous instability and Pb[2+] safety concerns. We construct a CsPbBr3@PDA-Au nanointerface that couples polydopamine (PDA) passivation with Au-assisted charge transfer, integrated into a peptide-to-CRISPR/Cas12a amplification scheme for ultrasensitive detection of matrix metalloproteinase-2 (MMP-2). PDA conformally coats CsPbBr3, suppressing trap-mediated quenching and mitigating Pb[2+] release, while providing catechol/amine anchors for a tetrahedral DNA scaffold bearing a ferrocene-labeled hairpin. Upon MMP-2 cleavage of a GPLG↓VRG peptide-DNA chimera, the released strand activates Cas12a trans-cleavage, removing the proximal quencher and switching the interface from "off" to "on." Under unified processing (baseline subtraction and renormalization), the platform achieves a 5.6 aM detection limit with high selectivity over non-target proteases and excellent inter-device reproducibility (triplicate error bars reported). TEM/HRTEM visualize uniform nanocubes; XPS/FTIR evidence PDA catechol/quinone/amine coordination; XRD confirms phase-pure CsPbBr3; and corrected CV/EIS analyses verify accelerated interfacial kinetics after Au decoration. The sensor retains ∼98 % intra-day and ≥91 % 7-day signal, supporting operational robustness. By integrating interfacial stabilization, catalytic electron transfer, and molecular amplification, this modular design advances eco-conscious, perovskite-based ECL diagnostics for low-abundance protease monitoring and broader clinical bioanalysis.},
}
@article {pmid41183412,
year = {2026},
author = {Liu, Y and Li, H and Wang, J and Liu, J and Zhou, X},
title = {Dual-acting CRISPR/Cas12a system enhanced hydrogel fluorescent aptasensor for one-pot detection of tetracycline in water.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118181},
doi = {10.1016/j.bios.2025.118181},
pmid = {41183412},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Tetracycline/analysis/isolation &amp; purification ; *Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems/genetics ; Hydrogels/chemistry ; Limit of Detection ; *Water Pollutants, Chemical/analysis/isolation &amp; purification ; *Anti-Bacterial Agents/analysis ; Water/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid yet ultrasensitive and accurate detection of tetracycline (TC) in aquatic environments subjected to composite pollution remains challenging. CRISPR-mediated biosensors have been extensively studied to achieve ultrasensitive detection. However, these biosensors have always been coupled with a time-consuming nucleic acid amplification process to improve the sensitivity, which may cause erratic signal due to the sophisticated biochemical reaction cascades, hence leading to inaccurate results. To address this issue, we proposed a novel amplification-free fluorescent aptasensor based on polyacrylamide-deoxyribonucleic acid hydrogel integrated with a dual-acting CRISPR/Cas12a system for ultrasensitive and rapid detection of TC in water. With this well-designed dual-acting CRISPR/Cas12a system, a single aptamer-target specific molecular binding event can synchronously initiate pairing between two distinct CRISPR RNA and their target nucleic acid modified on the hydrogel, enabling dual activation of Cas12a. The aptasensor instantly emitted quantifiable strong fluorescence due to the efficient cleavage of reporter probes by the twofold activated Cas12a, demonstrating a TC detection limit of 0.035 μg/L, with approximately 10.6-fold and 5.6-fold sensitivity improvement over the two corresponding single-crRNA systems. The entire detection process can be accomplished in one pot within 10 min. The one-step hydrogel aptasensor shows superior resistance to matrix interference over the conventional solution-phase system, achieving satisfactory recovery percentages (92 %-105 %) for TC in different water matrices. This study offers a new perspective on CRISPR/Cas12a biosensor design and advances the environmental antibiotic monitoring field.},
}
@article {pmid41175692,
year = {2026},
author = {Hao, X and Qin, C and He, C and Yuan, Q and Yang, P and Wen, Y and Wu, Y and Cui, R and Zhang, Y and Chen, W and Liu, Y and Ramadan, S and Ying, Y and Li, D and Xu, L},
title = {In situ detection of bacteria from skin interstitial fluid via CRISPR microneedles: An amplification-free platform for point-of-care diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118123},
doi = {10.1016/j.bios.2025.118123},
pmid = {41175692},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *Staphylococcus aureus/isolation &amp; purification/genetics ; Humans ; *Skin/microbiology ; *Extracellular Fluid/microbiology ; CRISPR-Cas Systems ; Gold/chemistry ; Graphite/chemistry ; Limit of Detection ; *DNA, Bacterial/genetics/isolation &amp; purification ; Wearable Electronic Devices ; Needles ; Point-of-Care Systems ; *Staphylococcal Infections/microbiology/diagnosis ; },
abstract = {We report a CRISPR/Cas12a-functionalized microneedle (MN) biosensor for in situ, amplification-free detection of pathogenic bacteria directly from skin interstitial fluid. The platform integrates conductive poly(styrene)/gold/graphene oxide MNs with a minimized electrochemical signal transducer, enabling real-time and highly specific sensing of bacterial DNA through CRISPR-mediated cleavage of ferrocene-labeled ssDNA reporters. Using Staphylococcus aureus as a model target, this strategy achieves direct detection limits of 0.69 pM in ex situ assays and 6.3 pM in situ, without the need for target amplification. The sensor also detects bacterial loads as low as 4.27 × 10[5] CFU/mL in interstitial fluid, which is below typical clinical thresholds. Engineered for biocompatibility and minimal invasiveness, the MNs effectively access interstitial fluid, capture targets and produce quantifiable electrochemical signals within 1 h. Paired with a portable reader and smartphone interface, this wearable system offers a user-friendly tool for point-of-care diagnostics and personalized infectious disease monitoring.},
}
@article {pmid41161233,
year = {2026},
author = {Shan, J and Wang, W and Sheng, Y and Hong, B and Luo, L and Liu, X and Ma, Y and Wang, J},
title = {Ultra-fast one-pot isothermal detection of respiratory virus: ADNA-initiated CRISPR/Cas12a-mediated RCA cycle.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118141},
doi = {10.1016/j.bios.2025.118141},
pmid = {41161233},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *SARS-CoV-2/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; Humans ; Limit of Detection ; *Molecular Diagnostic Techniques/methods ; *COVID-19/diagnosis/virology ; Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Respiratory virus detection is a prominent area of molecular diagnostics, yet current clinical diagnostic methods lack ultra-rapid and highly sensitive detection capabilities. Here, we reported an ultra-rapid, one-pot isothermal assay called "ADNA-initiated CRISPR-Cas12a-mediated RCA cycle" (ACRE). ACRE was developed through computational studying, engineered design of nucleic acid, and enzyme kinetics analysis, combining rolling circle amplification (RCA) with Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system for respiratory virus detection. The assay involved an engineered assistant DNA (ADNA) initiation reaction, followed by leveraging the cis-cleavage activity of Cas12a coupled with an engineered Padlock, which converted the linear RCA into the RCA cycle. When the RCA cycle is continuously in operation, the trans-cleavage activity of Cas12a facilitates both signal output and amplification. The limit of detection (LOD) for three respiratory viruses (SARS-CoV-2, Inf A, and Inf B) was as low as several hundred attomoles (751 aM, 3.7 fM, and 863 aM), with single-nucleotide specificity. Remarkably, the current assay can detect targets with concentrations above 10 pM within 2.5 min, without the reverse transcription step or specialized instrumentation. Given its exceptional speed, sensitivity, and specificity, ACRE could serve as a robust assay for detecting respiratory virus, enabling molecular diagnostics in clinical settings.},
}
@article {pmid41138445,
year = {2026},
author = {Hu, M and Liu, F and Zhang, J and Yang, L and Yan, Y and Wang, Y and Liu, M and An, H and Zhu, L and Ai, Y and Jiang, X},
title = {Integrated CRISPR/Cas12a-activated liposomal SERS amplification in microfluidic chips for ultrasensitive ampicillin detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118131},
doi = {10.1016/j.bios.2025.118131},
pmid = {41138445},
issn = {1873-4235},
mesh = {*Ampicillin/analysis/isolation &amp; purification ; Liposomes/chemistry ; CRISPR-Cas Systems/genetics ; *Spectrum Analysis, Raman/methods ; *Biosensing Techniques ; Limit of Detection ; *Anti-Bacterial Agents/analysis ; Animals ; Gold/chemistry ; Lab-On-A-Chip Devices ; Milk/chemistry ; Silver/chemistry ; Humans ; Metal Nanoparticles/chemistry ; },
abstract = {The misuse of antibiotics has created a vicious cycle: from excessive use in livestock breeding to elevated residue levels in food products, ultimately leading to heightened antibiotic resistance in humans, a phenomenon with significant public health consequences. Food-based antibiotic testing is crucial for preventing antibiotic misuse and protecting human health. However, traditional analytical methods exhibit high equipment dependency, restricting on-site detection in hills, plateaus, and other regions with inconvenient transportation. This study developed an integrated CRISPR/Cas12a-activated liposomal Surface-enhanced Raman scattering (SERS) amplification system within a microfluidic chip for ultrasensitive ampicillin detection. The method achieves primary signal amplification by converting ampicillin concentration into a DNA signal through catalytic hairpin assembly (CHA), followed by secondary signal amplification using an Au@Ag substrate coupled with SERS technology for 4-MPBA@liposome marker detection. The platform demonstrated a linear detection range of 1 fM-1 nM with a limit of detection as low as 740 aM. Notably, the method exhibited excellent stability, selectivity, and sensitivity, successfully detecting ampicillin residues in environmental water and milk samples. It provides a technological approach for environmental monitoring and food safety, demonstrating significant practical application value.},
}
@article {pmid41108536,
year = {2025},
author = {Siddiqui, S and Siddiqui, H and Riguene, E and Nomikos, M},
title = {Zebrafish: A Versatile and Powerful Model for Biomedical Research.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {47},
number = {12},
pages = {e70080},
pmid = {41108536},
issn = {1521-1878},
support = {//NPRP-Blue Skies Research Award/ ; NPRP-BSRA1-0504-210082//Qatar Research Development and Innovation Council (QRDI), Doha, Qatar/ ; },
mesh = {*Zebrafish/genetics ; Animals ; Humans ; *Biomedical Research/methods ; *Disease Models, Animal ; Precision Medicine/methods ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Zebrafish (Danio rerio) have become a versatile model in precision medicine, bridging fundamental biology with translational applications. Their optical transparency, rapid development, and high genetic conservation with humans enable real-time imaging and cost-efficient high-throughput screening. Advances in CRISPR/Cas9, prime editing, and morpholino approaches have expanded their utility for modeling diverse human diseases. In addition to well-established roles in cardiovascular, neurological, metabolic, oncological, and infectious disease research, emerging applications include non-invasive larval urine assays, functional validation of rare human variants, host-microbiome interactions, and automated behavioral profiling for neuropsychiatric conditions. Limitations such as species-specific lipid metabolism and limited antibody availability remain, yet recent integration of single-cell transcriptomics, computational modeling, and machine learning is enhancing translational relevance. Collectively, these innovations position zebrafish as a scalable and powerful platform for disease modeling and personalized therapeutic strategies, underscoring their growing impact in the evolving landscape of precision medicine.},
}
@article {pmid41101316,
year = {2025},
author = {Chitboonthavisuk, C and Martin, C and Huss, P and Peters, JM and Anantharaman, K and Raman, S},
title = {Systematic genome-wide mapping of host determinants of bacteriophage infectivity.},
journal = {Cell systems},
volume = {16},
number = {11},
pages = {101427},
doi = {10.1016/j.cels.2025.101427},
pmid = {41101316},
issn = {2405-4720},
mesh = {*Escherichia coli/virology/genetics ; *Bacteriophages/genetics/pathogenicity ; *Host-Pathogen Interactions/genetics ; CRISPR-Cas Systems/genetics ; Genome, Viral/genetics ; Bacteriophage T7/genetics/pathogenicity ; Host Microbial Interactions/genetics ; },
abstract = {Bacterial host factors regulate the infection cycle of bacteriophages. Except for some well-studied host factors (e.g., receptors or restriction-modification systems), the contribution of the rest of the host genome on phage infection remains poorly understood. We developed phage-host analysis using genome-wide CRISPR interference and phage packaging ("PHAGEPACK"), a pooled assay that systematically and comprehensively measures each host gene's impact on phage fitness. PHAGEPACK combines CRISPR interference with phage packaging to link host perturbation to phage fitness during active infection. Using PHAGEPACK, we constructed a genome-wide map of genes impacting T7 phage fitness in permissive E. coli, revealing pathways that affect phage packaging. When applied to the non-permissive E. coli O121, PHAGEPACK identified pathways leading to host resistance; their removal increased phage susceptibility up to a billion-fold. Bioinformatic analysis indicates that phage genomes carry homologs or truncations of key host factors, potentially for fitness advantage. In summary, PHAGEPACK offers insights into phage-host interactions, phage evolution, and bacterial resistance.},
}
@article {pmid41101079,
year = {2026},
author = {Ma, Q and Rong, Z and Shen, J and Yang, M and Han, Y},
title = {Integrated one-pot RPA-CRISPR/Cas13a platform enables ultrasensitive and field-deployable JAK2 V617F detection for myeloproliferative neoplasm diagnosis.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {268},
number = {},
pages = {117197},
doi = {10.1016/j.jpba.2025.117197},
pmid = {41101079},
issn = {1873-264X},
mesh = {Humans ; *Janus Kinase 2/genetics ; *CRISPR-Cas Systems/genetics ; *Myeloproliferative Disorders/genetics/diagnosis ; Mutation ; Sensitivity and Specificity ; Recombinases ; Real-Time Polymerase Chain Reaction/methods ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Myeloproliferative neoplasms (MPNs) are clonal hematopoietic malignancies driven by mutations like JAK2 V617F, a constitutively activating variant diagnostic for polycythemia vera and subsets of essential thrombocythemia and primary myelofibrosis. Sanger sequencing is the gold standard for detecting JAK2 mutations, but it faces limitations in terms of cost, sensitivity, and portability. CRISPR diagnostics generally requires preamplification, which increases the risk of contamination. There remains an unmet need for field-deployable, rapid MPNs diagnostics in resource-limited settings. We developed ONE-CASPR, a one-pot RPA-CRISPR/Cas13a system enabling ultrasensitive (0.1 % mutant allele frequency), rapid (30 min), portable JAK2 V617F detection. The platform synergizes recombinase polymerase amplification (RPA) with Cas13a trans-cleavage activity in a single-tube reaction at 37 °C, executed via a miniaturized wireless analysis device (CPod) to eliminate cross-contamination while preserving accuracy. Clinical validation across 36 patient samples demonstrated 100 % concordance with real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) in both sensitivity and specificity. ONE-CASPR provides a rapid, sensitive, field-deployable, and user-friendly solution for MPNs point-of-care diagnosis in resource-limited settings, with broad molecular screening application potential.},
}
@article {pmid41047505,
year = {2025},
author = {Rajalekshmi, S and Sathyan, KM},
title = {Direct Protein Degradation: Emerging Tools to Probe Biological Complexity in Mammalian Systems.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {47},
number = {12},
pages = {e70075},
doi = {10.1002/bies.70075},
pmid = {41047505},
issn = {1521-1878},
support = {//School of Medicine, Southern Illinois University/ ; },
mesh = {Animals ; *Proteolysis ; Humans ; Gene Editing/methods ; Mammals/metabolism ; *Proteins/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Conditional degron approaches for acute and reversible protein depletion have become standard tools for studying gene function in cells and model organisms. Traditional gene perturbation methods have advanced gene function studies but are limited by slow kinetics, potential irreversibility, and lethality when targeting essential genes. To overcome these limitations, tag-based and antibody-based direct protein degradation technologies have been developed. These direct protein degradation systems utilize endogenous protein degradation pathways to achieve rapid and reversible protein depletion. When combined with genome editing, these systems provide precise temporal-and in some cases, spatial-control over endogenous protein expression. In this review, we will discuss the current status of tag-based and antibody-based direct protein degron technologies. We aim to provide a comprehensive guide for selecting these tools, highlighting their context-dependent applications and potential improvements to enhance efficiency and reliability.},
}
@article {pmid41045937,
year = {2025},
author = {Koo, BM and Todor, H and Sun, J and van Gestel, J and Hawkins, JS and Hearne, CC and Banta, AB and Huang, KC and Peters, JM and Gross, CA},
title = {Comprehensive genetic interaction analysis of the Bacillus subtilis envelope using double-CRISPRi.},
journal = {Cell systems},
volume = {16},
number = {11},
pages = {101406},
doi = {10.1016/j.cels.2025.101406},
pmid = {41045937},
issn = {2405-4720},
mesh = {*Bacillus subtilis/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Gene Regulatory Networks/genetics ; *Cell Wall/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Peptidoglycan ; CRISPR-Cas Systems/genetics ; },
abstract = {Understanding bacterial gene function remains a major challenge. Double-mutant genetic interaction analysis addresses this challenge by uncovering the functional partners of targeted genes, enabling association of genes of unknown function with known pathways and unraveling of connections among well-studied pathways, but such approaches are difficult to implement at the genome scale. Here, we use double-CRISPR interference (CRISPRi) to systematically quantify genetic interactions at scale for the Bacillus subtilis cell envelope, including essential genes. We discover >1,000 genetic interactions, some known and others novel. Our analysis pipeline and experimental follow-ups reveal the shared and distinct roles of paralogous genes such as mreB and mbl in peptidoglycan and teichoic acid synthesis and identify additional genes involved in the well-studied process of cell division. Overall, our study provides valuable insights into gene function and demonstrates the utility of double-CRISPRi for high-throughput dissection of bacterial gene networks, providing a blueprint for future studies in diverse species. A record of this paper's transparent peer review process is included in the supplemental information.},
}
@article {pmid41045936,
year = {2025},
author = {Dénéréaz, J and Eray, E and Jana, B and de Bakker, V and Todor, H and van Opijnen, T and Liu, X and Veening, JW},
title = {Dual CRISPRi-seq for genome-wide genetic interaction studies identifies key genes involved in the pneumococcal cell cycle.},
journal = {Cell systems},
volume = {16},
number = {11},
pages = {101408},
doi = {10.1016/j.cels.2025.101408},
pmid = {41045936},
issn = {2405-4720},
mesh = {*Streptococcus pneumoniae/genetics ; *Cell Cycle/genetics ; *CRISPR-Cas Systems/genetics ; Genome, Bacterial/genetics ; Computational Biology/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Regulatory Networks/genetics ; Genome-Wide Association Study/methods ; },
abstract = {Uncovering genotype-phenotype relationships is hampered by genetic redundancy. For example, most genes in Streptococcus pneumoniae are non-essential under laboratory conditions. A powerful approach to unravel genetic redundancy is by identifying gene-gene interactions. We developed a broadly applicable dual CRISPRi-seq method and analysis pipeline to probe genetic interactions (GIs) genome-wide. A library of 869 dual single-guide RNAs (sgRNAs) targeting high-confidence operons was created, covering over 70% of the genetic elements in the pneumococcal genome. Testing these 378,015 unique combinations, 4,026 significant GIs were identified. Besides known GIs, we found previously unknown positive and negative interactions involving genes in fundamental cellular processes such as division and chromosome segregation. The presented methods and bioinformatic approaches can serve as a roadmap for genome-wide gene interaction studies in other organisms. All interactions are available for exploration via the Pneumococcal Genetic Interaction Network (PneumoGIN), which can serve as a starting point for new biological discoveries. A record of this paper's transparent peer review process is included in the supplemental information.},
}
@article {pmid40991130,
year = {2025},
author = {Kapoor, SA and Choudhary, P and Kasana, RC},
title = {Exploring CRISPR/Cas9-Mediated Gene Editing Advances in Conventional and Non-conventional Yeast Species.},
journal = {Applied biochemistry and biotechnology},
volume = {197},
number = {11},
pages = {7083-7122},
pmid = {40991130},
issn = {1559-0291},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Yeasts/genetics ; *Saccharomyces cerevisiae/genetics ; },
abstract = {In recent years, using modern technologies, researchers have harnessed the potential of yeast species for various industrial uses, such as the bioproduction of biopharmaceuticals, food additives, industrial biocatalysts, and biofuels. To improve the efficiency and potential of yeast species for industrial uses, genetic modification is carried out. Various genome engineering techniques, including Cre-loxP, homing endonucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), have been employed by different research groups for the genetic manipulation of yeast species. Among different genome engineering techniques, CRISPR/Cas9 has become popular because of its precise editing at targeted loci with increased efficiency. The ease of use, effectiveness, and adaptability of CRISPR/Cas9 make multiplexing possible for simultaneously targeting multiple genes, which was earlier very challenging through traditional methods. Moreover, the ability to perform marker-free editing is the significant advantage offered by CRISPR/Cas9. This review focuses on the applications of the CRISPR/Cas9 system in both conventional and non-conventional yeast species. Further, we discussed the advancements of CRISPR/Cas9, including the regulation of gene transcription-activation/repression and other genome engineering aspects. Additionally, innovations in CRISPR/Cas9, such as cloning-free CRISPR/Cas9 assembly, CRISPR-targeted in vivo editing (ACtive), CRISPR/Cas9-induced gene conversion, and selective ploidy ablation (CRI-SPA) are also discussed for enhancing the potential applications of CRISPR/Cas9 in diverse yeast species.},
}
@article {pmid41249581,
year = {2025},
author = {Turgeman-Grott, I and Golan, N and Neri, U and Naki, D and Altman-Price, N and Eizenshtein, K and Choudhary, DK and Levy, R and Navok, S and Cohen, L and Shalev, Y and Singla, H and Reshef, L and Gophna, U},
title = {A previously undescribed archaeal virus suppresses host immunity.},
journal = {EMBO reports},
volume = {},
number = {},
pages = {},
pmid = {41249581},
issn = {1469-3178},
support = {787514//EC | European Research Council (ERC)/ ; 1599/24//Israel Science Foundation (ISF)/ ; },
abstract = {Extremophilic archaea can have chronic viral infections that are well-tolerated by the hosts and may potentially protect against more lethal infections. Here we show that a natural Haloferax strain (48N), is chronically infected by a lemon-shaped virus. This viral infection is not cleared spontaneously, despite the multiple defense systems of the host. Curing 48N of its virus led to radical changes in the gene expression profile of 48N and a dramatic improvement in its growth rate. Remarkably, the cured 48N is the fastest-growing haloarchaeon reported to date, with a generation time of ~107 min at 45 °C, and faster than any known Haloferax species at this temperature. The virus subverts host defenses by reducing its transcription, including the CRISPR spacer acquisition machinery. Nonetheless, even in the virus-cured background, spacer acquisition is very low, indicating that another genetic element is disrupting CRISPR activity. Our results suggest that the slow growth of some halophilic archaea could be due to the effects of proviruses within their genomes that consume resources and alter the gene expression of their hosts.},
}
@article {pmid41249545,
year = {2025},
author = {Warkad, S and Kumar, A and Gampa, M and Goswami, S and T, V and Kumar, S and Dalal, M and Mishra, D and Jha, GK and C, V and Kumar, RR},
title = {Small RNAs big impact: a review on microRNA-mediated tolerance in wheat under terminal heat.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {245},
pmid = {41249545},
issn = {1438-7948},
support = {CABin//Indian Council of Agricultural Research/ ; NICRA//Indian Council of Agricultural Research/ ; },
mesh = {*Triticum/genetics/physiology ; *MicroRNAs/genetics/metabolism ; Gene Expression Regulation, Plant ; *Thermotolerance/genetics ; *Heat-Shock Response/genetics ; *RNA, Plant/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {MicroRNAs (miRNAs) are key regulators of gene expression in plant responses to abiotic stresses, including heat stress. High temperatures during the critical developmental stages of wheat (Triticum aestivum L.) drastically limit growth and production. Recent research has found that specific miRNAs regulate molecular complexes and physiological responses by targeting transcription factors, heat shock proteins, and signaling components, thereby modulating heat stress tolerance pathways. This review highlights current knowledge about heat-responsive miRNAs in wheat, including their validated targets and functional involvement in thermotolerance. In addition, we summarized the potential CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats /CRISPR associated protein 9) genome editing tool for precise alteration of miRNA genes or their targets, degradome profiling, the secondary structure of miRNA, and how interplay of miRNAs with HSFs and HSPs in target gene regulation to improve heat resilience. A comprehensive understanding of miRNA-regulated networks presents novel possibilities for developing climate-resilient wheat varieties, thereby ensuring food security in the face of global warming.},
}
@article {pmid41249169,
year = {2025},
author = {Ramadoss, GN and Namaganda, SJ and Kumar, MM and Hamilton, JR and Sharma, R and Chow, KG and Workley, LA and Macklin, BL and Sun, M and Ha, AS and Liu, JC and Fellmann, C and Watry, HL and Dierks, PH and Bose, RS and Jin, J and Perez, BS and Sandoval Espinoza, CR and Matia, MP and Lu, SH and Judge, LM and Shy, BR and Nussenzweig, A and Adamson, B and Murthy, N and Doudna, JA and Kampmann, M and Conklin, BR},
title = {Characterizing and controlling CRISPR repair outcomes in nondividing human cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9883},
pmid = {41249169},
issn = {2041-1723},
mesh = {Humans ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *DNA Repair/genetics ; *CRISPR-Cas Systems/genetics ; *Neurons/metabolism/cytology ; Myocytes, Cardiac/metabolism ; DNA Damage ; T-Lymphocytes/metabolism ; },
abstract = {Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, limiting the efficiency and precision of genome editing in many clinically relevant tissues. Here, we address this barrier by using induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. CRISPR editing outcomes differ dramatically in neurons compared to genetically identical dividing cells: neurons take longer to fully resolve this damage, and upregulate non-canonical DNA repair factors in the process. Manipulating this response with chemical or genetic perturbations allows us to direct DNA repair toward desired editing outcomes in nondividing human neurons, cardiomyocytes, and primary T cells. By studying DNA repair in clinically relevant cells, we reveal unforeseen challenges and opportunities for precise therapeutic editing.},
}
@article {pmid41248011,
year = {2025},
author = {Dereki, I and Chondrou, V and Lagoumintzis, G and Sgourou, A},
title = {Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {224},
pages = {},
doi = {10.3791/69328},
pmid = {41248011},
issn = {1940-087X},
mesh = {Humans ; *DNA Methylation/genetics ; K562 Cells ; DNA Methyltransferase 3A ; *DNA (Cytosine-5-)-Methyltransferases/genetics/biosynthesis ; Plasmids/genetics ; *Histone Deacetylase 1/genetics/metabolism ; Genetic Vectors/genetics ; Recombinant Fusion Proteins/genetics/biosynthesis ; *Epigenesis, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; },
abstract = {Investigating the precise role of DNA methylation in gene transcriptional regulation and developing therapies targeting specific gene methylation patterns presents significant challenges due to the scarcity of versatile tools capable of inducing site-specific and long-term epigenetic modifications for modulating gene expression. The study aimed to develop and validate an innovative episomal-based system to facilitate stable DNA methylation at a targeted gene locus, potentially useful for both basic epigenetic research and therapeutic applications. To achieve this, the K562 cell line was co-transfected with two distinct episomal vectors. Both vector types were engineered to express guide RNAs (gRNAs) targeting a 367 bp unmethylated region within the CpG island 326, located upstream of the ZBTB7A gene. Each vector encoded a deactivated form of endonuclease Cas9 (dead or dCas9) fused to either the catalytic domain of DNA methyltransferase DNMT3A (dCas-DNMT3A-CD) or the full-length histone deacetylase HDAC1 (dCas-HDAC1). The dCas sequence included two Nuclear Localization Signals (NLS) to ensure the protein's nuclear import. This dual system expression cassette promotes a chromatin state potentially conducive to long-term epigenetic silencing, promising robust and durable epigenetic results. This intervening approach to the host epigenome via utilization of self-replicating episomal vectors offers several advantages: maintenance and expression of vectors at low copy numbers throughout multiple cell divisions without integration into the host genome, hence minimizing off-target effects and preserving genome integrity. We report the precise and significant increase of DNA methylation at the targeted ZBTB7A CpG island 326. The findings validate that the engineered episomal CRISPR/dCas systems can elicit durable, site-specific DNA methylation. Therefore, this system is a valuable research tool for evaluating the functional effects of targeted methylation changes and a promising platform for developing future epigenetic treatments.},
}
@article {pmid41243026,
year = {2025},
author = {Menelih, A and Girma, A and Assamere, A},
title = {Mechanisms and applications of epigenome editing in plants: current status, challenges and future perspectives.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {243},
pmid = {41243026},
issn = {1438-7948},
mesh = {*Gene Editing/methods ; *Epigenome ; CRISPR-Cas Systems ; *Epigenesis, Genetic ; *Plants/genetics ; DNA Methylation ; *Genome, Plant ; Epigenome Editing ; },
abstract = {Epigenome editing has become a leading-edge technology of programmable, heritable and reversible control of gene expression in plants without changing the DNA sequence. CRISPR/dCas9 systems along with transcription activator-like effectors (TALEs) and zinc finger systems have made it possible to manipulate DNA methylation, histone modifications, and RNA epigenetic marks in a precise and locus-specific fashion. These tools have been used on major regulatory genes of flowering time, stress adjustment, and yield maximization in model and crop plants. This review synthesizes the current status of plant epigenome editing advances and highlights mechanistic innovations including SunTag, CRISPRoff/on and RNA m6A editing. It also emphasizes new paradigm shifts in chromatin reprogramming, including transcription-resistive chromatin states, locus-specific H3K27me3 demethylation, and nanobody-mediated chromatin targeting. Furthermore, it considers the consequences of these shifts in the context of trait stability and epigenetic inheritance. Moreover, the relative evaluation of dCas9-, TALE-, and ZFP-based platforms indicated that there are still enduring problems in the performance of delivery, off-target effects, and transgenerational stability. The review concludes with a conceptual framework connecting epigenome editing to climate-smart crop improvement and outlines future research priorities focused on combinatorial multi-omics integration and the development of environmentally responsive editing platforms.},
}
@article {pmid41242874,
year = {2025},
author = {Akter, T and Baishnab, SM and Damtie, M and Dumpati, S and Eshete, TB and Irfan, S and Kumar, M and Sara, M and Urmi, UL and Willcox, M},
title = {A review of technological advances in the management of bacterial, fungal, viral, and Acanthamoeba keratitis.},
journal = {Clinical &amp; experimental optometry},
volume = {},
number = {},
pages = {1-11},
doi = {10.1080/08164622.2025.2579169},
pmid = {41242874},
issn = {1444-0938},
abstract = {Microbial keratitis is a major cause of monocular blindness worldwide. Trauma and contact lens wear are major risk factors. Bacteria are a major cause, but fungi, viruses, and Acanthamoeba can also cause the disease. Therapeutic options are dwindling with the rise of antimicrobial resistance in all of these microbes, but especially bacteria. Infection with resistant strains results in worse clinical outcomes. New antimicrobial therapies are being developed but most have yet to be either used to treat ocular infections or become commercially available. For bacterial keratitis, antimicrobial peptides (AMPs) are a potential new option, as are bacteriophages and even bacteria that eat other bacteria or maggot secretions. For fungal keratitis, ocular natamycin, with oral ketoconazole or voriconazole treatments, offer improved outcomes for severe cases. For viral keratitis, AMPs again offer hope as new therapeutics, as do RNA interference and CRISPR-Cas systems. The recent release of a commercially available polyhexamethylene biguanide eye drop is a step forward, but new therapies that can kill both Acanthamoeba trophozoites and cysts are still needed. Beyond antibiotics, photoactivated chromophores for keratitis-corneal collagen cross-linking and photodynamic therapy have been successfully used. Therapies for large ulcers or scarring include corneal gluing, tissue scaffolds such as amniotic membrane or conjunctival flaps, and tissue replacement through penetrating or lamellar keratoplasty.},
}
@article {pmid41242543,
year = {2025},
author = {Hillary, VE and Rajan, V and Ceasar, SA},
title = {CRISPR's impact on cancer: From fundamental models to clinical solutions.},
journal = {Life sciences},
volume = {},
number = {},
pages = {124087},
doi = {10.1016/j.lfs.2025.124087},
pmid = {41242543},
issn = {1879-0631},
abstract = {CRISPR is a programmable genome editing method that has transformed genome engineering and advanced cancer research. Various engineering iterations of CRISPR, including base and prime editing, facilitate more accurate and adaptable genomic manipulations, opening new frontiers in understanding and combating cancer. This review emphasizes CRISPR's significant influence in cancer biology, from its ability to accurately recreate somatic mutations in cellular and animal models to its application in developing precise diagnostic tools. We also examine advancements in cancer therapies, particularly through the ex vivo engineering of robust chimeric antigen receptor (CAR) T cells that perform effectively in restrictive tumor microenvironments. Lastly, we consider the existing limitations of CRISPR-Cas systems and outline future directions that could further enhance the application of this powerful technology against cancer.},
}
@article {pmid41213415,
year = {2026},
author = {Xu, YY and Zhou, SM and Wang, LY and Zhang, R and Li, K and Qian, ZY and Xiao, L},
title = {Methods for detecting off-target effects of CRISPR/Cas9.},
journal = {Biotechnology advances},
volume = {86},
number = {},
pages = {108750},
doi = {10.1016/j.biotechadv.2025.108750},
pmid = {41213415},
issn = {1873-1899},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Animals ; },
abstract = {The CRISPR/Cas9 system has emerged as a revolutionary tool for gene editing, widely used in the biomedical field due to its simplicity, efficiency, and cost-effectiveness. However, evidence suggests that CRISPR/Cas9 can induce off-target effects, leading to unintended mutations that may compromise the precision of gene modifications. Consequently, predicting，detecting and evaluating these off-target effects is crucial for optimizing the accuracy and reliability of CRISPR/Cas9 system. This paper provides an overview of the various methodologies and strategies, used or to be used for identifying off-target effects in CRISPR/Cas9-based genome editing, offering insights to improve the precision and safety of CRISPR applications in research and therapeutics.},
}
@article {pmid41208769,
year = {2025},
author = {Luo, D and Wang, N and Major-Monfried, H and Ralls, J and Rha, S and Maitland, SA and Ponnienselvan, K and Yamada, M and Bauer, DE and Wolfe, SA and Kentsis, A},
title = {Modular Platform for Therapeutic Drug Delivery Using Trifunctional Bio-Orthogonal Macromolecular Conjugates.},
journal = {Bioconjugate chemistry},
volume = {36},
number = {11},
pages = {2472-2486},
doi = {10.1021/acs.bioconjchem.5c00440},
pmid = {41208769},
issn = {1520-4812},
mesh = {Humans ; *Cell-Penetrating Peptides/chemistry ; *Drug Delivery Systems/methods ; *Macromolecular Substances/chemistry ; Gene Editing ; Cell Line, Tumor ; Peptidomimetics/chemistry/pharmacology ; Ligands ; CRISPR-Cas Systems ; },
abstract = {Targeted delivery of macromolecular therapeutics holds great promise for overcoming the limitations of conventional small molecules, enabling the modulation of protein-protein interactions and precise genome editing. However, efficient, safe, and cell type-specific delivery remains a major challenge. To address this, we developed a modular platform for synthesizing heterotrifunctional bio-orthogonal macromolecular conjugates (BMCs) by engineering diverse combinations of targeting ligands, cell-penetrating peptides (CPPs), and bioactive cargos. We optimized facile bioconjugation chemistries to generate BMCs with improved yields, structural integrity, and activity. Modular BMCs accommodate diverse components, including antibodies and receptor ligands for targeting, CPPs for intracellular trafficking, and optical probes, therapeutic peptidomimetics, and CRISPR-Cas9 nuclease as cargo to confer specific biological activities. We assayed their utility across multiple applications: BMCs with fluorescently labeled cargo revealed endosomal escape and intracellular accumulation; peptidomimetic MYB transcription factor inhibitor BMCs exhibited potent antileukemic activity against acute myeloid leukemia cells; and Cas9 BMCs achieved rapid delivery and cell type-specific gene editing in human cells. The BMC approach enables the customizable delivery of functional macromolecules, nominating BMCs as a broadly applicable platform for biomedical applications.},
}
@article {pmid41186998,
year = {2025},
author = {Wang, W and Liu, H and Yang, Z and Wang, L and Li, W and He, H and Chen, S and Xu, M and Lu, Y},
title = {Enhancement of Spinosyn Production by Integrating a Static and Dynamic CRISPRi-Mediated Metabolic Switch in Saccharopolyspora spinosa.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {46},
pages = {29717-29728},
doi = {10.1021/acs.jafc.5c08276},
pmid = {41186998},
issn = {1520-5118},
mesh = {*Saccharopolyspora/genetics/metabolism/growth &amp; development ; *Macrolides/metabolism ; *Bacterial Proteins/metabolism/genetics ; Metabolic Engineering/methods ; CRISPR-Cas Systems ; Multigene Family ; Gene Expression Regulation, Bacterial ; *Insecticides/metabolism ; },
abstract = {Spinosyns, polyketide-derived macrolide insecticides produced by Saccharopolyspora spinosa, have broad market prospects. However, their large-scale production is still challenged by the low titers of industrial strains. Here, we developed an integrated static and dynamic CRISPRi strategy to redirect metabolic flux toward spinosyn overproduction without affecting cell growth. Initially, static CRISPRi-mediated repression of four genes (gltA1, fabH3, fabH4, and glgC) in three key nodes of primary metabolism boosted spinosyn titers by 19-55% without growth impairment. Simultaneous repression of these genes led to a further increase of spinosyn production. However, static repression of two other genes (pyc or gltA2) severely hindered bacterial growth and reduced spinosyn biosynthesis. To address this, two growth phase-dependent promoters from the spinosyn biosynthetic gene cluster (BGC) were characterized and employed for dynamic repression of pyc and gltA2, resulting in increased spinosyn titers by 44-68% while maintaining robust growth. Finally, combined static and dynamic repression of these six targets simultaneously achieved over a 2-fold increase of spinosyn production. Collectively, integrated static and dynamic CRISPRi-based reprogramming of primary metabolic pathways in S. spinosa achieved a balance between growth and secondary metabolism, thereby leading to spinosyn overproduction. This strategy has broad potential applicability in other actinomycetes for natural product overproduction.},
}
@article {pmid41170688,
year = {2025},
author = {Wang, S and Guan, X and Sun, S},
title = {A CRISPR/Cas12a-based aptasensor enhanced by functionalized AuNPs for sensitive full-range C-reactive protein detection.},
journal = {Journal of materials chemistry. B},
volume = {13},
number = {45},
pages = {14749-14760},
doi = {10.1039/d5tb01727k},
pmid = {41170688},
issn = {2050-7518},
mesh = {*C-Reactive Protein/analysis ; *Gold/chemistry ; *Aptamers, Nucleotide/chemistry/metabolism ; Humans ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; Particle Size ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {C-reactive protein (CRP) is a well-established biomarker whose plasma levels increase significantly during inflammatory and infectious episodes. It plays a vital role in the diagnosis of bacterial infections and autoimmune diseases, and cardiovascular risk assessment. However, conventional detection techniques often struggle to balance high sensitivity with a broad dynamic range. Here, we developed and validated a novel fluorescence-based aptasensor named AuCA (AuNP-enhanced CRISPR Aptasensor), which integrates nucleic acid aptamers, magnetic beads (MBs), gold nanoparticles (AuNPs), and the CRISPR/Cas12a system for robust CRP quantification. In this system, MBs functionalized with aptamer 1 selectively captured the target protein, while AuNPs co-modified with aptamer 2 and a Cas12a activation sequence (Trigger) enabled efficient signal transduction. Upon target binding, the Trigger activated the Cas12a/crRNA complex, initiating trans-cleavage of fluorescent reporters and producing a markedly amplified signal. The optimized AuCA platform achieved a low detection limit of 60 ng mL[-1] and a quantifiable range of 0.1-150 μg mL[-1]. It exhibited excellent specificity and resistance to biological interference, ensuring reliable measurements even in complex sample matrices. When applied to clinical human plasma, AuCA demonstrated results that are in strong concordance with results from a commercial immunoturbidimetric assay. AuCA allowed the simultaneous detection of both standard and hypersensitive CRP (hsCRP), supporting comprehensive full-range CRP (frCRP) analysis with strong potential for clinical applications.},
}
@article {pmid41151122,
year = {2025},
author = {Cea Salazar, VI and Boender, AJ and Seelke, AMH and Gaard, L and Mederos, SL and Rogers, S and Gutierrez, XZ and Bales, KL and Young, LJ and Trainor, BC},
title = {CRISPR-mediated knockdown of oxytocin receptor in extended amygdala reduces stress-induced social avoidance in female California mice.},
journal = {Hormones and behavior},
volume = {176},
number = {},
pages = {105845},
doi = {10.1016/j.yhbeh.2025.105845},
pmid = {41151122},
issn = {1095-6867},
mesh = {Animals ; *Receptors, Oxytocin/genetics/metabolism ; Female ; *Stress, Psychological/genetics/metabolism ; Peromyscus ; Mice ; *Amygdala/metabolism ; *Social Behavior ; Gene Knockdown Techniques ; Nucleus Accumbens/metabolism ; CRISPR-Cas Systems ; Septal Nuclei/metabolism ; *Avoidance Learning/physiology ; Behavior, Animal/physiology ; },
abstract = {Oxytocin receptors (OTRs) within the extended amygdala and nucleus accumbens (NAc) have been implicated in modulating social behaviors, particularly following stress. The effects of OTR could be mediated by modulating the activity of pre-synaptic axon terminals or via receptors in post-synaptic neurons or glia. Using a viral-mediated CRISPR/Cas9 gene editing system in female California mice (Peromyscus californicus), we selectively knocked down OTR in the anteromedial bed nucleus of the stria terminalis (BNST) or NAc to examine their roles modulating social approach and vigilance behaviors. Knockdown of OTR in the BNST attenuated stress-induced decreases of social approach and had less robust effects on vigilance when interacting with a target mouse behind a wire barrier. In this large arena, where mice could control their proximity to a target mouse, BNST OTR knockdown also increased investigation of a non-social stimulus (empty cage). Behavioral effects of BNST OTR knockdown were weaker in the small arena where focal mice physically interacted with target mice. Interestingly, OTR knockdown in the NAc, reduced stress-induced social vigilance without affecting social approach. These effects could mediate altered encoding of socially aversive experiences, as knockdown manipulations were performed before stress exposure. Together, these results highlight effects of local OTR on social behavior that are region-specific.},
}
@article {pmid41123407,
year = {2025},
author = {Xu, Z and Huang, Y and Dong, Y and An, X and Tong, Y and Li, M},
title = {Engineering broad-spectrum phage-resistant Escherichia coli via adaptive and programmable defense strategies.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {11},
pages = {e0159625},
doi = {10.1128/aem.01596-25},
pmid = {41123407},
issn = {1098-5336},
support = {2022FY101100//Science &amp; Technology Fundamental Resources Investigation Program/ ; 202423m10050004//Anhui Province Scientific and Technological Research Project/ ; 22322908D//S&amp;T Program of Hebei/ ; 82341119//National Natural Science Foundation of China/ ; XK2025-05//Interdisciplinary Research Center of Beijing University of Chemical Technology/ ; },
mesh = {*Escherichia coli/virology/genetics ; *Coliphages/genetics/physiology/isolation &amp; purification ; CRISPR-Cas Systems ; Genome, Viral ; Fermentation ; },
abstract = {UNLABELLED: Phage contamination, which impacts product quality and production efficiency, remains a major challenge in industrial fermentation. Although bacteria have evolved various defense systems to combat phage infection, these systems often suffer from narrow host specificity and limited efficacy. In this study, we isolated and characterized a novel lytic Escherichia coli phage, TR2, from a contaminated fermentation substrate. Its strong environmental stability, short latency period, and high lytic activity render it a significant threat to fermentation processes. Genomic sequencing revealed that phage TR2 has a linear, double-stranded DNA genome of 45,171 bp with a G+C content of 44% and 74 coding sequences. On the basis of the physiological characteristics and genomic features of this phage, we developed two strategies to generate phage-resistant E. coli strains: (i) selection of spontaneous mutations in bacterial surface receptors to prevent phage adsorption and infection and (ii) integration of an exogenous CRISPR/Cas9 system to confer sequence-specific immunity. Spontaneous mutation provides broad-spectrum resistance but at the cost of fitness and evolutionary stability, whereas CRISPR/Cas9 ensures long-term, programmable immunity with minimal growth defects. Importantly, both strategies successfully protected bacterial cultures from phage infection without compromising recombinant protein production, highlighting their potential for industrial application. Our findings provide a practical approach for mitigating phage contamination in industrial fermentation processes. This study also highlights the advantages and limitations of spontaneous mutations and natural phage defense systems, offering valuable insights for the design of more effective phage-resistant microbial platforms.<br><br>IMPORTANCE: Phage contamination is a significant challenge in industrial fermentation and severely impacts product quality and production efficiency. We systematically compared spontaneous mutation and CRISPR/Cas9-mediated immunity as two strategies for engineering phage-resistant E. coli strains. Both approaches effectively protected bacterial cultures from phage infection without compromising recombinant protein production, underscoring their potential for industrial applications. Notably, spontaneous mutation conferred broad-spectrum resistance but was associated with fitness costs and limited evolutionary stability. In contrast, CRISPR/Cas9-based immunity offered long-term, programmable protection with minimal growth impairment. By delineating the trade-offs between these two strategies, our work provides a framework for selecting tailored phage resistance solutions suited to diverse biomanufacturing scenarios.},
}
@article {pmid40962686,
year = {2025},
author = {Chen, H and Fan, S and Chen, K and Wang, F and Lu, M and Wu, Y and Lu, H and Li, J},
title = {CRISPR-edited iPSCs reveal BSN gene mutations induce neuronal hyperexcitability via astrocyte lipid accumulation.},
journal = {Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics},
volume = {22},
number = {6},
pages = {e00740},
doi = {10.1016/j.neurot.2025.e00740},
pmid = {40962686},
issn = {1878-7479},
mesh = {*Astrocytes/metabolism ; *Induced Pluripotent Stem Cells/metabolism ; Humans ; *Neurons/metabolism/physiology ; *Mutation/genetics ; *Lipid Metabolism/genetics/physiology ; Gene Editing/methods ; CRISPR-Cas Systems ; *Nerve Tissue Proteins/genetics ; Cells, Cultured ; Epilepsy/genetics ; },
abstract = {Mutations in the BSN gene, encoding the presynaptic protein Bassoon, are implicated in epilepsy, but their impact on astrocytes remains unclear. Using CRISPR/Cas9, we introduced patient-derived BSN mutations (p.M1903V and c.5672insCG) into human induced pluripotent stem cells (iPSCs) and differentiated them into astrocytes. We found that mutant astrocytes exhibited significant lipid accumulation, evidenced by elevated free cholesterol, reduced arginase activity, and increased lipid droplets. Proteomic analysis revealed upregulation of lipid metabolism regulators, such as APOE and FASN. Electrophysiological recordings showed impaired Kir4.1 potassium channel function, depolarized resting membrane potential, and increased capacitance in mutant astrocytes following kainic acid stimulation. Co-culture experiments with neurons demonstrated that BSN-mutant astrocytes led to reduced neurite outgrowth, elevated neuronal apoptosis, increased pro-inflammatory cytokines (IL-1β, TNF-α), and neuronal hyperexcitability. These findings demonstrate that BSN mutations disrupt astrocyte lipid homeostasis and impair neurosupportive functions, thereby driving neuronal hyperexcitability. This study establishes astrocytes as critical mediators of epilepsy pathogenesis in BSN-related disorders and highlights lipid metabolism as a potential therapeutic target.},
}
@article {pmid41242302,
year = {2025},
author = {Ishihara, K and Kitagawa, S and Adachi, N and Akutsu, M and Senda, T and Inanaga, H and Numata, T},
title = {Cryo-EM structure of Archaeoglobus fulgidus type III-B CRISPR-Cas effector and intermediate crRNA processing during effector assembly.},
journal = {Biochemical and biophysical research communications},
volume = {792},
number = {},
pages = {152978},
doi = {10.1016/j.bbrc.2025.152978},
pmid = {41242302},
issn = {1090-2104},
abstract = {Type III CRISPR-Cas effectors recognize target RNAs complementary to the crRNA guide, activating diverse downstream antiviral responses. In contrast to type III-A systems, the architecture of the type III-B effector (Cmr), comprising six proteins (Cmr1-Cmr6) and a crRNA, remains incompletely defined. Moreover, although maturation of the 3' region of type III crRNA has been attributed to polynucleotide phosphorylase (PNPase), an alternative maturation pathway has been suggested but remains to be elucidated. Here we determined the cryo-EM structure of the Cmr1-lacking Archaeoglobus fulgidus Cmr (AfCmrΔ1) bound to a target analog at 3.4 Å resolution. The complex forms a continuous basic channel that accommodates a crRNA-target heteroduplex. Comparative interface analysis explains why the previously reported cross-species Cmr assembly retains activity, revealing interface flexibility that enables compatible Cmr3-Cmr4 and Cmr2-Cmr5 interactions. Furthermore, we show the cooperative, site-specific processing of an intermediate crRNA that requires both AfCmrΔ1 and AfCmr1 and proceeds without divalent cations. In addition to identifying the cleavage site within the intermediate crRNA, mutational analysis of AfCmr1 reveals residues critical for the reaction. These findings suggest an alternative pathway for crRNA maturation during type III effector assembly that complements PNPase-mediated trimming of the intermediate crRNA, thereby expanding the mechanistic landscape of type III CRISPR-Cas systems.},
}
@article {pmid41242144,
year = {2025},
author = {Yu, ES and Jang, H and Kwon, JM and Jeong, H and Park, J and Kang, BH and Rho, D and Son, S and Kang, T and Jeong, KH},
title = {Nanoplasmonic real-time RT-RPA and CRISPR/Cas12a detection for rapid point-of-care molecular diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {294},
number = {},
pages = {118216},
doi = {10.1016/j.bios.2025.118216},
pmid = {41242144},
issn = {1873-4235},
abstract = {The rapid and precise detection of nucleic acids is crucial for effective disease diagnosis and management at the point-of-care (POC) level. Here we report a palm-sized plasmonic photothermal platform for real-time on-chip recombinase polymerase amplification (RPA) and CRISPR/Cas12a detection. An ultrathin photothermal nanoplasmonic cavity (PNC) of Au nanoislands (AuNIs) and an aluminum reflector delivers uniform and efficient photothermal heating under white LED illumination. The configuration drives isothermal amplification and CRISPR-mediated cleavage in a single microchamber while a fluorescence microlens array (FMLA) camera records real-time emission. The compact platform detects the SARS-CoV-2 E gene in 25 min at 25.7 copies per cartridge and achieves 100 % concordance with RT-qPCR across 42 clinical samples. This all-in-one platform can offer a robust and cost-effective solution for molecular diagnostics, facilitating scalable and real-time testing of infectious diseases in decentralized POC settings.},
}
@article {pmid41241907,
year = {2026},
author = {Treichel, AJ and Bazzini, AA and Tornini, VA},
title = {Functional Testing of Microproteins in a Vertebrate Model of Development.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2992},
number = {},
pages = {183-201},
pmid = {41241907},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; CRISPR-Cas Systems ; *Zebrafish Proteins/genetics/metabolism ; Ribosomes/metabolism/genetics ; Gene Expression Regulation, Developmental ; Embryonic Development/genetics ; Micropeptides ; },
abstract = {While thousands of putative microproteins have been identified through ribosome profiling, reporter assays, and mass spectrometry-based methods, their functional testing has remained challenging. Advances in genome sequencing and CRISPR/Cas technologies enable the prioritization and testing of candidate microprotein functions for roles in development, for example, in the maternal-to-zygotic transition or in neurodevelopment. Here, we describe the functional testing of microproteins in vivo using a vertebrate model of early development, Danio rerio (zebrafish).},
}
@article {pmid41241835,
year = {2025},
author = {Zhang, J and Li, L and Zhu, Y and Qian, K and Xu, Q and Qin, Y and Wu, L},
title = {Amplification-Free CRISPR-Cas System Integrated Centrifugal Digital Microfluidic Platform Developed for Multiplexed Respiratory Pathogen Nucleic Acid Analysis.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c04524},
pmid = {41241835},
issn = {1520-6882},
abstract = {In response to the urgent demand for highly sensitive and rapid multiplex detection technologies in the prevention and control of respiratory infectious diseases, this study presents the development of an integrated CRISPR-Cas9/Cas13a detection platform based on a centrifugal digital microfluidic chip. It aims to overcome the reliance of traditional real-time fluorescence quantitative PCR on specialized equipment and trained personnel. Additionally, it addresses the issue of false positives commonly associated with existing isothermal amplification technologies, while also meeting the requirement for preamplification in sensitive CRISPR-based detection methods. In this study, Methicillin-resistant Staphylococcus aureus (MRSA) and influenza A virus subtype H1N1 were selected as model pathogens. The off-chip CRISPR-Cas9/Cas13a dual nucleic acid detection system was initially developed and optimized to enable highly specific detection of MRSA-mecA DNA at a concentration of 173 pM and H1N1-HA RNA at a concentration of 117 pM. Subsequently, the optimal centrifugal digital chip structure was designed and screened to achieve a droplet filling rate of 99.6%. The optimized CRISPR system was finally integrated into the digital chip, resulting in significantly improved sensitivity, reaching 0.7 copies/μL for MRSA DNA and 1.2 copies/μL for H1N1 RNA within a 20 min reaction time at 37 °C. Furthermore, both the negative and positive detection rates achieved 100% accuracy across all 20 simulated clinical samples. The platform integrates centrifugal digital droplet segmentation technology with the CRISPR-Cas system in an innovative manner, enabling subcopy sensitivity detection without the need for nucleic acid preamplification. Therefore, this convenient, cost-effective, and contamination-resistant method provides a reliable solution for the rapid detection of respiratory pathogens in resource-constrained scenarios.},
}
@article {pmid41241617,
year = {2025},
author = {Kalogeropoulos, K and van Beljouw, SPB and Feldmann, D and van den Berg, DF and Brouns, SJJ},
title = {Proteases in bacteriophage defense systems and their potential in bioengineering.},
journal = {Trends in biochemical sciences},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibs.2025.10.006},
pmid = {41241617},
issn = {0968-0004},
abstract = {Novel phage defense systems featuring diverse enzymatic activities are continually being discovered. Among these, defense systems employing proteolytic enzymes have been identified, revealing a previously unrecognized enzymatic activity in phage defense. These protease-associated defense systems represent an untapped reservoir for new biotechnological tools and may serve as a springboard for the development of proteome editors. This review outlines recent advancements in the discovery and characterization of protease-containing defense systems, proposes methods for further exploration and investigation of protease activity, and considers the prospect of protease defense systems for modulating protein processing and cell fate.},
}
@article {pmid41220317,
year = {2025},
author = {Rayad, N and Chowdhury, EA and Meno-Tetang, GML},
title = {The Impact of QSP Modeling on the Design and Optimization of Gene Therapy Approaches.},
journal = {CPT: pharmacometrics &amp; systems pharmacology},
volume = {14},
number = {11},
pages = {1760-1764},
doi = {10.1002/psp4.70131},
pmid = {41220317},
issn = {2163-8306},
mesh = {*Genetic Therapy/methods ; Humans ; Gene Editing/methods ; Dependovirus/genetics ; Genetic Vectors ; Animals ; RNA, Messenger/administration &amp; dosage/genetics ; Nanoparticles/administration &amp; dosage ; CRISPR-Cas Systems ; *Models, Biological ; Lipids/chemistry ; Tissue Distribution ; Liposomes ; },
abstract = {Quantitative Systems Pharmacology (QSP) is increasingly utilized to support the design and translation of gene therapies. This perspective outlines the application of QSP modeling across three domains of gene therapy: mRNA-based therapeutics, adeno-associated virus (AAV) vectors, and genome editing systems. We highlight opportunities for dose optimization, biomarker interpretation, and mechanistic understanding, while addressing current limitations in model generalizability, data sparsity, and translational relevance. Examples include QSP platforms for lipid nanoparticle (LNP)-delivered mRNA, physiologically based pharmacokinetics (PBPK)-informed AAV biodistribution models, and CRISPR-Cas9-based editing systems. These case studies demonstrate QSP's value in de-risking development and personalizing therapies for rare and complex diseases.},
}
@article {pmid41191401,
year = {2025},
author = {Guo, Y and Xiong, Y and Tong, W and Huang, X and Xiong, Y},
title = {CRISPR/Cas12a-Based Dynamic Light Scattering Assay for Nucleic Acid Amplification-Free and Ultrasensitive Detection of Listeria monocytogenes.},
journal = {Analytical chemistry},
volume = {97},
number = {45},
pages = {25232-25244},
doi = {10.1021/acs.analchem.5c04740},
pmid = {41191401},
issn = {1520-6882},
mesh = {*Listeria monocytogenes/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Dynamic Light Scattering/methods ; Limit of Detection ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques ; *Biosensing Techniques/methods ; Particle Size ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Ultrasensitive signal transduction systems are essential for developing rapid, robust, and sensitive nucleic acid tests using the amplification-free CRISPR-Cas12a system. In this study, we introduce a novel platform termed CRISPR-DART (dynamic light scattering Assisted Rapid Test), which combines CRISPR-Cas12a with a dynamic light scattering (DLS) signal readout. We systematically evaluated the effects of nanoparticle size (20, 60, and 100 nm) and morphology (spherical, cubic, and flower-like) on DLS sensing performance. Results demonstrate that larger and more structurally complex nanoparticles significantly enhance scattering intensity, allowing stable DLS signals at lower concentrations and improving sensitivity for detecting low-abundance nucleic acid targets. By utilizing the high specificity and trans-cleavage activity of CRISPR-Cas12a, target-triggered cleavage of single-stranded DNA linkers modulates nanoparticle aggregation, enabling quantitative nucleic acid detection. Notably, CRISPR-DART based on 100 nm gold nanoflowers with large size and complex morphology achieves a detection limit (LOD) of 32 aM, an improvement of 5 orders of magnitude over conventional CRISPR-Cas12a assays using fluorophore-quencher as signal output. Furthermore, the amplification-free CRISPR-DART platform also achieves a LOD of 92 CFU/mL for Listeria monocytogenes in food samples, and after a brief preincubation step, successfully detects 1 CFU in 25 g of food sample. In summary, the CRISPR-DART platform provides a straightforward, highly sensitive, and specific tool for rapid on-site diagnostics and food safety monitoring.},
}
@article {pmid41052028,
year = {2025},
author = {Khan, S and Chen, L and Chou, CL and Khundmiri, SJ and Knepper, MA},
title = {Inducible Avp knockout mouse line.},
journal = {American journal of physiology. Renal physiology},
volume = {329},
number = {6},
pages = {F784-F795},
doi = {10.1152/ajprenal.00340.2025},
pmid = {41052028},
issn = {1522-1466},
support = {ZIA-HL001285//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; ZIA-HL006129//HHS | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; },
mesh = {Animals ; Mice, Knockout ; *Arginine Vasopressin/genetics/deficiency/metabolism ; Aquaporin 2/metabolism/genetics ; *Tamoxifen/pharmacology ; Mice ; Male ; *Kidney/metabolism/pathology ; Female ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; },
abstract = {Arginine vasopressin (AVP) is a peptide hormone synthesized in the hypothalamus and secreted by the posterior pituitary. Previous studies toward understanding AVP physiology relied heavily on Brattleboro rats, which have a spontaneous mutation in the Avp gene and lack circulating AVP. However, these rats are difficult to breed due to high neonatal death and behavioral issues, causing commercial breeders to stop production. To address this, we developed a mouse line with tamoxifen-inducible deletion of Avp. We used CRISPR/Cas9 to insert loxP sites into the Avp gene. These mice were then bred with mice expressing a tamoxifen-inducible Cre recombinase. The resulting conditional knockout mice (Avp[flx/flx]Cre[+]) are viable, fertile, and healthy before induction. Administration of tamoxifen in 8-12-wk-old mice successfully deleted Avp, as confirmed by Sanger sequencing. This deletion caused a significant decrease in urine osmolality, a hallmark of AVP deficiency. The kidney structure remained normal, with no signs of medullary atrophy. In addition, these mice exhibited a substantially decreased expression of the aquaporin 2 water channel (AQP2), which is involved in water reabsorption in the kidney inner medulla. We illustrate the use of this model by using RNA-seq to profile the consequences of Avp deletion on gene expression in the kidney. The curated RNA-seq data can be browsed, searched, or downloaded at https://esbl.nhlbi.nih.gov/Databases/AVP-KO/. In conclusion, we successfully created an inducible Avp knockout mouse line that has been made available to the research community. This model will be valuable for studying water balance regulation, polycystic kidney disease, and the neural, vascular, and metabolic functions of vasopressin.NEW &amp; NOTEWORTHY We developed an inducible Avp knockout mouse line that will be shared with the research community and is likely to be useful for further study of the regulation of water balance and polycystic kidney disease, as well as neural, vascular, and metabolic roles of vasopressin.},
}
@article {pmid41241097,
year = {2025},
author = {Hołubowicz, R and Gao, F and Du, SW and Menezes, CR and Zhang, J and Hołubowicz, MW and Chen, PZ and Armbrust, N and Geilenkeuser, J and Liu, DR and Jeffery Truong, DJ and Westmeyer, GG and Palczewska, G and Palczewski, K},
title = {Scalable purification enables high-quality virus-like particles for therapeutic translation.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {110946},
doi = {10.1016/j.jbc.2025.110946},
pmid = {41241097},
issn = {1083-351X},
abstract = {Emerging molecular therapies introduce enzymatic activity into cells by delivering genes, transcripts, or proteins. Owing to their robust cell-entry capacity, virus-like particles (VLPs) represent a technology of choice in genome editing, where low doses of heterologous proteins and nucleic acids are essential. However, clinical translation of VLP vectors is hindered by inadequate purification methods. Current approaches, relying primarily on ultracentrifugation, suffer from inconsistent product quality and poor scalability. Here, we report the development of a broadly applicable purification strategy that improves the purity and therapeutic efficacy of genome-editing VLPs. Considering the characteristic properties of murine leukemia virus (MLV)-derived engineered VLPs (eVLPs) and HIV-derived engineered nucleocytosolic vehicles for loading of programmable editors (ENVLPEs+), we developed a workflow that involves single- and multi-modal chromatographic steps, effectively removing host cell proteins and cell-culture contaminants while improving VLP integrity and biological activity. Our purified VLPs displayed superior protein composition, consistency, and enhanced functional delivery compared to VLPs partially purified by conventional ultracentrifugation methods. Mass spectrometric analysis revealed a substantial decrease in contaminants, with VLP-specific proteins comprising >90% of the final product. In vivo studies confirmed improved therapeutic outcomes when chromatographically purified VLPs were used. Our scalable purification platform addresses critical manufacturing bottlenecks and constitutes a starting point for further development of VLP therapeutics, enabling robust production of pure VLPs for diverse applications such as genome editing, vaccine development, and other uses that require intracellular protein delivery.},
}
@article {pmid41240805,
year = {2025},
author = {Ghoreshi, ZA and Ali-Hassanzadeh, M and Mashayekhi-Sardoo, H and Askarpour, H and Arefinia, N},
title = {The identification of tuberculosis using CRISPR technique: A systematic review and meta-analysis.},
journal = {Diagnostic microbiology and infectious disease},
volume = {114},
number = {2},
pages = {117183},
doi = {10.1016/j.diagmicrobio.2025.117183},
pmid = {41240805},
issn = {1879-0070},
abstract = {BACKGROUND: Rapid and precise detection of Mycobacterium tuberculosis (MTB) is essential for effective management and control of tuberculosis. The diagnostic challenge is particularly acute for extrapulmonary TB, which accounts for approximately 15-20% of cases and often presents with paucibacillary samples. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for pathogenic diagnosis across diverse sample types owing to its specificity and adaptability. This systematic review and meta-analysis aimed to appraise the diagnostic accuracy of CRISPR-based techniques in identifying MTB.<br><br>METHODS: A comprehensive search was conducted in Medline, Scopus, Embase, and ISI Web of Science to retrieve relevant studies, adhering to PRISMA guidelines. Quality was assessed using the Joanna Briggs comprehensive checklist. Data synthesis and analyses, including subgroup analyses, were performed with Meta-Disc 1.4, examining variables like CRISPR variants, gene targets, pre-amplification techniques, and signal readout methods.<br><br>RESULTS: From 341 identified studies, 13 met the inclusion criteria, encompassing 1,572 MTB strains. The pooled sensitivity and specificity of CRISPR-based techniques were 0.91 (95%CI: 0.89-0.92) and 0.97 (95%CI: 0.95-0.98), respectively. The pooled diagnostic odds ratio was 498.67 (95%CI: 255.1-974.7), with an AUC of 0.99 in the SROC curve, denoting excellent diagnostic accuracy. Subgroup analysis by sample type revealed that sputum samples achieved sensitivity and specificity of 0.92 (95%CI: 0.89-0.94) and 0.97 (95%CI: 0.94-0.99), while extrapulmonary samples (BALF, pus, CSF) showed comparable performance with sensitivity of 0.89 (95%CI: 0.84-0.93) and specificity of 0.98 (95%CI: 0.94-0.99).<br><br>CONCLUSION: CRISPR-based methods exhibit substantial diagnostic sensitivity and specificity for detecting MTB across both pulmonary and extrapulmonary samples, with notable variances across different CRISPR variants and methodological approaches.},
}
@article {pmid41240616,
year = {2025},
author = {Zong, W and Xie, S and Chu, H and Han, S and Zhang, X},
title = {Beyond traditional antibacterial agents: Novel approaches to combat resistant pathogens.},
journal = {European journal of medicinal chemistry},
volume = {302},
number = {Pt 2},
pages = {118362},
doi = {10.1016/j.ejmech.2025.118362},
pmid = {41240616},
issn = {1768-3254},
abstract = {As bacterial infections caused by antibiotic-resistant strains become increasingly prevalent, traditional antibacterial therapies face mounting challenges. These resilient pathogens not only complicate the treatment of common infections but also undermine the efficacy of therapies for major diseases. This growing threat underscores the urgent need for innovative therapeutic strategies. In recent years, the combinatorial use of antibacterial agents has emerged as a promising approach to enhance efficacy and combat resistant bacteria. This review first provides an overview of antibacterial classifications and their mechanisms of action against bacterial infections. It then explores two combined treatment strategies: antibacterial-antibacterial combinations and antibacterial-non-antibacterial pairings, alongside a drug delivery technology: antibacterial-loaded liposomes. Furthermore, we highlight emerging frontiers in antimicrobial strategies, including CRISPR-Cas technologies, AI-driven discovery platforms, nanomaterials beyond liposomes, microbiota-based therapies, and immunotherapeutic approaches. Finally, we offer a forward-looking perspective on the challenges and opportunities shaping the future of antibacterial development in the biomedical field.},
}
@article {pmid41178406,
year = {2025},
author = {Pei, C and Yan, B and Wang, Y and Chen, T and Du, K and Ma, L and Wang, J},
title = {TdT/Cas12a cascade amplification biosensor for sensitive ALP activity detection.},
journal = {The Analyst},
volume = {150},
number = {23},
pages = {5330-5337},
doi = {10.1039/d5an00938c},
pmid = {41178406},
issn = {1364-5528},
mesh = {Humans ; *Biosensing Techniques/methods ; *Alkaline Phosphatase/metabolism/analysis ; HeLa Cells ; CRISPR-Cas Systems ; Limit of Detection ; *DNA Nucleotidylexotransferase/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {This study presents a novel biosensor based on TdT and CRISPR-Cas12a, which integrates the catalytic activity of terminal deoxynucleotidyl transferase (TdT) with the trans-cleavage property of CRISPR-Cas12a to achieve ultra-sensitive biomolecular detection. The biosensor exhibited a broad linear detection range from 0 to 0.2 U L[-1] and a remarkably low detection limit of 1.7 × 10[-3] U L[-1], demonstrating high specificity and sensitivity. In practical validation, the biosensor successfully quantified alkaline phosphatase (ALP) activity in both cervical cancer cells and HeLa cell lysates, even at a dilution factor of up to 10[6]-fold. Its sensitivity allowed precise detection at the single-cell level. This technology offers a robust, simple, and cost-effective platform for cancer diagnosis, treatment monitoring, and enzyme inhibitor screening, while maintaining excellent detection performance in complex biological samples. This breakthrough establishes a foundation for serological tumor screening and early disease diagnosis, while also opening new avenues for enhanced cancer management and clinical translation, indicating significant potential in translational medicine.},
}
@article {pmid41171167,
year = {2025},
author = {Tharmatt, A and Guha, S and Kumeria, T and Yadav, S and Mittal, A and Chitkara, D},
title = {Lipopolymeric Nanoplex-Mediated CRISPR/Cas9 Delivery for VEGF-A Knockdown in Psoriatic Angiogenesis.},
journal = {ACS applied bio materials},
volume = {8},
number = {11},
pages = {10137-10157},
doi = {10.1021/acsabm.5c01478},
pmid = {41171167},
issn = {2576-6422},
mesh = {*CRISPR-Cas Systems ; *Psoriasis/drug therapy/metabolism/genetics/pathology ; Humans ; *Vascular Endothelial Growth Factor A/genetics/metabolism/antagonists &amp; inhibitors ; Animals ; *Biocompatible Materials/chemistry/pharmacology/chemical synthesis ; Particle Size ; *Neovascularization, Pathologic/drug therapy/genetics ; Mice ; Materials Testing ; *Polymers/chemistry ; Gene Knockdown Techniques ; *Nanoparticles/chemistry ; Angiogenesis ; },
abstract = {Psoriasis is a chronic, incurable inflammatory skin disease characterized by immune cell infiltration, aberrant keratinocyte differentiation, and enhanced angiogenesis. Overexpression of the vascular endothelial growth factor-A (VEGF-A) gene promotes angiogenesis and is essential for endothelial cell migration, adhesion, and proliferation. Therefore, downregulating VEGF-A represents a promising therapeutic strategy for angiogenesis-related disorders. We investigated the application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein complexes (sgRNA/eGFP-Cas9 RNPs) targeting VEGF-A in psoriasis. To enable efficient delivery in vitro and in vivo, we developed lipopolymeric nanoplexes (NPXs) encapsulating sgRNA/eGFP-Cas9 RNPs. These NPXs exhibited a particle size of 142.2 nm (polydispersity index: 0.144), a zeta potential of +4.27 mV, and achieved >70% transfection efficiency in HaCaT (human immortalized keratinocyte) cells. Ex vivo skin permeation studies demonstrated 66% of permeation after 24 h. The optimized NPX formulation was incorporated into a Carbopol-based gel, which displayed non-Newtonian, shear-thinning behavior with variable thixotropy and achieved 48% of skin permeation after 24 h. In vivo efficacy assessment in an imiquimod-induced psoriasis model in Swiss albino mice showed significantly improved Psoriasis Area and Severity Index (PASI) scores, reduced epidermal damage, and suppressed keratinocyte proliferation compared to naked RNPs and blank gel controls. Gene editing analysis revealed an indel frequency of 40.7% by T7 endonuclease I assay and 14% by Sanger sequencing. Enhanced cellular uptake, efficient skin permeation and retention, and improved therapeutic efficacy collectively highlight the potential of NPX-mediated CRISPR/Cas9 delivery as a noninvasive strategy for psoriasis treatment.},
}
@article {pmid40982615,
year = {2025},
author = {Kanwal, F and Aslam, A and Torriero, AAJ},
title = {Microalgae-based biodiesel: integrating AI, CRISPR and nanotechnology for sustainable biofuel development.},
journal = {Emerging topics in life sciences},
volume = {8},
number = {3},
pages = {131-143},
pmid = {40982615},
issn = {2397-8554},
mesh = {*Microalgae/metabolism/genetics ; *Biofuels ; *Nanotechnology/methods ; *Artificial Intelligence ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; },
abstract = {Microalgae are a promising feedstock for biodiesel due to their rapid growth, high lipid content and ability to use non-arable land and wastewater. This review synthesises recent advances in artificial intelligence (AI)-driven strain optimisation, engineering, nanotechnology-assisted processing, and life cycle and technoeconomic insights to evaluate pathways for industrialisation. Over the past decade (2015-2024), genetic engineering and, more recently, AI-guided strain selection have improved lipid productivity by up to 40%. Cultivation advances, including hybrid photobioreactor-open pond systems and precision pH/CO2 control, have enhanced biomass yields while reducing costs. Innovation in lipid extraction, such as supercritical CO2 and microwave-assisted methods, now achieves >90% yields with lower toxicity, while magnetic nanoparticle-assisted harvesting and electroflocculation have reduced energy inputs by 20-30%. Life cycle analyses (net energy ratio ~2.5) and integration of high-value co-products (e.g. pigments and proteins) underscore the need to align biological innovations with techno-economic feasibility. This review uniquely integrates advances in AI, CRISPR and nanotechnology with life cycle and techno-economic perspectives, providing a comprehensive framework that links laboratory-scale innovation to industrial feasibility and positions microalgal biodiesel as a viable contributor to global decarbonisation strategies.},
}
@article {pmid40447760,
year = {2025},
author = {Hebert, JD and Xu, H and Tang, YJ and Ruiz, PA and Detrick, CR and Wang, J and Hughes, NW and Donosa, O and Siah, VP and Andrejka, L and Karmakar, S and Aboiralor, I and Tang, R and Sotillo, R and Sage, J and Cong, L and Petrov, DA and Winslow, MM},
title = {Efficient and multiplexed somatic genome editing with Cas12a mice.},
journal = {Nature biomedical engineering},
volume = {9},
number = {11},
pages = {1982-1997},
pmid = {40447760},
issn = {2157-846X},
support = {T34FT8013//Tobacco-Related Disease Research Program (TRDRP)/ ; K00 CA234962/CA/NCI NIH HHS/United States ; DGE-2146755//National Science Foundation (NSF)/ ; R01 CA231253/CA/NCI NIH HHS/United States ; R35 HG011316/HG/NHGRI NIH HHS/United States ; R35-CA231997//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 CA234349/CA/NCI NIH HHS/United States ; R01-GM141627//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01-CA231253//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; K00CA234962//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; PF-21-112-01-MM//American Cancer Society (American Cancer Society, Inc.)/ ; P01 CA244114/CA/NCI NIH HHS/United States ; R35-HG011316//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P30-CA124435//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01-CA234349//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 CA230025/CA/NCI NIH HHS/United States ; R01 GM141627/GM/NIGMS NIH HHS/United States ; R35 CA231997/CA/NCI NIH HHS/United States ; P30 CA124435/CA/NCI NIH HHS/United States ; P01-CA244114//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; Mice, Transgenic ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Acidaminococcus/genetics/enzymology ; *Bacterial Proteins/genetics ; Genome ; *Endodeoxyribonucleases/genetics ; Genotype ; },
abstract = {Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations. However, existing models have a limited ability to create multiple targeted edits, hindering our understanding of complex genetic interactions. Here we generate transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a), which robustly generates compound genotypes, including diverse cancers driven by inactivation of trios of tumour suppressor genes or an oncogenic translocation. We integrate these modular CRISPR RNA (crRNA) arrays with clonal barcoding to quantify the size and number of tumours with each array, as well as the impact of varying the guide number and position within a four-guide array. Finally, we generate tumours with inactivation of all combinations of nine tumour suppressor genes and find that the fitness of triple-knockout genotypes is largely explainable by one- and two-gene effects. These Cas12a alleles will enable further rapid creation of disease models and high-throughput investigation of coincident genomic alterations in vivo.},
}
@article {pmid41240306,
year = {2026},
author = {Harada, A},
title = {Golgi Units as Modules in the Ribbon.},
journal = {Sub-cellular biochemistry},
volume = {110},
number = {},
pages = {35-42},
pmid = {41240306},
issn = {0306-0225},
mesh = {*Golgi Apparatus/metabolism/ultrastructure/genetics ; Glycosylation ; Humans ; Glycosaminoglycans/metabolism/biosynthesis ; CRISPR-Cas Systems ; HeLa Cells ; Animals ; },
abstract = {Using super-resolution microscopies, SCLIM and STORM, and CRISPR/Cas9 knockin technology, we show that the Golgi complex is assembled by a number of small "Golgi units" that are 1-3 μm in diameter. Each Golgi unit can be considered a functional and morphological module because it contains all sets of glycosylation enzymes that occupy small domains named "zones." Golgi units change shape dynamically. They attach, detach, fuse, and separate from each other. The zones of glycosylation enzymes rapidly move near the rim of the unit. However, the behaviors of N- and O-linked glycosylation enzyme zones differ from those of glycosaminoglycan (GAG)-synthesizing enzyme zones in localization and dynamics. Since depletion of giantin dissociates the Golgi units, it is responsible for their attachment. Giantin depletion also prevents the movement of GAG-synthesizing enzymes between units, leading to incomplete GAG synthesis. Here, we present the model of the Golgi and its glycosylation enzymes that can explain a number of characteristics of the glycosylation processes.},
}
@article {pmid41240134,
year = {2025},
author = {Gong, J and Soleimani Samarkhazan, H and Siavashi, M and Servatian, N and Pirsavabi, F},
title = {CRISPR-Cas9 in leukemia immunotherapy: precision engineering of CAR-T cells and tumor-microenvironment modulation.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {90},
pmid = {41240134},
issn = {1573-4978},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Leukemia/therapy/immunology/genetics ; Gene Editing/methods ; Receptors, Chimeric Antigen/genetics/immunology ; *Tumor Microenvironment/immunology/genetics ; *Immunotherapy, Adoptive/methods ; Precision Medicine/methods ; *Immunotherapy/methods ; T-Lymphocytes/immunology ; Animals ; },
abstract = {Together with base editors, prime editors, and alternative nuclease platforms, CRISPR-Cas9 technology has transformed the field of genetic engineering by providing unprecedented precision in genome editing and creating new opportunities for therapeutic interventions. This technology comprises a versatile genome-editing toolkit for achieving a variety of therapeutic objectives. In the context of leukemia, a group of life-threatening hematologic malignancies, CRISPR-Cas9 has emerged as a transformative tool for immunotherapy. By enabling targeted modifications of immune cells, such as T cells, this technology enhances their ability to recognize and eradicate leukemic cells. CRISPR-Cas9 facilitates the disruption of immune checkpoint inhibitors, the insertion of chimeric antigen receptors (CARs), and the correction of genetic mutations that drive leukemia progression. These advancements have led to the development of more potent and personalized immunotherapies, such as CAR-T cell therapies, with improved efficacy and reduced off-target effects. Moreover, CRISPR-Cas9 allows researchers to model leukemia in vitro, providing deeper insights into disease mechanisms and accelerating the discovery of novel therapeutic targets. Despite challenges such as delivery efficiency and potential immunogenicity, the integration of CRISPR-Cas9 into leukemia immunotherapy represents a paradigm shift, offering hope for durable remissions and potentially curative outcomes. As clinical trials progress, this technology promises to redefine the standard of care for leukemia patients, ushering in a new era of precision medicine. This narrative review explores the revolutionary applications of CRISPR-Cas9 in redefining therapeutic strategies for leukemia.},
}
@article {pmid41240131,
year = {2025},
author = {Behera, L and Samal, KC and Mishra, A and Sahoo, JP and Dash, M and Mishra, A},
title = {CRISPR-driven innovations in rice (Oryza sativa L.) breeding: precision development of male sterile lines.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {89},
pmid = {41240131},
issn = {1573-4978},
mesh = {*Oryza/genetics ; *Plant Infertility/genetics ; *Plant Breeding/methods ; Gene Editing/methods ; Pollen/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {Male sterility is a vital trait exploited in hybrid seed production to boost crop yield and improve quality. In rice, different male sterility systems have been developed, significantly advancing the production of high-yielding hybrids. The three main types of male sterility in rice are cytoplasmic male sterility (CMS), photoperiod-sensitive genic male sterility (PGMS), and genic male sterility (GMS). Among these, CMS is the most widely used, arising from interactions between mitochondrial (cytoplasmic) and nuclear genes, resulting in pollen dysfunction. PGMS, on the other hand, is influenced by environmental cues such as day length and temperature, while GMS is attributed to mutations in specific nuclear genes affecting anther or pollen development. A thorough understanding of the genetic and molecular mechanisms underlying these systems is essential for efficient hybrid rice breeding. CMS lines are typically crossed with maintainer and restorer lines carrying fertility-restoring genes to produce fertile F1 hybrids. Recent advancements in molecular biology, genomics, and genome editing technologies have accelerated the development of novel male-sterile and fertility-restoring lines, thereby enhancing the precision and scalability of hybrid breeding programs. These innovations are not only expanding the genetic base of hybrid rice but also making the production process more sustainable. As global food demand rises alongside climate uncertainties, the strategic use of male sterility in rice breeding holds immense potential for improving agricultural productivity.},
}
@article {pmid41239469,
year = {2025},
author = {Ma, J and Zhang, J and Guo, X},
title = {Harnessing CRISPR-Cas9 for Lactobacillus improvement in silage production: current knowledge and future perspectives.},
journal = {Journal of animal science and biotechnology},
volume = {16},
number = {1},
pages = {150},
pmid = {41239469},
issn = {1674-9782},
support = {U20A2002//National Natural Science Foundation of China/ ; },
abstract = {High-quality silage is the cornerstone to sustainable livestock development and animal food production. As the core fermentation bacteria of silage, Lactobacillus directly regulates silage fermentation by producing lactic acid, enzymes, and other bioactive molecules. However, traditional screening methods for functional strains are labor-intensive and time-consuming. Recent advances in synthetic biology, particularly the development of CRISPR-Cas genome editing technology, offer a revolutionary approach to designing Lactobacillus strains with customized traits. This review systematically reviewed the importance of silage in sustainable agricultural development and the limitations of current silage preparation and promotion. It also discussed the application of strain engineering approaches in optimizing the phenotypic performance of Lactobacillus for better silage. Building on this, we reviewed the research progress of CRISPR-Cas9 gene editing in Lactobacillus and discussed how to leverage its high efficiency and precision to optimize the strain's traits for improved silage quality and functionality. CRISPR-Cas9 toolkits are expected to achieve directed evolution of strain performance, ultimately yielding next-generation silage microbial inoculants with multiple functions, adaptability to multiple substrates, and eco-friendly characteristics. The use of such innovative biotechnologies would facilitate resource-efficient utilization, promote animal performance and health for sustainable development in livestock production.},
}
@article {pmid41239088,
year = {2025},
author = {Xu, H and Chen, H and Li, Y and Wang, Y and Zeng, H and Xu, Z and Chen, T and Kong, D and Huang, W and Cheng, H and Zhou, H and Jiang, X and Feng, J},
title = {CRISPR/Cas12a-SERS biosensor based on sea urchin-like AuNPs for the detection of β-thalassemia mutant gene CD31.},
journal = {Mikrochimica acta},
volume = {192},
number = {12},
pages = {810},
pmid = {41239088},
issn = {1436-5073},
mesh = {*Biosensing Techniques/methods ; *beta-Thalassemia/genetics/diagnosis ; *CRISPR-Cas Systems ; Animals ; *Spectrum Analysis, Raman/methods ; *Gold/chemistry ; *Metal Nanoparticles/chemistry ; Mutation ; Sea Urchins/chemistry ; Limit of Detection ; Humans ; *Endodeoxyribonucleases/genetics/metabolism ; DNA, Single-Stranded/chemistry/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Sulfhydryl Compounds/chemistry ; Bacterial Proteins ; Benzoates ; },
abstract = {Beta-thalassemia is a single-gene recessive disorder caused by mutations in the HBB gene, and approximately 1.5% of the global population are carriers of β-thalassemia. It is therefore vital to establish a rapid and sensitive method to detect the mutant genes of β-thalassemia. In this study, a CRISPR/Cas12a-mediated amplification-free surface-enhanced Raman spectroscopy (SERS) biosensor was developed. This biosensor uses sea urchin-shaped gold nanoparticles (SUGNPs) as the SERS enhancement substrate and 4-mercaptobenzoic acid (4-MBA) as the Raman reporter. It couples the SUGNPs/4-MBA with magnetic beads through single-strand DNA (ssDNA) to form an SERS probe with magnetic responsiveness. The presence of the β-thalassemia target mutation gene CD31 activates the cleavage activity of Cas12a, leading to non-specific cleavage of single-stranded DNA (ssDNA) on the probe. This results in a significant reduction in SERS intensity. This signal change enables quantitative detection of the target gene, thereby significantly enhancing the sensitivity of nucleic acid testing. We employed magnetic separation technology to enrich target nucleic acids in serum while eliminating matrix interference, enabling specific recognition and quantitative detection of the mutated CD31 gene. This method exhibits excellent linearity over a concentration range 0.1 fM to 10 pM, with a detection limit of 0.1 fM and a detection time of only 40 min. Compared to traditional qPCR and other CRISPR methods, this approach is simple, rapid, and offers advantages such as high sensitivity, high specificity, and cost-effectiveness. By simply replacing the crRNA, it can detect multiple β-thalassemia and other disease genes, demonstrating broad clinical application potential.},
}
@article {pmid41236708,
year = {2025},
author = {Zahedi, S},
title = {CRISPR-Based Functional Genomics in Pluripotent Stem Cells.},
journal = {Stem cell reviews and reports},
volume = {},
number = {},
pages = {},
pmid = {41236708},
issn = {2629-3277},
abstract = {The integration of CRISPR-based functional genomics with pluripotent stem cell (PSC) technologies has been recognized as a transformative approach for investigating gene function, modeling human disease, and advancing regenerative medicine. The aim of this review is to provide a comprehensive evaluation of recent developments in CRISPR-Cas platforms, including gene knockouts, base and prime editing, and CRISPR activation or interference (CRISPRa/i), as applied to PSC systems. Studies employing human PSCs, including embryonic stem cells and induced pluripotent stem cells, have been examined to summarize methodologies for genome-wide screening, lineage tracing, and therapeutic engineering. Advances in editing efficiency, delivery strategies, and genomic safety have been reported, while limitations persist in the form of off-target modifications, epigenetic variability, and cell-type-specific responses. Notable applications include the generation of immune-evasive PSC lines, the development of organoid models for physiological and pathological studies, and the implementation of phenotypic screening for disease-relevant traits. Collectively, these technological and methodological advancements have established functional genomics of PSC-CRISPRSPR as a versatile and powerful framework for elucidating fundamental aspects of human biology, dissecting complex traits, and accelerating the translation of discoveries from experimental research to clinical implementation.},
}
@article {pmid41130353,
year = {2026},
author = {Lee, SY and Kim, HS and Kim, YG and Shin, S and Kweon, S and Lee, JJ and Lee, GM},
title = {Genome-wide CRISPR screening identifies genes in recombinant human embryonic kidney 293 cells for increased ammonia resistance.},
journal = {Metabolic engineering},
volume = {93},
number = {},
pages = {184-193},
doi = {10.1016/j.ymben.2025.10.008},
pmid = {41130353},
issn = {1096-7184},
mesh = {Humans ; *Ammonia/pharmacology/metabolism ; HEK293 Cells ; *CRISPR-Cas Systems ; *Antibodies, Monoclonal/genetics/biosynthesis ; Gene Knockout Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genome, Human ; },
abstract = {Ammonia, a byproduct of glutamine metabolism, inhibits cell growth and reduces product yield and quality in mammalian cell culture. To identify novel genes associated with ammonia resistance, a genome-wide CRISPR knockout screening was conducted in monoclonal antibody (mAb)-producing human embryonic kidney 293 (HEK-mAb) cells using a virus-free, recombinase-mediated cassette exchange-based gRNA interrogation method. The knockout cell library was subcultured for five consecutive passages under 20 mM NH4Cl, enriching cells with a sgRNA that conferred a proliferation advantage under high-ammonia conditions. Next-generation sequencing analysis of the enriched population identified three target genes -WNT3, TSPAN1, and CYHR1-among 19,114 genes. Knockout of these genes in HEK-mAb cells resulted in a 1.33- to 1.56-fold increase in maximum viable cell concentration and a 1.28- to 1.58-fold increase in maximum mAb concentration under 20 mM NH4Cl. Notably, WNT3 knockout maintained N-glycan galactosylation proportions of mAb despite ammonia stress. These findings highlight the effectiveness of genome-wide CRISPR knockout screening in identifying novel gene targets for ammonia-resistant HEK293 cell, offering a promising strategy for improving mAb production.},
}
@article {pmid41110569,
year = {2025},
author = {Cui, Z and Liang, W and Li, J and Bai, Z},
title = {CRISPR/Cas9 gene editing strategy for cancer therapy: non-viral nanocarrier-mediated delivery of plasmids, RNA and ribonucleoprotein complexes.},
journal = {International journal of biological macromolecules},
volume = {331},
number = {Pt 1},
pages = {148389},
doi = {10.1016/j.ijbiomac.2025.148389},
pmid = {41110569},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Ribonucleoproteins/genetics ; *Neoplasms/therapy/genetics ; *Plasmids/genetics ; Animals ; *RNA/genetics ; *Gene Transfer Techniques ; Genetic Therapy/methods ; Nanoparticles/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing systems are among the most prevalent tools in modern genetic research and have been extensively studied in the context of cancer therapy. CRISPR/Cas9 systems can be categorized into three main delivery forms: plasmid DNA (pDNA) containing both Cas9 and guide RNA (sgRNA), messenger RNA (mRNA) of Cas9 along with sgRNA, and ribonucleoprotein (RNP) complexes consisting of Cas9 protein bound to sgRNA. This article will review the various CRISPR/Cas9 systems, focusing on the non-viral especially nanocarrier delivery strategies and common protocols used for their delivery and the challenges encountered during this process. This review will offer guidance on the potential applications of CRISPR/Cas9 gene editing technology for the treatment of malignant tumors in the future.},
}
@article {pmid41093194,
year = {2025},
author = {Yang, G and Rao, Q and Dong, W and Yin, X and Shen, R and Wang, R and Deng, X and Peng, X and Tao, Y and Li, S and Wang, X and Tang, Y and Du, D},
title = {Rapid and sensitive one-pot CRISPR-Cas12a detection platform for common fungal species in ocular infections using multiplex crRNAs.},
journal = {International journal of biological macromolecules},
volume = {331},
number = {Pt 1},
pages = {148279},
doi = {10.1016/j.ijbiomac.2025.148279},
pmid = {41093194},
issn = {1879-0003},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Fungi/genetics/isolation &amp; purification ; *Eye Infections, Fungal/microbiology/diagnosis/genetics ; DNA, Fungal/genetics ; },
abstract = {Corneal fungal infection is a major cause of visual impairment and can cause blindness if not diagnosed promptly. Traditional diagnostic methods are often time-consuming and require specialized expertise, UNDERSCORING the urgent need for rapid, sensitive, and user-friendly diagnostic tools. In this study, we utilized multiplex crRNAs in the Cas12a system to develop a highly sensitive and specific platform for the molecular detection of common fungal species causing corneal infections in a single reaction. We enhanced detection sensitivity through recombinase-aided amplification (RAA) and designed a streamlined tube with insert one-pot protocol to minimize aerosol contamination risk. Remarkably, our platform achieved a limit of detection of just 4 copies of fungal genomic DNA per reaction in only 32 min. It allows for direct detection from simulated tear samples, providing a potential for convenient and non-invasive testing option. Furthermore, our platform effectively tests clinical samples such as aqueous humor (AH) and vitreous humor (VH), demonstrating superior sensitivity compared to traditional PCR-based methods. These results highlight the potential of our platform as an on-site diagnostic solution for both clinical and non-clinical settings, contributing to the preservation of visual function.},
}
@article {pmid41043753,
year = {2025},
author = {Yang, Q and Zhao, H and Zhai, Y and Wang, Q and Zhu, Z and Rui, C and Yuan, H and Cui, L},
title = {Ryanodine receptor I4734M mutation confers diamide resistance but reduces invasiveness in Spodoptera frugiperda.},
journal = {International journal of biological macromolecules},
volume = {331},
number = {Pt 2},
pages = {148059},
doi = {10.1016/j.ijbiomac.2025.148059},
pmid = {41043753},
issn = {1879-0003},
mesh = {Animals ; *Spodoptera/genetics/drug effects ; *Ryanodine Receptor Calcium Release Channel/genetics ; *Mutation ; *Diamide/pharmacology ; *Insecticide Resistance/genetics ; ortho-Aminobenzoates/pharmacology ; Insecticides/pharmacology ; CRISPR-Cas Systems ; Gene Editing ; Pyrazoles ; },
abstract = {Although Spodoptera frugiperda populations with the ryanodine receptor (RyR) I4734M mutation occurred in their native regions, this mutation has not been detected in the Eastern Hemisphere. In order to clarify the functional role of this mutation, the first HDR-mediated RyR[I4734M] mutation was introduced into S. frugiperda using CRISPR/Cas9 technology and a homozygous SfRyR[I4734M] strain was successfully established. Meanwhile, a novel, non-invasive genotyping method based on insect fecal DNA to efficiently identify genome-edited individuals was developed. Compared with the wild type, the genome-edited SfRyR[I4734M] strain showing high-level resistance to chlorantraniliprole (396.7-fold) and tetraniliprole (149.1-fold), and moderate resistance to cyantraniliprole (32.3-fold) and flubendiamide (29.5-fold). Reciprocal crossing experiments indicated that resistance to chlorantraniliprole was inherited in an autosomally incompletely recessive mode. Furthermore, the SfRyR[I4734M] substitution adversely reduced the fitness and flight ability of S. frugiperda. The mutant strain exhibited significantly decreased fecundity and severely impaired flight distance and velocity. These findings provided in vivo genetic validation of SfRyR[I4734M] mutation in diamide resistance, demonstrating this mutation conferred subtle differences on the binding affinities of four diamides. Moreover, our results firstly demonstrated that SfRyR[I4734M] mutation could affect muscle function, thereby reducing the flight ability of S. frugiperda.},
}
@article {pmid41236489,
year = {2025},
author = {Wang, Y and Kong, Y and Zhang, Y and He, Y and Wang, W and Lu, Y and Meng, J and Yuan, H},
title = {Long-Term Attenuation of Vascular Hyperpermeability in a Hereditary Angioedema Mouse Model by Adenine Base Editing.},
journal = {Allergy},
volume = {},
number = {},
pages = {},
doi = {10.1111/all.70152},
pmid = {41236489},
issn = {1398-9995},
support = {//the National Natural Science Foundation of China/ ; //West China Hospital, Sichuan University/ ; },
abstract = {BACKGROUND: Hereditary angioedema (HAE) is a rare and potentially life-threatening disorder caused by dysregulated kallikrein-kinin signaling and bradykinin-induced vascular hyperpermeability. Current therapies targeting this pathway are effective but require lifelong administration, underscoring the need for durable and potentially curative interventions. Adenine base editors (ABEs), engineered from CRISPR/Cas systems, enable precise single-nucleotide modifications with minimal genomic disruption, offering a promising strategy for long-term gene silencing.<br><br>METHODS: NG-ABE8e was delivered via AAV8 or lipid nanoparticles (LNP) to disrupt the exon 2 splice donor site of Klkb1 in Serping1[+/-] mice. Editing outcomes were quantified by high-throughput sequencing; serum kallikrein levels were measured by ELISA; and vascular permeability was evaluated using Evans blue dye extravasation, fluorescent tracer leakage, and VE-cadherin immunostaining. Safety evaluations included off-target analysis, histopathology, serum biochemistry, activated partial thromboplastin time (aPTT), and systemic hemodynamic stability.<br><br>RESULTS: AAV8-NG-ABE8e induced >&#x2009;60% A&#x2022;T-to-G&#x2022;C conversion at the target site, promoting exon 2 skipping and reducing Klkb1 mRNA and serum kallikrein levels by >&#x2009;85%, an effect sustained for at least 1&#x2009;year. AAV8-NG-ABE8e also reversed bradykinin-driven vascular hyperpermeability and attenuated inflammatory gene signatures. Prolongation of aPTT was observed only when circulating kallikrein levels declined below 2&#x2009;&#x3bc;g/mL. Similarly, LNP-mediated delivery of NG-ABE8e mRNA resulted in >&#x2009;90% reductions in Klkb1 expression and serum kallikrein levels. No evidence of significant long-term toxicity was detected.<br><br>CONCLUSION: These findings demonstrate that, in a murine model, NG-ABE8e-mediated disruption of Klkb1 enables durable suppression of serum kallikrein and vascular stabilization, suggesting its potential as a promising single-intervention strategy for the treatment of HAE.},
}
@article {pmid41236435,
year = {2025},
author = {Chen, Y and Wang, Y and Liu, L and Yu, X and Zhang, Y and Xi, M and Xu, J and Yang, H and Xie, C and Wang, D},
title = {Targeting BnNAC038 improves drought tolerance with low yield penalty in Brassica napus.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {3},
pages = {e70571},
doi = {10.1111/tpj.70571},
pmid = {41236435},
issn = {1365-313X},
support = {241111112400//Key Research and Development Project of Henan Province/ ; 2023-YBNY-023//Key Research and Development Project of Shaanxi Province/ ; 32070324//National Natural Science Foundation of China/ ; 32301811//National Natural Science Foundation of China/ ; 2024YFD1200400//the National Key Research and Development Project from the Ministry of Agriculture and Rural Affairs of China/ ; },
mesh = {*Brassica napus/genetics/physiology/growth &amp; development ; Droughts ; *Plant Proteins/genetics/metabolism/physiology ; Gene Expression Regulation, Plant ; Photosynthesis/genetics ; CRISPR-Cas Systems ; Drought Resistance ; },
abstract = {Drought stress severely limits crop productivity in Brassica napus, yet strategies to enhance drought tolerance without compromising yield remain elusive. Here, we identify BnNAC038 as a negative regulator of drought responses in Brassica napus. CRISPR/Cas9-generated bnnac038 mutants exhibited improved drought survival, reduced water loss, and enhanced stomatal closure under drought conditions compared to wild-type (WT) plants. RNA-sequencing (RNA-seq) and DNA affinity purification sequencing (DAP-seq) analyses revealed that BnNAC038 directly represses drought-responsive genes, including BnSnRK2.6 (a key ABA signaling kinase), and genes involved in photosynthesis (BnPPC2) and gluconeogenesis (BnPGK). Field trials demonstrated that bnnac038 plants exhibit enhanced photosynthesis, accumulate more sucrose and glucose under drought, and exhibit increased biomass and seed yield compared to WT. Genetic interaction studies further showed that BnSnRK2.6 acts downstream of BnNAC038 to mediate drought tolerance. Our results indicate that targeted editing of BnNAC038 enhances drought tolerance while minimizing yield loss, providing a new strategy for developing drought-resilient Brassica napus varieties with minimal yield penalty.},
}
@article {pmid41236144,
year = {2025},
author = {Apsley, EJ and Riepsaame, J and Cheng, YC and Cowley, SA and Becker, EBE},
title = {Cerebellar organoids model cell type-specific FOXP2 expression during human cerebellar development.},
journal = {Disease models &amp; mechanisms},
volume = {18},
number = {11},
pages = {},
doi = {10.1242/dmm.052290},
pmid = {41236144},
issn = {1754-8411},
support = {BB/M011224/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //UK Research and Innovation/ ; },
mesh = {Humans ; *Forkhead Transcription Factors/metabolism/genetics ; *Organoids/metabolism ; *Cerebellum/metabolism/growth &amp; development/embryology/cytology ; Induced Pluripotent Stem Cells/metabolism/cytology ; Purkinje Cells/metabolism ; Gene Expression Regulation, Developmental ; Cell Differentiation/genetics ; *Models, Biological ; CRISPR-Cas Systems/genetics ; },
abstract = {Human cerebellar development is unique and cannot be fully replicated in animal models. Although human stem cell-derived cerebellar organoid models are increasingly being applied to model cerebellar diseases, their potential to provide insight into normal human cerebellar development remains underexplored. Here, we used CRISPR-based gene editing in cerebellar organoids as an approach for modelling specific features of early human cerebellar development. Forkhead box protein P2 (FOXP2) is a transcription factor associated with speech and language development that is highly expressed in the developing brain. However, little attention has been directed to the study of FOXP2 in the early developing cerebellum. We generated a fluorescent FOXP2 reporter line in human induced pluripotent stem cells to enable the characterisation of FOXP2-expressing cells during cerebellar organoid differentiation. Through transcriptomic profiling of FOXP2 reporter cerebellar organoids and cross-referencing with existing cerebellar datasets, we describe the expression and identify potential downstream targets of FOXP2 in the early developing human cerebellum. Our results highlight expression of FOXP2 in early human Purkinje cells and cerebellar nuclei neurons, and the vulnerability of these cell populations to neurodevelopmental disorders.},
}
@article {pmid41234307,
year = {2025},
author = {Tuncel, A and Kim, HU and Kim, MC},
title = {Editorial: Engineering future crops through genome editing.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1720325},
pmid = {41234307},
issn = {1664-462X},
}
@article {pmid41233858,
year = {2025},
author = {Mayer, J and Pack, M and Montenarh, M and Götz, C},
title = {Gene expression changes in pancreatic α-cell lines following knock-out Of either CK2α or CK2α'.},
journal = {Biological research},
volume = {58},
number = {1},
pages = {69},
pmid = {41233858},
issn = {0717-6287},
mesh = {*Casein Kinase II/genetics/metabolism ; *Glucagon-Secreting Cells/metabolism ; Animals ; Mice ; Cell Line ; Gene Knockout Techniques ; *Gene Expression/genetics ; },
abstract = {BACKGROUND: Protein kinase CK2 is known to exist as a tetramer of two catalytic α- or α'- subunits and two non-catalytic β-subunits, or as multimers of this tetramer. Moreover, CK2α (CSNK2A1) and CK2α' (CSNK2A2) are also active in the absence of CK2β (CSNK2B). Very little is known about specific functions of the individual subunits of protein kinase CK2.<br><br>RESULTS: In order to study the effects of CK2&#x3b1; and CK2&#x3b1;' on gene expression, we used the Mus musculus pancreatic &#x3b1;-cell line &#x3b1;TC1 and two derivatives with either CK2&#x3b1; (KO1 cells) or CK2&#x3b1;' (KO2 cells) expression knocked-out by CRISPR/Cas technology. We found numerous genes deregulated in both KO1 and KO2 cells compared to the parental cells. Applying stringent thresholds, 266 genes were found down-regulated and 153 genes up-regulated in KO1 cells, 233 genes were found down-regulated and 84 genes up-regulated in KO2 cells. Dozens of genes were found deregulated in a similar fashion in both KO1 and KO2 cells. We found altered expression of genes involved in the differentiation of pancreatic cells, including Hox genes, and in the regulation of glucagon synthesis or secretion. Moreover, many of the deregulated genes play an important role in developmental processes and in neuronal cell biology.<br><br>CONCLUSION: Our findings reveal individual and shared functions of the CK2&#x3b1; and CK2&#x3b1;' catalytic subunits, in particular regarding their involvement in regulating gene expression.},
}
@article {pmid41232012,
year = {2025},
author = {Maire, A and Bikard, D},
title = {Programmable DNA insertion in native gut bacteria.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6774},
pages = {676-677},
doi = {10.1126/science.aec3823},
pmid = {41232012},
issn = {1095-9203},
mesh = {Animals ; *Gastrointestinal Microbiome/genetics ; Mice ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Bacteria/genetics ; *Mutagenesis, Insertional ; DNA, Bacterial/genetics ; },
abstract = {A gene-editing approach enables modification of bacteria within the mouse gut.},
}
@article {pmid41231980,
year = {2025},
author = {Gelsinger, DR and Ronda, C and Ma, J and Kar, OB and Edwards, M and Huang, Y and Mavros, CF and Sun, Y and Perdue, T and Vo, PL and Ivanov, II and Sternberg, SH and Wang, HH},
title = {Metagenomic editing of commensal bacteria in vivo using CRISPR-associated transposases.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6774},
pages = {eadx7604},
doi = {10.1126/science.adx7604},
pmid = {41231980},
issn = {1095-9203},
mesh = {Animals ; Mice ; *Gastrointestinal Microbiome/genetics ; *Gene Editing/methods ; *Bacteroides/genetics/growth &amp; development ; Humans ; Metagenomics/methods ; *CRISPR-Cas Systems ; Symbiosis ; Mice, Inbred C57BL ; Metagenome ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Although metagenomic sequencing has revealed a rich microbial biodiversity in the mammalian gut, methods to genetically alter specific species in the microbiome are highly limited. Here, we introduce Metagenomic Editing (MetaEdit) as a platform technology for microbiome engineering that uses optimized CRISPR-associated transposases delivered by a broadly conjugative vector to directly modify diverse native commensal bacteria from mice and humans with new pathways at single-nucleotide genomic resolution. Using MetaEdit, we achieved in vivo genetic capture of native murine Bacteroides by integrating a metabolic payload that enables tunable growth control in the mammalian gut with dietary inulin. We further show in vivo editing of segmented filamentous bacteria, an immunomodulatory small-intestinal microbial species recalcitrant to cultivation. Collectively, this work provides a paradigm to precisely manipulate individual bacteria in native communities across gigabases of their metagenomic repertoire.},
}
@article {pmid41231855,
year = {2025},
author = {Liu, S and Bai, J and Zhan, B and Yao, J and Zhang, J and Yi, J and Dong, M and Li, Q and Shen, Y and Chen, Y and Zhao, Y},
title = {Development of an RNA aptamer-assisted CRISPR/Cas9 system for efficiently generating and isolating Cas9-free mutants in plant.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011931},
pmid = {41231855},
issn = {1553-7404},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Aptamers, Nucleotide/genetics ; Plants, Genetically Modified/genetics ; Mutation ; *Arabidopsis/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas9 gene-editing system is a powerful tool in plant genetic engineering; however, screening for Cas9-free edited plants remains complex and time-consuming. To address this limitation, we developed an RNA aptamer-assisted CRISPR/Cas9 system, termed 3WJ-4 × Bro/Cas9. In this system, the engineered RNA aptamer 3WJ-4 × Bro functions as a transcriptional reporter, serving as an alternative to traditional fluorescent proteins and thus avoiding their potential interference with Cas9 activity. Compared to the conventional GFP/Cas9 system, 3WJ-4 × Bro/Cas9 showed more efficient transformation and higher accuracy in fluorescence-based selection of positive T1 transformants, without significantly affecting plant growth. Furthermore, 3WJ-4 × Bro/Cas9 achieved a 78.6% increase in the T1 mutation rate compared to GFP/Cas9, with the homozygous mutation rate reaching 1.78%. In addition, 3WJ-4 × Bro/Cas9 enabled fluorescence-based visual screening in the T2 generation for rapid identification of Cas9-free mutants, improving sorting efficiency by 30.2% over the GFP-based method. Moreover, 3WJ-4 × Bro/Cas9 enabled more efficient generation of homozygous double-target mutants compared to GFP/Cas9. These results demonstrate that the 3WJ-4 × Bro/Cas9 system provides a non-transgenic, efficient, and broadly applicable strategy for plant genome editing and selection.},
}
@article {pmid40958664,
year = {2025},
author = {Yang, L and Zhao, Z and Lei, R and Zhang, Y and Wu, P},
title = {A one-pot RPA/CRISPR-bio-dCas9 lateral flow assay for rapid and on-site detection of Monilinia fructicola in stone and pome fruits.},
journal = {Pest management science},
volume = {81},
number = {12},
pages = {8564-8574},
doi = {10.1002/ps.70159},
pmid = {40958664},
issn = {1526-4998},
support = {//the National Key Research and Development Program of China (2021YFC2600400)/ ; //the Basic Scientific Research Foundation of the Chinese Academy of Quality and Inspection &amp; Testing (2024JK051)/ ; },
mesh = {*Plant Diseases/microbiology ; *Fruit/microbiology ; *Ascomycota/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Brown rot, a devastating fungal disease affecting stone and pome fruits, leads to substantial economic losses worldwide, impacting production, post-harvest storage, and transportation. Monilinia fructicola, the primary causal agent of brown rot, is especially challenging to manage because of its ability to establish latent infections in fruit, making early detection and control extremely difficult, enabling the disease to spread unnoticed. Therefore, it is essential to develop fast and accurate detection technologies.<br><br>RESULTS: In this study, a one-pot detection method for M. fructicola, integrating CRISPR-bio-dCas9, recombinase polymerase amplification (RPA), and a lateral flow assay (LFA) was developed. This method could detect the M. fruticola genome in less than 30&#x2009;min from sample collection to result, with a detection limit of 4 copies/&#x3bc;L of M. fructicola. The RPA/CRISPR-bio-dCas9 LFA method simplified M. fructicola detection by eliminating the need for probes, additional reporters, or specialized equipment, thereby reducing costs and complexity. Furthermore, the streamlined single-tube workflow minimized cross-contamination risks, enabling non-expert workers to perform efficient pathogen screening.<br><br>CONCLUSIONS: The RPA/CRISPR-bio-dCas9 LFA enabled advanced brown rot management by addressing the limitations of traditional detection (slow speed, high cost, complexity). With high sensitivity and a short detection time using an equipment-free design, it enabled practical on-site detection of M. fructicola, allowing timely interventions to reduce pre- and post-harvest losses in fruit production. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid40810834,
year = {2025},
author = {Xu, M and Chu, J and Li, M and Ren, X and Chen, X and Li, X and Cheng, H and Wang, C and Yang, F},
title = {A Streamlined One-Step Bioprocess for Isomaltulose Production in Bacillus subtilis Through Multicopy Genomic Integration of Sucrose Isomerase Gene.},
journal = {Applied biochemistry and biotechnology},
volume = {197},
number = {10},
pages = {6820-6833},
pmid = {40810834},
issn = {1559-0291},
support = {GXTZYKF202406//Opening Project of Guangxi Key Laboratory of Green Processing of Sugar Resources/ ; 2024-MSLH-037//Liaoning Provincial Science and Technology Program Joint Program/ ; 202410152013//Dalian Polytechnic University College Student Innovation and Entrepreneurship Training Program Project/ ; },
mesh = {*Bacillus subtilis/genetics/metabolism/enzymology ; *Isomaltose/analogs &amp; derivatives/biosynthesis ; *Glucosyltransferases/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Isomaltulose, a sucrose isomer with a low glycemic index and non-cariogenic properties, is extensively used in the food industry. The industrial production of this functional sugar relies on enzymatic biotransformation using sucrose isomerase (SIase). However, conventional bioprocesses involve expressing and isolating the SIase enzyme, followed by using the purified SIase to convert sucrose into isomaltulose, resulting in a multi-step and high-cost process that hindered the broader applications of isomaltulose. In this study, we reported a streamlined one-step bioprocess that integrates extracellular SIase secretion and direct isomaltulose biosynthesis in the culture medium of an engineered B. subtilis strain. Using CRISPR/Cas9 technology, we engineered B. subtilis to integrate multiple SIase expression cassettes into the genome while concurrently replacing genes within the sacP-sacA-ywdA and sacB-levB-yveA operons, which are crucial for sucrose hydrolysis in B. subtilis. This strategy synergistically increased the genomic copy number of SIase gene while limited sucrose consumption by native pathways, thereby maximizing substrate availability for SIase-mediated catalysis. The resulting engineered strain, containing four copies of the SIase expression cassettes, achieved an extracellular SIase activity of 8.2 U/mL in shake flasks. When cultured in a medium containing 200 g/L sucrose, this strain produced a maximum isomaltulose titer of 162.1 g/L with a yield of 0.81 g/g and a productivity of 13.5 g/L/h. These findings demonstrate an integrated bioprocess that eliminates costly enzyme isolation procedure and reduces fermentation complexity, presenting a commercially feasible strategy for sustainable isomaltulose production.},
}
@article {pmid40759871,
year = {2025},
author = {Luo, X and Su, B and Lai, P and Li, M and Deng, MR and Zhu, H},
title = {Development of an NADPH Regeneration System for L-threonine Production in Escherichia coli.},
journal = {Applied biochemistry and biotechnology},
volume = {197},
number = {10},
pages = {6575-6591},
pmid = {40759871},
issn = {1559-0291},
support = {2021YFC2100900//National Key Research and Development Program of China/ ; 2021JC06N628//Guangdong Special Support Program/ ; 2022GDASZH-2022010101//GDAS' Project of Science and Technology Development/ ; },
mesh = {*NADP/metabolism ; *Escherichia coli/metabolism/genetics ; *Threonine/biosynthesis ; Escherichia coli Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {NADPH is essential for the biosynthesis of L-threonine, and a deficiency in its supply significantly constrains L-threonine production. To address the challenge of inadequate NADPH availability that adversely affects L-threonine synthesis, we developed an NADPH regeneration system aimed at enhancing the NADPH supply and subsequently improving L-threonine production. Through overexpression of the zwf and gnd genes, which are involved in NADPH generation within the pentose phosphate pathway (PPP), the NADPH/NADP[+] ratio in the strain was elevated 4.1-fold compared with the control strain, resulting in a 2.0-fold increase in L-threonine production. Subsequently, integration of the asd and thrA1034 genes, which are linked to NADPH consumption, enhanced L-threonine production by 3.6-fold. Moreover, the application of promoter engineering facilitated a 7.1-fold increase in L-threonine production compared with the control strain. Finally, we employed the CRISPR-Cas12f1 system to delete the pgi gene to further examine its impact on L-threonine production. The results indicated an increase in the NADPH/NADP[+] ratio and a subsequent enhancement in L-threonine production following deletion of the pgi gene. Consequently, the NADPH regeneration system developed in this study demonstrates potential to effectively improve L-threonine production and may serve as a novel strategy for L-threonine synthesis.},
}
@article {pmid40751408,
year = {2025},
author = {Bao, Z and Chang, X and Cheng, L and Lin, W and Xu, W and Shi, J},
title = {A highly specific and ultrasensitive approach to detect Hylurgus ligniperda based on RPA-CRISPR-LbaCas12a-LFD system.},
journal = {Pest management science},
volume = {81},
number = {12},
pages = {7874-7884},
doi = {10.1002/ps.70099},
pmid = {40751408},
issn = {1526-4998},
support = {2023YFE0116200//National Key Research and Development Program of China/ ; 2023YFC2605200//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Weevils/genetics/classification ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; China ; Recombinases ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Hylurgus ligniperda is an invasive bark beetle that poses a serious threat to global coniferous forests and the timber trade. Its broad host range, high reproductive potential, and strong environmental adaptability enable it to establish and spread rapidly in newly invaded regions. In October 2020, H. ligniperda was first reported in Shandong Province, China. Developing a rapid, sensitive, and accurate field detection method is critical for early interception and effective management.<br><br>RESULTS: We developed a detection method for H. ligniperda based on recombinase polymerase amplification (RPA) coupled with CRISPR/Cas12a, with results monitored via fluorescence signals and lateral flow dipstick (LFD). The mitochondrial COI gene was selected as the target sequence, and key parameters-including incubation time, temperature, and concentrations of Cas12a protein and CRISPR RNA (crRNA)-were optimized. The RPA-CRISPR-LbaCas12a-LFD assay exhibited high specificity and sensitivity, successfully distinguishing H. ligniperda from five closely related species, and detecting target DNA at concentrations as low as 1 copy per &#x3bc;L. Finally, The field applicability of the detection system was validated using samples from global geographic populations.<br><br>CONCLUSION: This study establishes a portable, rapid, and sensitive visual detection system for H. ligniperda based on RPA-CRISPR-LbaCas12a-LFD, suitable for both laboratory and field applications. The method enables field detection without the need for specialized equipment, offering a robust tool for invasive pest surveillance, port quarantine, and early warning. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid41231321,
year = {2025},
author = {Kumari, S and Keshari, AK and Singh, SK and Pandey, S and Singh, A},
title = {CRISPR-Based genome editing in pulses: current approaches, challenges, and future prospects.},
journal = {Plant molecular biology},
volume = {115},
number = {6},
pages = {126},
pmid = {41231321},
issn = {1573-5028},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Crops, Agricultural/genetics ; *Fabaceae/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; },
abstract = {Legumes are the second most important food crop after cereals for the world population. It is a significant protein source for developing countries and integral to global food security. However, various agroecological constraints and biotic and abiotic factors often compromise the production of pulses. Legumes are long-term neglected crops worldwide and follow traditional breeding, leading to a time-consuming, labor-intensive, less economically feasible program associated with linkage drag. Recent sequencing attempts in the twenty-first century, with the development of an enormous repertoire of genetic and genomic resources, allowed scientists to accelerate the improvement of legumes with modern genome editing tools. One such promising tool is CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which has revolutionized and transformed the landscape of genetic engineering. The emergence of CRISPR/Cas systems has redefined precision breeding, offering unprecedented control over genome manipulation in legume crops. It has tremendous potential for crop improvement and can precisely make changes at genomic locations with incredible accuracy. Therefore, identifying the desired genes and their precise manipulation has enormous implications for legume crop improvement. This review will give an overview of the genome editing tools available for crop improvement and the efficiency of different transformation methods in legume crops. It will also discuss the current status of genome editing in legume crops, including challenges and future perspectives.},
}
@article {pmid41230228,
year = {2025},
author = {Park, S and Mani, V and Ha, K and Kim, JA and Lee, S},
title = {Plant synthetic biology: from knowledge to biomolecules.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1562216},
pmid = {41230228},
issn = {1664-462X},
abstract = {Plant synthetic biology is rapidly emerging as an innovative approach to solving complex problems in human health and agriculture. Although conventional metabolic engineering primarily focuses on microbial systems for large-scale biomolecules production, these platforms often face limitations in expressing plant-derived enzymes and synthesizing structurally complex molecules. In contrast, recent advances in plant synthetic biology have integrated multidisciplinary tools, from molecular biology and biochemistry to synthetic circuit design and computational modeling, to engineer plant systems with enhanced traits. These include improved yield, nutritional quality, environmental resilience, and synthesis of pharmaceutically relevant functional biomolecules. This review focuses on the fundamental technologies that have enabled such advances, which include DNA synthesis, programmable gene circuits, and CRISPR/Cas-based genome editing. We discussed recent applications of reprogramming plant metabolic pathways and existing obstacles, such as transformation efficiency, regulatory bottlenecks, and pathway instability. This review provides key case studies and a forward-looking perspective on the evolution of plant synthetic biology as a robust foundation for sustainable biomanufacturing and production of functional biomolecules.},
}
@article {pmid41229344,
year = {2025},
author = {Sures, K and Esser, SP and Bornemann, TLV and Moore, CJ and Soares, AR and Plewka, J and Figueroa-Gonzalez, PA and Ruff, SE and Moraru, C and Probst, AJ},
title = {Acquisition of Spacers from Foreign Prokaryotic Genomes by CRISPR-Cas Systems in Natural Environments.},
journal = {Genome biology and evolution},
volume = {17},
number = {11},
pages = {},
pmid = {41229344},
issn = {1759-6653},
mesh = {*CRISPR-Cas Systems ; *Genome, Archaeal ; *Archaea/genetics ; Genome, Bacterial ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics ; Metagenome ; Evolution, Molecular ; *DNA, Intergenic/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems of bacteria and archaea provide immunities against mobile genetic elements, like viruses. In addition, protospacer analyses revealed a very specific acquisition of CRISPR spacers derived from genomes of related species or from closely interacting episymbiont genomes as recently shown for subsurface archaea. However, the origin of most of the spacers that can be found in CRISPR-Cas systems from natural environments has not been deciphered. Here, by analyzing CRISPR-Cas systems of metagenome-assembled genomes (MAGs) from two subsurface environments spanning more than 1 Tb of sequencing data, we show that a substantial proportion of CRISPR spacers are acquired from DNA of other prokaryotes inhabiting the same environment. As such, we found that the number of respective spacers can be up to three times higher than the number of self-targeting spacers. Statistical analyses demonstrated that the acquisition of CRISPR spacers from other prokaryotic genomes is partly explained by the relative abundance of the MAG containing the protospacer, as well as by other factors, such as the total number of CRISPR arrays present in a MAG with the respective spacers. Further, we found that spacer acquisition from foreign prokaryotic DNA occurs in almost all types of CRISPR-Cas systems, but shows preferences for subtypes of CRISPR-Cas systems that differ across the investigated ecosystems. Taken together, our results shed new light on the diversity of CRISPR spacers in natural microbial communities and provide an explanation for some of the many unmatched spacers in public databases.},
}
@article {pmid41229123,
year = {2025},
author = {Ma, Y and Wei, W and Yang, Z and Zhou, Y and Dong, T and Wang, T and Xia, X and Ma, Y and Zhou, M and Gao, Y and Yu, B and Wang, C and Ruan, L and Hong, K and Gu, C},
title = {Exosomes as Nonviral Carrier for Targeted Delivery of CRISPR/Cas12a for Therapeutic HIV-1Proviral DNA Editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.11.012},
pmid = {41229123},
issn = {1525-0024},
abstract = {Current strategies to treat HIV infection including traditional cART and immunotherapy can effectively suppress viral replication but unable to eliminate the latent viral reservoir, particularly within circulating immune cells. Although genome editing by CRISPR-Cas provides a promising cure for HIV-1, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we developed an exosome-mediated targeted CRISPR-Cas12a delivery system (EMT-Cas12a), an engineered exosome system enabling targeted delivery of mRNA of Cas12a and crRNAs to CD4+ T cells. The EMT-Cas12a system uniquely optimizes cell-specific targeting, CRISPR-Cas12a expression, crRNAs maturation, nuclear entry efficiency, accuracy cleavage with major Delins and achieving dramatically HIV suppression in both cellular and humanized mouse models. Compared to single-crRNA approaches, the multiple crRNA arrays strategy demonstrates enhanced antiviral efficacy in HIV -infected mouse model, ex vivo expanded PBMCs from HIV+ subjects and especially in vitro cell line without detectable HIV DNA. Critically, the system exhibits no detectable off-target effects and restores CD4+ T cell counts in vivo and ex vivo PBMCs, indicating its dual therapeutic potential for viral clearance and immune reconstitution. Altogether, in vitro and in vivo excision of HIV-1 proviral DNA can be achieved via EMT-Cas12a delivery, which could advance efforts toward human clinical trials.},
}
@article {pmid41139833,
year = {2025},
author = {Neo, DM and Ben-Zion, I and Bagnall, J and Solomon, MY and Bond, AN and Gath, E and Zhang, S and Shoresh, N and Gomez, J and Hung, DT},
title = {A Multiplexed, Target-Based Phenotypic Screening Platform Using CRISPR Interference in Mycobacterium abscessus.},
journal = {ACS infectious diseases},
volume = {11},
number = {11},
pages = {3263-3275},
doi = {10.1021/acsinfecdis.5c00623},
pmid = {41139833},
issn = {2373-8227},
mesh = {*Mycobacterium abscessus/genetics/drug effects ; Microbial Sensitivity Tests ; Mycobacterium Infections, Nontuberculous/microbiology/drug therapy ; *Anti-Bacterial Agents/pharmacology ; Drug Discovery/methods ; Bacterial Proteins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; Phenotype ; Humans ; },
abstract = {The rise of difficult-to-treat Mycobacterium abscessus infections presents a growing clinical challenge due to the immense arsenal of intrinsic, inducible and acquired antibiotic resistance mechanisms that render many existing antibiotics ineffective against this pathogen. Moreover, the limited success in discovery of novel compounds that inhibit novel pathways underscores the need for innovative drug discovery strategies. Here, we report a strategic advancement in PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), which is an antimicrobial discovery strategy that measures chemical-genetic interactions between small molecules and a pool of bacterial mutants, each depleted of a different essential protein target, to identify whole-cell active compounds with high sensitivity. Applying this modified strategy to M. abscessus, in contrast to previously described versions of PROSPECT which utilized protein degradation or promoter replacement strategies for generating engineered hypomorphic strains, here we leveraged CRISPR interference (CRISPRi) to more efficiently generate mutants each depleted of a different essential gene involved in cell wall synthesis or located at the bacterial surface. We applied this platform to perform a pooled PROSPECT pilot screen of a library of 782 compounds using CRISPRi guides as mutant barcodes. We identified a range of active hits, including compounds targeting InhA, a well-known mycobacterial target but under-explored in the M. abscessus space. The unexpected susceptibility to isoniazid, traditionally considered to be ineffective in M. abscessus, suggested a complex interplay of several intrinsic resistance mechanisms. While further complementary efforts will be needed to change the landscape of therapeutic options for M. abscessus, we propose that PROSPECT with CRISPRi engineering provides an increasingly accessible, high-throughput target-based phenotypic screening platform and thus represents an important step toward accelerating early stage drug discovery.},
}
@article {pmid41086891,
year = {2025},
author = {Sun, J and Huang, Y and Bai, J and Wang, J and Langford, PR and Zhang, Y and Li, G},
title = {A CRISPR/Cas12a-based DNAzyme chemiluminescence platform for rapid detection of all Streptococcus suis and individually S. suis serotypes 7 and 9.},
journal = {International journal of biological macromolecules},
volume = {330},
number = {Pt 4},
pages = {148280},
doi = {10.1016/j.ijbiomac.2025.148280},
pmid = {41086891},
issn = {1879-0003},
mesh = {*Streptococcus suis/genetics/isolation &amp; purification/classification ; *DNA, Catalytic/metabolism/genetics ; Serogroup ; *CRISPR-Cas Systems/genetics ; *Luminescent Measurements/methods ; Swine ; Animals ; Biosensing Techniques/methods ; Luminescence ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Streptococcus suis is a zoonotic pathogen that can infect pigs and humans with causing meningitis, sepsis, endocarditis, and arthritis. S. suis serotypes 7 and 9 cause substantial economic losses to the swine industry and pose a major threat to public health, thus, accurate and rapid detection is important for the prevention and control of epidemic disease. In this study, we developed a platform, combining recombinase polymerase amplification (RPA) with a CRISPR/Cas12a detection system to rapidly detect all S. suis serotypes and individually differentiates serotypes 7 and 9. This was achieved by targeting recN of S. suis and serotype-specific cpsH genes of serotypes 7 and 9, respectively. Both fluorescence and G4 DNAzyme chemiluminescence visualization biosensing methods had high specificity and sensitivity, no cross-reaction was found with common pig pathogens, closely related Streptococcus spp., or other S. suis serotypes. Compared to traditional identification techniques, these two methods are rapid and convenient. Notably, the G4 DNAzyme chemiluminescence method provides a clear, direct visual interpretation of results without the need for specialized equipment, which is particularly advantageous for point-of-care testing. Thus, this platform has the potential to significantly enhance diagnostic capabilities and ultimately benefit both animal and public health.},
}
@article {pmid40865049,
year = {2025},
author = {Shang, Y and Li, S and Xu, T and Sun, Y and Li, F},
title = {An Optimized Platform Overcomes Excessive Tumor Immune Rejection Induced by the CRISPR/Cas9 Lentiviral System.},
journal = {Cancer research},
volume = {85},
number = {22},
pages = {4315-4328},
doi = {10.1158/0008-5472.CAN-25-0809},
pmid = {40865049},
issn = {1538-7445},
support = {2022YFA1103900//National Natural Science Foundation of China (NSFC)/ ; 82372794//National Natural Science Foundation of China (NSFC)/ ; 82172744//National Natural Science Foundation of China (NSFC)/ ; GZB20240544//China Postdoctoral Science Foundation (China Postdoctoral Foundation Project)/ ; 2024M752432//China Postdoctoral Science Foundation (China Postdoctoral Foundation Project)/ ; 24ZR1469100//Natural Science Foundation of Shanghai Municipality ()/ ; //Shanghai Municipal Education Commission ()/ ; },
mesh = {*CRISPR-Cas Systems ; Animals ; *Lentivirus/genetics ; Mice ; Humans ; Gene Knockout Techniques/methods ; Integrases/genetics ; *Neoplasms/immunology/genetics/therapy ; Genetic Vectors/genetics ; Adenoviridae/genetics ; Cell Line, Tumor ; Female ; },
abstract = {UNLABELLED: The clustered regularly interspaced short palindromic repeats/Cas9 lentiviral system has emerged as a powerful tool for gene knockout in cancer immunology research. However, the persistent expression of exogenous elements, such as Cas9 and resistance markers, often triggers excessive tumor immune rejection. This can lead to prolonged experimental timelines, increased data variability, biased outcomes, and even experimental failures. To address this challenge, several strategies have been investigated that offer partial solutions, but they have failed so far to comprehensively resolve the issue. In response, we developed the v2-Blast-lox2272 (VL)-adenovirus expressing Cre recombinase (AdCre) system, a strategy that enables efficient excision of exogenous expression elements following gene knockout. The VL-AdCre system effectively reduced tumor immune rejection in allograft models, streamlined experimental workflows, and improved the reliability of research outcomes. Overall, this optimized clustered regularly interspaced short palindromic repeats/Cas9 lentiviral gene knockout system offers a robust and practical solution for studying gene functions in vivo and advancing immunotherapeutic strategies.<br><br>SIGNIFICANCE: The VL-AdCre system enables effective removal of exogenous components following gene knockout, thus avoiding tumor immune rejection and enhancing the accuracy and reproducibility of in vivo cancer immunology research.},
}
@article {pmid41226739,
year = {2025},
author = {Arana, &#xc1;J and Veiga-Rua, S and Cora, D and Gónzalez-Gómez, MA and Seijas, A and Carballeda, M and Polo, D and Cuesta, A and Piñeiro, Y and Rivas, J and Novo, M and Al-Soufi, W and Martínez, P and Sánchez, L and Robledo, D},
title = {Comparative Analysis of CRISPR/Cas9 Delivery Methods in Marine Teleost Cell Lines.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
doi = {10.3390/ijms262110703},
pmid = {41226739},
issn = {1422-0067},
support = {CRISPR-NANOXEN//Universidade de Santiago de Compostela/ ; Optimizing CRISPR/Cas9 genome editing to improve disease resistance in aquaculture//Fundaci&#xf3;n Caixa Rural Galega Tom&#xe1;s Notario Vacas/ ; investment line n&#xba; 1 and component number 17, which includes the Complementary RTDI Plan for Marine Science//Spain's Recovery and Resilience Plan/ ; No 101076432&#x202f;(FishTRIM)//European Union ERC Starting Grant programme 2022/ ; (BBS/E/20002172, BBS/E/D/30002275, BBS/E/D/10002070 and BBS/E/RL/230002A)//Oportunius programme of the Axencia Galega the Innovaci&#xf3;n (GAIN, Xunta de Galicia)/ ; RYC2023-044793-I//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; 10.13039/501100011033//Agencia Estatal de Investigaci&#xf3;n/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Cell Line ; Electroporation/methods ; *Fishes/genetics ; Transfection/methods ; *Gene Transfer Techniques ; },
abstract = {Gene editing technologies such as CRISPR/Cas9 have revolutionized functional genomics, yet their application in marine fish cell lines remains limited by inefficient delivery. This study compares three delivery strategies-electroporation, lipid nanoparticles (LNPs), and magnetofection using gelatin-coated superparamagnetic iron oxide nanoparticles (SPIONs)-for CRISPR/Cas9-mediated editing of the ifi27l2a gene in DLB-1 and SaB-1 cell lines. We evaluated transfection and editing efficiency, intracellular Cas9 localization, and genomic stability of the target locus. Electroporation achieved up to 95% editing in SaB-1 under optimized conditions, but only 30% in DLB-1, which exhibited locus-specific genomic rearrangements. Diversa LNPs enabled intracellular delivery and moderate editing (~25%) in DLB-1 but yielded only minimal editing in SaB-1, while SPION-based magnetofection resulted in efficient uptake but no detectable editing, highlighting post-entry barriers. Confocal imaging and fluorescence correlation spectroscopy suggested that nuclear localization and Cas9 aggregation may influence editing success, highlighting the importance of intracellular trafficking in CRISPR/Cas9 delivery. Our findings demonstrate that CRISPR/Cas9 delivery efficiency is cell line-dependent and governed by intracellular trafficking and genomic integrity. These insights provide a practical framework for optimizing gene editing in marine teleosts, advancing both basic research and selective breeding in aquaculture.},
}
@article {pmid41226719,
year = {2025},
author = {Amiri, S and Adibzadeh, S and Khazaei Monfared, Y and Kaboli, S and Arashkia, A and Barkhordari, F and Mahmoudian, M and Kheirandish, MH and Trotta, F and Davami, F},
title = {Efficient Delivery of CRISPR-Cas9 RNP Complexes with Cyclodextrin-Based Nanosponges for Enhanced Genome Editing: TILD-CRISPR Integration.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
doi = {10.3390/ijms262110682},
pmid = {41226719},
issn = {1422-0067},
support = {BD-66002183//Pasteur Institute of Iran/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Cyclodextrins/chemistry ; CHO Cells ; Cricetulus ; *Ribonucleoproteins/genetics ; Green Fluorescent Proteins/genetics ; *Nanostructures/chemistry ; },
abstract = {The CRISPR-Cas9 system has transformed biomedical research by enabling precise genetic modifications. However, efficient delivery of CRISPR components remains a major hurdle for therapeutic applications. To address this, we employed a new modified cationic hyper-branched cyclodextrin-based polymer (Ppoly) system to deliver an integrating GFP gene using the TILD-CRISPR method, which couples donor DNA linearization with RNP complexes. The physicochemical properties, loading efficiency, and cellular uptake of RNP with Ppoly were studied. After transfection, antibiotic selection and single-cell cloning were performed. Junction PCR was then performed on the isolated clones, and we compared the knock-in efficiency of Ppoly with that of the commercial CRISPRMAX™ reagent (Thermo Fisher, Invitrogen™, Waltham, MA, USA). The results demonstrate the encapsulation efficiency of over 90% for RNP and Ppoly, and cell viability remaining above 80%, reflecting the minimal toxicity of this approach. These attributes facilitated successful GFP gene integration using the TILD-CRISPR with RNP delivered via cyclodextrin-based nanosponges. The present method achieved a remarkable 50% integration efficiency in CHO-K1 cells, significantly outperforming the 14% observed with CRISPRMAX™ while maintaining lower cytotoxicity. This study highlights a promising platform for precise and efficient genome editing, with strong potential for therapeutic and regenerative medicine applications.},
}
@article {pmid41226716,
year = {2025},
author = {Zhao, R and Zhu, J and Wang, J and Wang, D and Liu, X and Han, L and Li, S},
title = {Functional Characterization of Fp2Cas9, a Cold-Adapted Type II-C CRISPR Nuclease from Flavobacterium psychrophilum.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
doi = {10.3390/ijms262110681},
pmid = {41226716},
issn = {1422-0067},
support = {2023YFD2400300//National Key Research and Development Program of China/ ; 2024XT01, 2023TD45//the Central Public Interest Scientific Institution Basal Research Fund, CAFS/ ; Identification, Evaluation, and Breeding of Characteristic Aquatic Animal Germplasm Resources in Heilongjiang Province.//Heilongjiang Province Seed Industry Innovation and Development Fund Project/ ; },
mesh = {*Flavobacterium/enzymology/genetics ; Zebrafish/genetics ; Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Cold Temperature ; *CRISPR-Associated Protein 9/metabolism/genetics ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {Cas9 with specialized temperature adaptations are essential for broadening the application of CRISPR-based genome editing across diverse biological contexts. Although Cas9 orthologs from thermophilic and mesophilic organisms have been characterized for high- and moderate-temperature applications, cold-active variants remain largely unexplored, limiting genome engineering in low-temperature systems such as aquaculture species. Here, we report the functional characterization of Fp2Cas9, a cold-adapted Type II-C nuclease from Flavobacterium psychrophilum. In vitro assays showed that Fp2Cas9 efficiently cleaves double-stranded DNA with a refined PAM requirement of 5'-SNAAAG-3', and that its engineered sgRNA scaffold (sgRNA-V2) supports programmable DNA targeting. Notably, Fp2Cas9 retains 75% cleavage efficiency at 5 °C, approximately 2.5-fold higher than SpCas9 under the same conditions, but shows a marked reduction in activity at 35 °C. In vivo, a nuclear-localized variant (2NLS-Fp2Cas9) mediated efficient mutagenesis of the zebrafish slc45a2 gene, yielding ~60% indel frequencies and pigmentation-deficient phenotypes in ~43% of injected embryos. Collectively, these findings establish Fp2Cas9 as a cold-adapted Cas9 with reliable activity at low temperatures. This work adds a valuable tool to the CRISPR-Cas9 toolkit and may facilitate genome editing in cold-water organisms and other low-temperature systems.},
}
@article {pmid41226505,
year = {2025},
author = {Qian, Y and Liu, X and Wang, B and Li, D and Wu, Z and Tong, J},
title = {Genome-Wide Identification of the LsaPHR1 Gene Family and Preliminary Functional Validation of LsaPHR1.1 in Phosphorus Tolerance in Lactuca sativa.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
doi = {10.3390/ijms262110466},
pmid = {41226505},
issn = {1422-0067},
support = {KJCX20240508//Special Innovation Ability Construction Fund of Beijing Academy of Agricultural and Forestry Sciences/ ; QNJJ202248//Beijing Academy of Agriculture and Forestry Science Youth Research Fund/ ; KYCX202506//Innovation and Development Program of Beijing Vegetable Research Center/ ; CARS-24-B-02//China Agriculture Research System of the MOF and MARA/ ; KJCX20200205//Project for Scientific and Technological Innovation Capability Building of the Beijing Academy of Agriculture and Forestry Sciences/ ; },
mesh = {*Lactuca/genetics/metabolism ; *Phosphorus/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Stress, Physiological/genetics ; *Multigene Family ; CRISPR-Cas Systems ; Chlorophyll/metabolism ; Gene Editing ; },
abstract = {Phosphorus (P) is a limiting nutrient for plant growth and productivity. Improving P use efficiency is important for crop production. In Lactuca sativa (lettuce), five phosphate starvation response 1 (PHR1) genes were identified and characterized through a bioinformatics approach. The expression patterns of LsaPHR1s were examined using qRT-PCR under various treatments, including devoid phosphorus (DP), low phosphorus (LP), high phosphorus (HP), darkness, ABA, IAA, and MeJA. The results indicate that LsaPHR1s in lettuce responded to phosphorus stress, hormones, and darkness. Furthermore, we engineered LsaPHR1.1 knock-out mutants via CRISPR/Cas9-mediated genome editing. Then, the mutants were subjected to phosphorus stress (DP, LP, and HP). In contrast to WT, the mutants improved nitrate and ammonium contents, increased antioxidant enzyme activity, and elevated antioxidant and chlorophyll contents. Our results offer a potential strategy for improving phosphorus stress tolerance in lettuce, which holds great significance for maintaining yield and quality.},
}
@article {pmid41225502,
year = {2025},
author = {Huang, S and Zhou, F and Jiang, Z and Jiang, S and Yang, Q and Yang, L and Huang, J and Shi, J and Ding, Y and Li, E and Li, Y},
title = {Development and validation of PmMAD7 for efficient gene editing in Penaeus monodon.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {125},
pmid = {41225502},
issn = {1472-6750},
support = {2022YFD2401900//National Key R &amp; D Program of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Penaeidae/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based gene editing has become a promising approach for enhancing traits in aquaculture species. Nevertheless, traditional CRISPR-Cas systems encounter challenges, including significant off-target effects and strict protospacer adjacent motif (PAM) requirements, which constrain their use in crustaceans such as Penaeus monodon. To address these limitations, this research has developed PmMAD7, a codon-optimized CRISPR system specifically designed for P. monodon, which incorporates nuclear localization signals to improve editing efficiency and precision.<br><br>RESULTS: This research successfully synthesized and delivered PmMAD7 mRNA and crRNAs targeting the ECH1 and AQP4 genes into the hemocytes of P. monodon. Quantitative PCR analysis demonstrated that PmMAD7 achieved significant gene silencing, reducing the expression levels of ECH1 and AQP4 by 81.5% and 78.33%, respectively. Next-generation sequencing confirmed targeted insertions and deletions at the gene loci, with knockout efficiencies of 14.81% for ECH1 and 20.57% for AQP4, which were significantly higher than those obtained with LbCas12a (7.14% and 12.43%, respectively). Furthermore, functional analysis indicated that ECH1 knockout resulted in increased cell volume and mortality, while AQP4 knockout led to decreased cell volume and reduced viability. These specific results highlight the first successful demonstration of MAD7-based genome editing in shrimp. The broader PAM compatibility and enhanced editing efficiency of PmMAD7 provide a versatile platform for gene editing in shrimp.<br><br>CONCLUSION: PmMAD7 constitutes an enhanced CRISPR editing tool specifically designed for P. monodon, exhibiting superior precision, expanded PAM compatibility, and enhanced editing efficacy relative to conventional Cas12a systems. These results lay the groundwork for the advancement of gene editing applications in crustaceans and contribute to sustainable genetic improvements in aquaculture.},
}
@article {pmid41223249,
year = {2025},
author = {Pan, X and Huang, P and Ali, SS and Renslo, B and Greenberg, Z and Erwin, N and Li, Y and Ding, Z and Hutchinson, TE and Warnecke, A and Fernandez, NE and Staecker, H and He, M},
title = {Extracellular vesicle-mediated gene editing for the treatment of nonsyndromic progressive hearing loss in adult mice.},
journal = {Science translational medicine},
volume = {17},
number = {824},
pages = {eadn3993},
doi = {10.1126/scitranslmed.adn3993},
pmid = {41223249},
issn = {1946-6242},
mesh = {Animals ; *Extracellular Vesicles/metabolism ; *Gene Editing/methods ; *Hearing Loss/therapy/genetics ; Mice ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Genetic Therapy/methods ; Ribonucleoproteins/metabolism ; Hair Cells, Auditory/metabolism ; Electroporation ; Humans ; RNA, Guide, CRISPR-Cas Systems/metabolism ; },
abstract = {The clinical translation of gene therapy has been challenging in part because of the limitations of current delivery approaches. Herein, we report an efficient nonviral genome editor delivery approach using extracellular vesicles (EVs) carrying single-guide RNA (sgRNA): CRISPR-Cas9 ribonucleoprotein (RNP) complexes for in vivo gene therapy. By leveraging a high-throughput microfluidic droplet-based electroporation system (μDES), we achieved a 10-fold enhancement in loading efficiency and more than 1000-fold increase in processing throughput for loading RNP complexes into EVs compared with conventional high-voltage pulsed electroporation. μDES generated uniform microdroplets containing EVs and RNPs by applying direct current-controlled low voltage (up to 60 V) to transiently permeabilize membranes and enable efficient cargo encapsulation while maintaining EV integrity at both the protein and morphological levels. In the Myo7a[WT/Sh1] mouse model of autosomal dominant progressive hearing loss, which may model MYO7A-associated DFNA11 hearing loss in humans, we demonstrated the effective delivery of RNPs by EVs into cochlear hair cells by cross-sectional and whole-mount confocal imaging. The injection of RNP-EVs via the posterior semicircular canal in 4-week-old Myo7a[WT/Sh1] mice resulted in a reduction in Myo7a[Sh1] messenger RNA expression and evidence of hearing preservation, as measured by auditory brainstem responses, compared with untreated ears and EV only-injected mice. This study highlights the potential of μDES-produced RNP-EVs for gene editing as a treatment for progressive nonsyndromic hearing loss in patients.},
}
@article {pmid41223195,
year = {2025},
author = {Kimata, K and Satou, K},
title = {Improved CRISPR/Cas9 off-target prediction with DNABERT and epigenetic features.},
journal = {PloS one},
volume = {20},
number = {11},
pages = {e0335863},
pmid = {41223195},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Epigenesis, Genetic ; *Gene Editing/methods ; Genome, Human ; Deep Learning ; },
abstract = {CRISPR/Cas9 is a powerful genome editing tool, but its clinical application is hindered by off-target effects. Accurate computational prediction of these unintended edits is crucial for ensuring the safety and efficacy of therapeutic applications. While various deep learning models have been developed, most are trained only on task-specific data, failing to leverage the vast knowledge embedded in entire genomes. To address this limitation, we introduce a novel approach that integrates DNABERT, a deep learning model pre-trained on the human genome, with epigenetic features (H3K4me3, H3K27ac, and ATAC-seq). We conducted a comprehensive benchmark of our model, DNABERT-Epi, against five state-of-the-art methods across seven distinct off-target datasets. Our results demonstrate that the pre-trained DNABERT-based models achieve competitive or even superior performance. Rigorous ablation studies quantitatively confirmed that both genomic pre-training and the integration of epigenetic features are critical factors that significantly enhance predictive accuracy. Furthermore, by applying advanced interpretability techniques (SHAP and Integrated Gradients), we identified the specific epigenetic marks and sequence-level patterns that influence the model's predictions, offering insights into its decision-making process. This study is the first to establish the significant potential of a pre-trained DNA foundation model for CRISPR/Cas9 off-target prediction. Our findings underscore that leveraging both large-scale genomic knowledge and multi-modal data is a key strategy for advancing the development of safer genome editing tools.},
}
@article {pmid41222688,
year = {2025},
author = {Yuan, A and Sun, T and Deng, Z and Ye, L and Shang, Y and Xie, W and Peng, H},
title = {A multifunctional approach: merging CRISPR/Cas technology with DNA nanomachines for advanced biosensing.},
journal = {Analytical and bioanalytical chemistry},
volume = {},
number = {},
pages = {},
pmid = {41222688},
issn = {1618-2650},
support = {2023YFA0915102//National Key Research and Development Program of China/ ; 22276199//National Natural Science Foundation of China/ ; 22306195//National Natural Science Foundation of China/ ; XDB0750100//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
abstract = {CRISPR/Cas systems have revolutionized nucleic acid recognition, offering unparalleled programmability for biosensing. Simultaneously, DNA nanomachines have emerged as powerful bioanalytical tools due to their structural precision, biocompatibility, and multifunctional capabilities. The strategic integration of these platforms synergistically enhances sensitivity, specificity, and multiplexing potential, creating next-generation bioanalytical systems. While promising, this convergence presents unique engineering challenges and necessitates critical evaluation. This review systematically analyzes the target recognition mechanisms of CRISPR/Cas systems and critically evaluates prevalent signal readout modalities. A core focus is the critical assessment of innovative biosensing strategies that leverage DNA nanomachines-particularly dynamic walker systems-coupled with CRISPR/Cas activation. We highlight representative integrated platforms, detailing their operational principles and objectively examining their demonstrated advantages against inherent limitations such as signal leakage, complex assembly, and in vivo applicability constraints. By providing a critical analysis of the synergistic potential and current constraints of CRISPR/Cas-DNA nanomachine integration, this review aims to guide rational design towards robust, clinically translatable precision diagnostics and therapeutics.},
}
@article {pmid41221123,
year = {2025},
author = {van Karnebeek, CDM and Gailus-Durner, V and Engelke, UF and Seisenberger, C and Marschall, S and Dragano, NRV and da Silva-Buttkus, P and Leuchtenberger, S and Fuchs, H and Hrabě de Angelis, M and Wevers, RA and Coughlin, CR and Lefeber, DJ},
title = {New treatment for pyridoxine-dependent epilepsy due to ALDH7A1 deficiency: first proof-of-principle of upstream enzyme inhibition in the mouse.},
journal = {Brain communications},
volume = {7},
number = {6},
pages = {fcaf397},
pmid = {41221123},
issn = {2632-1297},
abstract = {Pyridoxine-dependent epilepsy (PDE) due to recessive ALDH7A1 mutations is characterized by intractable epilepsy that is often unresponsive to antiseizure medications. Irrespective of pyridoxine (vitamin B6) supplementation and lysine reduction therapy, patients present severe residual neurocognitive deficits. We evaluated upstream inhibition of 2-aminoadipic semialdehyde synthase (AASS) as a novel therapeutic strategy to reduce the accumulating metabolites (α-aminoadipic semialdehyde, Δ[1]-piperideine-6-carboxylate, pipecolic acid, 6-oxo-pipecolic acid and 2S,6S-/2s,6R-oxopropylpiperidine-2-carboxylic acid) considered neurotoxic. We utilized an existing mouse knockout model of hyperlysinaemia (Aass-knockout) and generated a PDE model, a Aldh7a1 single knockout model via CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) and generated the double-knockout Aass/Aldh7a1 mice. Next-generation metabolomics screening was performed to measure all known biomarkers in brain, liver and plasma of wild-type and mutant mice. Metabolomics confirmed the known metabolite markers for Aldh7a1-knockout and Aass knockout mice in all samples. The potentially neurotoxic metabolites (Δ[1]-piperideine-6-carboxylate, pipecolic acid, 6-oxo-pipecolic acid and 2S,6S-/2s,6R-oxopropylpiperidine-2-carboxylic acid) significantly decreased in double-knockout Aass/Aldh7a1 mice brain and liver tissues compared to Aldh7a1-knockout mice. Plasma analysis revealed a significant reduction of known biomarkers, suggesting a reliable monitoring option in human patients. We demonstrate the first mammalian evidence that AASS inhibition is a viable strategy to rescue abnormal brain metabolism associated with PDE. This may target the intellectual disability and neurologic deficits caused by persistent lysine catabolic-related neurotoxicity despite adequate vitamin B6 supplementation.},
}
@article {pmid41220980,
year = {2025},
author = {Yuan, X and Yu, X and Zhao, W and Sun, J},
title = {A Practical CRISPR-Based Method for Rapid Genome Editing in Caulobacter crescentus.},
journal = {Bio-protocol},
volume = {15},
number = {21},
pages = {e5490},
pmid = {41220980},
issn = {2331-8325},
abstract = {The RNA-guided Cas enzyme specifically cuts chromosomes and introduces a targeted double-strand break, facilitating multiple kinds of genome editing, including gene deletion, insertion, and replacement. Caulobacter crescentus and its relatives, such as Agrobacterium fabrum and Sinorhizobium meliloti, have been widely studied for industrial, agricultural, and biomedical applications; however, their genetic manipulations are usually characterized as time-consuming and labor-intensive. C. crescentus and its relatives are known to be CRISPR/Cas-recalcitrant organisms due to intrinsic limitations of SpCas9 expression and possible CRISPR escapes. By fusing a reporting gene to the C terminus of SpCas9M and precisely manipulating the expression of SpCas9M, we developed a CRISPR/SpCas9M-reporting system and achieved efficient genome editing in C. crescentus and relatives. Here, we describe a protocol for rapid, marker-less, and convenient gene deletion by using the CRISPR/SpCas9M-reporting system in C. crescentus, as an example. Key features • CRISPR-SpCas9M-reporting system overcomes the limitation of CRISPR escape and achieves a high apparent editing efficiency. • The method enables multiple kinds of genome editing, generating in-frame and marker-less chromosomal modifications. • The method completes a cycle of genome editing within one week. • The method could be readily applied for genome editing in C. crescentus, A. fabrum, and S. meliloti.},
}
@article {pmid41219964,
year = {2025},
author = {George, NA and Zhou, Z and Anantharaman, K and Hug, LA},
title = {Discarded diversity: novel megaphages, auxiliary metabolic genes, and virally encoded CRISPR-Cas systems in landfills.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {370},
pmid = {41219964},
issn = {1743-422X},
support = {R35GM143024/GM/NIGMS NIH HHS/United States ; 2016-03686//Natural Sciences and Engineering Research Council of Canada/ ; Tier II chair//Canada Research Chairs/ ; },
mesh = {*Bacteriophages/genetics/classification/isolation &amp; purification ; *CRISPR-Cas Systems ; *Waste Disposal Facilities ; Metagenomics ; Archaea/virology/genetics ; *Bacteria/virology/genetics ; Genome, Viral ; Host Microbial Interactions ; Computational Biology ; North America ; },
abstract = {BACKGROUND: Viruses are the most abundant microbial entities on the planet, impacting microbial community structure and ecosystem services. Despite outnumbering bacteria and archaea by an order of magnitude, viruses have been comparatively underrepresented in reference databases. Metagenomic examinations have illustrated that viruses of bacteria and archaea have been specifically understudied in engineered environments. Here we employed metagenomic and computational biology methods to examine the diversity, host interactions, and genetic systems of viruses predicted from 27 samples taken from three municipal landfills across North America.<br><br>RESULTS: We identified numerous viruses that are not represented in reference databases, including the third largest bacteriophage genome identified to date (~&#x2009;678 kbp), and noted a large diversity of viruses in landfills that has limited overlap across landfills and is distinct from viromes in other systems. Host-virus interactions were examined via host CRISPR spacer to viral protospacer mapping which captured hyper-targeted viral populations and six viral populations predicted to infect hosts across multiple phyla. Auxiliary metabolic genes (AMGs) were identified with the potential to augment hosts' methane, sulfur, and contaminant degradation metabolisms, including AMGs not previously reported in the literature. CRISPR arrays and CRISPR-Cas systems were identified from predicted viral genomes, including the two largest bacteriophage genomes to contain these genetic features. Some virally encoded Cas effector-like proteins appear distinct relative to previously reported Cas effectors and are interesting targets for potential genome editing tools.<br><br>CONCLUSIONS: Our observations indicate landfills, as heterogeneous contaminated sites with unique selective pressures, are key locations for diverse viruses and atypical virus-host dynamics.},
}
@article {pmid41219611,
year = {2025},
author = {Javaid, MM and Ahmed, J and Ahmed, M and Awan, MJA and Waqas, MAB and Ali, Z and Saeed, NA},
title = {CRISPR-Cas9 based editing of the susceptibility allele TaLr34 enhances leaf rust resistance in bread wheat without yield penalty.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {236},
pmid = {41219611},
issn = {1438-7948},
mesh = {*Triticum/genetics/microbiology/growth &amp; development ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Diseases/genetics/microbiology ; *Gene Editing ; Alleles ; *Plant Proteins/genetics/metabolism ; Basidiomycota/pathogenicity ; Plant Leaves/microbiology/genetics ; Plant Breeding ; },
abstract = {Wheat (Triticum aestivum L.) is widely grown and consumed cereal crop around the world, but most wheat-producing regions suffer from rust diseases, especially stripe and leaf rust, which has caused a devastating global pandemic and severely reduced grain yields. The most effective way to control rust problem in wheat is to sow and breed durable, rust resistant wheat varieties. Conventional breeding for disease-resistant crops primarily relies on resistance (R) genes; however, the effectiveness of R gene-mediated resistance is often compromised by mutations in the pathogen. In this study, we employed CRISPR-Cas9-based genome editing as an advanced breeding tool to enhance rust resistance in the bread wheat cultivar Galaxy-13 by knocking out the homologs of the susceptibility allele TaLr34, specifically targeting the conserved regions within exon 11. Out of 21 transformed plants, five carried successful editing and exhibited resistance to moderate resistance against leaf rust. The TaLr34 mutants were evaluated for leaf rust resistance under both glasshouse and field conditions over three consecutive growing seasons at multiple geographical locations. Our results demonstrate that CRISPR-Cas9-mediated knockout of TaLr34 provides a robust strategy for achieving durable leaf rust resistance in the high-yielding elite wheat cultivar Galaxy-13 without compromising grain yield and agronomic performance.},
}
@article {pmid41218116,
year = {2025},
author = {Wang, X and Cramer, Z and Leu, NA and Monaghan, K and Durning, K and Adams-Tzivelekidis, S and Rhoades, JH and Heintz, J and Tian, Y and Rico, J and Mendez, D and Petroni, R and King, AC and Kim, MS and Matsuda, R and Hanselman, O and Shin, AE and Carrera Rodríguez, MF and Brodsky, IE and Rustgi, A and Li, N and Lengner, CJ and Andrés Blanco, M},
title = {Forward genetic screening in engineered colorectal cancer organoids identifies regulators of metastasis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {46},
pages = {e2510910122},
doi = {10.1073/pnas.2510910122},
pmid = {41218116},
issn = {1091-6490},
support = {P30-DK050306/GF/NIH HHS/United States ; F31CA250267-02/GF/NIH HHS/United States ; F31AI160741-01/GF/NIH HHS/United States ; R01 CA279317-01/GF/NIH HHS/United States ; },
mesh = {*Colorectal Neoplasms/genetics/pathology ; Animals ; *Organoids/pathology/metabolism ; Mice ; Humans ; *Neoplasm Metastasis/genetics ; CRISPR-Cas Systems ; *Genetic Testing/methods ; Cell Movement/genetics ; Cell Line, Tumor ; Neoplasm Invasiveness ; *Adenocarcinoma/genetics/pathology ; },
abstract = {Metastatic outgrowth requires that cancer cells delaminate from the primary tumor, intravasate, survive in circulation, extravasate, migrate to, and proliferate at a distal site. Recurrent genetic drivers of metastasis remain elusive, suggesting that unlike the early steps of oncogenesis, metastasis drivers may be variable. We develop a framework for identifying metastasis regulators using CRISPR/Cas9-based screening in a genetically defined organoid model of colorectal adenocarcinoma. We conduct in vitro screens for invasion and migration alongside orthotopic, in vivo screens for gain of metastasis in a syngeneic mouse model. We identify CTNNA1 and BCL2L13 as bona fide metastasis-specific suppressors which do not confer any selective advantage in primary tumors. CTNNA1 loss promotes cell invasion and migration, and BCL2L13 loss promotes anchorage-independent survival and non-cell-autonomous changes to macrophage polarization. This study demonstrates proof of principle that large-scale genetic screening can be performed in tumor-organoid models in vivo and identifies novel regulators of metastasis.},
}
@article {pmid41215577,
year = {2025},
author = {Uombe, NPI and Velikkakam, T and Silveira, ACA and Rodrigues, CC and Borges, BC and Teixeira, TL and Pereira, CL and Servato, JPS and Melo, NS and Mortara, RA and Silveira, JFD and Silva, CVD},
title = {Trypanosoma cruzi P21 Is a Pleiotropic Protein That Is Involved in Parasite Host Cell Invasion and Intracellular Parasitism.},
journal = {MicrobiologyOpen},
volume = {14},
number = {6},
pages = {e70154},
pmid = {41215577},
issn = {2045-8827},
support = {//This study was supported by grants and fellowships from FAPESP, FAPEMIG, CAPES, and CNPq. FAPESP Grant number: 2016/15000-4 and 2019/05049-4 (Postdoctoral fellowship); FAPEMIG - RED-00198-23./ ; },
mesh = {*Trypanosoma cruzi/genetics/pathogenicity/growth &amp; development/physiology/metabolism ; Animals ; *Chagas Disease/parasitology ; Vero Cells ; Chlorocebus aethiops ; Mice ; *Host-Parasite Interactions ; *Protozoan Proteins/genetics/metabolism ; Gene Knockout Techniques ; Virulence ; Disease Models, Animal ; CRISPR-Cas Systems ; },
abstract = {We characterized the secreted Trypanosoma cruzi P21 protein and hypothesized its role in parasite invasion and multiplication. To investigate the role of T. cruzi P21 protein in host-parasite interactions, specifically focusing on the low-virulence G strain. P21 knockout parasites were generated using CRISPR/Cas9. Cell invasion, multiplication, egress, and tissue parasitism were assessed in vitro and in vivo, comparing knockout and control parasites. P21 knockout significantly reduced parasite invasion and multiplication in Vero cells. In vivo, knockout parasites also showed reduced heart tissue parasitism in infected mice, despite no observable systemic parasitemia. Accordingly, P21 knockout trypomastigote egress was reduced in Vero cells. P21 plays a pleiotropic role in T. cruzi infection, differentially impacting parasite biology in the low-virulent G strain. In the G strain, P21 promotes invasion and persistence, potentially through mechanisms distinct from its role in the Y strain previously described. This highlights its potential as a therapeutic target for Chagas disease, warranting further investigation into strain-specific functions.},
}
@article {pmid41185939,
year = {2025},
author = {Kong, H and Liu, X and Xia, K and Guan, Y and Zhang, J and Zhang, Y and Liu, B and Ge, Z and Li, Q and Zuo, X and Wang, L and Li, J and Zhu, Y and Fan, C},
title = {Magnetically Driven High-Speed Rolling Nanoclusters for Enhanced CRISPR/Cas9 Genome Editing.},
journal = {ACS applied materials &amp; interfaces},
volume = {17},
number = {45},
pages = {61707-61717},
doi = {10.1021/acsami.5c15498},
pmid = {41185939},
issn = {1944-8252},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Magnetite Nanoparticles/chemistry ; Plasmids/genetics/chemistry/metabolism ; Jurkat Cells ; Protein Serine-Threonine Kinases/genetics ; Cell Cycle Proteins/genetics ; Viscosity ; },
abstract = {Despite the tremendous potential of the CRISPR/Cas9 gene-editing technology in precision therapeutics, intracellular delivery remains a major challenge. High cytoplasmic viscosity and lysosomal entrapment significantly impair the cytosolic transport and gene-editing efficiency. In this study, we demonstrate that both the size and magnetic responsiveness of Fe3O4 nanoclusters can be finely tuned by modulating ionic strength, enabling their rapid propulsion under external magnetic fields. Leveraging this property, we develop magnetic nanoparticle cluster nanorobots (MagCbots) of approximately 200 nm in size by electrostatically assembling Fe3O4 nanoclusters with CRISPR-Cas9 plasmids. Under magnetic actuation, MagCbots exhibit rapid rotation in highly viscous intracellular environments, achieving a linear velocity of ∼0.41 μm/s. MagCbots reduce intracellular viscosity by approximately 50% and enhance lysosomal escape efficiency by 3-fold compared to nonactuated counterparts. Their porous architecture not only offers high payload capacity but also protects plasmid DNA from enzymatic degradation. Notably, MagCbots enable efficient genome editing of both PD1 and PLK1 genes across various cell lines including hard-to-transfect Jurkat T cells. This magnetically driven nanorobot platform presents a promising strategy for active intracellular delivery and holds significant potential for advancing gene therapy and related biomedical applications.},
}
@article {pmid41183022,
year = {2025},
author = {Rajamani, S and Vilchez, L and Cracovia, N and Dule, D and Vata, A and Landaverde, S and Iyengar, A and Dubrovsky, EB},
title = {Mitochondrial-specific perturbation of Drosophila RNase Z in neurons leads to motor impairments, disrupted learning and neurodegeneration.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011938},
doi = {10.1371/journal.pgen.1011938},
pmid = {41183022},
issn = {1553-7404},
mesh = {Animals ; *Mitochondria/genetics/metabolism/pathology ; *Neurons/metabolism/pathology ; *Drosophila Proteins/genetics/metabolism ; Drosophila melanogaster/genetics ; *Endoribonucleases/genetics/metabolism ; Mutation ; Learning/physiology ; CRISPR-Cas Systems ; *Neurodegenerative Diseases/genetics/pathology ; Animals, Genetically Modified ; Reactive Oxygen Species/metabolism ; },
abstract = {Clinical studies have linked a rare form of neurological disorder to the highly conserved RNase Z gene, which encodes an endoribonuclease responsible for the processing of nuclear and mitochondrial primary tRNA transcripts. Patients harboring mutant variants of this gene exhibit a spectrum of neurological dysfunction; however, no studies to date have established the causality of RNase Z-linked neuropathology. We employed CRISPR/Cas9 technology to create flies with a neuron-specific knockout of the RNase Z gene, which is rescued with transgenes encoding a wild-type or a mutant copy of RNase Z. Neuronal activity of RNase Z is vital, as mutants display striking morphological abnormalities in central and peripheral neurons, along with attenuated motor circuit function and associative learning performance. Neuron-specific mutations of RNase Z also led to mitochondrial fragmentation and elevated ROS production. By employing the rescue transgene encoding RNase Z devoid of a mitochondrial targeting signal (MTS), we segregated the mitochondrial activity of RNase Z from that in other compartments, allowing us to assess organelle-specific contributions to disease etiology and progression. We found that mutating mitochondrial RNase Z was sufficient to induce the neuropathology in flies, as they recapitulate the salient phenotypes observed in the pan-neuronal mutants. Collectively, our study validates the pathogenicity of mutant RNase Z and establishes mitochondrial-specific contributions to neuropathology.},
}
@article {pmid41170612,
year = {2025},
author = {Pu, J and Zhou, S and Ma, W and Liu, C and Guo, L and Yan, B and Huo, Z},
title = {Light-controlled CRISPR-Cas12a one-pot platform for ultrasensitive cell-free DNA detection in systemic lupus erythematosus diagnosis.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {44},
pages = {8916-8922},
doi = {10.1039/d5ay01286d},
pmid = {41170612},
issn = {1759-9679},
mesh = {*Lupus Erythematosus, Systemic/diagnosis/blood/genetics ; Humans ; *Cell-Free Nucleic Acids/blood/genetics/analysis ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Limit of Detection ; },
abstract = {Systemic lupus erythematosus (SLE), as a complex autoimmune disease with heterogeneous clinical manifestations, presents significant challenges for early diagnosis. Circulating cell-free DNA (cfDNA) has emerged as a promising disease monitoring biomarker due to its correlation with SLE pathological progression in terms of concentration and fragmentation patterns. However, existing detection methods lack sufficient sensitivity and practicality for clinical application. To address this, we developed a spatiotemporally resolved light-controlled biosensor by integrating a photoactivatable CRISPR-Cas12a system with TdT-mediated poly-A tail extension, achieving three major innovations: (1) implementation of NPOM-dt modified crRNA for precise regulation; (2) optimization of 365 nm UV activation protocol to eliminate interference in one-pot reactions; and (3) establishment of a three-phase "Extension-Activation-Detection" workflow. The platform demonstrates outstanding performance with a detection limit of 0.42 pM, excellent linearity (R[2] = 0.9956) in the 0-0.1 nM range, and the novel DNA Integrity Index (DII) as a diagnostic indicator - showing significantly higher values in SLE patients (9.82 × 10[3] nmol g[-1]) versus healthy controls (4.2 × 10[3] nmol g[-1], P < 0.0001) with an AUC of 0.8947. This study provides an innovative detection platform combining high sensitivity with clinical utility for early SLE diagnosis.},
}
@article {pmid41165493,
year = {2025},
author = {Zhang, J and Li, X and Wang, M and Ren, S and Li, M and Liu, Y and Lu, F and Li, Q and Li, Y},
title = {Rational Construction of a Robust Bacillus amyloliquefaciens Cell Factory for Acid-Stable α Amylase Production.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {45},
pages = {28883-28895},
doi = {10.1021/acs.jafc.5c04227},
pmid = {41165493},
issn = {1520-5118},
mesh = {*Bacillus amyloliquefaciens/genetics/metabolism/enzymology ; *Bacterial Proteins/genetics/metabolism/chemistry ; *alpha-Amylases/genetics/chemistry/metabolism/biosynthesis ; Fermentation ; Gene Editing ; Prophages/genetics/metabolism ; Enzyme Stability ; CRISPR-Cas Systems ; Multigene Family ; },
abstract = {With the development of synthetic biology and biotechnology, chassis engineering has become the main means of industrial protein production, but it has been limited by the lack of efficient gene editing methods and effective engineering strategies. Bacillus amyloliquefaciens shows potential for expressing heterologous proteins, but its cells undergo early autolysis, hindering further application. In this study, an autolysis-related prophage gene cluster was rationally deleted by establishing an efficient CRISPR-nCas9 editing process, and the prophage mutant strain was constructed, which prevented cell lysis. Based on the prophage mutant strain, we screened secondary metabolite biosynthetic gene clusters that hindered the expression of heterologous proteins, and we made reasonable deletions to further improve their efficient expression. Finally, an optimized yield of acid-stable α amylase (2,46,089.21 U/mL) was obtained in a 5-L fed-batch fermentation. Therefore, we successfully constructed an ideal candidate strain for the expression of heterologous proteins, which provides an important research basis for the development of more chassis strains.},
}
@article {pmid41144643,
year = {2025},
author = {Huang, C and Guo, LJ and Li, JY and Chen, YW and Wu, B and Liu, YW and Zhang, CZ and Lin, WW and Yang, Y and Chen, JY and Liu, ZJ},
title = {A CRISPR-Cas12a Fluorescent Aptasensor for Point-of-Care Drug Concentration Detection: A Synergistic Regulation by DNA Aptamer and RNA Blocker.},
journal = {Analytical chemistry},
volume = {97},
number = {44},
pages = {24415-24424},
doi = {10.1021/acs.analchem.5c03744},
pmid = {41144643},
issn = {1520-6882},
mesh = {*Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Point-of-Care Systems ; *Vancomycin/analysis/blood ; Limit of Detection ; *RNA/chemistry/antagonists &amp; inhibitors ; Humans ; *Fluorescent Dyes/chemistry ; *Anti-Bacterial Agents/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The combination of CRISPR-Cas12a with aptamers can potentially improve the sensitivity, specificity, and speed of non-nucleic acid target detection. Nevertheless, current CRISPR-Cas12a aptasensors, solely dependent on aptamer affinity and overlooking the enzymatic regulation of CRISPR-Cas12a, may produce false-positive signals. We proposed a CRISPR-Cas12a aptasensor synergistically regulated by an aptamer and RNA, which incorporated aptamer-mediated drug recognition and CRISPR-powered signal amplification in a one-pot format. Herein, a regulatory RNA probe synergized with the conformational switching of a drug-induced aptamer, enabling precise regulation of Cas12a trans-cleavage activity via toehold-mediated strand displacement (TMSD). This dual regulatory mechanism transformed the aptamer-activated CRISPR-Cas12a sensing process into a TMSD-driven conditional reaction, avoiding false-positive signals and thus achieving better detection performance with a lower detection limit. With the vancomycin (VCM) aptamer as a model, the aptasensor can detect VCM within 30 min from 2% serum, 1% synovial fluid, and 1% cerebrospinal fluid, with a detection limit of 13.62, 7.56, and 6.75 nM, respectively. The proposed aptasensor was incorporated into a custom 3D-printed portable fluorometer and underwent clinical validation in 22 VCM serum samples, reporting no significant difference when compared with the enzyme-multiplied immunoassay technique, confirming the reliability for point-of-care quantification. It further received cross-validation with a quinine aptamer, suggesting universality [a linear range of 10-250 nM (R[2] = 0.985) and a detection limit of 0.42 nM]. By integration of aptamer selectivity with CRISPR programmability, this work presents a novel robust biosensing paradigm for point-of-care drug concentration detection.},
}
@article {pmid41122778,
year = {2025},
author = {Hasselbeck, S and Wang, J and Bai, Z and Hüfner, T and Hummer, G and Grote, P and Cheng, X},
title = {A Chemically Induced CRISPR/dCas13[FCPF] Platform for Precise and Programmable RNA Regulation.},
journal = {Journal of medicinal chemistry},
volume = {68},
number = {21},
pages = {22633-22649},
doi = {10.1021/acs.jmedchem.5c01609},
pmid = {41122778},
issn = {1520-4804},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *RNA/genetics/metabolism ; Alternative Splicing/drug effects ; Survival of Motor Neuron 2 Protein/genetics ; HEK293 Cells ; Exons ; },
abstract = {Alternative splicing enhances proteomic diversity, yet its dysregulation drives cancer, neurodegeneration, and inherited disease. Small-molecule splicing modulators, while clinically validated, like risdiplam, often lack locus specificity, producing off-target effects. CRISPR/Cas13 enables programmable transcript-level targeting, but dCas13 fusion effectors are bulky and can hamper delivery and RNA homeostasis. Building on our previous Chem-CRISPR/dCas9[FCPF] system for epigenome editing, we now introduce Chem-CRISPR/dCas13[FCPF], a modular platform that covalently tethers a perfluorobiphenyl-tagged small molecule to dCas13 via a four-residue FCPF π-clamp tag. Guided by crRNAs, dRfxCas13d[FCPF] recruits a risdiplam-derived conjugate to the SMN2 exon 7 splice region, inducing exon inclusion at ligand doses ∼500-fold lower than those of free risdiplam and with no detectable effects at known risdiplam-sensitive transcripts in our assays. The approach generalizes to additional transcripts by crRNA redesign. By coupling CRISPR addressability with dose-sparing chemical action, Chem-CRISPR/dCas13[FCPF] establishes a proximity-induced, chemically controllable route to precise RNA modulation suitable for therapeutic exploration.},
}
@article {pmid41108054,
year = {2025},
author = {Mariki, A and Kohlmeier, KA and Mousavi, SM and Shabani, M},
title = {A systematic review of CRISPR applications in demyelinating peripheral nervous system disorders.},
journal = {Regenerative medicine},
volume = {20},
number = {11},
pages = {653-662},
pmid = {41108054},
issn = {1746-076X},
mesh = {Humans ; Animals ; *Demyelinating Diseases/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; *Peripheral Nervous System Diseases/therapy/genetics ; Charcot-Marie-Tooth Disease/therapy/genetics ; Schwann Cells/metabolism/pathology ; Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {AIM: This review evaluates CRISPR-based strategies for myelin regeneration in peripheral demyelinating disorders, with a focus on Guillain - Barré syndrome (GBS) and Charcot - Marie - Tooth disease type 1A (CMT1A). It aims to identify current therapeutic approaches, delivery systems, and gaps in the literature.<br><br>MATERIALS &amp; METHODS: A systematic literature search was conducted across PubMed, Scopus, Web of Science, and Google Scholar for studies published from 2010 onward, following PRISMA guidelines. Study quality was assessed using OHAT and SYRCLE tools, and 14 articles met the inclusion criteria.<br><br>RESULTS: In GBS, CRISPR interventions primarily targeted antiviral immune regulation (AXL, IFI6, IFNL2), inhibition of viral entry mechanisms (Integrin &#x3b1;v&#x3b2;5, SPCS1), and Schwann cell repair. In CMT1A, therapeutic approaches focused on correcting PMP22 overexpression. Lentiviral transduction was the most frequently used delivery method, with no major adverse effects reported. However, most studies were in vitro, and only two were in vivo, highlighting the need for further validation in animal models.<br><br>CONCLUSIONS: CRISPR technology shows considerable potential for addressing peripheral nerve demyelination through precise genetic modifications that may enhance Schwann cell function and support myelin repair. Nevertheless, the field remains at an early discovery stage, with no near-term clinical applicability demonstrated.},
}
@article {pmid41061783,
year = {2025},
author = {Ma, Y and Zhao, Q and Yuan, J and Wang, D and Chen, X and Yu, Y and Li, J and Yu, M and Yuan, J and Lou, J and Du, S and Wen, Y and Wang, Y and Wu, R and Yan, QG and Huang, X and Zheng, Y and Zhao, F and Cao, SJ},
title = {RIPK4-p53 interaction drives aflatoxin B1-induced renal mitochondrial apoptosis via Ser15 phosphorylation: A CRISPR-Cas9 mechanistic study.},
journal = {International journal of biological macromolecules},
volume = {330},
number = {Pt 3},
pages = {148130},
doi = {10.1016/j.ijbiomac.2025.148130},
pmid = {41061783},
issn = {1879-0003},
mesh = {*Apoptosis/drug effects ; Animals ; *CRISPR-Cas Systems ; *Aflatoxin B1/toxicity ; Phosphorylation/drug effects ; *Mitochondria/metabolism/drug effects ; *Tumor Suppressor Protein p53/metabolism ; *Kidney/metabolism/drug effects/pathology ; Swine ; Cell Line ; *Protein Serine-Threonine Kinases/metabolism/genetics ; Protein Binding ; },
abstract = {Aflatoxin B1 (AFB1), recognized as a highly toxic and carcinogenic mycotoxin, contaminates more than 25 % of the global grain supply, thereby presenting a substantial public health threat and posing significant risks to renal health. However, the host factors that mediate the associated damage remain inadequately understood. This study aimed to identify key host factors in AFB1-induced cytotoxicity using a genome-wide CRISPR/Cas9 screen and elucidate the underlying molecular mechanisms. We developed a porcine kidney epithelial (PK15) cell model, followed by knockout validation, CCK-8 assays, qRT-PCR, western blotting, AO-EB staining, flow cytometry, and co-immunoprecipitation to dissect mechanistic pathways. Receptor-Interacting Protein Kinase 4 (RIPK4) was identified as a critical pro-apoptotic factor. RIPK4 knockout increased PK15 cell viability by ~50 % (P < 0.001) and reduced apoptosis by ~44 %(P < 0.001), accompanied by downregulation of APAF1, Cyt-c, cleaved-Caspase-9/-3, and p53 Ser15 phosphorylation, and upregulation of Bcl-2. Mechanistically, RIPK4 directly interacted with p53 via its N-terminal 1-490 aa region, enhancing its phosphorylation and pro-apoptotic activity. In conclusion, RIPK4 promotes AFB1 nephrotoxicity by activating p53-mediated mitochondrial apoptosis, identifying it as a novel therapeutic target. Future studies should validate these findings in vivo models and explore the potential of RIPK4-specific inhibitors for mitigating nephrotoxicity.},
}
@article {pmid41025776,
year = {2025},
author = {Messina, G and Goerner, A and Bennett, C and Brennan, E and Carruthers, VB and Martorelli Di Genova, B},
title = {Impact of equilibrative nucleoside transporters on Toxoplasma gondii infection and differentiation.},
journal = {mBio},
volume = {16},
number = {11},
pages = {e0220725},
pmid = {41025776},
issn = {2150-7511},
support = {P20 GM125498/GM/NIGMS NIH HHS/United States ; },
mesh = {*Toxoplasma/genetics/growth &amp; development/metabolism ; Animals ; Mice ; *Toxoplasmosis/parasitology ; *Protozoan Proteins/genetics/metabolism ; *Nucleoside Transport Proteins/genetics/metabolism ; *Equilibrative Nucleoside Transport Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {UNLABELLED: Toxoplasma gondii cannot synthesize purines de novo and must import them; however, the functional interplay among its four equilibrative nucleoside transporters (ENTs) homologs remains unclear. We systematically deconstructed this network by combining CRISPR-Cas9 knockouts with an auxin-inducible degron. Across all phenotypic assays, tachyzoite replication, nucleoside-analog sensitivity, alkaline-stress-induced differentiation, and murine cyst formation, the ΔTgENT2 strain was indistinguishable from the parental line, indicating that TgENT2 is dispensable under the conditions tested. In contrast, the double mutant ΔTgAT1ΔTgENT3 exhibited delayed bradyzoite differentiation in vitro and produced smaller brain cysts in vivo. This double deletion triggered approximately threefold transcriptional upregulation of TgENT1, whose product we partially localized to the plant-like vacuolar compartment (PLVAC). Conditional depletion of TgENT1 caused complete intracellular growth arrest, PLVAC swelling, and a purine-starvation-like transcriptomic program enriched for nucleoside phosphatases and cyclic-nucleotide phosphodiesterases. These findings reveal a compensatory salvage pathway in which the parasite reroutes purine acquisition through a vacuolar route when plasma-membrane import is compromised. Although this response sustains tachyzoite proliferation, it fails during the energetically demanding transition to bradyzoites, creating a metabolic bottleneck that impairs chronic infection. Our work reveals an adaptable yet ultimately limited purine-import network and identifies TgENT1, along with the vacuolar salvage axis it mediates.<br><br>IMPORTANCE: In this manuscript, we demonstrate that Toxoplasma gondii employs a flexible transporter network that redirects to a vacuolar salvage route when primary transporters are compromised. Disrupting this backup pathway disrupts parasite growth, exposing an Achilles' heel in purine homeostasis. Because nucleoside transporters are druggable, these findings suggest that the purine import machinery and TgENT1 may be potential targets for therapies against T. gondii infections.},
}
@article {pmid40985725,
year = {2025},
author = {Sayson, SG and Ashbaugh, A and Bauer, LC and Smulian, AG},
title = {Extracellular vesicle-mediated delivery of genetic material for transformation and CRISPR/Cas9-based gene editing in Pneumocystis murina.},
journal = {mBio},
volume = {16},
number = {11},
pages = {e0182525},
pmid = {40985725},
issn = {2150-7511},
support = {R61 AI187097/AI/NIAID NIH HHS/United States ; 1R61AI187097//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Extracellular Vesicles/metabolism/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Pneumocystis/genetics ; Animals ; Mice ; *Transformation, Genetic ; *Gene Transfer Techniques ; Lung/microbiology ; Plasmids/genetics ; },
abstract = {Pneumocystis species are obligate fungal pathogens that cause severe pneumonia, particularly in immunocompromised individuals. The absence of robust genetic manipulation tools has impeded our mechanistic understanding of Pneumocystis biology and the development of novel therapeutic strategies. Herein, we describe a novel method for the stable transformation and CRISPR/Cas9-mediated genetic editing of Pneumocystis murina utilizing extracellular vesicles (EVs) as a delivery vehicle. Building upon our prior investigations demonstrating EV-mediated delivery of exogenous material to Pneumocystis, we engineered mouse lung EVs to deliver plasmid DNA encoding reporter genes and CRISPR/Cas9 components. Our initial findings demonstrated successful in vitro transformation and subsequent expression of mNeonGreen and Dhps[ARS] in P. murina organisms. Subsequently, we established stable in vivo expression of mNeonGreen in mice infected with transformed P. murina for a duration of up to 5 weeks. Furthermore, we designed and validated a CRISPR/Cas9 system targeting the P. murina Dhps gene, confirming DNA cleavage efficiency in vitro. Ultimately, we achieved successful in vivo CRISPR/Cas9-mediated homologous recombination, precisely introducing a Dhps[ARS] mutation into the P. murina genome, which was confirmed by Sanger sequencing across all tested animals. Here, we establish a foundational methodology for genetic manipulation in Pneumocystis, thereby opening avenues for functional genomics, drug target validation, and the generation of genetically modified strains for advanced research and potential therapeutic applications.IMPORTANCEPneumocystis species are obligate fungal pathogens and major causes of pneumonia in immunocompromised individuals. However, their strict dependence on the mammalian lung environment has precluded the development of genetic manipulation systems, limiting our ability to interrogate gene function, study antifungal resistance mechanisms, or validate therapeutic targets. Here, we report the first successful approach for stable transformation and CRISPR/Cas9-based genome editing of Pneumocystis murina, achieved through in vivo delivery of engineered extracellular vesicles containing plasmid DNA and encoding CRISPR/Cas9 components. We demonstrate sustained transgene expression and precise modification of the dhps locus via homology-directed repair. This modular, scalable platform overcomes a long-standing barrier in the field and establishes a foundation for functional genomics in Pneumocystis and other obligate, host-adapted microbes.},
}
@article {pmid40980888,
year = {2025},
author = {Danskog, K and Petersen, F and Frängsmyr, L and Gonzalez, G and Becker, M and Lenman, A and Arnberg, N},
title = {CD46 is a cellular receptor for species D human adenovirus.},
journal = {mBio},
volume = {16},
number = {11},
pages = {e0158725},
pmid = {40980888},
issn = {2150-7511},
support = {101098647//HORIZON EUROPE European Innovation Council/ ; 2023-01831//Vetenskapsr&#xe5;det/ ; 2019-01472//Vetenskapsr&#xe5;det/ ; 22 2005 Pj//Cancerfonden/ ; CAN 2018/771//Cancerfonden/ ; },
mesh = {Humans ; *Membrane Cofactor Protein/metabolism/genetics ; *Adenoviruses, Human/physiology/genetics/classification ; *Receptors, Virus/metabolism/genetics ; A549 Cells ; Viral Tropism ; CRISPR-Cas Systems ; Desmoglein 2/genetics/metabolism ; Virus Attachment ; Coxsackie and Adenovirus Receptor-Like Membrane Protein/genetics/metabolism ; },
abstract = {UNLABELLED: Human adenovirus species D (HAdV-D) contains two-thirds of all known HAdV types (116 in total) and is important as a vector in clinical applications. However, the broad tropism exhibited by several HAdV-D types poses challenges for their use as targeted gene delivery vectors. Since adenoviral tropism is largely governed by receptor usage, we aimed to determine the relative importance of known adenovirus receptors in mediating infection by different HAdV-D types. Here, we generated A549 single-cell CRISPR/Cas9 knockout clones of desmoglein 2 (DSG2), CD46, the coxsackievirus and adenovirus receptor (CAR), and cytidine monophosphate N-acetylneuraminic acid synthetase (CMAS; needed for biosynthesis of sialic acid-containing glycans), and assessed their relative importance for infection by 18 different HAdV-D types. We show that CD46 is the most important receptor for a majority of species D HAdVs. Minor changes in infection levels were noted with A549-ΔCAR and A549-ΔDSG2 cells, whereas A549-ΔCMAS cells displayed an increased susceptibility to infection. We proceed to show that HAdV-D types require CD46 for efficient attachment to A549 cells, and surface plasmon resonance analysis demonstrates that their hexon proteins bind CD46 in an avidity-dependent manner. Strategies to retarget HAdV-D vectors should thus consider hexon-CD46 interactions as a critical determinant of tropism, as CD46 is broadly expressed in vivo. These results increase our understanding of adenovirus-host interactions and will guide the development and targeting of vectors based on HAdV-D types.<br><br>IMPORTANCE: Several human adenovirus species D (HAdV-D) types are currently used, or under development, as viral vectors for vaccines and gene delivery. However, the unusually broad tropism observed in many HAdV-D types limits their specificity and effectiveness as targeted vectors. Since tropism is largely governed by receptor usage, and previous studies have reported conflicting findings on receptor preferences within this species, clarifying receptor usage is essential. In this study, we systematically investigated receptor usage in 18 different HAdV-D types and identified CD46 as the primary receptor. Since CD46 is widely expressed across human tissues, our findings explain the broad cellular tropism of these viruses and provide valuable insight for the rational design and refinement of HAdV-D-based vectors.},
}
@article {pmid40973826,
year = {2025},
author = {Petri, K and D'Ippolito, E and Künkele, A and Köhl, U and Busch, DH and Einsele, H and Hudecek, M},
title = {Next-generation T cell immunotherapies engineered with CRISPR base and prime editing: challenges and opportunities.},
journal = {Nature reviews. Clinical oncology},
volume = {22},
number = {12},
pages = {902-923},
pmid = {40973826},
issn = {1759-4782},
mesh = {Humans ; *Gene Editing/methods ; *T-Lymphocytes/immunology/transplantation ; *CRISPR-Cas Systems/genetics ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology/genetics ; Receptors, Antigen, T-Cell/genetics ; },
abstract = {T cells can be reprogrammed with transgenic antigen recognition receptors, including chimeric antigen receptors and T cell receptors, to selectively recognize and kill cancer cells. Such adoptive T cell therapies are effective in patients with certain haematological cancers but challenges persist, including primary and secondary resistance, a lack of efficacy in patients with solid tumours, a narrow range of targetable antigens, and time-consuming and complex manufacturing processes. CRISPR-based genome editing is a potent strategy to enhance cellular immunotherapies. Conventional CRISPR-Cas9 systems are useful for gene editing, transgene knock-in or gene knockout but can result in undesired editing outcomes, including translocations and chromosomal truncations. Base editing and prime editing technologies constitute a new generation of CRISPR platforms and enable highly precise and programmable installation of defined nucleotide variants in primary T cells. Owing to their high precision and versatility, base editing and prime editing systems, hereafter collectively referred to as CRISPR 2.0, are advancing to become the new standard for precision-engineering of cellular immunotherapies. CRISPR 2.0 can be used to augment immune cell function, broaden the spectrum of targetable antigens and facilitate streamlined production of T cell therapies. Notably, CRISPR 2.0 is reaching clinical maturity, with multiple clinical trials of CRISPR 2.0-modified cellular therapies currently ongoing. In this Review, we discuss emerging CRISPR 2.0 technologies and their progress towards clinical translation, highlighting challenges and opportunities, and describe strategies for the use of CRISPR 2.0 to advance cellular immunotherapy for haematological malignancies and solid tumours in the future.},
}
@article {pmid40973646,
year = {2025},
author = {Dinneen, E and Dasgupta, P and Sharma, A and Nisaa, K and Silva-García, CG},
title = {A single-copy knock-in system: one plasmid to target all chromosomes in C. elegans.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {11},
pages = {},
pmid = {40973646},
issn = {2160-1836},
support = {//NIA/ ; R00AG065508/GF/NIH HHS/United States ; A24058//American Federation for Aging Research/ ; //Brown University Division of Research Seed Award/ ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *Plasmids/genetics ; CRISPR-Cas Systems ; *Chromosomes/genetics ; *Gene Knock-In Techniques/methods ; Transgenes ; },
abstract = {Successful transgenesis in model organisms has significantly helped us understand gene function, regulation, genetic networks, and potential applications. Here, we introduce a single-copy knock-in system that uses 1 plasmid to target all chromosomes in Caenorhabditis elegans (SKI PLACE), designed for inserting a transgene by CRISPR/Cas9. The SKI PLACE system uses the pSKI plasmid to insert a desired transgene at specific harbor loci on each chromosome. The pSKI plasmid contains multiple restriction sites for cloning and serves as a CRISPR/Cas9-based insertion repair template because it has 2 synthetic and long homology arms that recombine with the SKI PLACE cassettes. This system also uses a single crRNA guide, which acts as a Co-CRISPR enrichment marker. Overall, the SKI PLACE system is flexible; with the same SKI PLACE cassette on each chromosome, researchers can select the insertion site, work with 1 plasmid, and streamline tracking using standard primers.},
}
@article {pmid40972573,
year = {2025},
author = {Pfefferle, A and Malmberg, KJ},
title = {Rewiring natural killer cells for next-generation cancer therapies.},
journal = {Cancer cell},
volume = {43},
number = {11},
pages = {1980-1982},
doi = {10.1016/j.ccell.2025.08.007},
pmid = {40972573},
issn = {1878-3686},
mesh = {*Killer Cells, Natural/immunology ; Humans ; *Neoplasms/therapy/immunology/genetics ; *Immunotherapy/methods ; CRISPR-Cas Systems ; Animals ; },
abstract = {Harnessing the cytotoxic potential of natural killer (NK) cells for cancer immunotherapy has proven challenging. In this issue of Cancer Cell, Biederstädt et al. and Nikolic et al. utilize genome-wide CRISPR screening to uncover novel regulators of NK cell function, paving the way for developing next-generation NK cell therapies.},
}
@article {pmid40902837,
year = {2025},
author = {Huang, M and Ewadi, A and Servatian, N and Noormohamadi, H and Aminov, Z and Muzammil, K and Esfahani, MA and Abak, N and Soleimani Samarkhazan, H},
title = {Epigenetic mechanisms and next-gen editing platforms in hematology: From molecular basis to therapeutic frontiers.},
journal = {Critical reviews in oncology/hematology},
volume = {215},
number = {},
pages = {104916},
doi = {10.1016/j.critrevonc.2025.104916},
pmid = {40902837},
issn = {1879-0461},
mesh = {Humans ; *Gene Editing/methods ; *Epigenesis, Genetic ; *Hematologic Diseases/therapy/genetics ; CRISPR-Cas Systems ; Animals ; *Hematology/methods ; Genetic Therapy/methods ; },
abstract = {Epigenetic regulation is fundamental to hematopoiesis, influencing stem cell fate, lineage commitment, and the development of hematologic diseases. Recent technological innovations have transitioned from traditional genetic editing towards programmable, reversible epigenetic modulation without altering the DNA sequence. This review explores the evolution of epigenetic editing platforms, from zinc finger proteins and TALEs to the transformative CRISPR-dCas9 system, and introduces next-generation technologies leveraging dCas12, dCas13, and modular RNA-guided systems. By fusing catalytically inactive CRISPR variants with chromatin or RNA-modifying enzymes, these tools enable precise control of gene expression and epitranscriptomic landscapes. In hematology, these advances offer novel strategies to modulate oncogenes, reactivate silenced tumor suppressors, and correct epigenetic dysregulation in malignancies such as leukemia, lymphoma, and myelodysplastic syndromes, as well as in inherited disorders like β-thalassemia and sickle cell disease. The integration of epigenetic editing into immune engineering, particularly in enhancing CAR-T and NK cell therapies, underscores its growing clinical impact. Together, these next-generation approaches herald a paradigm shift, enabling safer, more dynamic, and tunable interventions for blood disorders. This review highlights the current landscape and future directions of epigenetic editing, positioning it as a cornerstone of precision hematologic therapy.},
}
@article {pmid40845844,
year = {2025},
author = {Biederstädt, A and Basar, R and Park, JM and Uprety, N and Shrestha, R and Reyes Silva, F and Dede, M and Watts, J and Acharya, S and Xiong, D and Liu, B and Daher, M and Rafei, H and Banerjee, P and Li, P and Islam, S and Fan, H and Shanley, M and Jin, J and Kumar, B and Woods, V and Lin, P and Tiberti, S and Nunez Cortes, AK and Jiang, XR and Biederstädt, I and Zhang, P and Li, Y and Rawal, S and Liu, E and Muniz-Feliciano, L and Deyter, GM and Shpall, EJ and Fowlkes, NW and Chen, K and Rezvani, K},
title = {Genome-wide CRISPR screens identify critical targets to enhance CAR-NK cell antitumor potency.},
journal = {Cancer cell},
volume = {43},
number = {11},
pages = {2069-2088.e11},
pmid = {40845844},
issn = {1878-3686},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; R01 CA280827/CA/NCI NIH HHS/United States ; P50 CA281701/CA/NCI NIH HHS/United States ; R01 CA211044/CA/NCI NIH HHS/United States ; P01 CA148600/CA/NCI NIH HHS/United States ; P50 CA127001/CA/NCI NIH HHS/United States ; S10 OD024977/OD/NIH HHS/United States ; R01 CA288617/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Killer Cells, Natural/immunology/transplantation/metabolism ; *Immunotherapy, Adoptive/methods ; Animals ; Gene Editing/methods ; Mice ; *CRISPR-Cas Systems ; *Receptors, Chimeric Antigen/genetics/immunology ; *Neoplasms/therapy/immunology/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Tumor Microenvironment/immunology ; },
abstract = {Adoptive cell therapy using engineered natural killer (NK) cells is a promising approach for cancer treatment, with targeted gene editing offering the potential to further enhance their therapeutic efficacy. However, the spectrum of actionable genetic targets to overcome tumor and microenvironment-mediated immunosuppression remains largely unexplored. We performed multiple genome-wide CRISPR screens in primary human NK cells and identified critical checkpoints regulating resistance to immunosuppressive pressures. Ablation of MED12, ARIH2, and CCNC significantly improved NK cell antitumor activity against multiple treatment-refractory human cancers in vitro and in vivo. CRISPR editing augmented both innate and CAR-mediated NK cell function, associated with enhanced metabolic fitness, increased secretion of proinflammatory cytokines, and expansion of cytotoxic NK cell subsets. Through high-content genome-wide CRISPR screening in NK cells, this study reveals critical regulators of NK cell function and provides a valuable resource for engineering next-generation NK cell therapies with improved efficacy against cancer.},
}
@article {pmid40845150,
year = {2025},
author = {Carballar-Lejarazú, R and Pham, TB and Tushar, T and James, AA},
title = {Mutant allele formation and inheritance during Cas9/guide RNA-mediated gene drive in a population modification mosquito strain for human malaria control.},
journal = {Genetics},
volume = {231},
number = {3},
pages = {},
pmid = {40845150},
issn = {1943-2631},
support = {//University of California Irvine Malaria Initiative/ ; INV-043645/GATES/Gates Foundation/United States ; AI170692/NH/NIH HHS/United States ; //AAJ/ ; },
mesh = {Animals ; *Anopheles/genetics/parasitology ; *Gene Drive Technology/methods ; Alleles ; *Malaria/prevention &amp; control/parasitology ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Mutation ; Mosquito Vectors/genetics ; *CRISPR-Cas Systems ; Female ; Male ; },
abstract = {Gene-drive systems are under development for the population modification of anopheline vectors of human malaria parasites. The key to their success is the fixation of genes in target mosquito populations that encode molecules preventing parasite transmission. High-efficiency Cas9/guide RNA (gRNA)-based gene-drive systems can facilitate this objective. A potential challenge to these systems is the presence of naturally-occurring or drive system-induced sequence polymorphisms in the genomic target site that could impede Cas9/gRNA-mediated cleavage and negatively impact gene-drive dynamics and fixation. Careful choice of the target site can mitigate the impact of natural variation, and here we analyze drive system-mediated, target-site mutagenesis in the outcross and testcross progeny of an Anopheles gambiae strain homo- and hemizygous for the TP13-based gene-drive system. The resulting data allow for estimation of the rates at which drive-system activity generates mutant target-site alleles in the germline and the impact of inherited paternal- and maternal-effect mutations. Functional and nonfunctional mutant alleles are recovered from the germlines at average rates per target gene/generation of 0.08% for paternal and 0.33% for maternal testcross lineages, with an overall average rate of 0.21%. Clustering effects amplify the potential inheritance frequencies of the mutant alleles. Mutations originating in the germlines represent 47% of the total inherited in testcross progeny, with the balance coming from mutant alleles generated by paternal and maternal effects inherited through the respective parental lineages. This approach allows the estimation of potential cleavage-resistant allele formation and inheritance for this drive system in this species and provides empirically derived values to inform more realistic data-driven gene-drive modeling.},
}
@article {pmid40827355,
year = {2025},
author = {Spilsbury, K and Wu, J and Reidy, M and Kropp, PA},
title = {The mitochondrial trans-2-enoyl-CoA reductase is necessary for mitochondrial homeostasis in C. elegans.},
journal = {Genetics},
volume = {231},
number = {3},
pages = {},
doi = {10.1093/genetics/iyaf166},
pmid = {40827355},
issn = {1943-2631},
support = {//Intramural Research Program/ ; //Digestive and Kidney Diseases/ ; //National Heart, Blood, and Lung Institute/ ; //Kenyon College/ ; },
mesh = {Animals ; *Caenorhabditis elegans/genetics/metabolism ; *Mitochondria/metabolism/genetics/enzymology ; *Homeostasis ; *Caenorhabditis elegans Proteins/genetics/metabolism ; Fatty Acids/metabolism/biosynthesis ; CRISPR-Cas Systems ; *Acyl-CoA Dehydrogenases/genetics/metabolism ; Oxidoreductases Acting on CH-CH Group Donors ; },
abstract = {Fatty acids function not only as signaling molecules and for energy storage but also as essential cofactors for mitochondrial enzymes. These fatty acid cofactors are produced by the mitochondrial fatty acid synthesis (mtFAS) pathway, the terminal enzyme of which is mitochondrial trans-2-enoyl-CoA reductase (MECR). Dysfunction of MECR prevents the synthesis of fatty acids and is the monogenic cause of Mitochondrial Enoyl-CoA Protein Associated Neurodegeneration (MEPAN) syndrome, a rare mitochondrial disease characterized by dystonia, basal ganglia degeneration, and optic nerve atrophy. Given the necessity of mtFAS products for mitochondrial function, MECR should be essential. Yet, evidence from MEPAN individuals and model organisms with MECR loss of function indicate that mitochondrial function is not as severely impaired as would be expected. However, many of these studies have been limited to single cells or cell types. To better understand the role of MECR and its products in a multicellular system, we used CRISPR/Cas9 to knock out its 2 orthologs in Caenorhabditis elegans, MECR-1 and MECR-2. We found that only MECR-1 is necessary for normal mitochondrial function, germline development, and neuromuscular function. We thus establish a model in which further studies of MECR/MECR-1 can clarify its biochemical, developmental, and physiological roles.},
}
@article {pmid40784261,
year = {2026},
author = {Wang, Y and Fu, L and Li, S and Tao, D and Gong, P and Yang, Y and Ruan, J and Xie, S and Wang, C and He, D},
title = {A rapid and highly sensitive CRISPR assay utilizing Cas12a orthologs for the detection of Novel Duck Reovirus.},
journal = {Talanta},
volume = {297},
number = {Pt B},
pages = {128680},
doi = {10.1016/j.talanta.2025.128680},
pmid = {40784261},
issn = {1873-3573},
mesh = {Nucleic Acid Amplification Techniques/methods ; Animals ; *Orthoreovirus, Avian/genetics/isolation &amp; purification ; Ducks/virology ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; *Reoviridae Infections/diagnosis/veterinary/virology ; Poultry Diseases/virology/diagnosis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; },
abstract = {The Novel Duck Reovirus (NDRV) seriously threatens the global poultry industry due to the lack of effective therapies. Preventive measures for NDRV heavily depend on early disease detection, highlighting the need for rapid and sensitive diagnostic methods. This study used Cas12a orthologs Gs12-18 to develop a visual CRISPR-based detection assay targeting the NDRV S3 gene. Comparative analysis of candidate Cas12a proteins, Gs12-16 and Gs12-18, showed that Gs12-18 has significantly better trans-cleavage activity, making it especially suitable for highly sensitive nucleic acid detection. We integrated Gs12-18 with loop-mediated isothermal amplification (LAMP) technology to create a LAMP-CRISPR/Gs12-18 detection platform. This method enables visual detection of the NDRV S3 gene with high specificity and sensitivity, with a detection limit of 38 copies per reaction. It does not require complex equipment and is suitable for point-of-care testing. This research provides a reliable diagnostic tool for the early prevention and control of NDRV.},
}
@article {pmid40591237,
year = {2025},
author = {Sharma, J and Biswas, R and Khare, P},
title = {Design principle of successful genome editing applications using CRISPR-based toolkits.},
journal = {Journal of applied genetics},
volume = {66},
number = {4},
pages = {1065-1077},
pmid = {40591237},
issn = {2190-3883},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Streptococcus pyogenes/genetics/enzymology ; Plasmids/genetics ; Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Synthetic Biology/methods ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins are the most promising toolkit of synthetic biology for genetic engineering applications across species. Essentially, the Type II CRISPR system, featuring Cas9 nuclease from Streptococcus pyogenes complexed with sgRNA, introduces targeted DNA cleavage, enabling modifications with exceptional precision. This technology can be utilized for not only editing but also modulating gene expressions, thereby finding widespread utility in various biotechnological applications. Here we discuss strategies to construct a consolidated platform aiming at developing a CRISPR-based gene editing system in microbial hosts such as yeast. Employing the well-known gene editing enzymes, i.e., Cpf1 and dCas9, two independent strategies to develop a one-pot plasmid system have been proposed. Furthermore, approaches to reduce off-target cleavages introduced by non-specific targeting of CRISPR complex have been discussed. Finally, an overarching discussion on advanced strategies to design robust CRISPR components is provided for streamlining future genome editing applications.},
}
@article {pmid40579953,
year = {2025},
author = {Ye, Z and Fan, G and Wei, Y and Li, L and Liu, F},
title = {CRISPR/Cas9-mediated germline mutagenesis in the subsocial parasitoid wasp, Sclerodermus guani.},
journal = {Insect molecular biology},
volume = {34},
number = {6},
pages = {939-947},
doi = {10.1111/imb.13014},
pmid = {40579953},
issn = {1365-2583},
support = {Qian Ke He Ji Chu ZK [2024] General 423//Guizhou Provincial Science and Technology Department/ ; 32401587//National Natural Science Foundation of China/ ; QSXM [2022] B09//Guizhou Normal University/ ; Qian Jiao Ji [2024] 54//Department of Education of Guizhou Province/ ; },
mesh = {Animals ; *Wasps/genetics/growth &amp; development ; *CRISPR-Cas Systems ; Female ; Male ; *Mutagenesis ; *Kynurenine 3-Monooxygenase/genetics/metabolism ; Insect Proteins/genetics/metabolism ; Phylogeny ; Gene Editing ; },
abstract = {The ectoparasitoid wasp Sclerodermus guani (Hymenoptera: Bethylidae), as a subsocial insect, is widely applied in biological control against beetle vectors of pine wood nematodes. Despite significant advances in behavioural research, functional genetics in S. guani remains underdeveloped due to the absence of efficient gene manipulation tools. In this study, we employed CRISPR-mediated mutagenesis to achieve germline gene knockout targeting the eye pigment-associated gene kynurenine 3-monooxygenase (KMO). Phylogenetic analysis revealed that S. guani KMO shares a close relationship with its homologue in Prorops nasuta (Hymenoptera: Bethylidae). Two single-guide RNAs (sgRNAs), coupled with Cas9 protein with and without nuclear localisation signal (NLS) were tested. Both sgRNAs induced specific in vitro DNA cleavage and in vivo heritable indels at the target genomic loci. Homozygous null mutant females and males exhibit a white-eye phenotype, which was identified during pupal stage. Optimal editing efficiency in vivo was achieved using the Cas9-NLS variant. Given the complication of germline gene editing in eusocial Hymenopterans, the application of CRISPR in the subsocial parasitoid wasp S. guani provides an accessible research platform for the molecular evolution of insect sociality.},
}
@article {pmid39681701,
year = {2025},
author = {Uijttewaal, ECH and Lee, J and Sell, AC and Botay, N and Vainorius, G and Novatchkova, M and Baar, J and Yang, J and Potzler, T and van der Leij, S and Lowden, C and Sinner, J and Elewaut, A and Gavrilovic, M and Obenauf, A and Schramek, D and Elling, U},
title = {CRISPR-StAR enables high-resolution genetic screening in complex in vivo models.},
journal = {Nature biotechnology},
volume = {43},
number = {11},
pages = {1848-1860},
pmid = {39681701},
issn = {1546-1696},
support = {TFRI Project #1107//Terry Fox Research Institute (Institut de Recherche Terry Fox)/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Genetic Testing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Gene Editing/methods ; },
abstract = {Pooled genetic screening with CRISPR-Cas9 has enabled genome-wide, high-resolution mapping of genes to phenotypes, but assessing the effect of a given genetic perturbation requires evaluation of each single guide RNA (sgRNA) in hundreds of cells to counter stochastic genetic drift and obtain robust results. However, resolution is limited in complex, heterogeneous models, such as organoids or tumors transplanted into mice, because achieving sufficient representation requires impractical scaling. This is due to bottleneck effects and biological heterogeneity of cell populations. Here we introduce CRISPR-StAR, a screening method that uses internal controls generated by activating sgRNAs in only half the progeny of each cell subsequent to re-expansion of the cell clone. Our method overcomes both intrinsic and extrinsic heterogeneity as well as genetic drift in bottlenecks by generating clonal, single-cell-derived intrinsic controls. We use CRISPR-StAR to identify in-vivo-specific genetic dependencies in a genome-wide screen in mouse melanoma. Benchmarking against conventional screening demonstrates the improved data quality provided by this technology.},
}
@article {pmid39604565,
year = {2025},
author = {Cullot, G and Aird, EJ and Schlapansky, MF and Yeh, CD and van de Venn, L and Vykhlyantseva, I and Kreutzer, S and Mailänder, D and Lewków, B and Klermund, J and Montellese, C and Biserni, M and Aeschimann, F and Vonarburg, C and Gehart, H and Cathomen, T and Corn, JE},
title = {Genome editing with the HDR-enhancing DNA-PKcs inhibitor AZD7648 causes large-scale genomic alterations.},
journal = {Nature biotechnology},
volume = {43},
number = {11},
pages = {1778-1782},
pmid = {39604565},
issn = {1546-1696},
support = {855741-DDREAMM-ERC-2019-SyG//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; DFG CA 311/4-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Gene Editing/methods ; Humans ; *DNA-Activated Protein Kinase/antagonists &amp; inhibitors ; *Pyrazoles/pharmacology ; CRISPR-Cas Systems/genetics ; *Recombinational DNA Repair/drug effects/genetics ; Protein Kinase Inhibitors/pharmacology ; Pyrimidines ; },
abstract = {The DNA-PKcs inhibitor AZD7648 enhances CRISPR-Cas9-directed homology-directed repair efficiencies, with potential for clinical utility, but its possible on-target consequences are unknown. We found that genome editing with AZD7648 causes frequent kilobase-scale and megabase-scale deletions, chromosome arm loss and translocations. These large-scale chromosomal alterations evade detection through typical genome editing assays, prompting caution in deploying AZD7648 and reinforcing the need to investigate multiple types of potential editing outcomes.},
}
@article {pmid41219913,
year = {2025},
author = {Muttappagol, M and Sharma, M and Adhikari, DN and Suma, R and Borgohain, T and Saikia, B and Keithellakpam, A and Dutt, A and Laskar, A and Hiremath, S and Chikkaputtaiah, C},
title = {Towards Climate-Resilient Crops: Integrative Strategies for Reproductive Stage Abiotic Stress Tolerance.},
journal = {Physiologia plantarum},
volume = {177},
number = {6},
pages = {e70635},
doi = {10.1111/ppl.70635},
pmid = {41219913},
issn = {1399-3054},
support = {MMP025301//Council of Scientific and Industrial Research, India/ ; CRG/2022/007073 TSA//Anusandhan National Research Foundation (ANRF), Government of India/ ; BT/PR38410/GET/119/310/2020//Department of Biotechnology, Government of India (DBT)/ ; },
abstract = {Abiotic stresses such as drought, heat, cold, and salinity, threaten global crop productivity, particularly during the highly sensitive reproductive phase. These stresses disrupt female gametophyte formation, ovule development, fertilization, and seed maturation, resulting in reduced fertility and yield losses. While the mechanisms of vegetative stress tolerance are relatively well studied, reproductive-stage resilience, especially the resilience of the female gametophyte, remains insufficiently understood, despite its decisive role in determining yield stability. Recent advances provide an opportunity to close this gap through an integrative perspective. Physiological assessments and high-throughput phenotyping offer reliable tools to evaluate gametophyte and embryo performance under stress. Multi-omics approaches, including genomics, transcriptomics, proteomics, and metabolomics, are covering regulatory pathways underlying reproductive success. Emerging technologies such as CRISPR/Cas genome editing, biomarker discovery and machine learning further accelerate trait identification and their application in breeding pipelines. The novelty of this review lies in its focus on the female gametophyte as an overlooked determinant of stress resilience and in presenting a systems-level framework that integrates physiology, omics, breeding, and AI. This integration does more than catalogue stress effects; it connects mechanistic insights with breeding strategies and scales them with computational tools. The ultimate outcome is climate-resilient varieties that sustain productivity under stress, which support farmer livelihoods and contribute to global food security.},
}
@article {pmid41219189,
year = {2025},
author = {Uchigashima, M and Iguchi, R and Fujii, K and Shiku, K and Kumar, P and Liu, X and Isogai, M and Hoshino, C and Abe, M and Nozumi, M and Okamura, Y and Igarashi, M and Sakimura, K and Bise, R and Lavis, LD and Mikuni, T},
title = {Single-cell synaptome mapping of endogenous protein subpopulations in mammalian brain.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9705},
pmid = {41219189},
issn = {2041-1723},
support = {JP19dm0207080, JP21wm0525014, and JP24wm0625117//Japan Agency for Medical Research and Development (AMED)/ ; JPMJPR16F9//MEXT | JST | Precursory Research for Embryonic Science and Technology (PRESTO)/ ; CDA00043/2019-C//Human Frontier Science Program (HFSP)/ ; N/A//Uehara Memorial Foundation/ ; JP20K21461, JP20H03349, JP20H05918, JP23K18160, and JP24K02130//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPMJFR231M//MEXT | Japan Science and Technology Agency (JST)/ ; N/A//Kowa Life Science Foundation/ ; },
mesh = {Animals ; *Synapses/metabolism ; *Brain/metabolism/cytology ; *Single-Cell Analysis/methods ; Mice ; *Neurons/metabolism ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; },
abstract = {Different spatial or temporal protein populations, such as cell-surface/intracellular or pre-existing/nascent subpopulations, determine the basal and activity-induced functions of individual synapses within a neuron in vivo. Here, we developed a simple and generalizable platform to image different spatial and temporal subpopulations of endogenous proteins at thousands of synapses in single neurons in the mammalian brain. The platform is based on the development, improvement and integration of CRISPR-Cas9-mediated protein labeling methods, chemical tag labeling techniques, and a semi-automatic analytical pipeline. The combined platform enables whole-cell mapping of total, cell-surface, intracellular, pre-existing, nascent or nascent-and-surface populations of endogenous proteins, such as receptor, scaffold and signaling proteins, at thousands of synapses in individual neurons in living or fixed mouse brain. Our single-cell "synaptome" mapping of endogenous protein subpopulations comprehensively visualizes the spatial representation of synapse diversity in protein localization, trafficking and turnover, providing valuable insights into single-cell organization and computations in the brain.},
}
@article {pmid41218598,
year = {2025},
author = {Nazarudeen, A and Aswathy, VA and Rauf, AA and Aswathy, UV and Mahesh Chandran, VR and Abhirami, N and Sudhina, S and Chandran, A and Ayyappan, JP},
title = {Targeting Circular RNAs (circRNAs) in Atherosclerosis Using CRISPR Technology.},
journal = {The journal of gene medicine},
volume = {27},
number = {11},
pages = {e70048},
doi = {10.1002/jgm.70048},
pmid = {41218598},
issn = {1521-2254},
support = {//University of Kerala/ ; //Start-up Research Grant from the Science and Engineering Research Board (SERB), India/ ; //Start-up grant for new faculty from the University Grants Commission (UGC), India/ ; //University of Kerala research support in the form of Plan and Nonplan funds/ ; },
mesh = {*RNA, Circular/genetics ; Humans ; *Atherosclerosis/genetics/therapy/metabolism ; *CRISPR-Cas Systems/genetics ; Animals ; Gene Editing/methods ; Genetic Therapy/methods ; Lipid Metabolism/genetics ; },
abstract = {Atherosclerosis is a chronic inflammatory condition that remains a major global cause of cardiovascular morbidity and death. Circular RNAs (circRNAs), emerging as key regulators of biological processes, have been linked to atherosclerosis because of their functions in inflammation, lipid metabolism, and plaque stability. This review explores the biogenesis and cellular functions of circRNAs, highlighting specific circRNAs, such as circANRIL, circHIPK, and circRSF1, which influence atherosclerosis progressions and development. CRISPR-Cas technology, specifically Cas9 and Cas13, has transformed the way atherosclerosis is studied and potentially treated. Targeting PCSK9, LDLR, and APOB to modify lipid metabolism, including lowering LDL cholesterol and repairing mutations in familial hypercholesterolemia, has been made possible using CRISPR-Cas9 in atherosclerosis models. In parallel, CRISPR-Cas13 offers a novel approach for RNA-level intervention by selectively editing circRNAs, providing a dynamic approach to regulate atherosclerosis-related pathways. In order to convert these findings into therapeutic treatments, future research should focus on elucidating the mechanics of circRNA, which in turn determines CRISPR-Cas13, and designing specific delivery systems. This review paper demonstrates the revolutionary promise of circRNA research and CRISPR innovation in the treatment of atherosclerosis and underscores the need for extensive preclinical validation to bridge the gap towards clinical use.},
}
@article {pmid41217051,
year = {2025},
author = {Alam, SS and Mehdi, A and Zafar, A and Ali, S and Rehman, AU and Liaqat, I and Peng, L and Kanwal, F and Afzal, S and Haq, IU and Aftab, MN},
title = {Advances in microbial biofuel production by metabolic and enzyme engineering, synthetic biology, metagenomics, and genome editing applications.},
journal = {Emerging topics in life sciences},
volume = {},
number = {},
pages = {},
doi = {10.1042/ETLS20240002},
pmid = {41217051},
issn = {2397-8554},
abstract = {Microorganisms are the primary source of genetic diversity on earth due to their unparalleled metabolic and functional variability. With the depletion of fossil fuels, a sustainable alternative approach is the use of biofuels, where plant biomass as feedstock is essentially degraded to sugars with the aid of microbe-derived enzymes, followed by the conversion of those sugars to biofuels. Several cellulolytic and non-cellulolytic enzymes are involved in biofuel synthesis. Molecular cloning, along with the advancements in genetic and metabolic engineering in microbial cells, plays a significant contribution to biofuel overproduction. Advanced molecular technologies such as metagenomics and synthetic biology approaches are also being used to construct effective microorganisms for biofuel manufacturing. Obtaining novel enzymes from undiscovered microbial consortia and functional gene analysis is possible through a metagenomics approach. While synthetic biology provides engineered biological systems to generate required biofuel productivity, the CRISPR-Cas genome editing tool is another revolutionary tool being utilized for efficient biofuel production. This article provides a brief overview of different methods of biofuel production using microorganisms.},
}
@article {pmid41216030,
year = {2025},
author = {Naki, D and Gophna, U},
title = {Evolutionary insights into provirus-encoded CRISPR-Cas systems in halophilic archaea.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf033},
pmid = {41216030},
issn = {2633-6693},
abstract = {Prokaryotic microorganisms coexist with mobile genetic elements (MGEs), which can be both genetic threats and evolutionary catalysts. In Haloferax lucentense, a halophilic archaeon, we have recently identified an unusual genomic arrangement: a complete type I-B CRISPR-Cas system encoded on a megaplasmid and an incomplete type I-B system within an integrated provirus in the main chromosome. The provirus-encoded system lacks the adaptation genes (cas1, cas2, and cas4), suggesting its potential reliance on the megaplasmid-encoded CRISPR-Cas module for the acquisition of new spacers. This arrangement suggests a potential instance of "adaptive outsourcing," where a provirus might leverage a co-resident MGE for a key function. Through comparative genomics, we show that similar proviral CRISPR-Cas systems are found in distantly related haloarchaea (e.g. Natrinema and Halobacterium), indicating probable virus-mediated horizontal transfer and suggesting they may function as mobile defense modules. Phylogenetic analysis highlights distinct evolutionary origins of the two systems: the plasmid system clusters with other Haloferax CRISPR-Cas systems, while the proviral system clusters with those from other genera, consistent with horizontal acquisition. Interestingly, spacer analysis reveals that the proviral systems predominantly target viral sequences, while the plasmid system appears to target both plasmids and viral sequences, a distribution mirroring broader trends observed in other plasmid- and chromosome-encoded CRISPR systems. This observed targeting preference suggests a potential for complementarity that could support a model of cooperative immunity, where each system may protect its genetic "owner" from competition and, indirectly, the host.},
}
@article {pmid41214117,
year = {2025},
author = {Bakthavachalam, V and Sanborn, MA and Mathayan, M and Salunkhe, SS and Wood, JM and Maienschein-Cline, M and Setty, S and Kabeer, BSA and Rehman, J and Prabhakar, BS},
title = {CRISPR/Cas9-mediated deletion of MADD induces cell cycle arrest and apoptosis in anaplastic thyroid cancer cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {39264},
pmid = {41214117},
issn = {2045-2322},
support = {VA Merit Review, Award # 5 I01 BX004697//United States (U.S.) Department of Veteran's Affairs Biomedical Laboratory Research and Development Services (BLRD)/ ; },
mesh = {*Apoptosis/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Thyroid Carcinoma, Anaplastic/genetics/pathology/metabolism ; Animals ; Cell Line, Tumor ; *Cell Cycle Checkpoints/genetics ; Mice ; *Thyroid Neoplasms/genetics/pathology ; Cell Proliferation/genetics ; *Death Domain Receptor Signaling Adaptor Proteins/genetics ; Gene Expression Regulation, Neoplastic ; Cell Movement/genetics ; Gene Deletion ; Guanine Nucleotide Exchange Factors ; },
abstract = {Anaplastic thyroid cancer (ATC) is an aggressive malignancy with a poor prognosis and limited treatment options. Previous studies have shown that selective downregulation of the MADD (MAP-kinase-activating death domain-containing protein) gene isoform increases ATC cell susceptibility to TRAIL-induced apoptosis. However, the existence of multiple MADD gene isoforms raised the possibility of functional compensation. This study aimed to definitively evaluate the role of MADD in ATC by employing CRISPR-Cas9 to Cas9 to target exon 3, a conserved exon expressed in all known MADD isoforms, resulting in functional knockout of MADD expression. CRISPR-Cas9-mediated MADD knockout, performed in three ATC cell lines (8505 C, C643, and HTH7) with distinct mutational backgrounds, significantly impaired ATC cell function in vitro, as indicated by reduced viability, increased apoptosis, decreased migration, and G0/G1 cell cycle arrest. RNA-seq analysis revealed alterations in genes related to cell survival, proliferation, and metastasis. In the orthotopic ATC mouse model, MADD deletion dramatically suppressed tumor growth, reduced lung metastases, and prolonged survival. Our findings demonstrate that MADD plays a crucial role in ATC cell survival, proliferation, and metastasis. The consistent effects observed across multiple cell lines and in vivo models suggest that MADD may represent a promising therapeutic target for this aggressive malignancy.},
}
@article {pmid41213972,
year = {2025},
author = {Chisholm, CG and Bartlett, R and Brown, ML and Proctor, EJ and Farrawell, NE and Gorman, J and Delerue, F and Ittner, LM and Vine-Perrow, KL and Ecroyd, H and Cashman, NR and Saunders, DN and McAlary, L and Lum, JS and Yerbury, JJ},
title = {Development of a targeted BioPROTAC degrader selective for misfolded SOD1.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9713},
pmid = {41213972},
issn = {2041-1723},
mesh = {Animals ; *Superoxide Dismutase-1/metabolism/genetics/chemistry ; *Amyotrophic Lateral Sclerosis/genetics/metabolism/pathology/drug therapy ; Mice, Transgenic ; Mice ; Protein Folding ; Humans ; Disease Models, Animal ; Proteolysis ; Motor Neurons/metabolism/pathology ; Ubiquitin-Protein Ligases/metabolism/genetics ; CRISPR-Cas Systems ; Disease Progression ; HEK293 Cells ; },
abstract = {The accumulation of misfolded proteins underlies a broad range of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Due to their dynamic nature, these misfolded proteins have proven challenging to target therapeutically. Here, we specifically target misfolded disease variants of the ALS-associated protein superoxide dismutase 1 (SOD1), using a biological proteolysis targeting chimera (BioPROTAC) composed of a SOD1-specific intrabody and an E3 ubiquitin ligase. Screening of intrabodies and E3 ligases for optimal BioPROTAC construction reveals a candidate capable of degrading multiple disease variants of SOD1, preventing their aggregation in cells. Using CRISPR/Cas9 technology to develop a BioPROTAC transgenic mouse line, we demonstrate that the presence of the BioPROTAC delays disease progression in the SOD1[G93A] mouse model of ALS. Delayed disease progression is associated with protection of motor neurons, a reduction of insoluble SOD1 accumulation and preservation of innervated neuromuscular junctions. These findings provide proof-of-concept evidence and a platform for developing BioPROTACs as a therapeutic strategy for the targeted degradation of neurotoxic misfolded species in the context of neurodegenerative diseases.},
}
@article {pmid41212595,
year = {2025},
author = {Wu, Y and Su, J and Sun, J and Li, Z and Yang, J and Wu, C and Liu, Z and Wang, J and Wang, Y and Liu, J and Duan, L and Wang, C and Zhu, G and Wen, X},
title = {Rapid detection of Seneca Valley virus based on reverse transcription recombinase polymerase amplification and CRISPR-Cas13a.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2585719},
pmid = {41212595},
issn = {2150-5608},
mesh = {Animals ; Swine ; *CRISPR-Cas Systems ; *Swine Diseases/diagnosis/virology ; Sensitivity and Specificity ; *Picornaviridae/isolation &amp; purification/genetics ; *Picornaviridae Infections/diagnosis/veterinary/virology ; Recombinases/genetics ; *Nucleic Acid Amplification Techniques/methods ; Reverse Transcription ; },
abstract = {The Seneca Valley virus (SVV) is an emerging novel etiological agent that triggers vesicular stomatitis in pigs. SVV leads to considerable financial detriment to the global swine industry. Availability of expeditious, simple, and precise detection techniques would aid in the diagnosis and control of Seneca Valley disease. In this study, we developed a novel SVV detection assay based on reverse transcription recombinase polymerase amplification (RT-RPA)/CRISPR-Cas13a. This technique can specifically identify SVV with no cross-reactivity with other viruses, such as the porcine reproductive and respiratory syndrome virus, pseudorabies virus, classical swine fever virus, and foot-and-mouth disease virus. It boasts a detection threshold as sensitive as 2.43 copies/μL, matching the proficiency of reverse transcriptase quantitative PCR in clinical diagnostics. The streamlined RT-RPA/CRISPR-Cas13a assay for SVV minimizes procedural complexity and the likelihood of contamination. Integrating this method with nucleic acid acquisition via nucleic acid releaser enables a turnaround time of only 40 min from sample acquisition to result determination, underscoring its suitability for on-site diagnostics and affirming its role as a robust instrument for the monitoring and detection of SVV.},
}
@article {pmid41208113,
year = {2025},
author = {Hermann, MD and Fernandez Perez, N and Yeroslaviz, A and Murray, PJ},
title = {Normal macrophage signaling and gene expression in Rosa26 Cas9-expressing mice.},
journal = {ImmunoHorizons},
volume = {9},
number = {11},
pages = {},
pmid = {41208113},
issn = {2573-7732},
support = {//Deutsche Forschungsgemeinschaft/ ; //Max-Planck-Gesellschaft/ ; },
mesh = {Animals ; Mice ; *Macrophages/metabolism/immunology ; *Signal Transduction/genetics ; CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *RNA, Untranslated/genetics ; Mice, Inbred C57BL ; Toll-Like Receptor 3/metabolism ; Mice, Transgenic ; Toll-Like Receptor 4/metabolism ; },
abstract = {Cas9-expression from the Rosa26 "safe harbor" locus are widely used for gene manipulation and Crispr-based screening. Recently, experimental evidence suggested that macrophages isolated from Rosa26-Cas9 mice may have signaling differences compared to control mice in terms of TRIF signaling downstream of TLR3 and TLR4. As we frequently use the Rosa26-Cas9 mice made by Feng Zhang (Cas9-FZ, Jackson Laboratory stock No. 026179), arguably the strain with the widest distribution and utilization, we were motivated to test macrophage signaling in these mice under our conventional conditions. We used different macrophage polarization and signaling conditions combined with RNA sequencing and measurement of TLR signaling by immunoblotting. Our results suggest that the Cas9-FZ mice bear no obvious defects in any commonly used macrophage signaling pathway. We document the differences in our macrophage culture techniques compared to Raychowdhury et al., which may aid in how individual laboratories use Cas9-expressing macrophages, especially for focused or genome-wide screening.},
}
@article {pmid41206041,
year = {2025},
author = {Liu, Q and Pan, TT and Wang, LJ and Zhang, CY},
title = {Mirror-synchronized asymmetric CRISPR nanoswitch for single-molecule profiling of multiple circRNAs in different stages of breast cancer.},
journal = {Nucleic acids research},
volume = {53},
number = {20},
pages = {},
pmid = {41206041},
issn = {1362-4962},
support = {22474019//National Natural Science Foundation of China/ ; },
mesh = {*RNA, Circular/genetics ; *Breast Neoplasms/genetics/pathology ; Humans ; *CRISPR-Cas Systems/genetics ; Female ; *RNA/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Single Molecule Imaging/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Circular RNAs (circRNAs) represent a class of endogenous noncoding RNAs characterized by their covalently closed circular structures. They have been implicated in significant transcriptional and post-transcriptional regulation of gene expression. Here, we present a one-pot method for the detection of circRNAs based on engineered DNA hairpins and CRISPR-Cas12a signal amplification, which involves signal pre-amplification via coupled probe-mediated hairpin amplification of two palindromic hairpins and Cas12a signal generation via trans-cleavage. We demonstrate that this platform is sensitive (detection limit of 1.07 aM), specific (capable of single-mismatch discrimination), and fast (reaction time of 25 min) and can be used to detect different circRNAs from RNase R-treated RNA (both in vitro and in clinically relevant samples, including correct classification of disease progression). This method enables single-molecule profiling and can be extended to detect other types of nucleic acids.},
}
@article {pmid41205588,
year = {2025},
author = {Kumar, KRR},
title = {Plant genome editing goes viral: balancing innovation and biosafety.},
journal = {Trends in biotechnology},
volume = {43},
number = {11},
pages = {2684-2685},
doi = {10.1016/j.tibtech.2025.09.006},
pmid = {41205588},
issn = {1879-3096},
mesh = {*Gene Editing/methods ; *Genome, Plant/genetics ; *Plants, Genetically Modified/genetics ; Genetic Vectors/genetics ; CRISPR-Cas Systems ; Containment of Biohazards ; },
abstract = {Weiss and colleagues demonstrated a breakthrough in transgene-free heritable genome editing using viral vectors. While promising for controlled laboratory applications, the open-field use envisaged by Sajjad and colleagues raises ecological and biosafety concerns. Rigorous risk assessment is essential to harness innovation responsibly while safeguarding biodiversity and public trust.},
}
@article {pmid41190788,
year = {2025},
author = {Orzechowski, M and Hoikkala, V and Chi, H and McMahon, S and Gloster, T and White, MF},
title = {A viral SAVED protein with ring nuclease activity degrades the CRISPR second messenger cA4.},
journal = {The Biochemical journal},
volume = {482},
number = {22},
pages = {},
doi = {10.1042/BCJ20253271},
pmid = {41190788},
issn = {1470-8728},
mesh = {*Viral Proteins/metabolism/genetics/chemistry ; *Adenine Nucleotides/metabolism ; *CRISPR-Cas Systems ; *Second Messenger Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Oligoribonucleotides ; },
abstract = {Type III CRISPR systems typically generate cyclic oligoadenylate second messengers such as cyclic tetra-adenylate (cA4) on detection of foreign RNA. These activate ancillary effector proteins which elicit a diverse range of immune responses. The Calp (CRISPR associated Lon protease) system elicits a transcriptional response to infection when CalpL (Calp Lon protease) binds cA4 in its SAVED (SMODS associated and fused to various effectors domain) sensor domain, resulting in filament formation and activation of the Lon protease domain, which cleaves the anti-Sigma factor CalpT, releasing the CalpS (Calp Sigma factor) for transcriptional remodelling. Here, we show that thermophilic viruses have appropriated the SAVED domain of CalpL as an anti-CRISPR, AcrIII-2 (second anti-CRISPR of type III systems), which they use to degrade cA4. AcrIII-2 dimers sandwich cA4, degrading it in a shared active site to short linear products, using a mechanism highly reminiscent of CalpL. This results in inhibition of a range of cA4 activated effectors in vitro. This is the first example of a virally encoded SAVED domain with ring nuclease activity, highlighting the complex interplay between viruses and cellular defences.},
}
@article {pmid41187216,
year = {2025},
author = {Treaster, MJ and McCann, J and Solovei, KS and Palmieri, RJ and White, MA},
title = {A Y-linked duplication of anti-Mullerian hormone is the sex determination gene in threespine stickleback.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011932},
pmid = {41187216},
issn = {1553-7404},
mesh = {Animals ; *Smegmamorpha/genetics ; Male ; *Anti-Mullerian Hormone/genetics ; *Sex Determination Processes/genetics ; Female ; *Y Chromosome/genetics ; *Gene Duplication ; CRISPR-Cas Systems ; Evolution, Molecular ; Sex Differentiation/genetics ; },
abstract = {Many taxa have independently evolved genetic sex determination where a single gene located on a sex chromosome controls gonadal differentiation. The gene anti-Mullerian hormone (amh) has convergently evolved as a sex determination gene in numerous vertebrate species, but how this gene has repeatedly evolved this novel function is not well understood. In the threespine stickleback (Gasterosteus aculeatus), amh was duplicated onto the Y chromosome (amhy) ~22 million years ago. To determine whether amhy is the primary sex determination gene, we used CRISPR/Cas9 and transgenesis to show that amhy is necessary and sufficient for male sex determination, consistent with the function of a primary sex determination gene. We find that amhy contributes to a higher total dosage of amh early in development and likely contributes to differential germ cell proliferation key to sex determination. The creation of sex-reversed lines also allowed us to investigate the genetic basis of secondary sex characteristics. Threespine stickleback have striking differences in behavior and morphology between sexes. Here we show one of the classic traits important for reproductive success, blue male nuptial coloration, is controlled by both sex-linked genetic factors as well as hormonal factors independent of sex chromosome genotype. This research establishes stickleback as a model to investigate how amh regulates gonadal development and how this gene repeatedly evolves novel function in sex determination. Analogous to the "Four Core Genotypes" model in house mice, sex-reversed threespine stickleback offer a new vertebrate model for investigating the separate contributions of gonadal sex and sex chromosomes to sexual dimorphism.},
}
@article {pmid41170863,
year = {2025},
author = {Liu, J and Feng, G and Guo, C and Liu, D and Li, Y},
title = {Opportunities and Challenges of CRISPR-Cas9 in the Sustainable Development of Animal Husbandry.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {45},
pages = {28575-28587},
doi = {10.1021/acs.jafc.5c09076},
pmid = {41170863},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; Animals ; Gene Editing ; Sustainable Development ; *Animal Husbandry/methods ; *Livestock/genetics/metabolism/growth &amp; development ; Animals, Genetically Modified/genetics/metabolism ; },
abstract = {Food security is of vital importance to the destiny of the entire globe. However, the realization of the United Nations' Sustainable Development Goals, which advocate for the eradication of hunger and the improvement of nutrition, appears somewhat bleak. The advent of gene editing has made precise modification of biological genomes a reality. Among these technologies, CRISPR-Cas9, due to its convenience and high efficiency, has been widely applied in fields such as agriculture. The application of CRISPR-Cas9 in genetically engineered animals can rapidly improve animal traits, reduce resource consumption, and has great potential in addressing food security issues. Therefore, this review focuses on the editing principles of CRISPR-Cas9, its developmental trajectory, and the opportunities and challenges it presents in animal husbandry, aiming to provide a theoretical reference for the further application of CRISPR-Cas9 to promote the sustainable development of animal husbandry.},
}
@article {pmid41162785,
year = {2025},
author = {Coda, DM and Watt, L and Glauser, L and Batiuk, MY and Burns, AM and Stahl, CL and Wong, LY and Gräff, J},
title = {Cell-type- and locus-specific epigenetic editing of memory expression.},
journal = {Nature genetics},
volume = {57},
number = {11},
pages = {2661-2668},
pmid = {41162785},
issn = {1546-1718},
support = {101043457//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 310030_219342//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 310030_197752//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Epigenesis, Genetic/genetics ; *Memory/physiology ; Animals ; *Gene Editing/methods ; Mice ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic ; Nerve Tissue Proteins/genetics ; Neuronal Plasticity/genetics ; Male ; Cytoskeletal Proteins/genetics ; Neurons/metabolism/physiology ; Epigenome Editing ; },
abstract = {Epigenetic mechanisms have long been proposed to act as molecular mnemonics[1-3], but whether the epigenetic makeup of a single genomic site can guide learnt behaviors remains unknown. Here we combined CRISPR-based epigenetic editing tools[4,5] with c-Fos-driven engram technologies[6,7] to address this question in memory-bearing neuronal ensembles. Focusing on the promoter of Arc, which encodes a master regulator of synaptic plasticity[8], we found that its locus-specific and temporally controlled epigenetic editing is necessary and sufficient to regulate memory expression. Such effects occurred irrespective of the memory phase-during the initially labile period after learning and for fully consolidated memories-and were reversible within subject, testifying to their inherent plasticity. These findings provide a proof-of-principle that site-specific epigenetic dynamics are causally implicated in memory expression.},
}
@article {pmid41087678,
year = {2025},
author = {Kaplan, EG and Steger, RJ and Shah, ST and Drepanos, LM and Griffith, AL and Reint, G and Doench, JG},
title = {Activity-based selection for enhanced base editor mutational scanning.},
journal = {Nature genetics},
volume = {57},
number = {11},
pages = {2920-2929},
pmid = {41087678},
issn = {1546-1718},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Base editing is a CRISPR-based technology that enables high-throughput, nucleotide-level functional interrogation of the genome that is essential for understanding the genetic basis of human disease and informing therapeutic development. Base editing screens have emerged as a powerful experimental approach, yet significant cell-to-cell variability in editing efficiency introduces noise that may obscure meaningful results. Here we develop a co-selection method that enriches for cells with high base editing activity, substantially increasing editing efficiency at a target locus. We evaluate this activity-based selection method against a traditional screening approach by tiling guide RNAs across TP53, demonstrating its enhanced capacity to pinpoint specific mutations and protein regions of functional importance. We anticipate that this modular selection method will enhance the resolution of base editing screens across many applications.},
}
@article {pmid40996032,
year = {2025},
author = {Mofed, D and Gowripalan, A and Berrigan, J and Das, PK and Pujari, N and Ajasin, D and Haldar, S and McCullough, J and Zhang, Y and Kalpana, GV and Bresnick, A and Kielian, M and Wilson, DW and Zhang, J and Chandran, K and Prasad, VR},
title = {Influence of CCL2-mediated modulation of ALIX in the budding and replication of viruses from multiple families.},
journal = {mBio},
volume = {16},
number = {11},
pages = {e0224125},
doi = {10.1128/mbio.02241-25},
pmid = {40996032},
issn = {2150-7511},
support = {R01 AI125244/AI/NIAID NIH HHS/United States ; R01 AI153008/NH/NIH HHS/United States ; R01 AI125244/NH/NIH HHS/United States ; R01 AI075647/NH/NIH HHS/United States ; R01 AI185073/NH/NIH HHS/United States ; P30CA013330/NH/NIH HHS/United States ; S10OD026833-01/NH/NIH HHS/United States ; S10OD032169-01/NH/NIH HHS/United States ; },
mesh = {Humans ; HeLa Cells ; *Virus Replication ; *Endosomal Sorting Complexes Required for Transport/metabolism/genetics ; *Calcium-Binding Proteins/metabolism/genetics ; *Chemokine CCL2/genetics/metabolism ; *Virus Release ; HIV-1/physiology/genetics ; *Cell Cycle Proteins/metabolism/genetics ; Gene Knockout Techniques ; Signal Transduction ; CRISPR-Cas Systems ; Animals ; },
abstract = {UNLABELLED: Signaling by C-C motif ligand 2 (CCL2), a β-chemokine, modulates HIV-1 budding and release by mobilizing ALG-2-interacting protein X (ALIX) from the F-actin cytoskeleton to the cytosol. Immunodepleting CCL2 in the medium sequesters ALIX to F-actin. We developed a novel tool to study HIV budding and release without mutating viral late domains or silencing ESCRT genes, but by blocking CCL2 signaling using CRISPR-Cas9 knockout (KO) of the CCL2 or CCR2 genes. We knocked out CCL2 (CCL2KO) and CCR2 (CCR2KO) singly or together (double knockout) in HeLa cells and confirmed that knockout was associated with the absence of CCL2 or CCR2 expression. In KO cells, ALIX was associated with the F-actin cytoskeleton, while in control cells, it was associated with the cytosolic soluble fraction. In KO cells, HIV-1 production was profoundly reduced (10-fold). Strikingly, for CCL2KO cells, the addition of CCL2 mobilized ALIX to the soluble fraction, and virus production was stimulated to levels higher than those of untreated HeLa cells. We utilized these cells to test the involvement of ALIX in the budding and/or replication of several viruses, including Simian Immunodeficiency Virus (SIV), Equine Infectious Anemia Virus (EIAV), Herpes Simplex Virus type 1 (HSV-1), Dengue virus (DENV), and Hazara virus (HAZV). Budding and release of SIV and EIAV were both inhibited in CCL2KO cells and rescued by CCL2 addition. Replication of HSV-1 and DENV was unaffected in CCL2KO cells, confirming that ALIX is not involved in their replication. Finally, HAZV replication was affected by CCL2 signaling. Our studies indicate that CCL2 signaling and ALIX mobilization are important for several viral families.<br><br>IMPORTANCE: C-C motif ligand 2 (CCL2) plays a regulatory role in the budding and release of HIV-1 in macrophages and HeLa cells. CCL2 signaling mobilizes ALG-2-interacting protein X (ALIX) from the F-actin cytoskeleton to the soluble cytosol, where it is accessible for recruitment by the HIV-1 Gag polyprotein in the assembling virions at the plasma membrane. In previous studies, CCL2 immunodepletion, which blocks CCL2 signaling, resulted in ALIX sequestration to the F-actin cytoskeleton and inhibited virus production. Here, we developed a HeLa CCL2 gene knockout cell line and found that abrogation of CCL2 signaling can be restored by CCL2 addition, as evidenced by the restoration of ALIX to the cytosolic fraction and rescue of HIV-1 release. Employing such a system, we tested Simian Immunodeficiency Virus, Equine Infectious Anemia Virus, Herpes Simplex Virus type 1, Dengue, and Hazara virus for their dependence on ALIX for virus replication. The results indicate that CCL2 signaling and ALIX release from F-actin may play a role in the replication of several viruses.},
}
@article {pmid40980903,
year = {2025},
author = {Khurram, I and Choudhery, MS and Ghani, MU and Arif, T and Naeem, A and Mahmood, R and Niaz, A and Khan, MU},
title = {Gene Editing for Cystic Fibrosis: Advances and Prospects of CRISPR-Cas9 Therapy.},
journal = {Cell biology international},
volume = {49},
number = {12},
pages = {1564-1578},
doi = {10.1002/cbin.70082},
pmid = {40980903},
issn = {1095-8355},
mesh = {*Cystic Fibrosis/genetics/therapy/diagnosis ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Genetic Therapy/methods ; Mutation/genetics ; Animals ; },
abstract = {Cystic fibrosis (CF) is an inherited, autosomal recessive disorder that is caused by mutations in the gene encoding cystic fibrosis transmembrane conductance regulator (CFTR). CFTR maintains the balance between water and salts by transporting chloride ions along various epithelial surfaces. CFTR impairment affects the function of several organs, including the lungs. Newborn screening, prenatal diagnosis, and pharmacological interventions have altered the prevalence and incidence of cystic fibrosis. Although CFTR modulators are a promising treatment option, their ability to target and correct only one mutation at a time restricts their therapeutic potential. The development of genome editing technologies such as Clustered Regularly Interspaced Short Palindromic Repeats-Cas(CRISPR-Cas9) has the potential to correct genetic mutations, including those associated with CF, thereby offering a permanent treatment by fixing the root cause of CF. This article summarizes cystic fibrosis development, prognosis, and diagnosis, as well as possibilities for correcting various types of CFTR gene mutations. The review focuses on the potential of gene editing technologies to repair CFTR mutations and their applications in the advancement of CF treatment.},
}
@article {pmid40975652,
year = {2025},
author = {Sajjad, MW and Naqvi, RZ and Amin, I},
title = {Viral genome editing: striking a balance between promises and precautions.},
journal = {Trends in biotechnology},
volume = {43},
number = {11},
pages = {2686-2687},
doi = {10.1016/j.tibtech.2025.09.010},
pmid = {40975652},
issn = {1879-3096},
mesh = {*Genome, Viral/genetics ; *Genetic Vectors/genetics ; *Genetic Engineering/methods ; Humans ; *Viruses/genetics ; *Gene Editing ; CRISPR-Cas Systems ; Agriculture ; },
abstract = {Viral vectors can provide transgene-free genome editing. The forward-looking frameworks for their use must be biosafety and freedom from ecological risk. The responsible innovations, showing a balance between promise and caution following regulatory frameworks, can guarantee that viral genome editing develops into a revolutionary but environmentally conscious strategy for sustainable agricultural improvement.},
}
@article {pmid40962831,
year = {2025},
author = {Su, X and Zhang, H and Hong, Y and Yang, Q and Wang, L and Le, T and Liu, J and Cheruvu, L and Labour, E and Zhang, S and Mendez-Maldonado, K and Kreimer, A and Song, H and Ming, GL and Duan, J and Pang, ZP},
title = {Mutations of schizophrenia risk gene SETD1A dysregulate synaptic function in human neurons.},
journal = {Molecular psychiatry},
volume = {30},
number = {12},
pages = {5680-5693},
pmid = {40962831},
issn = {1476-5578},
support = {ASF_23-004/ASF/Autism Science Foundation/United States ; R01MH125528//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R01 MH106575/MH/NIMH NIH HHS/United States ; 74260//Robert Wood Johnson Foundation (RWJF)/ ; R01 AG081374/AG/NIA NIH HHS/United States ; R01 AG063175/AG/NIA NIH HHS/United States ; RM1 MH133065/MH/NIMH NIH HHS/United States ; ASF_23-004/ASF/Autism Science Foundation/United States ; },
mesh = {Humans ; *Schizophrenia/genetics/metabolism ; *Histone-Lysine N-Methyltransferase/genetics/metabolism ; Neurons/metabolism/physiology ; Induced Pluripotent Stem Cells/metabolism ; Mutation/genetics ; Synapses/genetics/metabolism ; Genetic Predisposition to Disease ; Haploinsufficiency/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Schizophrenia (SCZ) is a complex neuropsychiatric disorder associated with both common risk variants of small effect sizes and rare risk variants of high penetrance. Rare premature open reading frame (ORF) termination variants in SETD1A (SET Domain Containing 1A) show a strong association with SCZ; however, it remains largely unclear how rare premature ORF termination variants in SETD1A contribute to the pathophysiology of SCZ. To understand the impact of SETD1A rare premature ORF termination variants in human neurons, we CRISPR/Cas9-engineered five isogenic pairs of human induced pluripotent stem cells (iPSCs), with a recurrent heterozygous patient-specific premature ORF termination mutation c.4582-2delAG in two donor lines and a heterozygous frameshift mutation c.4596_4597insG (p. Leu1533fs) in three donor lines. These two mutations are predicted to cause a premature stop codon in exon 16 of SETD1A, leading to SETD1A haploinsufficiency. We found that these presumably loss-of-function (LoF) mutations caused the SETD1A mRNAs to be degraded by nonsense-mediated decay (NMD), accompanied by a reduction of full-length SETD1A protein level in iPSCs. We then characterized the morphological, electrophysiological, and transcriptomic impacts of SETD1A[+/-] LoF mutations in iPSC-derived human excitatory neurons induced by NGN2. We found that the SETD1A[+/-] exon-16 LoF mutations altered dendrite complexity, dysregulated synaptic transmission, and synaptic plasticity, likely by dysregulating genes involved in synaptic function. These results provide mechanistic insights into how SETD1A[+/-] exon-16 patient-specific LoF mutations affect neuron phenotypes that may be relevant to the pathophysiology of SCZ.},
}
@article {pmid40817029,
year = {2025},
author = {Vidal, P and Giménez-Dejoz, J and Fernandez-Lopez, L and Romero, S and Nazemi, SA and Luengo, M and Gonzalez-Alfonso, JL and Martinez-Sugrañes, M and Robles-Martín, A and Almendral, D and Roda, S and Pérez-García, P and Kruse, L and Jaeger, KE and Streit, WR and Plou, FJ and Floor, M and Shahgaldian, P and Bargiela, R and Guallar, V and Ferrer, M},
title = {Computationally guided genome rewiring of Escherichia coli and its application for nanopolyethylene terephthalate (PET) biodegradation and upcycling.},
journal = {Trends in biotechnology},
volume = {43},
number = {11},
pages = {2874-2903},
doi = {10.1016/j.tibtech.2025.07.008},
pmid = {40817029},
issn = {1879-3096},
mesh = {*Polyethylene Terephthalates/metabolism/chemistry ; *Escherichia coli/genetics/metabolism ; Biodegradation, Environmental ; *Metabolic Engineering/methods ; *Genome, Bacterial ; *Nanoparticles/chemistry/metabolism ; CRISPR-Cas Systems ; Escherichia coli Proteins/genetics/metabolism ; Gene Editing/methods ; },
abstract = {Numerous strategies for the biodegradation and upcycling of polyethylene terephthalate (PET) are under investigation. Here, we present a proof-of-concept study for reprogramming the Escherichia coli BL21(DE3) strain to degrade PET nanoparticles (nPET) without introducing foreign DNA and compromising native cellular fitness. In brief, native proteins selected in silico from the genome were repurposed to acquire artificial PETase activity without compromising their function and were subsequently replaced via CRISPR/Cas9 editing. A variant of the transport protein LsrB, selected for its ability to bind PET, was engineered to degrade PET powder (at 37-60°C). Building on LsrB periplasmic localization, we engineered a strain that degrades nPET at 37°C. The strain was further engineered to grow on nPET degradation products and produce valuable compounds. Our method, which is applicable across diverse genomes and microbial chassis, expands the potential of metabolic engineering to address plastic biodegradation and upcycling while reducing reliance on foreign DNA.},
}
@article {pmid40473489,
year = {2025},
author = {Sen, MK and Sellamuthu, G and Mondal, SK and Varshney, RK and Roy, A},
title = {Epigenome editing for herbicide-resistant crops.},
journal = {Trends in plant science},
volume = {30},
number = {11},
pages = {1201-1210},
doi = {10.1016/j.tplants.2025.05.003},
pmid = {40473489},
issn = {1878-4372},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics/drug effects ; *Herbicide Resistance/genetics ; CRISPR-Cas Systems/genetics ; *Epigenome/genetics ; *Herbicides/pharmacology ; Plants, Genetically Modified/genetics ; Epigenome Editing ; },
abstract = {Herbicide resistance (HR) is fundamental for sustainable agriculture as global food security increasingly relies on efficient and eco-friendly weed management. Recent advances in CRISPR/dCas9-based epigenome editing offer a promising, non-genetic approach by precisely targeting regulatory regions of genes involved in herbicide sensitivity and detoxification. While CRISPR/Cas9 has successfully been used to develop HR crops, CRISPR/dCas9 remains underexplored in this field. We propose that CRISPR/dCas9-driven epigenome editing could enable time- and tissue-specific control of gene expression, allowing for the introduction of heritable HR traits without altering DNA sequences. This innovative approach could transform sustainable HR development, offering a powerful solution to enhance agricultural resilience and food security while aligning with eco-friendly weed management strategies.},
}
@article {pmid41215733,
year = {2025},
author = {Claisse, O and Mosterd, C and Marrec, CL and Samot, J},
title = {Defense Systems and Prophage Detection in Streptococcus mutans Strains.},
journal = {Molecular oral microbiology},
volume = {},
number = {},
pages = {e70014},
doi = {10.1111/omi.70014},
pmid = {41215733},
issn = {2041-1014},
support = {//Association France Parkinson/ ; },
abstract = {Although the species is extensively studied, limited data are available on antiphage defense systems (APDSs) in Streptococcus mutans. The present study aimed to explore the diversity and the occurrence of APDSs and to search for prophages in the genomes of clinical isolates of S. mutans using bioinformatics tools. Forty-four clinical isolates of S. mutans were obtained from saliva samples of people with Parkinson's disease. Genomic DNA was extracted, sequenced using Illumina MiSeq technology, and analyzed for the presence of defense systems using DefenseFinder and PADLOC. CRISPR-Cas systems were characterized using CRISPRCasFinder, and prophages were detected by the PhiSpy pipeline from RAST. AcrFinder and AcrHub were used to identify anti-CRISPR proteins. Each strain harbored between 6 and 12 APDS, with restriction-modification systems being the most prevalent, followed by the MazEF toxin-antitoxin system and CRISPR-Cas systems. Type II-C CRISPR-Cas systems were not identified here in S. mutans. Novel variations in type II-A signature protein Cas9 were identified, allowing their classification into four distinct groups. Variability in direct repeat sequences within the same CRISPR array was also observed, and 80% of the spacers were classified as targeting "dark matter". A unique prophage, phi_37bPJ2, was detected, showing high similarity with previously described phages. The AcrIIA5 protein encoded by phi_37bPJ2 was conserved and suggested to remain functionally active. This study reveals the diversity of APDSs in S. mutans and the limited presence of prophages. The findings provide a foundation for future research on the evolutionary dynamics of these systems and their role in S. mutans adaptation to phage pressure.},
}
@article {pmid41215480,
year = {2025},
author = {Ali, RM and Arshad, H and Zafar, AQ and Gull, S and Gul, A and Mansoor, F and Zahid, R},
title = {A Comprehensive Review on Engineering Lactic Acid Bacteria: Emerging Genetic Tools and Synthetic Biology Strategies.},
journal = {Biotechnology and applied biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1002/bab.70079},
pmid = {41215480},
issn = {1470-8744},
abstract = {Lactic acid bacteria (LAB) are pivotal in food, pharmaceutical, and environmental applications due to their metabolic versatility and probiotic potential. This review explores the advancements in genetic engineering and synthetic biology strategies to enhance LAB functionality. We examine the genomic architecture of key LAB species, such as Lactobacillus and Lactococcus, highlighting their natural genetic traits and metabolic constraints. Emerging genetic tools, including electroporation, conjugation, and CRISPR-Cas systems, have revolutionized LAB modification, enabling precise gene editing and expression control. Synthetic biology approaches, such as genetic circuits, riboswitches, and biosensor development, offer novel pathways for optimizing LAB for functional foods, mucosal therapeutics, and industrial biotechnology. We discuss applications in probiotic delivery, bioremediation, and agricultural enhancement, emphasizing LAB's role in producing bioactive metabolites and combating pathogens. Challenges, including plasmid instability, metabolic burden, and regulatory hurdles, are addressed alongside socio-ethical considerations for genetically modified LAB. The integration of genome-scale engineering and CRISPR-based technologies holds promise for overcoming these barriers, paving the way for next-generation LAB with enhanced stress tolerance and tailored functionalities. This review synthesizes current knowledge and future prospects, underscoring the transformative potential of engineered LAB in addressing global health, environmental, and industrial needs while navigating biosafety and public perception challenges.},
}
@article {pmid41213945,
year = {2025},
author = {Hoetzel, J and Wang, T and Suess, B},
title = {Beyond the niche - unlocking the full potential of synthetic riboswitches.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9897},
pmid = {41213945},
issn = {2041-1723},
support = {SU402/12-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; WA 5722/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
abstract = {Synthetic riboswitches have undergone great development in the past decade, evolving into valuable regulatory tools. Operating entirely at the RNA level and independently of auxiliary proteins, they offer a promising alternative to protein-based systems such as TetON/OFF or CRISPR-Cas. As compact, modular RNA elements they unite sensing and regulatory functions within a single molecule, giving them the advantages of high modularity, portability and low metabolic burden. Here, we explore the unique features of synthetic riboswitches, highlight key applications, assess current bottlenecks and limitations and put them in context with emerging solutions, to emphasise the potential of synthetic riboswitches.},
}
@article {pmid41212235,
year = {2025},
author = {Yilmaz-Çolak, &#xc7;},
title = {The CRISPR-cas repertoire of Kluyvera ascorbata: insights from genomic data.},
journal = {Current genetics},
volume = {71},
number = {1},
pages = {28},
pmid = {41212235},
issn = {1432-0983},
abstract = {The genus Kluyvera has gained increasing attention due to their emerging role as opportunistic pathogens and their antibiotic resistance determinants. Various approaches have been employed to reveal genomic insights into the evolution and pathogenicity of Kluyvera species. However, detailed knowledge about Kluyvera-specific clustered regularly interspaced short palindromic repeats (CRISPR) is still missing. In this study, a genome-mining approach was employed for the characterization of CRISPR-Cas loci in a total of 13 complete Kluyvera genomes using CRISPRCasFinder and related tools. Out of 13 species, only K. ascorbata displayed multiple CRISPR-Cas arrays and a complete set of cas genes characteristics of a type I-E system. Spacer analysis revealed potential targets within phage and plasmid sequences, indicating historical exposure to mobile genetic elements. Furthermore, a phylogenetic tree constructed using the Cas3 protein sequence positioned K. ascorbata closely with other enteric bacteria, including Salmonella spp. and Citrobacter spp. This study provides the first detailed insight into the CRISPR-Cas architecture of K. ascorbata. Although there is no significant diversity of the CRISPR-Cas system identified in this species, it can emphasize a role as active immune defenses against invaders and offer a foundation for future functional and evolutionary investigations. Moreover, difficulties in identification of the genus Kluyvera can be overcome through the CRISPR-Cas system using next-generation diagnostics tools.},
}
@article {pmid41211267,
year = {2025},
author = {Ramesh, B and Fakoya, AO},
title = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas Unleashed: Transforming Gene Editing With Breakthroughs, Applications, and Ethical Dilemmas.},
journal = {Cureus},
volume = {17},
number = {11},
pages = {e95908},
pmid = {41211267},
issn = {2168-8184},
abstract = {The most significant breakthrough in gene editing is the advent of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. This innovative technology enables scientists to insert or delete genes using specific enzymes, facilitating modifications to genomes that can influence an organism's phenotype. The Cas9 enzyme is the most widely used within the CRISPR framework and has already received approval for treating sickle cell disease, with many other applications likely to follow. As this rapidly evolving field continues to advance, it holds great promise for addressing genetic disorders and diseases. This article will explore the various enzymes available in the CRISPR system, the range of diseases and conditions that could be treated using this technology, alternative gene therapy methods, and the ethical considerations surrounding its use.},
}
@article {pmid41211185,
year = {2025},
author = {Allmang, S and Witzel, HR and Hausen, A and Marquard, S and Eckert, C and Marnet, N and Hörner, N and Mayer, P and Heinrich, S and Dang, H and Roth, W and Gaida, MM},
title = {Aberrant Expression of A Disintegrin and Metalloproteinase With Thrombospondin Motifs 13 (ADAMTS13) in Pancreatic Cancer Leads to Dichotomic Functions.},
journal = {MedComm},
volume = {6},
number = {11},
pages = {e70462},
pmid = {41211185},
issn = {2688-2663},
abstract = {Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers characterized by highly invasive growth into the surrounding peripancreatic fat tissue, where tumor cells can directly interact with adipocytes. Due to poor response to the currently available (radio)chemotherapies, there is an urgent need for advanced therapy concepts. The present study shows that ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13), a key factor in blood coagulation, is significantly overexpressed in human PDAC. Immunohistochemical analysis revealed that ADAMTS13 expression is associated with prolonged survival and negatively correlated with vascular density. In vitro and in vivo experiments demonstrate its partial induction by leptin. Mechanistically, CRISPR/Cas-mediated ADAMTS13 knockout in PDAC cells resulted in reduced migration and invasion. In an avian xenograft tumor model, ADAMTS13 loss led to increased vascularization, decreased vascular length, and diminished tumor growth, accompanied by reduced expression of multiple key angiogenic and angioplastic factors. Furthermore, loss of ADAMTS13 was associated with decreased expression of mesenchymal markers. In conclusion, we identified an aberrant expression and alternative function of ADAMTS13 in PDAC linked to tumor progression, plasticity, and angiogenesis, partly induced by the peripancreatic fat tissue, making this metalloproteinase an interesting target for personalized therapies.},
}
@article {pmid41210586,
year = {2025},
author = {Joo, JH and Lee, S and Kim, KP},
title = {Precision gene editing: The power of CRISPR-Cas in modern genetics.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102733},
pmid = {41210586},
issn = {2162-2531},
abstract = {Gene editing has transformed molecular biology by enabling precise modifications to genomic DNA across a wide variety of organisms. Gene editing technologies make it possible to add, remove, or modify specific DNA sequences, with a range of applications including gene knockouts, therapeutic gene correction, and the design of targeted genetic traits. These techniques depend on two main DNA repair mechanisms: homology-directed repair (HDR), which facilitates precise changes to the genome, and non-homologous end joining (NHEJ), which often results in mutations such as deletions or frameshift errors. Among the diverse gene-editing platforms, the CRISPR-Cas system has emerged as the most extensively employed, owing to its simplicity, low cost, and efficiency. This review presents the evolution of gene-editing technologies, with a particular emphasis on the CRISPR-Cas system and its expanding applications in genetics, biotechnology, agriculture, and medicine. Furthermore, advanced gene editing approaches are discussed, offering an overview of emerging trends.},
}
@article {pmid41209346,
year = {2025},
author = {Wei, Y and Sun, J and Zhu, R},
title = {CRISPR-epigenetic crosstalk: From bidirectional regulation to therapeutic potential.},
journal = {Computational and structural biotechnology journal},
volume = {27},
number = {},
pages = {4496-4504},
pmid = {41209346},
issn = {2001-0370},
abstract = {Recent advances in epigenetics have elucidated the pivotal roles of epigenetic modifications in genomic regulation and disease pathogenesis. Concurrently, CRISPR-based technologies have transcended conventional gene-editing applications and have emerged as powerful tools for target gene screening, chromatin imaging, and epigenetic modulation. Notably, epigenetic landscapes substantially influence the CRISPR editing efficiency, whereas CRISPR itself can reshape epigenetic states, forming a dynamic CRISPR-Epigenetics Regulatory Circuit. This review systematically examines the bidirectional interplay between CRISPR systems and epigenetic modifications, emphasizing their collective impact on genome-editing precision, disease progression, and therapeutic development. Existing studies have predominantly focused on the application of CRISPR in epigenetic modifications or the impact of epigenetic landscapes on CRISPR, exhibiting unidirectional characteristics. However, accumulating evidence suggests a bidirectional interaction between the two. Here, a transformative "CRISPR-Epigenetics Regulatory Circuit" model is synthesized and presented, supported by three pivotal breakthroughs: demonstrating CRISPR as an active epigenetic programmer, synthesizing the epigenetic preconditioning therapeutic paradigm, and elucidating the first predictive mathematical model (EPIGuide). Further exploration of this circuit is expected to enhance CRISPR performance, optimize sgRNA design via epigenetic predictive models, and pioneer sequential epigenetic or gene editing therapies.},
}
@article {pmid41206864,
year = {2025},
author = {Docherty, JAD and Cook, R and Kiu, R and Dyball, X and Brown, TL and Kujawska, M and Smith, RL and Phillips, S and Watt, R and Telatin, A and Tiwari, SK and Hall, LJ and Adriaenssens, EM},
title = {Diverse defense systems and prophages in human-associated Bifidobacterium species reveal coevolutionary "arms race" dynamics.},
journal = {Cell reports},
volume = {44},
number = {11},
pages = {116542},
doi = {10.1016/j.celrep.2025.116542},
pmid = {41206864},
issn = {2211-1247},
abstract = {Bacteria of the genus Bifidobacterium are pivotal for human health, especially in early life, where they dominate the gut microbiome in healthy infants. Bacteriophages, as drivers of gut bacterial composition, can affect bifidobacterial abundance. Here, we use a bioinformatics approach to explore direct interactions between human-associated Bifidobacterium spp. and prophages, as evidenced by their genomes. Analysis of 1,086 bifidobacterial genomes reveals the presence of complex systems that prevent viral invasion, with 34 defense systems and 56 subtypes detected, including several different CRISPR-Cas systems. CRISPR spacers target almost three-quarters of bifidobacteria-derived prophages, indicating dynamic interactions. At least one prophage is present in ∼67% of strains, with phages exhibiting high genomic diversity and evidence of historical recombination. These prophages encode various defense and anti-defense systems, such as anti-CRISPR genes and restriction-modification mechanisms. Overall, this investigation reveals that coevolutionary "arms race" dynamics drive genomic diversity in both bifidobacteria and their phages.},
}
@article {pmid41206491,
year = {2025},
author = {Landesman, WJ and Hudson, TR and Bedore, SE and Suarez, MC and Hayden, MS},
title = {A CRISPR/LbCas12a system for Borrelia burgdorferi sensu stricto detection in blacklegged ticks.},
journal = {Journal of medical entomology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jme/tjaf163},
pmid = {41206491},
issn = {1938-2928},
support = {P20GM103449//Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health/ ; U01CK000661/ACL/ACL HHS/United States ; /CC/CDC HHS/United States ; },
abstract = {CRISPR/Cas systems have the potential to revolutionize DNA detection of vector-borne pathogens with highly specific and user-friendly assays. One such system, named DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR), uses a guide RNA (gRNA) and Cas enzyme to bind to and cut DNA targets. Following cutting, Cas12a exhibits non-specific collateral cleavage of single-stranded DNA (ssDNA). A ssDNA reporter in the reaction allows the trans-cleavage activity to be harnessed as an amplified output signal upon recognition of the target by the Cas12a/gRNA complex. We developed a DETECTR assay to detect Borrelia burgdorferi sensu stricto, the primary Lyme disease pathogen in the United States, in blacklegged ticks (Ixodes scapularis) collected from forests in southern Vermont. We compared DETECTR to gel electrophoresis of PCR-amplified products and used quantitative real-time PCR (qPCR) of a different B. burgdorferi primer set for independent confirmation. We found that 123/125 of the samples had identical results for DETECTR and gel electrophoresis. Both assays identified the same 33 B. burgdorferi-positive samples and the same 90 B. burgdorferi-negative samples. On a subset of eight samples, we tested DETECTR using lateral flow test strips and obtained identical results to those obtained with the fluorescence-based DETECTR. The sensitivity of DETECTR was lower than qPCR, which detected nine additional B. burgdorferi-positive samples. When qPCR is not available, the DETECTR assay offers a robust alternative to gel electrophoresis that is more user-friendly and requires less time. Due to the highly specific nature of the assay, DETECTR provides additional confidence that a B. burgdorferi target is present.},
}
@article {pmid41206049,
year = {2025},
author = {Wu, JN and Li, C and Liu, Z and Li, X and Wang, J and Lou, X and Xia, F and Dai, J and Wu, T},
title = {Photocontrolled dissociation and toehold-mediated strand displacement-based synergistic regulation of CRISPR-Cas12a.},
journal = {Nucleic acids research},
volume = {53},
number = {20},
pages = {},
pmid = {41206049},
issn = {1362-4962},
support = {22&#x2009;474&#x2009;045//National Natural Science Foundation of China/ ; 82&#x2009;172&#x2009;372//National Natural Science Foundation of China/ ; HUST: 2024JYCXJ010//Central Universities/ ; S202410487577//Undergraduates of Hubei Province/ ; },
abstract = {The trans-cleavage activity of the CRISPR-Cas system holds broad potential across diverse fields, yet precise spatiotemporal regulation remains challenging due to the predominantly single-direction control strategies available. Here, we present a bidirectional, multi-round modulation strategy for CRISPR-Cas12a trans-cleavage activity, utilizing toehold-mediated strand displacement and photocontrolled dissociation. This approach enables dynamic transitions between on and off states: Cas12a activity is activated by an activator, inhibited by a photosensitive blocker, and reactivated via UV light. We further integrated this system with DNA cryptography, establishing a hierarchical temporal authorization system that enhanced cryptographic security. The method supported multi-round modulation, achieving restoration of 95.4% activity after multiple cycles in the on state while maintaining suppression to 12.4% in the off state. This precise control strategy provides a versatile tool for spatiotemporal regulation in CRISPR-based applications, with significant implications for advanced gene editing, diagnostics, and bioengineering.},
}
@article {pmid41206045,
year = {2025},
author = {Nakamura, J and Shiraishi, M and Yamamoto, J and Suzuki, K},
title = {Development of programmable RNA imaging with RNA-guided GFP via click chemistry.},
journal = {Nucleic acids research},
volume = {53},
number = {20},
pages = {},
pmid = {41206045},
issn = {1362-4962},
support = {25K21779//The Uehara Memorial Foundation and the Japan Society/ ; JPMJSP2138//JST/ ; //Japan Society for the Promotion of Science/ ; },
abstract = {The CRISPR-Cas system revolutionized molecular biology by guiding Cas proteins to target nucleic acid sequences using customizable guide RNAs, offering unparalleled precision and versatility. Inspired by this innovation, we developed RNA-guided green fluorescent protein (RGG), a simple and programmable platform for targeting nucleic acid. Using a streamlined click chemistry approach, known for its high efficiency and specificity, we conjugated dibenzocyclooctyne (DBCO)-modified guide nucleic acids, designed to complement target sequences, with azide-exposed proteins to construct RGG. Systematic optimization identified 30-nt RNA with 3'-DBCO modifications as the most effective configuration for RGG, enabling precise visualization of nuclear-localized RNAs, including NEAT1 and Satellite III RNA, in living cells. This establishes RGG as a customizable and efficient system for RNA imaging and molecular analysis, underscoring the potential of direct conjugation between guide nucleic acids and proteins to enable precise nucleic acid recognition and dynamic molecular modification in living cells.},
}
@article {pmid41205654,
year = {2025},
author = {Yu, L and Niu, M and Dong, Z and Dong, X and Han, Y and An, J and Jiang, T and Chen, Y and Feng, Y and Sun, Y and Li, H},
title = {High-efficiency inhibition of human adenovirus type 55 replication by CRISPR-Cas12a.},
journal = {The Journal of infection},
volume = {},
number = {},
pages = {106650},
doi = {10.1016/j.jinf.2025.106650},
pmid = {41205654},
issn = {1532-2742},
abstract = {OBJECTIVES: Human adenovirus 55 (HAdV-55), a highly pathogenic double-stranded DNA virus, presents a serious global public health challenge due to its rapid transmission and complex pathogenesis. Current antiviral treatment options for HAdV-55 are limited, with no specific antiviral drugs available. The CRISPR-Cas system, capable of precisely targeting viral genomes, has emerged as a novel approach for antiviral therapy. This study aimed to leverage targeted DNA cleavage activity of the CRISPR-Cas12a system to develop a therapeutic strategy for effectively inhibiting HAdV-55 replication.<br><br>METHODS: We developed a rapid and efficient screening platform for identifying antiviral targets by integrating CRISPR-Cas12a fluorescence detection technology with bioinformatics analysis. Using this platform, we systematically screened 194 candidate targets against HAdV-55.<br><br>RESULTS: The E1B-crRNA6-Cas12a system was identified, demonstrating a highly potent antiviral activity with 99.17% inhibitory efficiency and a selectivity index (SI) of 2482.80. This target significantly outperformed the clinical broad-spectrum anti-adenovirus drug cidofovir in both inhibitory efficacy and duration.<br><br>CONCLUSIONS: This study not only holds promise for providing safe and highly effective antiviral candidate targets for HAdV-55 therapy but also, through the construction of an interdisciplinary technical platform, is expected to enhance the translational potential of CRISPR antiviral technology for preclinical applications.},
}
@article {pmid41203870,
year = {2025},
author = {Chen, X and Xiao, L and Wang, Q and Zhou, L and Xu, Y and Zhou, C and Dai, M and Wang, F and Xu, H and Luo, D and Sirois, P and Li, K and Liao, D and Zhang, J},
title = {Coloring target and off-target effects of genetically modified nucleases by blue &amp; white colony assays.},
journal = {Biotechnology letters},
volume = {47},
number = {6},
pages = {128},
pmid = {41203870},
issn = {1573-6776},
support = {SKY2021047//the Suzhou Science and Technology Planning Project/ ; ND2022B04//Program of Clinical Research Center of Neurological Disease/ ; NS2021016//Funda&#xe7;&#xe3;o de Apoio ao Desenvolvimento do Ensino, Ci&#xea;ncia e Tecnologia do Estado de Mato Grosso do Sul/ ; },
mesh = {*Gene Editing/methods ; Escherichia coli/genetics ; *CRISPR-Cas Systems ; *Endonucleases/genetics/metabolism ; Humans ; beta-Galactosidase/genetics/metabolism ; },
abstract = {More sensitive evaluation of the off-target effects of gene editing nucleases is crucial for human gene therapy. Here we report chromogenic assays designed for sensitive evaluation of gene editing activities using CRISPR/Cas9 test system. Based on beta-galactosidase alpha complementation, qualitative and quantitative evaluations of the target and off-target effects of CRISPR/Cas9 were well established through the color alteration of the E.coli colonies. In addition to target effect analysis, these new assays provide extremely sensitive and efficient tool to profile the off-target effects with one or more bases mismatched between the targets and the gRNAs. Moreover, these assays allow the identification of gene editing effects for off-targets with one base mismatched PAM sites.},
}
@article {pmid41203686,
year = {2025},
author = {Sun, Y and Qu, K and Corsi, GI and Anthon, C and Pan, X and Xiang, X and Jensen, LJ and Lin, L and Luo, Y and Gorodkin, J},
title = {Deep learning models simultaneously trained on multiple datasets improve base-editing activity prediction.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9821},
pmid = {41203686},
issn = {2041-1723},
support = {NNF21OC0068988//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; },
mesh = {*Deep Learning ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Neural Networks, Computer ; Humans ; Software ; },
abstract = {CRISPR-derived base editors (BE) enable precise single nucleotide substitution without introducing double-stranded DNA breaks. Apart from the base editing enzymes, efficient base editing strongly depends on both the CRISPR guide RNA (gRNA) efficiency and the edited position. Here, we show that the accuracy of BE gRNA design can be significantly improved by generating more data and by introducing deep neural networks trained on multiple different datasets simultaneously. Generating ~20,000 gRNAs for A•T to G•C and C•G to T•A conversions, we present such deep learning models, which also allow users to do dataset-aware predictions. The methods are available online and as stand-alone software.},
}
@article {pmid41203361,
year = {2025},
author = {Xia, N and Gupta, N},
title = {Function and therapeutic potential of transketolase enzymes in parasitic protists.},
journal = {Methods in enzymology},
volume = {722},
number = {},
pages = {97-115},
doi = {10.1016/bs.mie.2025.07.010},
pmid = {41203361},
issn = {1557-7988},
mesh = {*Toxoplasma/enzymology/genetics/pathogenicity ; *Transketolase/genetics/metabolism ; Animals ; Mice ; *Protozoan Proteins/genetics/metabolism ; *Toxoplasmosis/parasitology/therapy ; CRISPR-Cas Systems ; Recombinant Proteins/genetics/metabolism ; Gene Editing ; Humans ; },
abstract = {Transketolase (TKT), the rate-limiting enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP), catalyzes the reversible transfer of glycolaldehyde moieties between sugar phosphates. Here, we present wide-ranging methods to study TKT function in a clinically-relevant and globally-prevalent model intracellular pathogen, Toxoplasma gondii. The experimental framework utilizes heterologous expression of recombinant TgTKT proteins in a bacterial system, anti-TgTKT antibody production, CRISPR/Cas9-assisted genome editing to generate TgTKT-null mutants, and in-depth phenotyping, including [13]C-based metabolomic analysis to determine the carbon flux in the acutely-infectious tachyzoite stage of T. gondii. The phenotypic studies comprise the parasite proliferation in vitro and virulence assessment in a murine model of acute toxoplasmosis. The data suggest TgTKT as a metabolic rheostat regulating nucleotide biosynthesis and enabling adaptation of this highly promiscuous parasite in different nutritional environments. Last but not least, the work presents the therapeutic potential of the TgTKT-null mutant as a metabolically-attenuated vaccine, highlights the evolutionary divergence of TgTKT proteins, and provides a robust foundation to investigate TgTKT orthologs in other pathogenic protozoan organisms.},
}
@article {pmid41203292,
year = {2025},
author = {Yu, D and Duan, C},
title = {[Epigenetics and precise crop breeding for resistance].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {10},
pages = {3918-3938},
doi = {10.13345/j.cjb.250513},
pmid = {41203292},
issn = {1872-2075},
mesh = {*Epigenesis, Genetic/genetics ; *Crops, Agricultural/genetics ; *Plant Breeding/methods ; DNA Methylation/genetics ; Gene Editing ; *Disease Resistance/genetics ; CRISPR-Cas Systems ; },
abstract = {Epigenetics refers to a heritable phenomenon that dynamically modulates gene expression without altering the DNA sequence, through molecular mechanisms such as DNA methylation, histone modification, non-coding RNA, chromatin remodeling, and RNA modifications. In plants, these modifications are extensively involved in key biological processes, including flowering time, gametogenesis, stress responses, and immune defenses. Over the past few decades, the research on epigenetics has gradually shifted from fundamental studies primarily conducted in Arabidopsis thaliana to investigations in various crop species such as rice and tomato. This transition has revealed the multifaceted roles of epigenetic regulation in shaping agronomic traits. This review integrates current knowledge of epigenetic regulatory mechanisms and their functions in plant responses to both biotic and abiotic stresses. Epigenetic editing tools such as CRISPR-dCas9 enable targeted DNA methylation or histone acetylation. Emerging transformation technologies, including magnetic nanoparticles and virus-based delivery systems, have the potential to overcome the bottlenecks of plant regeneration, offering new possibilities for precise epigenetic editing. In future agriculture, it is essential to further elucidate multi-layered epigenetic regulatory mechanisms at the single-cell level, develop efficient delivery systems, and leverage artificial intelligence to advance the application of epigenetic breeding for sustainable agricultural development.},
}
@article {pmid41203276,
year = {2025},
author = {Ding, Y and Guo, W},
title = {[Advances in the application of genome editing technologies in plant pathogenic fungi].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {10},
pages = {3683-3700},
doi = {10.13345/j.cjb.250539},
pmid = {41203276},
issn = {1872-2075},
mesh = {*Gene Editing/methods ; *Fungi/genetics/pathogenicity ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology ; *Plants/microbiology ; *Genome, Fungal/genetics ; },
abstract = {Filamentous fungi represent an important group of eukaryotic microorganisms with diverse ecological functions and ubiquitous distribution in various ecosystems. Among them, many species are closely associated with agriculture, functioning as major plant pathogens that cause yield losses and produce mycotoxins to compromise both the quality and safety of agricultural products. In recent years, the CRISPR/Cas system has emerged as a powerful and programmable genome editing tool, and it has been extensively applied to the genetic study of plant pathogenic fungi. This technology has greatly facilitated the investigation of pathogenic mechanisms, mycotoxin biosynthetic pathways and key gene functions, antifungal resistance, and rapid pathogen detection. This review summarizes the development of CRISPR/Cas systems and the key strategies for their application in plant pathogenic fungi and makes an outlook on the practical deployment. With the continuous advancement of gene editing technologies, emerging fungal-adapted editing systems hold great promise for advancing functional genomics and enabling innovations in disease-resistant breeding and sustainable crop protection.},
}
@article {pmid40178656,
year = {2025},
author = {Gao, C and Cai, Y and Wu, X and Song, J and Zheng, Q and Wang, M and Luo, Y and Luo, Y and Fei, X and Zhang, Y and Yang, Y and Kuai, L and Ru, Y and Hong, S and Tian, N and Li, B and Zhang, Z},
title = {CRISPR/Cas9-Mediated Knockout and Overexpression Studies Unveil the Role of PD-L1 in Immune Modulation in a Psoriasis-like Mouse Model.},
journal = {Inflammation},
volume = {48},
number = {5},
pages = {3519-3528},
pmid = {40178656},
issn = {1573-2576},
support = {20224Y0373, 20234Y0269, 20234Y0075//Shanghai Municipal Health Commission Health Industry Clinical Research Special Project/ ; No. 82174383, 82204954, 82304819, 82305232, 82374458, W2433194//Shanghai Municipal Health Commission Health Industry Clinical Research Special Project/ ; 22YF1441300//Shanghai Science and Technology Development Funds (Sailing Program)/ ; lczh2021-05, lcfy2022-04, lczh2023-01//Clinical Transformation Incubation Program in Hospital/ ; zyyzdxk-2023065//Evidence-based dermatology base sponsored by State Administration of Traditional Chinese Medicine, High-level Chinese Medicine Key Discipline Construction Project (Integrative Chinese and Western Medicine Clinic) of National Administration of TCM/ ; 2023ZZ02017//Shanghai Dermatology Research Center/ ; SHDC2023CRW009//Shanghai Dermatology Hospital Demonstration Research Ward Project/ ; shzyyzdxk-2024104//Shanghai Key Discipline Construction Project of Traditional Chinese Medicine/ ; 2024-QNRC2-B04//Youth Medical Talents-Specialist Program of Shanghai "Rising Stars of Medical Talents" Youth Development Program, and Youth Talent Promotion Project of China Association of Traditional Chinese Medicine (2024-2026) Category B/ ; },
mesh = {Animals ; *B7-H1 Antigen/genetics/immunology ; *Psoriasis/immunology/chemically induced/genetics/pathology/metabolism ; Mice ; *CRISPR-Cas Systems ; Disease Models, Animal ; Mice, Knockout ; Imiquimod ; },
abstract = {The role of programmed death-ligand 1 (PD-L1), an essential immune checkpoint protein, has garnered considerable interest in recent years due to its influence on immune responses, particularly inhibiting immature Th cells into Th17 cells. This study aims to examine the effect of PD-L1 on psoriasis progress, which is the condition characterized by an immune response dominated by Th17 cells. We constructed the PD-L1 knockout (PD-L1[KO]) and overexpression (PD-L1[OE]) mice through CRISPR/Cas9 technology to assess the impact of PD-L1 in an imiquimod (IMQ)-induced psoriasis-like mouse model. In comparison to IMQ, the ear thickness exhibited a reduction, the PASI score decreased, and HE sections revealed a thinning of the epidermal spines in PD-L1[OE] mice. PD-L1[KO] mice, however, showed opposite results. Moreover, immunohistochemical assessments of the skin lesion tissues demonstrated heightened epidermal proliferation and inflammatory infiltration in the PD-L1[KO] group, accompanied by elevated tissue expression of proliferating cell nuclear antigen (PCNA), Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p50, and F4/80 in comparison to IMQ-treated and WT mice. The absence of PD-L1 in IMQ-induced mice was found to intensify the immune response, as evidenced by heightened expression of phosphorylated signal transducers and activators of transcription 3 (pSTAT3) and CD3 in the affected tissues compared to both IMQ-treated and WT mice. According to our findings, PD-L1 plays important roles in inhibiting inflammation, proliferation, and regulating immune responses. Targeting PD-L1 may present a promising therapeutic strategy for the management of psoriasis.},
}
@article {pmid41202215,
year = {2025},
author = {Wang, T and Brogan, DJ and Zawalick, NM and Chen, F and Mkrtchian, Y and Komor, AC and Akbari, OS},
title = {CTRL Enables Gene-Specific RNA Regulation Using CRISPR-Cas7-11.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00658},
pmid = {41202215},
issn = {2161-5063},
abstract = {Advancements in synthetic biology have enabled the development of precision gene expression technologies for comprehensive investigations of biological and biochemical networks. Here, we describe the development of a refined and innovative tool, CRISPR-Cas Transgenic Repressible eLement (CTRL), which utilizes the direct repeat processing activity of the recently discovered CRISPR-Cas7-11 effector to site-specifically target synthetic mRNA molecules. We demonstrate that CTRL exhibits high efficiency, tunable regulation of expression, and gene-specific repression of mRNA and protein expression. We engineered multiple permutations of the Cas7-11 effector that differ in their ability to reduce gene expression, suggesting flexibility for the application of choice. CTRL is a novel variation on gene repression technology that exhibits broad applicability across multiple model systems.},
}
@article {pmid41202131,
year = {2025},
author = {Liu, Y and Ban, Y and Gao, D},
title = {Oligo-CALL: A next-generation barcoding platform for studying resistance to targeted therapy.},
journal = {Science advances},
volume = {11},
number = {45},
pages = {eadw9990},
doi = {10.1126/sciadv.adw9990},
pmid = {41202131},
issn = {2375-2548},
mesh = {Humans ; *Drug Resistance, Neoplasm/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Lung Neoplasms/genetics/drug therapy/pathology ; Single-Cell Analysis ; Molecular Targeted Therapy ; Proto-Oncogene Proteins p21(ras)/genetics/antagonists &amp; inhibitors ; },
abstract = {Understanding therapy resistance requires deconvolving heterogeneous cell populations and tracking clonal trajectories. While CRISPR-based cellular barcoding is powerful for lineage tracing, many platforms suffer from low efficiency and limited compatibility with single-cell transcriptomics. We developed Oligo-CALL (Oligonucleotide-inducible CRISPR transcriptional activator-Assisted Lineage Labeling), an advanced barcoding system enabling precise lineage tracing, live clone isolation, and seamless integration with single-cell RNA sequencing. Applied to lung cancer cells treated with a KRAS[G12C] inhibitor, Oligo-CALL identified clones consistently enriched posttreatment, supporting a model of predestined resistance. Oligo-CALL achieved >95% efficiency in linking lineage identity to transcriptomes, uncovering diverse clone-specific pathways with underlying resistance. Paired analysis of barcode-matched clones from naïve and resistant populations revealed transient and fixed resistance phenotypes. Notably, DNA repair pathways are recurrently altered in resistant clones, and inhibition of poly(adenosine 5'-diphosphate-ribose) polymerase synergizes with KRAS G12C inhibition to overcome resistance. Together, Oligo-CALL provides a versatile platform for dissecting lineage evolution and molecular dynamics of targeted therapy resistance.},
}
@article {pmid41202069,
year = {2025},
author = {Verkuyl, C and Belotserkovsky, A and Zerbes, T and Williams, D and Krishnan, MR and Zhu, S and Grunnesjӧ, S and Eid, S and Zhang, C and Zhao, W and Xu, L and Lin, E and O'Shea, T and Draper, B and Jungman, A and Most, P and Schmitt-Ulms, G},
title = {Toward an all-in-one recombinant adeno-associated virus vector for functionally ablating the prion gene using CRISPR-Cas technology.},
journal = {PloS one},
volume = {20},
number = {11},
pages = {e0336578},
doi = {10.1371/journal.pone.0336578},
pmid = {41202069},
issn = {1932-6203},
mesh = {*Dependovirus/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; *Genetic Vectors/genetics ; Mice ; Humans ; Gene Editing/methods ; Genetic Therapy/methods ; Prion Diseases/genetics/therapy ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Any strategy that can selectively and persistently lower the brain levels of the cellular prion protein (PrPC) is expected to extend survival in prion diseases. Recent advances in the virus-mediated delivery of gene therapies prompted us to explore if a recombinant adeno-associated virus (rAAV) vector delivering a CRISPR-Cas-based gene editor can be devised that induces a functional knockout of the prion gene. Whereas the eventual objective is to assess the therapeutic potency of an optimized vector in prion-infected mice, in this proof-of-concept study, we evaluated tools and methods that are suited to achieve this goal. The result of these efforts is a first-generation all-in-one rAAV vector that codes for a prion gene-specific guide RNA and a small Cas9 endonuclease, whose expression is controlled by a truncated neural cell adhesion molecule 1 (NCAM1) promoter that is active in PrPC expressing cells. We also constructed a second rAAV vector coding for a prion gene-specific 'traffic light reporter' (TLR). The TLR can be used to monitor prion gene-editing efficacy by coding for red and green fluorescent proteins separated by a segment of the prion gene that is targeted by the gene editor. For the purification of AAVs, we adopted a robust and scalable rAAV vector assembly pipeline and undertook proof-of-concept prion gene editing experiments in human cells and mice, which to date yielded prion gene editing rates of approximately 20% and 5%, respectively. Finally, we compared brain distributions of rAAV vectors following intrathalamic versus retro-orbital injection, and selected the 9P31 capsid for future studies based on a 7.5-fold higher heterologous gene expression level as compared to the PHP.eB capsid.},
}
@article {pmid41201953,
year = {2025},
author = {Fan, J and Huang, L and Chen, J and Jiang, Y and Du, X and Wang, L and Ding, K and Huang, J and Qiu, S and Liu, H and Song, H},
title = {Highly Sensitive Field Detection Technology for Anthrax Based on the CRISPR/Cas13a System.},
journal = {Microbial biotechnology},
volume = {18},
number = {11},
pages = {e70240},
doi = {10.1111/1751-7915.70240},
pmid = {41201953},
issn = {1751-7915},
support = {32141003//National Science Foundation of China/ ; 32300080//National Science Foundation of China/ ; },
mesh = {*Bacillus anthracis/genetics/isolation &amp; purification ; *Anthrax/diagnosis/microbiology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Humans ; Point-of-Care Testing ; },
abstract = {In this study, we established a highly sensitive on-site detection technology for Bacillus anthracis. Firstly, we integrated Multiple Enzyme Isothermal Rapid Amplification (MIRA) with the clustered regularly interspaced short palindromic repeats (CRISPR) /associated protein 13a (CRISPR/Cas13a) detection system to develop a highly sensitive CRISPR/Cas13a assay. After testing crRNA selection, MIRA primers, reaction temperature, and CRISPR detection conditions, the CRISPR/Cas13a detection system employing dual crRNAs achieved a detection limit of 1000 copies/mL for B. anthracis. Quantitative analysis was additionally attempted. Compared with other common respiratory pathogens, the assay demonstrated high specificity. In clinically simulated samples, all 20 positive specimens were correctly identified, and all 13 negatives were unambiguously classified as negative. Based on these findings, we established a CRISPR point-of-care testing technology. By developing a CRISPR point-of-care testing device together with a tested lyophilised reagent system, the device achieved a detection limit of 250 copies/mL and delivered results within 30 min. All positive samples were accurately identified, and every negative sample was classified as negative. Consequently, this study presents a highly sensitive and portable technology for on-site detection of B. anthracis. It holds significant value for on-site detection of emerging infectious diseases.},
}
@article {pmid41200999,
year = {2025},
author = {Moreira, R and Nascimento, H and Martins, TMDM and Barbieri, G and Pires, P and Carvalho, LN and Rosa, LR and Almeida, A and Pessuti, CL and Ferrer, H and Gomes, J&#xc1;P and Goulart, E and Raia, S and Belfort, R},
title = {Porcine Corneal Tissue and Xenozoonotic Risks: A Review of the Current Evidence.},
journal = {Xenotransplantation},
volume = {32},
number = {6},
pages = {e70068},
doi = {10.1111/xen.70068},
pmid = {41200999},
issn = {1399-3089},
support = {//by FAPESP/ ; },
mesh = {Animals ; *Transplantation, Heterologous/methods/adverse effects ; Swine ; Humans ; *Corneal Transplantation/methods/adverse effects ; *Cornea/microbiology/virology ; Heterografts/microbiology ; },
abstract = {Corneal opacities affect millions worldwide, with corneal transplantation as the primary treatment. However, donor shortages remain a challenge, leaving thousands waiting for transplants. Xenotransplantation using porcine corneas has emerged as a promising alternative due to anatomical and physiological similarities with human corneas. Advances in CRISPR/Cas technology enable genetic modifications to address immune rejection and zoonotic risks. A key concern is xenozoonosis, the transmission of infectious agents from pigs to humans. Pathogens such as porcine endogenous retroviruses (PERVs), porcine cytomegalovirus (PCMV), and bacteria pose potential risks. While PERVs can infect human cells in vitro, no transmission has been documented in vivo. Regulatory bodies, including the WHO and IXA, have established guidelines for monitoring and clinical trials. The first human corneal xenotransplantation trials in South Korea and China are underway. Enhanced biosecurity measures in tissue banks have reduced microbial contamination, improving safety. Corneal xenotransplantation presents lower risks compared to solid organ xenotransplants. With ongoing research, stringent regulations, and improved pathogen-free animal models, this technique could become a viable clinical option. Future human trials will provide crucial insights into its long-term safety and effectiveness.},
}
@article {pmid41199512,
year = {2025},
author = {Friess, L and van Sinderen, D and Lee, C},
title = {A CRISPRi Gene Regulation System for Bifidobacteria.},
journal = {Microbial biotechnology},
volume = {18},
number = {11},
pages = {e70260},
doi = {10.1111/1751-7915.70260},
pmid = {41199512},
issn = {1751-7915},
support = {883766//Horizon 2020 Framework Programme/ ; SFI/12/RC/2273-412 P1/SFI_/Science Foundation Ireland/Ireland ; SFI/12/RC/2273 P2/SFI_/Science Foundation Ireland/Ireland ; //APC Microbiome Institute/ ; },
mesh = {*CRISPR-Cas Systems ; Streptococcus thermophilus/genetics/enzymology ; *Gene Expression Regulation, Bacterial ; *Bifidobacterium/genetics ; Plasmids ; *Bifidobacterium breve/genetics ; },
abstract = {This work describes the development of a CRISPR interference (CRISPRi) system for targeted gene repression in bifidobacteria. We first validated the CRISPRi-based approach using Bifidobacterium breve strains engineered to express nuclease-dead orthologs of Cas9 and demonstrated that the CRISPR-Cas system from Streptococcus thermophilus is efficient at targeting both reporter and endogenous genes through the use of single guide RNAs corresponding to the gene of interest. We also developed a one-plasmid system for targeted gene repression in bifidobacteria and demonstrated its utility by targeting genes involved in nucleotide metabolism and carbohydrate metabolism in several species of bifidobacteria. Efficient gene repression was achieved across all tested bifidobacterial species without the requirement for extensive optimization of transformation parameters or sequence optimization to avoid restriction modification systems thus removing the key barriers to genetic manipulation in this genus. This CRISPRi system provides a novel approach to functional genomics in bifidobacteria which facilitates future mechanistic studies in these commercially important microbes.},
}
@article {pmid41199069,
year = {2025},
author = {Singh, S and Chaudhary, R and Tiwari, S},
title = {CRISPR/Cas9-mediated gene targeting at BBM2 locus demonstrates HDR-assisted precise knock-in in banana cv. Grand Naine.},
journal = {Plant cell reports},
volume = {44},
number = {12},
pages = {265},
pmid = {41199069},
issn = {1432-203X},
support = {BRIC-NABI Core Research Grant//BRIC-National Agri-Food and Biomanufacturing Institute (BRIC-NABI)/ ; BIRAC/Tech Transfer/08/I2/QUT-BBF//Biotechnology Industry Research Assistance Council/ ; },
mesh = {*Musa/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; Gene Editing/methods ; Green Fluorescent Proteins/genetics/metabolism ; Plants, Genetically Modified ; *Gene Targeting/methods ; *Plant Proteins/genetics/metabolism ; *Recombinational DNA Repair/genetics ; },
abstract = {The present study demonstrates the first CRISPR/Cas-mediated precise knock-in of the eGFP gene at the BABYBOOM2 (GN-BBM2) locus in banana cv. Grand Naine, facilitating the detection of editing events in early embryogenic developmental stages. Genome editing has accelerated crop improvement programs by introducing targeted and precise genetic modifications. Among different tools, CRISPR/Cas-based genome editing has been widely used for enabling mutations through double-stranded breaks (DSBs), repaired either by non-homologous end joining (NHEJ) for gene knockouts or homology-directed repair (HDR) to generate knock-in events. While gene knockouts are well established in banana, efficient knock-in remains a major challenge due to low HDR activity, sterility, and the vegetatively propagated nature of banana. In the present study, we report the first successful CRISPR/Cas-based gene knock-in editing in banana by targeting the BABYBOOM2 (BBM2) gene, which encodes a transcription factor involved in somatic embryogenesis. The enhanced green fluorescent protein (eGFP) gene was precisely inserted at the BBM2 locus in banana cv. Grand Naine to enable visual detection during embryogenesis. In vitro validation showed ~ 95% target cleavage efficiency of the selected gRNA. The PCR-based screening and shift-in amplicon size analyses confirmed three edited lines (#3, #11, and #14) harboring eGFP knock-in at the targeted locus. Sequencing of the amplicon from these lines further confirmed the precise knock-in events. Hence, this study establishes a foundation for precise knock-in-based genome modification in banana and opens new avenues for targeted trait improvement in this important clonally propagated crop.},
}
@article {pmid41198952,
year = {2025},
author = {Makarova, KS and Shmakov, SA and Wolf, YI and Mutz, P and Altae-Tran, H and Beisel, CL and Brouns, SJJ and Charpentier, E and Cheng, D and Doudna, J and Haft, DH and Horvath, P and Moineau, S and Mojica, FJM and Pausch, P and Pinilla-Redondo, R and Shah, SA and Siksnys, V and Terns, MP and Tordoff, J and Venclovas, &#x10c; and White, MF and Yakunin, AF and Zhang, F and Garrett, RA and Backofen, R and van der Oost, J and Barrangou, R and Koonin, EV},
title = {An updated evolutionary classification of CRISPR-Cas systems including rare variants.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {41198952},
issn = {2058-5276},
support = {Intramural funds//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
abstract = {The known diversity of CRISPR-Cas systems continues to expand. To encompass new discoveries, here we present an updated evolutionary classification of CRISPR-Cas systems. The updated CRISPR-Cas classification includes 2 classes, 7 types and 46 subtypes, compared with the 6 types and 33 subtypes in our previous survey 5 years ago. In addition, a classification of the cyclic oligoadenylate-dependent signalling pathway in type III systems is presented. We also discuss recently characterized alternative CRISPR-Cas functionalities, notably, type IV variants that cleave the target DNA and type V variants that inhibit the target replication without cleavage. Analysis of the abundance of CRISPR-Cas variants in genomes and metagenomes shows that the previously defined systems are relatively common, whereas the more recently characterized variants are comparatively rare. These low abundance variants comprise the long tail of the CRISPR-Cas distribution in prokaryotes and their viruses, and remain to be characterized experimentally.},
}
@article {pmid41198678,
year = {2025},
author = {Nukaga, S and Shiraishi, K and Hamabe, K and Mochizuki, A and Hamaguchi, Y and Ogawa, E and Le, NT and Shimada, Y and Ono, H and Nishinakamura, H and Kobayashi, Y and Hamamoto, J and Ui, A and Araki, M and Sagae, Y and Ohgino, K and Sugihara, K and Endo, S and Miyakoshi, J and Shiraishi, Y and Yasuda, H and Okuno, Y and Yoshida, T and Goto, Y and Ohe, Y and Watanabe, SI and Yatabe, Y and Nishikawa, H and Hamamoto, R and Kohno, T and Nakaoku, T},
title = {Mutation of CMTR2 in Lung Adenocarcinoma Alters RNA Alternative Splicing and Reveals Therapeutic Vulnerabilities.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9754},
pmid = {41198678},
issn = {2041-1723},
support = {JP23ak0101205//Japan Agency for Medical Research and Development (AMED)/ ; JP24ama221233//Japan Agency for Medical Research and Development (AMED)/ ; JP24ck0106905//Japan Agency for Medical Research and Development (AMED)/ ; 25K02539//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 22KJ3158//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 20H00545//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPMJCR1689//MEXT | Japan Science and Technology Agency (JST)/ ; JPMJCR18Y4//MEXT | Japan Science and Technology Agency (JST)/ ; },
mesh = {Humans ; *Adenocarcinoma of Lung/genetics/drug therapy ; *Lung Neoplasms/genetics/drug therapy/pathology ; *Alternative Splicing/genetics ; *Mutation ; Cell Line, Tumor ; *Methyltransferases/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; },
abstract = {RNA splicing dysregulation has emerged as a hallmark of cancer and a promising therapeutic target; however, its full landscape in human solid cancer remains poorly characterized. To address this, we perform alternative splicing analyses using RNA-sequencing data from 751 lung adenocarcinoma samples from our cohort integrated with 519 samples from The Cancer Genome Atlas. Visualization of splicing patterns using t-distributed stochastic neighbor embedding reveals substantial inter-tumor heterogeneity driven by distinct molecular subtypes and histological differentiation. We identify a unique molecular subtype associated with inactivating mutations in CMTR2, which encodes Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 2. CMTR2 mutations are observed in 3.8% of cases and are predominantly truncating mutations, which form an isolated cluster within the splicing landscape. Intrinsic and CRISPR-Cas9-engineered CMTR2 mutations disrupt alternative splicing and sensitize cancer cells to sulfonamide-based RNA splicing modulators and immune checkpoint blockade therapy. Retrospective patient data confirm the increased sensitivity of CMTR2-deficient tumors to immune checkpoint blockade therapy. These findings uncover a previously unrecognized RNA splicing deficiency in human cancers and define a molecular subtype of lung adenocarcinoma driven by RNA splicing dysregulation, suggesting targets for therapeutic intervention in lung cancer.},
}
@article {pmid41197724,
year = {2025},
author = {Ito, C and Yamamoto, M and Yokota, N and Nakamura, N and Shiomi, M and Kizu, M and Ichinose, M and Fujii, S and Fujii, T and Fukunaga, R},
title = {Dissecting stress-activated protein kinase (SAPK)-signaling pathways using multiplex gene knockout HeLa cells.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {110901},
doi = {10.1016/j.jbc.2025.110901},
pmid = {41197724},
issn = {1083-351X},
abstract = {The stress-activated protein kinase (SAPK) family consists of three JNK and four p38 members. To explore the isotype-specific or overlapping roles of SAPK members, HeLa-derived multiplex SAPK-knockout (KO) cells, such as JNK1/2/3-triple KO, p38α/β/γ/δ-quadruple KO, and JNK1/2/3/p38α/β/γ/δ-septuple KO cells were generated using the CRISPR/Cas9 method. Also, 'sole survivor' hextuple KO (ss-HKO) cells, in which only one of seven SAPK genes remains intact, were generated. Western blot analyses using phospho-specific antibodies for SAPK substrates showed that serum or anisomycin-induced phosphorylation of MAPKAPK2, MSK1, Mnk1, and CREB/ATF-1 largely depended on p38, while anisomycin-induced phosphorylation of c-Jun/JunD depended on JNK. Similar analyses using the ss-HKO cell lines revealed that JNK1 rather than JNK2 contributed to the c-Jun/JunD phosphorylation, while p38α was the primary species phosphorylating the examined p38 substrates. Expression analyses of three typical immediate-early genes, c-Jun, EGR1, and c-Fos, demonstrated that JNK1 and 2 are responsible for c-Jun expression induced by interleukin-1β, tumor necrosis factor-α, ultraviolet-C (UV), and heat shock (HS), whereas p38 is predominant in EGR1 expression induced by UV and HS, and in c-Fos expression induced by the cytokines, UV, and HS. On the other hand, neither JNK nor p38 contributed significantly to the cytokine-induced EGR1 expression, suggesting complicated SAPK-signaling mechanisms that regulate immediate-early gene expression. Together, these results demonstrate the utility of the comprehensive multigene KO and sole-survivor KO strategy in dissecting intracellular signaling pathways consisting of multiple family members.},
}
@article {pmid41193960,
year = {2025},
author = {Van Goethem, MW and Vikram, S and Cowan, DA and Makhalanyane, TP},
title = {Comparative genomics reveals adaptive traits in novel Antarctic lithic cyanobacteria.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {994},
pmid = {41193960},
issn = {1471-2164},
abstract = {BACKGROUND: Terrestrial polar cyanobacteria persist at the cold limits of life, enduring a suite of extreme stressors including sub-zero temperatures, frequent freeze–thaw cycles, oligotrophic soils, variable light regimes with long periods of darkness, and desiccation. To survive, cyanobacteria have evolved diverse physiological strategies. A key adaptation among Antarctic terrestrial cyanobacteria is niche colonization: inhabiting the undersides of translucent quartz rocks (hypoliths) and the interior spaces of porous rocks (endoliths), which buffer environmental extremes and sustains the potential for photoautotrophic carbon fixation. However, the full genomic repertoire facilitating their resilience is incomplete.<br><br>RESULTS: We cultivated cyanobacteria from endolithic and hypolithic niches in Victoria Valley, Eastern Antarctica, and recovered four near-complete genomes (100% completeness,&#x2009;<&#x2009;1% contamination). Three hypolithic genomes showed near-identical sequence similarity (whole genome average nucleotide identity&#x2009;=&#x2009;99.98%) and phylogenomic proximity to the genus Coleofasciculus, yet represent a novel species, Coleofasciculus caryii H7-2. The fourth genome, derived from an endolith, showed moderate similarity to Aliterella antarctica (whole genome average nucleotide identity&#x2009;=&#x2009;79.1%), and is proposed as a new species, Aliterella bergstromii E5.1. C. caryii H7-2 possessed a larger genome (~&#x2009;6.1 Mbp) than A. bergstromii E5.1 (~&#x2009;5.4 Mbp). Both genomes encoded complete pathways for carbon fixation via oxygenic photosynthesis (RuBisCO and phosphoribulokinase), extensive phycobilisomes, and multiple photoprotective mechanisms. Predicted optimal growth temperatures were 21.7&#xa0;&#xb0;C and 23.2&#xa0;&#xb0;C, respectively. Shared stress-mitigation genes included those for osmotic, thermal and oxidative (superoxide dismutase) stress response. All genomes contained biosynthetic gene clusters associated with stress-adaptive secondary metabolites, including heterocyst glycolipids, siderophores, phenazines, compounds related to nostopeptolide and merocyclophane. The C. caryii H7-2 genome encoded multiple CRISPR-Cas systems, suggesting adaptive immunity and historical phage exposure. In contrast, A. bergstromii E5.1 harboured a single prophage and an array of 24 plasmids.<br><br>CONCLUSIONS: These finding reveal that the newly-described cyanobacteria possess a rich genomic repertoire of adaptations to withstand Antarctic extremes, emphasizing the resilience and ecological importance of lithobiontic cyanobacteria in polar deserts.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-12203-7.},
}
@article {pmid41191214,
year = {2025},
author = {Choi, S and Lee, JM and Kim, KK},
title = {Biomolecular condensates: molecular structure, biological functions, diseases, and therapeutic targets.},
journal = {Molecular biomedicine},
volume = {6},
number = {1},
pages = {99},
pmid = {41191214},
issn = {2662-8651},
support = {RS-2025-00513647//National Research Foundation of Korea/ ; 20240678//Bisa Research Grant of Keimyung University/ ; },
mesh = {Humans ; *Biomolecular Condensates/chemistry/metabolism ; Animals ; Neurodegenerative Diseases/metabolism ; },
abstract = {Cells constantly encounter environmental and physiological fluctuations that challenge homeostasis and threaten viability. In response to these cues, specific proteins and nucleic acids engage in multivalent interactions and undergo phase separation to form membraneless assemblies known as biomolecular condensates. Nuclear condensates include paraspeckles, nuclear speckles, and Cajal bodies, while cytoplasmic condensates include stress granules, processing bodies, RNA transport granules, U-bodies, and Balbiani bodies. These assemblies regulate transcription, splicing fidelity, RNA stability, translational reprogramming, and integration of signaling pathways, thereby serving as dynamic platforms for metabolic regulation and physiological adaptation. However, dysregulation of these condensates has been increasingly recognized as a central pathogenic mechanism in neurodegenerative diseases, cancers, and viral infections, contributing to toxic protein aggregation, nucleic acid dysregulation, and aberrant cell survival signaling. This review provides a comprehensive synthesis of the molecular mechanisms governing condensation, delineates the diverse types and functions of major biomolecular condensates, and examines therapeutic approaches based on their pathophysiological relevance to disease development and progression. Furthermore, we highlight the cutting-edge technologies, including CRISPR/Cas-based imaging, optogenetic manipulation, and AI-driven phase separation prediction tools, which enable the real-time monitoring and precision targeting of cytoplasmic biomolecular condensates. These insights underscore the emerging potential of biomolecular condensates as both biomarkers and therapeutic targets, paving the way for precision medicine approaches in condensate-associated diseases.},
}
@article {pmid41058551,
year = {2025},
author = {Dear, HS and Franks, NE and Markley, EE and Holtz, AM and Song, JY and Johnson, CN and Medina-Cabrera, PI and Hernandez, D and Joel, P and Pasca di Magliano, M and Wellik, DM and Allen, BL},
title = {GliFHV mice: a tool to investigate GLI processing and localization.},
journal = {Development (Cambridge, England)},
volume = {152},
number = {21},
pages = {},
doi = {10.1242/dev.204251},
pmid = {41058551},
issn = {1477-9129},
support = {R01 CA275182/CA/NCI NIH HHS/United States ; R01 CA275182/CA/NCI NIH HHS/United States ; //Rogel Cancer Center, University of Michigan/ ; },
mesh = {Animals ; Mice ; *Zinc Finger Protein GLI1/metabolism/genetics ; Cilia/metabolism ; Fibroblasts/metabolism ; Hedgehog Proteins/metabolism ; Zinc Finger Protein Gli3/metabolism/genetics ; Gene Editing ; Signal Transduction ; Alleles ; CRISPR-Cas Systems ; Female ; Mice, Transgenic ; Nerve Tissue Proteins ; },
abstract = {GLI proteins (GLI1-3) are the transcriptional effectors of mammalian hedgehog (HH) signaling. However, studies of GLI function have been hampered by the lack of robust GLI antibodies. To address this, we utilized CRISPR-based gene editing to generate endogenous epitope-tagged Gli alleles for each Gli gene (Gli1FLAG, Gli2HA, Gli3V5). Through breeding, we established a novel mouse model, Gli1FLAG/FLAG;Gli2HA/HA;Gli3V5/V5, referred to as GliFHV. Importantly, GliFHV animals are viable and fertile with no overt phenotypes. Sanger and long-range DNA sequencing confirmed proper editing of each Gli allele, while qPCR and western blot analysis confirmed similar gene expression and protein levels, respectively, between wild-type and GliFHV animals. We utilized these mice to assess GLI localization in the developing limb, finding that all three GLIs localize to primary cilia with distinct distributions. Finally, we generated immortalized GliFHV mouse embryonic fibroblasts, demonstrating that these cells are HH responsive and that GLIs localize to primary cilia and nuclei in a HH-dependent fashion. These animals and cell lines provide a valuable resource for analyses of GLI processing, localization and function throughout embryogenesis, postnatal development, and in adults.},
}
@article {pmid40944362,
year = {2025},
author = {Hillebrand, GH and Carlin, SC and Giacobe, EJ and Stephenson, HA and Collins, J and Hooven, TA},
title = {A Cas12a Toolbox for Rapid and Flexible Group B Streptococcus Genomic Editing and CRISPRi.},
journal = {Molecular microbiology},
volume = {124},
number = {5},
pages = {449-461},
pmid = {40944362},
issn = {1365-2958},
support = {P20GM125504//Centers of Biomedical Research Excellence/ ; P20 GM125504/GM/NIGMS NIH HHS/United States ; R01 AI182835/AI/NIAID NIH HHS/United States ; //UPMC Children's Hospital of Pittsburgh Research Advisory Council/ ; R01AI177991/NH/NIH HHS/United States ; R01 AI177991/AI/NIAID NIH HHS/United States ; R21AI178067/NH/NIH HHS/United States ; R01AI182835/NH/NIH HHS/United States ; R21 AI178067/AI/NIAID NIH HHS/United States ; R01AI177991/NH/NIH HHS/United States ; R01AI182835/NH/NIH HHS/United States ; R21AI178067/NH/NIH HHS/United States ; },
mesh = {*Streptococcus agalactiae/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Genome, Bacterial ; *Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Mutagenesis ; Enterococcus faecium/genetics ; },
abstract = {Streptococcus agalactiae (group B Streptococcus; GBS) is a leading cause of neonatal sepsis and meningitis. Despite advances in molecular microbiology, GBS genome engineering remains laborious due to inefficient mutagenesis protocols. Here, we report a versatile and rapid Cas12a-based toolkit for GBS genetic manipulation. We developed two shuttle plasmids-pGBSedit for genome editing and pGBScrispri for inducible CRISPR interference-derived from an Enterococcus faecium system and optimized for GBS. Using these tools, we achieved targeted gene insertions, markerless deletions, and efficient, template-free mutagenesis via alternative end-joining repair. Furthermore, a catalytically inactive dCas12a variant enabled inducible gene silencing, with strand-specific targeting effects. The system demonstrated broad applicability across multiple GBS strains and minimal off-target activity, as confirmed by whole-genome sequencing. In benchmarking, template-less Cas12a mutagenesis yielded sequence-confirmed mutants in ~7 days and homology-directed edits in ~7-14 days; aTC-resistant colonies arose at ~10[-4] of uninduced CFU, and 27%-65% of resistant clones carried the intended homology-directed edit depending on locus and homology arm length (e.g., ~27% markerless deletion; ~35% insertion; 65% with 1 kb arms). These workflows provide a rapid alternative to temperature-sensitive plasmid mutagenesis protocols that typically require ≥ 4 weeks. This Cas12a-based platform offers an efficient, flexible, and scalable approach to genetic studies in GBS, facilitating functional genomics and accelerating pathogenesis research.},
}
@article {pmid40675807,
year = {2025},
author = {Bykonya, AG and Guschin, DY and Barlev, NA},
title = {Modern approaches to engineering human reporter cell lines using CRISPR within Safe Harbor loci and endogenous genes.},
journal = {Critical reviews in biotechnology},
volume = {45},
number = {8},
pages = {1816-1832},
doi = {10.1080/07388551.2025.2515834},
pmid = {40675807},
issn = {1549-7801},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genes, Reporter/genetics ; Cell Line ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Reporter systems are gaining increasing popularity in modern molecular biology as they provide reliable and clear readouts for various types of assays, both in cellulo and in vivo. The generation of reporter cell lines is instrumental for screening activators and inhibitors of signaling pathways to develop new therapeutic approaches. Reporter cell lines are those with stably integrated reporter constructs containing signaling genes (often luciferase or fluorescent proteins), enabling the visualization and tracking of protein expression. Although seemingly harmless and straightforward, untargeted genomic integration of reporter genes may severely affect the expression of neighboring genes, causing unwanted and unpredictable effects. Unlike the untargeted approach, the CRISPR/Cas9 system provides a more precise method of reporter integration, especially when reporters are integrated into Safe Harbor loci. This ensures minimal influence on neighboring genomic regions. This review discusses recent advancements in creating reporter lines using the CRISPR/Cas9 system and experimental approaches for identifying suitable Safe Harbor loci.},
}
@article {pmid41191093,
year = {2025},
author = {Sun, Y and Han, H and Zhao, R and Liu, X and Liu, Z and Liang, J and Wang, B and Zhao, B and Pang, Y and Xiao, R},
title = {CRISPR-assisted MoS2@TQD nanosheets-based lateral flow assay for dual-mode and sensitive diagnosis of SARS-CoV-2.},
journal = {Mikrochimica acta},
volume = {192},
number = {12},
pages = {792},
pmid = {41191093},
issn = {1436-5073},
mesh = {Humans ; *SARS-CoV-2/isolation &amp; purification/genetics ; *COVID-19/diagnosis ; *Molybdenum/chemistry ; *Quantum Dots/chemistry ; Limit of Detection ; *Disulfides/chemistry ; Colorimetry/methods ; CRISPR-Cas Systems ; Nanostructures/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; COVID-19 Testing/methods ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) assembled lateral flow assay (LFA) have been developed for rapid virus diagnosis, however, the single-mode visual half-quantitative characteristics limit their applications in accurate quantitative diagnosis. Herein, a CRISPR assisted MoS2@TQD (MoS2 nanosheet assembled ternary quantum dot) based colorimetric and fluorescent dual-mode LFA was fabricated for the ultrasensitive and flexible detection of the SARS-CoV-2. Compared with single QDs, MoS2@TQD can provide stable and superior colorimetric/fluorescent signals in various bio-samples. RT-RAA/CRISPR-Cas13 was adopted to couple with MoS2@TQD so that the detection sensitivity can be further enhanced. Based on the dual-mode LFA, the colorimetric visual detection limit was 400 copies/mL, and for fluorescence quantification mode, as low as 250 copies/mL can be obtained. For clinic diagnosis, 35 SARS-CoV-2 positive throat swab samples and 18 negative samples were detected by this LFA, 100% accuracy and 100% consistency with quantitative polymerase chain reaction (qPCR) results can be obtained. It indicated that MoS2@TQD-based dual-mode LFA can be a rapid, sensitive, and accurate detection platform for SARS-CoV-2 and other infectious pathogens in the point-of-care diagnosis.},
}
@article {pmid41188812,
year = {2025},
author = {Hussain, T and Awasthi, S and Shahid, F and Yi, SS and Sahni, N and Aldaz, CM},
title = {Therapeutic potential of PRMT1 as a critical survival dependency target in multiple myeloma.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {1704},
pmid = {41188812},
issn = {1471-2407},
support = {R35GM133658/GM/NIGMS NIH HHS/United States ; R35GM137836/NH/NIH HHS/United States ; 7016-18//Leukemia and Lymphoma Society/ ; },
mesh = {*Protein-Arginine N-Methyltransferases/antagonists &amp; inhibitors/genetics/metabolism ; Humans ; *Multiple Myeloma/drug therapy/genetics/pathology/metabolism ; Cell Survival/drug effects ; Cell Line, Tumor ; *Repressor Proteins/antagonists &amp; inhibitors/genetics/metabolism ; Arginine/metabolism ; Gene Expression Regulation, Neoplastic/drug effects ; CRISPR-Cas Systems ; Cell Cycle/drug effects ; DNA Damage ; },
abstract = {Multiple myeloma (MM) is a neoplasm of antibody-producing plasma cells and is the second most prevalent hematological malignancy worldwide. Development of drug resistance and disease relapse significantly impede the success of MM treatment, highlighting the critical need to discover novel therapeutic targets. In a custom CRISPR/Cas9 screen targeting 197 DNA damage response-related genes, Protein Arginine N-Methyltransferase 1 (PRMT1) emerged as a top hit, revealing it as a potential therapeutic vulnerability and survival dependency in MM cells. PRMT1, a major Type I PRMT enzyme, catalyzes the asymmetric transfer of methyl groups to arginine residues, influencing gene transcription and protein function through post-translational modification. Dysregulation or overexpression of PRMT1 has been observed in various malignancies including MM and is linked to chemoresistance. Treatment with the Type I PRMT inhibitor GSK3368715 resulted in a dose-dependent reduction in cell survival across a panel of MM cell lines. This was accompanied by reduced levels of asymmetric dimethylation of arginine (ADMA) and increased arginine monomethylation (MMA) in MM cells. Cell cycle analysis revealed an accumulation of cells in the G0/G1 phase and a reduction in the S phase upon GSK3368715 treatment. Additionally, PRMT1 inhibition led to a significant downregulation of genes involved in cell proliferation, DNA replication, and DNA damage response (DDR), likely inducing genomic instability and impairing tumor growth. This was supported by Reverse Phase Protein Array (RPPA) analyses, which revealed a significant reduction in levels of proteins associated with cell cycle regulation and DDR pathways. Overall, our findings indicate that MM cells critically depend on PRMT1 for survival, highlighting the therapeutic potential of PRMT1 inhibition in treating MM.},
}
@article {pmid41188277,
year = {2025},
author = {Yeh, TY and Chiu, IM and Huang, YJ and Wu, SC and Hsieh, CC and Sung, HH and Hwu, WL and Wang, GJ and Ju, JC},
title = {Generation of exogenous kidneys via CRISPR/Cas9 mediated blastocyst complementation targeting Osr1 gene in mice.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {38648},
pmid = {41188277},
issn = {2045-2322},
support = {108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; *Blastocyst/metabolism/cytology ; *Kidney/metabolism/embryology ; Mice, Knockout ; Female ; Gene Targeting ; RNA, Guide, CRISPR-Cas Systems/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {Blastocyst complementation has been reported to produce exogenous mouse organs including the pancreas, lungs, and kidneys, but the complemented kidneys still failed to rescue the host animals. In the present study, we generated mouse complemented kidneys through a two-step procedure: using CRISPR/Cas9 ribonucleoproteins (RNP) to knockout Osr1 alleles, followed by injecting mouse embryonic stem (ES) cells that express enhanced green fluorescent protein (EGFP). When two different sgRNAs targeting the exon 2 of Osr1 were microinjected into the pronucleus of a mouse zygote, 34% of the embryos had deletions on both alleles, and these Osr1-knockouts died with no mesonephric duct development shown by histochemical staining. With three sgRNA injections, the knockout efficiencies increased, and mesonephric duct development with EGFP-positive cells was observed in ES cell-injected E12.5 embryos. Most of the ES cell-injected Osr1-knockout embryos degenerated from E13.5 to E15.5. Four of the 264 ES cell-injected embryos were born alive and survived to the second day, with strong EGFP signals observed in both the kidneys and the heart. Therefore, complementation of the Osr1-knockout blastocyst is a potential method to produce exogenous kidneys, although further modification is still needed to increase the efficiency.},
}
@article {pmid41187082,
year = {2025},
author = {Avaro, AS and Mirjalili, S and Griffiths, AD and Santiago, JG},
title = {Microfluidic networks using isotachophoresis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {45},
pages = {e2511724122},
doi = {10.1073/pnas.2511724122},
pmid = {41187082},
issn = {1091-6490},
support = {//Swedish e-Science Research Centre (SeRC)/ ; //Kungliga Tekniska H&#xf6;gskolan (KTH)/ ; },
abstract = {The development of microfluidic technologies has enabled chemical and biological analysis systems with increased functionality, complexity, and parallelization. These functionalities often drive the creation and control of complex and dynamic fluidic architectures. Introduced here is a class of microfluidic network based on isotachophoresis (ITP), an electrokinetic process that can extract and purify samples, selectively transport, mix, and aliquot (split) samples in a system with no moving parts. Presented is a theoretical framework to describe these networks. The framework relies on the coupling between a one-dimensional description of ITP and two-dimensional, transient graphs to describe the dynamic evolution of ITP networks. We leverage this framework to create numerical simulations of branched ITP circuits. We build, control, and experimentally study a variety of ITP networks. These systems automatically split and merge ITP zones, enabling complex sample manipulation with minimal external control. The model captures the experimentally observed sample dynamics. We demonstrate an example system where an ITP network is used to control and quantify parallel CRISPR-Cas enzymatic reactions. The methods described here are generally applicable to highly complex topologies and may offer a basis for easily reconfigurable, electric field-driven microfluidic systems. Networks generally offer broad potential for automated chemical and biochemical analysis and lab-on-a-chip integration.},
}
@article {pmid41171899,
year = {2025},
author = {Gil-Campillo, C and Mignolet, J and Domínguez-San Pedro, A and Rapún-Araiz, B and Janssen, AB and de Bakker, V and Veening, JW and Garmendia, J},
title = {CRISPRi-seq in Haemophilus influenzae reveals genome-wide and medium-specific growth determinants.},
journal = {PLoS pathogens},
volume = {21},
number = {10},
pages = {e1013650},
pmid = {41171899},
issn = {1553-7374},
mesh = {*Haemophilus influenzae/genetics/growth &amp; development ; *Genome, Bacterial ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation, Bacterial ; Genomics/methods ; },
abstract = {Work in the human pathobiont Haemophilus influenzae has pioneered functional genomics in bacteria such as genome-wide transposon mutagenesis combined with deep sequencing. These approaches unveiled a large set of likely essential genes, but functional studies are hampered due to a limited molecular toolbox. To bridge this gap, we engineered a titratable anhydrotetracycline-inducible CRISPRi (Clustered Regularly Interspaced Short Palindromic Repeats interference) platform for efficient regulation of gene expression in H. influenzae. Genome-wide fitness analyses in two different in vitro culture media by CRISPRi-seq revealed growth medium-dependent fitness cost for a panel of H. influenzae genes. We demonstrated that CRISPRi-programmed fitness defects can be rescuable, and we refined previous Tn-seq based essentialome studies. Finally, we introduce HaemoBrowse, an extensive user-friendly online resource for visual inspection of H. influenzae genome annotations, including sgRNA spacers. The inducible CRISPRi platform described here represents a valuable tool enabling functional genomics and the study of essential genes, thereby contributing to the identification of therapeutic targets for developing drugs and vaccines against H. influenzae.},
}
@article {pmid41109141,
year = {2026},
author = {Song, L and Bu, S and Ding, Y and Chai, Y and Fu, Y and Zhang, P and Yuan, R},
title = {CRISPR-enhanced low-potential ECL sensor based on AuPt-functionalized porphyrin AlOG for ultrasensitive aflatoxin B1 detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {292},
number = {},
pages = {118089},
doi = {10.1016/j.bios.2025.118089},
pmid = {41109141},
issn = {1873-4235},
mesh = {*Aflatoxin B1/analysis/isolation &amp; purification/chemistry ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; Luminescent Measurements/methods ; Electrochemical Techniques/methods ; *Porphyrins/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Aluminum/chemistry ; Food Contamination/analysis ; },
abstract = {Herein, an ultrasensitive low-potential electrochemiluminescence (ECL) sensor was developed by integrating an AuPt nanoparticle-functionalized porphyrin aluminum-organic gel (P-AlOG@AuPt) emitter with an enhanced CRISPR/Cas12a amplification module for the detection of aflatoxin B1 (AFB1) related to food safety. Impressively, the P-AlOG@AuPt emitter exhibited strong ECL emission and high signal-to-noise ratio at a low triggering potential (-0.8 V), addressing key limitations of previous high-potential aluminum-organic gels. Meanwhile, the in situ enriched AuPt as a coreaction accelerator catalyzed the reduction of coreactant S2O8[2-] to produce abundant SO4[•-] for facilitating its interactions with P-AlOG@AuPt[•-], resulting in a significant ECL enhancement. Furthermore, a synergistic catalytic hairpin assembly (CHA) pre-amplification and tetrahedral DNA nanostructure (TDN)-anchored ssDNA reporters significantly improved the cleavage efficiency of CRISPR/Cas12a, boosting the detection sensitivity of the ECL sensor for AFB1. The established ECL sensor exhibited ultrasensitive detection of AFB1 with a linear range of 0.1 pg mL[-1] - 500 ng mL[-1] and detection limit of 0.044 pg mL[-1], outperforming most reported ECL- and CRISPR-based AFB1 sensors. The practical usability of the proposed ECL sensor was verified by applying it for the quantification of AFB1 in rice samples. This work offers a promising low-potential ECL sensing platform for practical mycotoxin monitoring and broader biosensing applications.},
}
@article {pmid41052497,
year = {2026},
author = {Huang, X and Wang, A and Lin, Z and Xu, Y and Zheng, J},
title = {Novel electrochemiluminescence resonance energy transfer biosensor driven by CRISPR-Cas12a system for ctDNA detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {292},
number = {},
pages = {118067},
doi = {10.1016/j.bios.2025.118067},
pmid = {41052497},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Circulating Tumor DNA/genetics/blood/isolation &amp; purification ; *Lung Neoplasms/genetics/blood/diagnosis ; *Luminescent Measurements/methods ; Limit of Detection ; Electrochemical Techniques/methods ; Gold/chemistry ; *Carcinoma, Non-Small-Cell Lung/genetics/blood/diagnosis ; Energy Transfer ; Mutation ; },
abstract = {Efficient energy donor-acceptor pairing and distance control are critical in resonance energy transfer-based electrochemiluminescence (ECL-RET) biosensor. In this work, a novel ECL-RET system with Ru(phen)3[2+] as ECL energy donor and Au nanocages as energy acceptor was constructed based on the characteristic that Ru(phen)3[2+] can be stably embedded into double-stranded DNA. On this basis, a homogeneous ECL biosensor based on target activated CRISPR-Cas12a system and driving ECL-RET effect was developed. The ECL biosensor has been successfully applied to the detection of circulating tumor DNA (ctDNA) and demonstrated efficient recognition of L858R mutation in ctDNA of non-small cell lung cancer patients. The presented ECL-RET biosensor achieves a wide linear detection range from 10 fM to 1 nM and an excellent detection limit as low as 3.0 fM for ctDNA due to its combination of the highly efficient and specific cleavage of the CRISPR-Cas12a system.},
}
@article {pmid40883986,
year = {2025},
author = {Jenny Gao, and Bamidele, N and Pires-Ferreira, D and Zheng, C and Destefano, A and Cheng, H and Tang, Q and Cao, Y and Xie, J and Gao, G and Gruntman, A and Sontheimer, E and Flotte, TR and Xue, W},
title = {A compact base editor rescues AATD-associated liver and lung disease in mouse models.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {11},
pages = {5817-5828},
pmid = {40883986},
issn = {1525-0024},
support = {UH3 HL147367/HL/NHLBI NIH HHS/United States ; R01 CA275945/CA/NCI NIH HHS/United States ; P01 HL158506/HL/NHLBI NIH HHS/United States ; R01 GM150273/GM/NIGMS NIH HHS/United States ; F30 HL176024/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; Mice ; Disease Models, Animal ; *Gene Editing/methods ; *alpha 1-Antitrypsin/genetics ; *alpha 1-Antitrypsin Deficiency/genetics/therapy/complications ; *Liver Diseases/therapy/genetics/etiology ; *Lung Diseases/therapy/genetics/etiology ; Mice, Transgenic ; Humans ; *Genetic Therapy/methods ; Mutation ; CRISPR-Cas Systems ; Dependovirus/genetics ; Genetic Vectors/genetics ; },
abstract = {Alpha-1 antitrypsin deficiency (AATD) is commonly caused by a G-to-A mutation in the SERPINA1 gene (the PiZ mutation). The mutant PiZ AAT protein is sequestered in hepatocytes, causing lung emphysema due to insufficient AAT protein to inhibit neutrophil elastase in the lung. Here, we show that a compact adenine base editor (ABE) with an evolved Cas9 nickase derived from Neisseria meningitidis (eNme2.C) can be packaged in a single AAV and correct the PiZ mutation in mouse models of AATD. An all-in-one eNme2.C-TadA8e/guide 2 plasmid achieved approximately 20% on-target editing in PiZ reporter cells. TadA9e demonstrated sequence-specific reduction in bystander editing compared to TadA8e, without significantly affecting the on-target edit. In PiZ transgenic mice, eNme2.C-TadA9e AAV showed approximately 23% editing efficiency after 8 weeks and reduced liver disease burden in treated mice. In a new AAT-null;PiZ transgenic mouse model, ABE restored serum levels of AAT to beyond the 570 μg/mL therapeutic level. Moreover, ABE treatment was able to significantly correct lung functions in AAT-null;PiZ animals with emphysema. This study demonstrates the feasibility of an eNme2.C-based ABE in a single AAV to treat both AATD-associated liver and lung disease.},
}
@article {pmid40842155,
year = {2025},
author = {Ibel, A and Bhardwaj, R and Yilmaz, DE and Kong, S and Wendlinger, S and Cordero, C and Papaioannou, D and Papazian, M and Schönauer, R and Meng, Q and Eckardt, KU and Hassan, F and Volpe, I and Klämbt, V and Halbritter, J and Fedeles, S and Krappitz, M and Kaminski, MM},
title = {In vivo base editing reduces liver cysts in autosomal dominant polycystic kidney disease.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {11},
pages = {5373-5382},
doi = {10.1016/j.ymthe.2025.08.026},
pmid = {40842155},
issn = {1525-0024},
mesh = {*Polycystic Kidney, Autosomal Dominant/genetics/therapy/pathology ; Animals ; *Gene Editing/methods ; Mice ; Humans ; TRPP Cation Channels/genetics ; *Cysts/genetics/therapy/pathology/etiology ; *Liver Diseases/genetics/therapy/etiology/pathology ; Disease Models, Animal ; CRISPR-Cas Systems ; Genetic Therapy/methods ; Liver/pathology/metabolism ; Mutation, Missense ; },
abstract = {Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent genetic kidney disorder, affecting over 10 million individuals worldwide. Cystic expansion typically progresses to kidney failure and also involves the liver with limited treatment options. Pathogenic variants in PKD1 or PKD2 account for 85%-90% of cases. Genetic re-expression of Pkd1 or Pkd2 has been shown to partially reverse key characteristics of the disease phenotype in mice. Despite advancements in understanding the genetic basis, it remains unclear whether correcting pathogenic variants can effectively prevent, modify, or reverse the disease. Additionally, the feasibility of genome editing as a treatment remains largely unexplored. In this study, we employed CRISPR base editing to correct representative pathogenic PKD1 variants selected from a patient cohort, achieving precise and efficient editing in vitro. Correction of a murine missense variant (c.6646C>T (R2216W)) in primary renal epithelial cells increased polycystin-1 expression and reduced the endoplasmic reticulum stress marker sXBP1. In vivo, base editor delivery to the c.6646C>T (R2216W) knockin mouse enabled correction of the pathogenic variant, resulting in a significant reduction in liver cysts. These findings provide the first evidence that genome editing may ameliorate key features of ADPKD, opening promising therapeutic perspectives for affected patients and their families.},
}
@article {pmid40833860,
year = {2025},
author = {Yazdani, R and Nasr Esfahani, MH and Eghbalsaied, S and Karamali, F},
title = {Extracellular Vesicle-Liposome Hybrid Nanoparticles Delivery of CRISPR/Cas9 Induces a Unique DNA Repair Pattern in the HGF Gene of Stem Cells from Apical Papilla.},
journal = {DNA and cell biology},
volume = {44},
number = {11},
pages = {618-627},
doi = {10.1177/10445498251370554},
pmid = {40833860},
issn = {1557-7430},
mesh = {*CRISPR-Cas Systems/genetics ; *Liposomes/chemistry ; *Nanoparticles/chemistry ; *Extracellular Vesicles/metabolism/chemistry ; *Hepatocyte Growth Factor/genetics ; Humans ; *Stem Cells/metabolism ; Gene Editing/methods ; },
abstract = {Extracellular vesicles (EVs) have been investigated due to their natural biocompatibility and targeting capabilities. The specific approach of combining EVs with liposomes to create hybrid nanoparticles (ELNPs) for the delivery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) system for deletion of the HGF gene in stem cells, but their effectiveness in encapsulating large nucleic acids is limited due to their small size. This study aimed to knock out the HGF gene by the CRISPR/Cas9 system by ELNPs, and it was expected that the efficiency of the CRISPR/Cas9 system transfer would increase compared to the usual methods of using lipofectamine in stem cells from apical papilla (SCAPs). In this study, gRNA suitable for the HGF gene is designed first, and after insertion into the CRISPR/Cas9 vector, it enters Lipofectamine 2000. In the next step, ELNPs are prepared after collecting EVs and hybridizing them with liposomes containing CRISPR/Cas9 vector. Then, these integrated nanoparticles were presented to SCAPs, and the removal of HGF gene expression was evaluated at the level of RNA and protein. This study showed that the CRISPR/Cas9 system can be efficiently transferred to SCAP cells using ELNPs. Genomic DNA sequencing analyses of SCAP cells showed a unique pattern of mutation, highly likely mediated through EVs. Quantitative PCR and protein staining further showed a decrease in HGF gene expression in the knockout cells. Moreover, cell proliferation analysis showed a decrease in cell proliferation in KO-HGF adipose cells compared to the nonedited counterpart. In summary, this study highlights the supportive role of EVs in facilitating cell transfection and promoting a dominant DNA repair pattern, likely through an RNA-mediated mechanism, rather than the random insertions and deletions typically induced during CRISPR editing of the HGF gene in SCAPs.},
}
@article {pmid40808259,
year = {2025},
author = {Mamia, K and Kolbeinsdottir, S and Labun, K and Li, Z and Komisarczuk, A and Keskitalo, S and Reint, G and Loe Haugen, F and Olaug Lindestad, B and Skundberg Jensen, S and Gjerdingen, TJ and Tuhkala, A and Wieczorek Ervik, C and Kopcil, P and Fatkhutdinov, N and Bronken Martinsen, KH and Erichsen, HC and Szymanska, M and Tölö, E and Glumoff, V and Saarela, J and Melbye Michelsen, T and Schalin-Jäntti, C and Olweus, J and Leinonen, E and Varjosalo, M and Valen, E and Hautala, T and Enge, M and Martelius, T and Dahal-Koirala, S and Haapaniemi, E},
title = {Precision T cell correction platform for inborn errors of immunity.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {11},
pages = {5624-5643},
doi = {10.1016/j.ymthe.2025.08.018},
pmid = {40808259},
issn = {1525-0024},
mesh = {*Gene Editing/methods ; *T-Lymphocytes/metabolism/immunology ; Humans ; CRISPR-Cas Systems ; Animals ; Mice ; STAT1 Transcription Factor/genetics ; Genetic Therapy/methods ; Disease Models, Animal ; },
abstract = {CRISPR-Cas9 gene editing is a promising tool to correct pathogenic variants for autologous cell therapies targeting inborn errors of immunity (IEI). Current strategies, such as gene knockout or cDNA knockin, address many single-gene defects but can disrupt gene expression, highlighting the need for precise correction platforms. While transplanting corrected autologous hematopoietic stem cells is a curative approach, it is unsuitable for patients with advanced disease, inflammation, or acute infections. As correcting T cells is an alternative therapeutic strategy for lymphoid IEIs, we present an efficient T cell single-nucleotide variant (SNV) correction platform based on homology-directed repair (HDR). By using STAT1 gain-of-function, cartilage hair hypoplasia, deficiency of ADA2, and autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy as IEI models, we demonstrate that our platform achieves up to 80% correction, with resultant functional correction of the disease phenotype in the selected models. Furthermore, we performed safety profiling using GUIDE-seq, single-cell RNA sequencing, long-read genome sequencing, and proteomics analysis and detected no genomic, transcriptomic, or proteomic aberrations. This study establishes HDR-based SNV editing as a portable method for developing clinical autologous T cell therapies and represents a promising step toward a broad-spectrum gene correction platform for treating diverse monogenic immune disorders.},
}
@article {pmid40739756,
year = {2025},
author = {Skov, TW and Wolff, JH and Haslund, D and Revenfeld, AL and van de Venn, L and Dorset, SR and Ravendran, S and Jørgensen, SE and Thomsen, MK and Holm, M and Corn, JE and Møller, BK and Mogensen, TH and Bak, RO and Mikkelsen, JG},
title = {Treatment of GATA2 deficiency by allele-specific CRISPR-Cas9-directed gene correction in hematopoietic stem cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {11},
pages = {5644-5660},
doi = {10.1016/j.ymthe.2025.07.038},
pmid = {40739756},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; *Hematopoietic Stem Cells/metabolism ; Humans ; Alleles ; *Gene Editing/methods ; *GATA2 Transcription Factor/genetics/deficiency ; *GATA2 Deficiency/therapy/genetics ; Dependovirus/genetics ; *Genetic Therapy/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; Mice ; Genetic Vectors/genetics ; Recombinational DNA Repair ; },
abstract = {GATA2 deficiency is a rare inborn error of immunity caused by monoallelic variants in the GATA2 gene, leading to dysfunction of hematopoietic stem and progenitor cells (HSPCs). Here, we investigate a potential therapeutic strategy for GATA2 deficiency based on CRISPR-Cas9-based gene correction, utilizing recombinant adeno-associated virus serotype 6 (rAAV6) as a template for homology-directed repair (HDR). For a 7-base pair (bp) deletion giving rise to GATA2 deficiency, we identify a single guide RNA (sgRNA) supporting allele-specific cleavage in the disease allele. Initially, we observe high cytotoxicity in HSPCs upon Cas9/sgRNA ribonucleoprotein nucleofection and rAAV6 transduction, but this is mitigated by co-administering mRNA-based modulators of the DNA damage response combined with a 10-fold reduction in rAAV6 dose. Using this protocol, we achieve efficient HDR (>80%) in HSPCs derived from a patient carrying the 7-bp deletion and show increased engraftment potential after GATA2 correction. Using DISCOVER-seq, we find limited off-target activity. However, with PCR-free long-read sequencing, we detect frequent large aberrations at the on-target site in HSPCs, primarily attributed to the integration of AAV concatemers identified in 15% of the targeted alleles. Our findings describe the effect of gene correction on GATA2 deficiency and highlight potential on-target aberrations following HDR-mediated gene correction.},
}
@article {pmid40652526,
year = {2025},
author = {Oksenych, V and Petakh, P and Kainov, D and Kamyshnyi, O},
title = {A CRISPR-Cas9-based tool for dose-dependent DNA damage detection.},
journal = {The FEBS journal},
volume = {292},
number = {21},
pages = {5625-5628},
doi = {10.1111/febs.70186},
pmid = {40652526},
issn = {1742-4658},
mesh = {*CRISPR-Cas Systems/genetics ; *Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; *DNA Breaks, Double-Stranded ; *DNA Damage/genetics ; Retroelements/genetics ; Protein Serine-Threonine Kinases/genetics/metabolism ; Intracellular Signaling Peptides and Proteins ; },
abstract = {Auboiron et al. developed a CRISPR-Cas9-based system in yeast Saccharomyces cerevisiae that allows precise, dose-dependent induction of DNA double-strand breaks by targeting Ty retrotransposons. This system combines sequence specificity with control over break numbers (×1, ×15, or ×59), enabling a more detailed and precise study of DNA damage response. It revealed that the key DNA damage response kinase, Tel1, localizes to the nuclear periphery and forms multiple foci after induction of DNA double-strand breaks. The system also exposed limitations in Cas9 availability at higher break levels and offers a scalable platform for studying genome stability across organisms.},
}
@article {pmid40631397,
year = {2025},
author = {Auboiron, M and Coiffard, J and Kumanski, S and Santt, O and Pardo, B and Moriel-Carretero, M},
title = {A CRISPR-Cas9-based system for the dose-dependent study of DNA double-strand break sensing and repair.},
journal = {The FEBS journal},
volume = {292},
number = {21},
pages = {5629-5658},
doi = {10.1111/febs.70143},
pmid = {40631397},
issn = {1742-4658},
support = {Laureate 2018//ATIP-Avenir Program/ ; ANR-21-CE12-0004-01//Agence Nationale de la Recherche/ ; ARCPJA22020060002119//Fondation ARC pour la Recherche sur le Cancer/ ; PLBIO19-098 INCA_13832//Institut National Du Cancer/ ; },
mesh = {*DNA Breaks, Double-Stranded ; *Saccharomyces cerevisiae/genetics/metabolism ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *DNA Repair/genetics ; Protein Serine-Threonine Kinases/genetics/metabolism ; Intracellular Signaling Peptides and Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; DNA Transposable Elements/genetics ; },
abstract = {The integrity of DNA is put at risk by different lesions, among which double-strand breaks (DSBs) occur at a lower frequency but have the most life-threatening consequences. The study of DSB repair requires tools that can induce the accumulation of these breaks and includes the use of chemical genotoxins, ionizing radiation, or the expression of sequence-specific nucleases. While genotoxins and irradiation allow for dose-dependent studies, nuclease expression permits assessments at precise locations. In this work, we have leveraged the repetitive nature of the Ty transposon elements in the genome of Saccharomyces cerevisiae and the cutting activity of the RNA-guided Cas9 nuclease to create a tool that combines sequence specificity and dose-dependency. In particular, we can achieve the controlled induction of 0, 1, 15, or 59 DSBs in cells with an otherwise identical genetic background. We make the first application of this tool to better understand the behavior of the apical kinase of the DNA damage response Tel1 in the nuclear space. We found that Tel1 is capable of forming nuclear foci, which are clustered by condensin when DSBs occur in Ty elements. In striking contrast with other DSB-related protein foci, Tel1 foci are in tight contact with the nuclear periphery, therefore suggesting a role for the nuclear membrane in their congregation.},
}
@article {pmid40501348,
year = {2025},
author = {Rafiq, S and Assad, A},
title = {RNAS-sgRNA: Recurrent Neural Architecture Search for Detection of On-Target Effects in Single Guide RNA.},
journal = {Journal of computational biology : a journal of computational molecular cell biology},
volume = {32},
number = {11},
pages = {1041-1059},
doi = {10.1089/cmb.2025.0031},
pmid = {40501348},
issn = {1557-8666},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Neural Networks, Computer ; Computational Biology/methods ; Algorithms ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 is a leading genomic editing tool, but its effectiveness is limited by considerable heterogeneity in target efficiency among different single guide RNAs (sgRNA). This study presents RNAS-sgRNA, a hybrid model that integrates neural architecture search (NAS) with recurrent neural networks (RNN) to evaluate the on-target efficacy of CRISPR/Cas9 sgRNA. The RNAS-sgRNA model automates architectural discovery, improving sgRNA sequence categorization without considerable manual adjustment. The NAS component improves the RNN architecture, which analyzes sgRNA sequences represented as binary matrices and produces a classification score. Upon evaluation across several datasets, RNAS-sgRNA exhibits substantial performance enhancements with multiple cell lines, comparing its area under the receiver operating characteristic curve (AUROC) performance to the baseline CRISPRpred(SEQ) and DeepCRISPR models. RNAS-sgRNA demonstrated substantial improvements in AUROC performance in several cell lines compared with existing models. Notable improvements include enhancements of 8.62% for HCT116, 121.57% for HEK293T, 13.40% for HeLa, and 20.78% for HL60 cell lines, resulting in an overall improvement of 13.46%. Compared with DeepCRISPR, the model achieved additional AUROC gains in all cell lines tested, with an average improvement of 14.74%. The study also highlighted the ability of the model to deliver superior performance on smaller datasets through transfer learning, underscoring its potential applications in personalized medicine and genetic research. RNAS-sgRNA introduces a novel integration of NAS with RNN to evaluate the efficacy of CRISPR/Cas9 sgRNA. Unlike traditional methods that require significant manual adjustments, this model automates architectural discovery, optimizing the RNN structure for sgRNA sequence analysis. Furthermore, the application of transfer learning to fine-tune the pretrained model on small cell-line datasets represents a pioneering approach in the domain. The model's demonstrated ability to significantly outperform existing algorithms, including CRISPRpred(SEQ) and DeepCRISPR, across multiple cell lines highlights its innovative contribution to genomic editing research and personalized medicine.},
}
@article {pmid41184760,
year = {2025},
author = {Guo, F and Wang, D and Wu, H and Zhao, X and Xu, F and Cui, Y and Shao, Y and Zhang, Y and Bu, W and Wen, T},
title = {Genomic characteristics, antimicrobial resistance profiles and virulence factors of Gallibacterium anatis isolates from layer chickens in Northern China.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {706},
pmid = {41184760},
issn = {1471-2180},
support = {31802193//National Natural Science Foundation of China/ ; XMS202505//the R &amp; D Foundation of Institute of Animal Husbandry and Veterinary Medicine/ ; KJCX20230427//the Special Program on Science and Technology Innovation Capacity Building of BAAFS/ ; },
mesh = {Animals ; *Chickens/microbiology ; China ; *Virulence Factors/genetics ; *Poultry Diseases/microbiology ; Phylogeny ; *Pasteurellaceae/genetics/drug effects/pathogenicity/isolation &amp; purification/classification ; *Genome, Bacterial ; Anti-Bacterial Agents/pharmacology ; *Pasteurellaceae Infections/veterinary/microbiology ; Drug Resistance, Multiple, Bacterial/genetics ; Virulence ; Genomics ; *Drug Resistance, Bacterial ; },
abstract = {BACKGROUND: Gallibacterium anatis is an emerging pathogen causing substantial economic losses in global poultry production. Despite its growing clinical significance, the genomic basis of pathogenicity and antimicrobial resistance in this species remains poorly understood, particularly in China.<br><br>RESULTS: We sequenced and analyzed five clinical G. anatis isolates from Chinese layer chickens alongside 31 global strains, their genomes ranged from 2.25 to 2.81&#xa0;Mb with 39.8% average GC content. Phylogenetic analysis revealed that Chinese isolates cluster according to historical breeding stock importation patterns, reflecting international trade influences on pathogen distribution. Contemporary isolates showed extensive multidrug resistance compared to the antimicrobial-sensitive historical strain F149, with resistance profiles correlating directly with documented antibiotic usage in Chinese poultry production. Virulence analysis identified universal conservation of the RTX toxin system (97-100% prevalence) across all strains, contrasting sharply with variable distribution of other factors including fimbriae (30-35%) and secretion systems (50-60%). All strains harbored CRISPR-Cas systems, predominantly types I and III, indicating strain-specific phage defense adaptations.<br><br>CONCLUSIONS: The RTX toxin system represents a core virulence mechanism and potential universal vaccine target for G. anatis. The rapid evolution from antimicrobial-sensitive to extensively resistant phenotypes demonstrates how global poultry trade accelerates both pathogen spread and resistance development. These findings provide molecular insights for targeted interventions against an increasingly problematic poultry pathogen.},
}
@article {pmid41184307,
year = {2025},
author = {White, N and Chalk, JA and Hu, YT and Pins, SM and Joseph, CR and Antoniou, P and Wimberger, S and Svensson, S and Caetano-Silva, SP and Mudde, ACA and Rai, R and Selvaraj, S and Feist, WN and Romito, M and Sienski, G and Nitsch, R and Booth, C and Santilli, G and Cavazza, A and Porteus, MH and Maresca, M and Thrasher, AJ and Turchiano, G},
title = {Unveiling the cut-and-repair cycle of designer nucleases in human stem and T cells via CLEAR-time dPCR.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9571},
pmid = {41184307},
issn = {2041-1723},
support = {217112/Z/19/Z//Wellcome Trust (Wellcome)/ ; },
mesh = {Humans ; *DNA Repair/genetics ; *T-Lymphocytes/metabolism ; Gene Editing/methods ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; Induced Pluripotent Stem Cells/metabolism ; *Endonucleases/metabolism/genetics ; Mutation ; },
abstract = {DNA repair mechanisms in human primary cells, including error-free repair, and, recurrent nuclease cleavage events, remain largely uncharacterised. We elucidate gene-editing related repair processes using Cleavage and Lesion Evaluation via Absolute Real-time dPCR (CLEAR-time dPCR), an ensemble of multiplexed dPCR assays that quantifies genome integrity at targeted sites. Utilising CLEAR-time dPCR we track active DSBs, small indels, large deletions, and other aberrations in absolute terms in clinically relevant edited cells, including HSPCs, iPSCs, and T-cells. By quantifying up to 90% of loci with unresolved DSBs, CLEAR-time dPCR reveals biases inherent to conventional mutation screening assays. Furthermore, we accurately quantify DNA repair precision, revealing prevalent scarless repair after blunt and staggered end DSBs and recurrent nucleases cleavage. This work provides one of the most precise analyses of DNA repair and mutation dynamics, paving the way for mechanistic studies to advance gene therapy, designer editors, and small molecule discovery.},
}
@article {pmid41184277,
year = {2025},
author = {Nguyen, C and Omotayo, AI and Sanz Juste, S and Feng, X and López Del Amo, V},
title = {A temperature-sensitive CRISPR-Cas12a system for sterile insect technique.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9677},
pmid = {41184277},
issn = {2041-1723},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Male ; Female ; Temperature ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals, Genetically Modified ; *Infertility, Male/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Gene Editing/methods ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {The sterile insect technique (SIT) reduces population numbers by releasing sterile males that produce non-viable progeny. Specifically, CRISPR/Cas9-based precision-guided SIT (pgSIT) generates sterile males through genetic crosses of two transgenic lines: a Cas9 strain and a guide RNA (gRNA) strain targeting male sterility and female viability or infertility. However, pgSIT requires separate maintenance of the two lines and sorting to obtain sterile males, creating possible challenges for scaling. To overcome this, we propose using Cas12a nuclease, which is inoperative at lower temperatures but active at higher temperatures. Here, we develop a Cas12a-based pgSIT system involving a single strain containing both the Cas12a nuclease and gRNAs to induce male sterility and female lethality. This strain can be maintained as a mixed stock of both sexes and only activated by increasing temperature, producing sterile males after just one generation. By reducing the challenges that arise with maintaining two separate lines, this system could offer a scalable alternative for vector control in combating vector-borne diseases.},
}
@article {pmid41106454,
year = {2025},
author = {Xie, D and Wang, J and Huang, L and Zhao, Z and Wang, R and Long, H and Ni, Q and Liu, H and Hu, M and Huang, J},
title = {A novel X-linked retinitis pigmentosa beagle dog model for ophthalmic research established rapidly by editing the RPGR gene in zygotes.},
journal = {Experimental eye research},
volume = {261},
number = {},
pages = {110697},
doi = {10.1016/j.exer.2025.110697},
pmid = {41106454},
issn = {1096-0007},
mesh = {Animals ; *Retinitis Pigmentosa/genetics/metabolism/physiopathology ; Dogs ; *Disease Models, Animal ; *Eye Proteins/genetics ; *Gene Editing/methods ; *Genetic Diseases, X-Linked/genetics ; Female ; *Zygote/metabolism ; Electroretinography ; CRISPR-Cas Systems ; Male ; Mutation ; },
abstract = {Inherited retinal diseases (IRDs) constitute several ocular disorders leading to progressive and severe visual impairment. While significant progress has been made in understanding the genetic basis of IRDs, preclinical animal models are crucial for advancing therapeutic development. Although well-established mouse models exist, the scarcity of large animal models represents a significant limitation. Dogs (Canis familiaris) exhibit numerous physiological and anatomical similarities to humans, rendering them as potential models. X-linked retinitis pigmentosa (XLRP) is a severe, early-onset form of IRDs, primarily caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. Here, we efficiently generated a stably inherited RPGR-knockout beagle model of XLRP in one-step via CRISPR/Cas9 gene editing tool with multiple gRNAs and zygote microinjection technology. Two F0 generation beagle dogs were successfully obtained with high knockout efficiency in the RPGR gene and the F0 female exhibited characteristic XLRP phenotypes, including progressive deterioration of both rod and cone photoreceptor function, a gradual reduction in the outer nuclear layer thickness from 7 to 35 months, and attenuated retinal arteries with minimal pigmentation. Notably, the F1 male of hemizygous RPGR-knockout beagle dogs displayed more pronounced disease phenotypes by 7 months of age. In conclusion, we successfully generated a stably inherited beagle dog model of XLRP exhibiting an intermediate characteristic disease onset, which is highly suitable for therapeutic research in gene therapy and stem cell transplantation for determining optimal intervention timing and therapeutic dosing.},
}
@article {pmid41081521,
year = {2025},
author = {Wu, H and Lin, X and Tian, C and Xiong, D},
title = {Establishment of an RAA-CRISPR/Cas12a assay based on CpSge1 for rapid detection of Cryphonectria parasitica.},
journal = {Microbiology spectrum},
volume = {13},
number = {11},
pages = {e0107925},
pmid = {41081521},
issn = {2165-0497},
support = {2023YFD1401301//National Key Research and Development Program/ ; },
mesh = {*Ascomycota/genetics/isolation &amp; purification ; *Plant Diseases/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Fagaceae/microbiology ; Fungal Proteins/genetics ; Recombinases/metabolism/genetics ; China ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Chestnut blight disease caused by Cryphonectria parasitica is a serious branch disease that occurs worldwide, especially in Europe and North America. In recent years, chestnut blight disease has also been severe and even posed a great threat to the healthy development of chestnut orchards in some areas of China. Accurate and rapid detection of C. parasitica during the initial stages of the disease is helpful to take corresponding prevention and control measures in advance. In this study, we selected the CpSge1 (Gti1/Pac2 transcription factor family) of C. parasitica as the detection target and established a rapid and visual detection system of C. parasitica that combined the recombinase-aided amplification (RAA) and CRISPR/Cas12a, called CpSge1-RAA-CRISPR/Cas12a. The system allows for the specific detection of C. parasitica in approximately 60 mins, with visualization of results. The detection sensitivity of this system was found to be 1 pg/µL. We combined the RAA-CRISPR/Cas12a with a lateral flow dipstick, which also showed specific, high sensitivity, and fast characters. In conclusion, the RAA-CRISPR/Cas12a assay has great potential to be a method for early diagnosis and on-site detection of C. parasitica, especially for areas where specialized equipment is lacking.IMPORTANCEA rapid, highly sensitive, and visualized detection system of Cryphonectria parasitica was established by using the RAA-CRISPR/Cas12a method based on the C-terminal variable regions of a fungal-specific transcription factor CpSge1. The detection system was performed at a constant temperature condition of 37°C, which provides important support for the diagnosis of chestnut blight diseases in the field.},
}
@article {pmid41075360,
year = {2026},
author = {Rahimi, M and Schreiber, M and Habermann, H and Haake, L and Miskel, D and Tesfaye, D and Held-Hoelker, E and Brenig, B and Benedetti, C and Hoelker, M},
title = {Efficient genome editing of bovine in vitro derived zygotes via Cas9 RNP-electroporation using extended-stored bovine ovaries.},
journal = {Theriogenology},
volume = {250},
number = {},
pages = {117703},
doi = {10.1016/j.theriogenology.2025.117703},
pmid = {41075360},
issn = {1879-3231},
mesh = {Animals ; Cattle/embryology ; Female ; *Gene Editing/veterinary/methods ; *Electroporation/veterinary/methods ; *Zygote/physiology ; *Ovary/physiology ; *CRISPR-Cas Systems ; Fertilization in Vitro/veterinary ; CRISPR-Associated Protein 9 ; Ribonucleoproteins/genetics ; },
abstract = {Bovine embryos serve as a relevant model for human preimplantation development and are increasingly used in genome editing research. Although CRISPR-Cas9 electroporation in zygotes offers a promising approach, challenges with respect to editing efficiency and mosaicism remain a challenge. However, traditional in vitro fertilization (IVF) workflows enforce highly inconvenient electroporation times of zygotes usually around midnight. This study therefore aimed to investigate the feasibility of using bovine zygotes derived from extended-stored slaughterhouse derived ovaries (Phosphate buffered saline, 14-18 °C, 20-22 h) with particular attention to the strength of the voltage field during electroporation and the Cas9-RNP concentration with respect to embryonic development and genome editing efficiency. Oocytes obtained under these conditions demonstrated in vitro developmental competence similar to those derived from fresh ovaries, with comparable cleavage and blastocyst formation rates. Electroporation conducted 10 h post-insemination at voltages of 15V, 20V, and 25V revealed that 25V yielded the highest blastocyst editing rate (40.7 %) while maintaining acceptable developmental rates. Increasing voltage to 30V and 35V further enhanced blastocyst editing rate (66.7 % and 67.9 %, respectively), with a greater proportion of blastocyst showing a full edit. While higher voltages reduced developmental rates, the optimal voltage for electroporation was found to be 30V for bovine zygotes derived from extended stored ovaries. Using that setting, contrasting Cas9-RNP concentrations (6 μM vs. 3 μM) were tested. A concentration of 6 μM resulted in higher editing rates compared to 3 μM (81.5 % vs. 60.0 %), although the distribution of monoallelic, biallelic, and mosaic modifications did not differ significantly. Taken together, this study confirmed for the first time that zygotes from extend-stored bovine ovaries are suitable for genome editing via CRISPR Cas9-RNP electroporation. Mosaicism, however, remained prevalent, underscoring the need for strategies to improve the precision and consistency of embryo editing.},
}
@article {pmid41036840,
year = {2025},
author = {Diaz, B and Krishna, R and Schoeniger, JS and Mageeney, CM},
title = {Exploring phage-host interactions in Burkholderia cepacia complex bacterium to reveal host factors and phage resistance genes using CRISPRi functional genomics and transcriptomics.},
journal = {Microbiology spectrum},
volume = {13},
number = {11},
pages = {e0193625},
pmid = {41036840},
issn = {2165-0497},
support = {LDRD//Sandia National Laboratories/ ; InCoGenTEC//U.S. Department of Energy/ ; },
mesh = {*Bacteriophages/physiology/genetics ; *Burkholderia cepacia complex/virology/genetics ; Genomics/methods ; Transcriptome ; Gene Expression Profiling ; CRISPR-Cas Systems ; Host Specificity ; Humans ; Host-Pathogen Interactions/genetics ; },
abstract = {Complex interactions of bacteriophages with their bacterial hosts determine phage host range and infectivity. While phage defense systems and host factors have been identified in model bacteria, they remain challenging to predict in non-model bacteria. In this paper, we integrate functional genomics and transcriptomics to investigate phage-host interactions, revealing active phage resistance and host factor genes in Burkholderia cenocepacia K56-2. Burkholderia cepacia complex species are commonly found in soil and are opportunistic pathogens in immunocompromised patients. We studied infection of B. cenocepacia K56-2 with Bcep176, a temperate phage isolated from Burkholderia multivorans. A genome-wide dCas9 knockdown library targeting B. cenocepacia K56-2 was constructed, and a pooled infection experiment identified 63 novel genes or operons coding for candidate host factors or phage resistance genes. The activities of a subset of candidate host factor and resistance genes were validated via single-gene knockdowns. Transcriptomics of B. cenocepacia K56-2 during Bcep176 infection revealed that expression of genes coding for host factor and resistance candidates identified in this screen was significantly altered during infection by 4 h post-infection. Identifying which bacterial genes are involved in phage infection is important to understand the ecological niches of B. cenocepacia and its phages, and for designing phage therapies.IMPORTANCEBurkholderia cepacia complex bacteria are opportunistic pathogens inherently resistant to antibiotics, and phage therapy is a promising alternative treatment for chronically infected patients. Burkholderia bacteria are also ubiquitous in soil microbiomes. To develop improved phage therapies for pathogenic Burkholderia bacteria, or engineer phages for applications, such as microbiome editing, it's essential to know the bacterial host factors required by the phage to kill bacteria, as well as how the bacteria prevent phage infection. This work identified 65 genes involved in phage-host interactions in Burkholderia cenocepacia K56-2 and tracked their expression during infection. These findings establish a knowledge base to select and engineer phages infecting or transducing Burkholderia bacteria.},
}
@article {pmid40996288,
year = {2025},
author = {Boucher, MJ and Madhani, HD},
title = {CRISPR/Cas9-compatible plasmids enabling seven dominant genetic selection methods for the human fungal pathogen Cryptococcus neoformans.},
journal = {Microbiology spectrum},
volume = {13},
number = {11},
pages = {e0193525},
pmid = {40996288},
issn = {2165-0497},
support = {R01AI100272//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Cryptococcus neoformans/genetics/drug effects ; *CRISPR-Cas Systems ; Humans ; *Plasmids/genetics ; *Selection, Genetic ; Drug Resistance, Fungal/genetics ; Nucleosides ; },
abstract = {Cryptococcus neoformans is the most common cause of human fungal meningitis and an important model system for studying fundamental eukaryotic biology. Genetic manipulation of this organism relies on three dominant drug resistance markers (nourseothricin acetyltransferase [NAT], neomycin phosphotransferase II [NEO], and hygromycin B phosphotransferase [HYG]) and the recyclable dominant prototrophic marker amdS. With ongoing technological advances that are expanding our ability to explore cryptococcal gene function, contemporary studies often require multiple genetic manipulations in the same strain. Additional dominant selection methods would maximize the utility of these tools by facilitating their combinatorial use. Here, we identify blasticidin S resistance via the blasticidin S deaminase (BSD) or blasticidin S resistance (BSR) markers as a novel dominant selection method for C. neoformans. We further validate phleomycin resistance via the bleomycin resistance gene (BLE) marker as an additional selection method, confirming a study that first established this marker 25 years ago (J. Hua, J. D. Meyer, and J. K. Lodge, Clin Diagn Lab Immunol 7:125-128, 2000, https://doi.org/10.1128/cdli.7.1.125-128.2000). To enable highly efficient CRISPR/Cas9-mediated genome modification, we incorporated these markers, as well as the newly established dominant prototrophic marker ptxD (M. Khongthongdam, T. Phetruen, and S. Chanarat, Microbiol Spectr 13:e01618-24, 2025, https://doi.org/10.1128/spectrum.01618-24), into a vector series that enables the construction of fused marker-sgRNA products via PCR. Altogether, this work expands the number of dominant genetic selection methods for C. neoformans to seven, including five drug selection regimes and two prototrophic methods. The vector series has been deposited at Addgene. IMPORTANCE Cryptococcus neoformans is the top-ranked World Health Organization priority fungal pathogen due to its widespread distribution and inadequate treatment options. Additionally, as a basidiomycete yeast occupying an underexplored branch of the fungal kingdom, this organism is a powerful system for deciphering core eukaryotic biology that is absent in classic model fungi. Defining functions for novel cryptococcal genes is a crucial priority, and the availability of additional genetic selection methods would facilitate these efforts. In this study, we establish blasticidin S resistance as a novel genetic selection method for C. neoformans, and we validate a previous report using phleomycin resistance as such. This work expands the number of reliable dominant selection methods to seven, providing flexibility for the introduction of sequential genetic modifications into single strains.},
}
@article {pmid40089804,
year = {2025},
author = {Zhu, J and Zhang, Y and Zhang, M and Hong, Y and Sun, C and Guo, Y and Yin, H and Lv, C and Guo, B and Wang, F and Xu, R},
title = {Natural variation and CRISPR/Cas9 gene editing demonstrate the role of a group VII ethylene response factor, HvERF62, in regulation of barley waterlogging tolerance.},
journal = {Journal of experimental botany},
volume = {76},
number = {17},
pages = {5071-5085},
doi = {10.1093/jxb/eraf101},
pmid = {40089804},
issn = {1460-2431},
support = {32301871//National Natural Science Foundation of China/ ; 2022YFD2301302//National key research and development plant/ ; },
mesh = {*Hordeum/genetics/physiology/metabolism ; *Plant Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Editing ; Ethylenes/metabolism ; Genome-Wide Association Study ; Quantitative Trait Loci ; *Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Plant ; },
abstract = {Waterlogging stress is a serious problem for plants throughout the world, inhibiting plant growth and development and even leading in severe circumstances to plant death. Ethylene-responsive VII (ERFVII) is a key transcription factor regulating plant waterlogging tolerance. In this study, a major quantitative trait locus influencing chlorophyll content under waterlogging stress was detected on chromosome 4H through genome-wide association study using a natural population of barley (Hordeum vulgare). Using RNA-Seq analysis, the group VII ethylene response factor HvERF62 was identified as the candidate gene. CRISPR/Cas9-guided knockout HvERF62 mutants showed sensitivity to waterlogging, with lower chlorophyll content, fewer adventitious roots, and lower root activity. Physiological and transcriptomic analyses showed that HvERF62 plays an important role in aerenchyma formation, reactive oxygen species homeostasis, and carbohydrate accumulation under waterlogging stress, and regulates waterlogging tolerance through starch and sucrose metabolism, the mitogen-activated protein kinase signaling pathway, plant hormone signal transduction, and glycolysis/gluconeogenesis signaling. Haplotype analysis showed that the amino acid sequence of the waterlogging-intolerant haplotype Hap3 was terminated prematurely. This study provides a new genetic resource and a relevant marker for identification of waterlogging-sensitive material and deepens our understanding of the molecular mechanisms of waterlogging response in barley.},
}
@article {pmid41184108,
year = {2026},
author = {Abir, AH and Benz, J and Frey, B and Bruns, H and Krönke, G and Gaipl, US and Schober, K and Mougiakakos, D and Mielenz, D},
title = {Glycolytic flux sustains human Th1 identity and effector function via STAT1 glycosylation.},
journal = {Life science alliance},
volume = {9},
number = {1},
pages = {},
doi = {10.26508/lsa.202503315},
pmid = {41184108},
issn = {2575-1077},
mesh = {Humans ; *Glycolysis ; Glycosylation ; *STAT1 Transcription Factor/metabolism/genetics ; *Th1 Cells/metabolism/immunology ; Interferon-gamma/metabolism ; Phosphorylation ; CRISPR-Cas Systems ; Cell Differentiation ; Gene Editing ; },
abstract = {T helper (Th) cell lineages are linked to metabolism, but precise mechanisms in human Th1 cells remain unclear. We addressed this question by in vitro stimulation and CRISPR/Cas9-mediated gene editing. Metabolic profiling revealed enhanced glycolytic activity in Th1 versus non-polarized cells, evidenced by increased extracellular acidification rate, ATP production via glycolysis, lactate secretion, NADH abundance, and elevated glycolysis-dependent anabolic activity. Inhibition of glycolysis reduced IFNγ production and STAT1 phosphorylation independent of JAK1/2 or SHP2 activity and STAT1 abundance, implicating glycolysis directly in sustaining STAT1-mediated Th1 functionality. O-glycosylation of STAT1 via O-glycosyltransferase was pivotal in modulating STAT1 activity. Pharmaceutical O-glycosyltransfer-ase inhibition prevented Th1 differentiation as well as STAT1 O-glycosylation. CRISPR/Cas9 mediated mutation of the O-glycosylation Ser499 and Thr510 sites diminished STAT1 Ser727 phosphorylation and IFNγ synthesis. Together, this study highlights glycolysis as key regulator of human Th1 cell identity and effector function, with STAT1 O-glycosylation selectively maintaining Th1 effector capacity. This mechanism could be explored to safeguard Th1 cells.},
}
@article {pmid41183413,
year = {2025},
author = {Chen, H and Zeng, Z and Wei, Y and Huang, H and Negahdary, M and Han, X and Lin, Y and He, L and Song, F and Wan, Y},
title = {Dynamic biosensing enables amplifier-collateral-cleavage enhancement for pathogen diagnostic.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118158},
doi = {10.1016/j.bios.2025.118158},
pmid = {41183413},
issn = {1873-4235},
abstract = {While CRISPR-Cas system and dynamic DNA nanotechnology have been extensively applied to mainstream biomedical domains, including gene editing, biochemical analysis, and molecular imaging-current approaches remain constrained by limitations in addressing increasingly nuanced and specialized experimental scenarios. Here, we report that CRISPR-CasΦ possesses a unique collateral-cleavage blockade characteristic: CasΦ is unable to recognize the "TTN" sequence in the loop region at the 3' end of stem-loop DNA, resulting in the blockade of collateral-cleavage activity. Leveraging this discovery, we successfully designed and customized two back-end signal amplifiers for biosensing by integrating dynamic DNA sensing studies of CasΦ. Based on these two specialized stem-loop amplifiers, we further developed the Amplifier-collateral-cleavage enhancement (ACE) method, achieving exponential signal amplification. Clinical validation using 112 urine samples demonstrated that ACE exhibits 98.8 % sensitivity and 90 % specificity. These findings highlight the potential of CasΦ dynamic sensing and establish a bridge for future integration of dynamic DNA technology and CRISPR systems.},
}
@article {pmid41124025,
year = {2025},
author = {Su, F and Zhang, H and Ren, C and Jiang, Y and Qiao, Y and Zhang, S and Liu, J and Li, S and Li, Y},
title = {MnO2@Mn3O4 Heterojunction as a Coreactant Catalyst Collaborated with T-Shaped DNA Cycling-CRISPR/Cas12a Cascade Amplification for Locus-Specific N6-Methyladenosine RNA Modification Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {43},
pages = {24165-24175},
doi = {10.1021/acs.analchem.5c04791},
pmid = {41124025},
issn = {1520-6882},
mesh = {*Adenosine/analogs &amp; derivatives/analysis ; *Manganese Compounds/chemistry ; *Oxides/chemistry ; *Biosensing Techniques/methods ; Humans ; *RNA/analysis/chemistry ; Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *DNA/chemistry/genetics ; Metal Nanoparticles/chemistry ; Catalysis ; Gold/chemistry ; Nucleic Acid Amplification Techniques ; CRISPR-Associated Proteins/genetics ; Limit of Detection ; Luminescent Measurements ; HeLa Cells ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {As the predominant RNA modification, N6-methyladenosine (m[6]A) is recognized to play pivotal regulatory roles in fundamental cellular functions and oncogenic processes. However, the precise analysis of site-specific m[6]A modifications continues to present significant challenges. In this work, a unique electrochemiluminescence (ECL) biosensor for the locus-specific detection of m[6]A in RNA was developed in the first utilization of a MnO2@Mn3O4 heterojunction as a coreaction catalyst in collaboration with a T-shaped DNA cycling-CRISPR/Cas12a cascade amplification strategy. The MnO2@Mn3O4 heterojunction was observed to significantly enhance coreactant catalytic activity, yielding a 7.3-fold increase in the ECL intensity of the gold nanoparticles (AuNPs)/MnO2@Mn3O4/(2,2'-bipyridine) dichlororuthenium(II) (Ru(bpy)3[2+])/Nafion/GCE compared to the AuNPs/Ru(bpy)3[2+]/Nafion/GCE. Sequentially, the T-shaped DNA cycling amplification strategy effectively converted the target m[6]A RNA into an amplified biosignal, further enhanced by a CRISPR/Cas12a signal amplification system mediated by framework nucleic acid (FNA)-based photocontrollable nucleic acid protection, ensuring the sensitivity and specificity of m[6]A RNA. The integration of the triple signal amplification strategy achieved detection limits as low as 0.05 pM (S/N = 3) for a linearity spanning from 100 fM to 100 nM. The proposed ECL biosensor has been applied in detecting site-specific m[6]A modifications in total real RNA samples extracted from HeLa cells, demonstrating its promising applications for clinical diagnosis.},
}
@article {pmid41084820,
year = {2025},
author = {Ma, AX and Zhang, Q and Zhang, MY and Liu, SL and Wang, ZG and Pang, DW},
title = {One-step naked-eye fluorescence detection of viruses using quantum dot-magnetic beads coupled with CRISPR/Cas12a.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {89},
pages = {17432-17435},
doi = {10.1039/d5cc03645c},
pmid = {41084820},
issn = {1364-548X},
mesh = {*Quantum Dots/chemistry ; *CRISPR-Cas Systems ; Humans ; Fluorescence ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Simplexvirus/isolation &amp; purification ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {This work constructs quantum dot-magnetic beads coupled with CRISPR/Cas12a for naked-eye detection of herpes simplex virus. The system integrates the high brightness of quantum dots and trans-cleavage activity of Cas12a, enabling rapid, simple, and accurate viral detection.},
}
@article {pmid40954319,
year = {2025},
author = {Wei, D and Cheng, P and Song, Z and Liu, Y and Xu, X and Huang, X and Wang, X and Zhang, Y and Shu, W and Wei, Y},
title = {AI-guided Cas9 engineering provides an effective strategy to enhance base editing.},
journal = {Molecular systems biology},
volume = {21},
number = {11},
pages = {1563-1580},
pmid = {40954319},
issn = {1744-4292},
support = {12331018//MOST | National Natural Science Foundation of China (NSFC)/ ; 32401220//MOST | National Natural Science Foundation of China (NSFC)/ ; PT2025-03-01//Shanghai institute for Biomedical and Pharmaceutical Technologie/ ; RC2025-01//Shanghai institute for Biomedical and Pharmaceutical Technologie/ ; CXPJJH122006-1014//CHEN XIAO-PING FOUNDATION FOR THE DEVELOPMENT OF SCIENCE AND TECHNOLOGY OF HUBEI PROVINCE/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Protein Engineering/methods ; *Artificial Intelligence ; Point Mutation ; Cell Line, Tumor ; HEK293 Cells ; },
abstract = {Precise genome editing is crucial for functional studies and therapies. Base editors, while powerful, require optimization for efficiency. Meanwhile, emerging protein design methods and protein language models have driven efficient and intelligent protein engineering. In this study, we employed the Protein Mutational Effect Predictor (ProMEP) to predict the effects of single-site saturated mutations in Cas9 protein, using AncBE4max as the prototype to construct and test 18 candidate point mutations. Based on this, we further predicted combinations of multiple mutations and successfully developed a high-performance variant AncBE4max-AI-8.3, achieving a 2-3-fold increase in average editing efficiency. Introducing the engineered Cas9 into CGBE, YEE-BE4max, ABE-max, and ABE-8e improved their editing performance. The same strategy also substantially improves the efficiencies of HF-BEs. Stable enhancement in editing efficiency was also observed across seven cancer cell lines and human embryonic stem cells. In conclusion, we validated that AI models can serve as more effective protein engineering tools, providing a universal improvement strategy for a series of gene editing tools.},
}
@article {pmid40878190,
year = {2025},
author = {Tian, L and Gao, Y and Lu, Y and Xu, F and Feng, Z and Zi, L and Deng, Z and Yang, J},
title = {Modular RCA-CRISPR/Cas12a amplification on a multi-volume SlipChip for ultrafast, single-copy quantification of circRNA and miRNA in ovarian cancer.},
journal = {Lab on a chip},
volume = {25},
number = {22},
pages = {5762-5776},
doi = {10.1039/d5lc00585j},
pmid = {40878190},
issn = {1473-0189},
mesh = {Humans ; *MicroRNAs/genetics/analysis ; *RNA, Circular/genetics/analysis ; Female ; *Ovarian Neoplasms/genetics ; *Nucleic Acid Amplification Techniques/instrumentation ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Lab-On-A-Chip Devices ; *CRISPR-Associated Proteins/metabolism/genetics ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The aberrant expression of RNAs in ovarian cancer (OC) progression highlights their potential as clinical biomarkers. However, rapid and accurate quantification of these RNAs in biosamples remains a significant challenge. In this study, we develop a modular isothermal rolling circle amplification (RCA)-activated Cas12a loop-enhanced (MIRACLE) amplification method for circRNA and miRNA quantification without the need of reverse transcription. In this design, isothermal amplification of modular DNA can be initiated by target-specific RCA primers or miRNAs, with the amplification products subsequently recognized by the Cas12a system to generate measurable signals. When integrated with a multi-volume sliding chip (SlipChip) platform, this MIRACLE method enables portable, rapid and ultra-sensitive quantification of these two types of RNA. Under optimized conditions, this platform exhibits detection limits of 0.125 copies per μL for circRNA and 0.326 copies per μL for miRNA, covering a 5-log dynamic range from 10[-1] to 10[3] copies per μL within 35 min. The platform was validated using OC cell lines and clinical blood samples. It successfully profiled OC RNA biomarkers (hsa_circ_0049101 and hsa-miR-338-3p) and effectively distinguished between early and advanced stages of OC. These results show a strong correlation with RT-qPCR (R[2] = 0.953 for circRNA and R[2] = 0.947 for miRNA). This work establishes a versatile CRISPR-microfluidic platform for cancer diagnosis. Its modular design allows for adaptation to detect other cancer-related RNA biomarkers, thereby addressing critical needs in precision oncology.},
}
@article {pmid41183015,
year = {2025},
author = {Keuthen, H and Pozhydaieva, N and Höfer, K},
title = {Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {224},
pages = {},
doi = {10.3791/69022},
pmid = {41183015},
issn = {1940-087X},
mesh = {*CRISPR-Cas Systems/genetics ; *Bacteriophages/genetics ; *Mixed Function Oxygenases/genetics/metabolism ; Mutagenesis ; DNA, Viral/genetics/chemistry ; *Mutagenesis, Site-Directed/methods ; },
abstract = {Bacteriophages, viruses that specifically target their bacterial hosts, hold significant potential for biotechnology and medicine, especially in combating multidrug-resistant infections. However, the molecular mechanisms underlying phage infection remain largely underexplored. Precise, site-specific mutagenesis of phages is a powerful tool to elucidate gene functions and phage-host interactions. However, a major challenge in phage genome mutagenesis is the presence of phage DNA modifications that interfere with conventional genome editing tools like CRISPR-Cas. While CRISPR-Cas systems have been used successfully for targeted mutagenesis in various organisms, their effectiveness in phage mutagenesis is often limited by DNA modifications such as cytosine glycosylation. To overcome this barrier, we developed an efficient method that temporarily reduces the abundance of phage DNA modifications, enabling efficient CRISPR-Cas targeting and precise mutation introduction into phage genomes. Specifically, we use the Ten Eleven Translocation (TET) methylcytosine dioxygenase from Naegleria gruberi (NgTET), which iteratively demodifies methylated and hydroxymethylated cytosines in DNA. By oxidizing hydroxymethylated cytosines within phage DNA, NgTET prevents subsequent cytosine modification like glycosylation and significantly enhances the efficiency of Cas-mediated DNA cleavage. In conclusion, the scarless and precise genome-editing approach presented here enables the efficient introduction of point mutations while maintaining the native gene architecture in phage genomes. By preserving intact transcriptional and translational frameworks, this method minimizes unintended disruptions to complex regulatory networks. This is particularly important for investigating essential or multifunctional phage proteins. The ability to generate targeted genetic modifications without introducing extraneous sequences significantly expands the experimental toolkit for phage biology. This strategy not only facilitates detailed functional studies but also enhances the potential for rational engineering of phages for therapeutic and biotechnological applications.},
}
@article {pmid41182907,
year = {2025},
author = {Pantoja-Alonso, MA and Camas-Reyes, JA and Cano-Segura, R and Cárdenas-Aquino, MDR and Martínez-Antonio, A},
title = {A comprehensive review of genomic-scale genetic engineering as a strategy to improve bacterial productivity.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {11},
pages = {},
doi = {10.1099/mic.0.001628},
pmid = {41182907},
issn = {1465-2080},
mesh = {*Bacteria/genetics/metabolism ; *Genetic Engineering/methods ; CRISPR-Cas Systems ; *Genome, Bacterial ; Genomics/methods ; Gene Editing ; Metabolic Engineering/methods ; Synthetic Biology/methods ; },
abstract = {Bacterial genome engineering has evolved to provide increasingly precise, robust and rapid tools, driving the development and optimization of bacterial production of numerous compounds. The field has progressed from early random mutagenesis methods, labour-intensive and inefficient, to rational and multiplexed strategies enabled by advances in genomics and synthetic biology. Among these tools, CRISPR/Cas has stood out for its versatility and its ability to achieve precision levels ranging from 50% to 90%, compared to the 10-40% obtained with earlier techniques, thereby enabling remarkable improvements in bacterial productivity. Nevertheless, like its predecessors, it still demands continuous refinement to reach full maturity. In this context, the present review addresses the lack of a unified overview by summarizing historical milestones and practical applications of genomic engineering tools in bacteria. It integrates diverse approaches to provide a comprehensive perspective on the evolution and prospects of these fundamental biotechnological tools.},
}
@article {pmid41182905,
year = {2025},
author = {Lamb, CH and Riesle-Sbarbaro, S and Prescott, JB and Te Velthuis, AJW and Myhrvold, C and Nilsson-Payant, BE},
title = {Amplification-free detection of zoonotic viruses using Cas13 and multiple CRISPR RNAs.},
journal = {The Journal of general virology},
volume = {106},
number = {11},
pages = {},
doi = {10.1099/jgv.0.002169},
pmid = {41182905},
issn = {1465-2099},
mesh = {Animals ; *RNA, Viral/genetics ; Humans ; *Influenza A virus/genetics/isolation &amp; purification ; Sensitivity and Specificity ; *Orthohantavirus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Zoonoses/virology/diagnosis ; },
abstract = {Zoonotic viruses such as hantaviruses and influenza A viruses present a threat to humans and livestock. There is thus a need for methods that are rapid, sensitive and relatively cheap to detect infections with these pathogens early. Here, we use an amplification-free clustered regularly interspaced short palindromic repeats-associated protein 13 (CRISPR-Cas13)-based assay, which is simple, cheap and field-deployable, to detect the presence or absence of genomic hantavirus or influenza A virus RNA. In addition, we evaluate whether the use of multiple CRISPR RNAs (crRNAs) can improve the sensitivity of this amplification-free method. We demonstrate that for the hantaviruses Tula virus (TULV) and Andes virus (ANDV), a combination of two or three crRNAs provides the best sensitivity for detecting viral RNA, whereas for influenza virus RNA detection, additional crRNAs provide no consistent benefit. We also show that the amplification-free method can be used to detect TULV and ANDV RNA in tissue culture infection samples, ANDV from hamster lung samples and influenza A virus RNA in clinical nasopharyngeal swabs. In clinical samples, the Cas13 assay has an 85% agreement with RT-qPCR for identifying a positive sample. Overall, these findings indicate that amplification-free CRISPR-Cas13 detection of viral RNA has potential as a tool for rapidly detecting zoonotic virus infections.},
}
@article {pmid41182611,
year = {2026},
author = {Chiurillo, MA and Ahmed, M and González, C and Rosón, JN and Das, A and Lander, N},
title = {Cloning-Free Genome Editing by CRISPR/T7RNAP/Cas9 in Trypanosoma cruzi.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2982},
number = {},
pages = {59-76},
pmid = {41182611},
issn = {1940-6029},
mesh = {*Trypanosoma cruzi/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *DNA-Directed RNA Polymerases/genetics ; *Viral Proteins/genetics ; Cloning, Molecular ; Humans ; *Genome, Protozoan ; Gene Knockout Techniques ; },
abstract = {The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 technology for genome editing in this organism. Initially, the system was successfully used for gene knockout and endogenous C-terminal tagging in T. cruzi. Recently, an updated version of this technology has been used for gene complementation, site-directed mutagenesis, and N-terminal tagging in trypanosomatids. This cloning-free strategy, called CRISPR/T7RNAP/Cas9, is extremely useful for identifying essential genes when null mutants are not viable. Mutant cell lines obtained by this new system have been used for the functional characterization of proteins in different developmental stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter, we describe the methodology to achieve genome editing by CRISPR/T7RNAP/Cas9 in T. cruzi. Our method involves the generation of T. cruzi epimastigotes that constitutively express the T7 RNA polymerase (T7RNAP) and SpCas9, and their co-transfection with an sgRNA template and donor DNA(s) as polymerase chain reaction (PCR) products. Using this strategy, we have generated genetically modified parasites in 2-3 weeks without the need for gene cloning, cell sorting, or having to perform several transfection attempts to verify the sgRNA efficiency for targeting the gene of interest. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines, and endogenous (N- or C-terminal) tagging in T. cruzi.},
}
@article {pmid41182029,
year = {2025},
author = {Ashworth, KE and Zhang, J and D'Amata, C and Héon, E and Ballios, BG},
title = {USH2A-Mutated Human Retinal Organoids Model Rod-Cone Dystrophy.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {66},
number = {14},
pages = {2},
doi = {10.1167/iovs.66.14.2},
pmid = {41182029},
issn = {1552-5783},
mesh = {Humans ; *Organoids/pathology/metabolism ; Induced Pluripotent Stem Cells/pathology/metabolism ; *Mutation ; *Extracellular Matrix Proteins/genetics/metabolism ; *Cone-Rod Dystrophies/genetics/pathology/metabolism ; Retinal Rod Photoreceptor Cells/pathology ; *Retinitis Pigmentosa/genetics/pathology ; CRISPR-Cas Systems ; },
abstract = {PURPOSE: USH2A mutations are the leading cause of autosomal recessive retinitis pigmentosa (RP), a progressive blinding disease marked by photoreceptor degeneration. Animal models fail to recapitulate the features of USH2A RP seen in humans, and its earliest pathogenic events remain unknown. Here, we established a human model of USH2A RP using retinal organoids derived from patient induced pluripotent stem cells and CRISPR-Cas9-engineered isogenic-USH2A-/- induced pluripotent stem cells.<br><br>METHODS: We assessed organoids for cellular, molecular, and morphological defects using serial live imaging and whole organoid and fixed section analyses.<br><br>RESULTS: Both patient-derived and isogenic-USH2A-/- organoids showed preferential rod photoreceptor loss followed by widespread degeneration, consistent with the clinical phenotype. Additionally, isogenic-USH2A-/- organoids showed early defects in proliferation and structure.<br><br>CONCLUSIONS: Our findings suggest that molecular changes precede overt photoreceptor loss in USH2A RP, and pathogenesis may begin before clinical symptoms emerge. By defining early and late disease features, we provide new insight on the developmental origins of USH2A RP to guide therapeutic strategies.},
}
@article {pmid41178730,
year = {2025},
author = {Avaro, AS and Santiago, JG},
title = {Engineering guidelines for CRISPR diagnostics.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc04206b},
pmid = {41178730},
issn = {1364-548X},
abstract = {This Feature Article reviews engineering guidelines for the design of CRISPR assays, including experimentally validated theoretical models and recommendations for experimental research practice and reporting. First, the state of the art of CRISPR kinetics studies is reviewed. Then presented is a summary of the existence and persistence of widespread gross errors in reports of kinetic rate constants of CRISPR-Cas enzymes, as well as the fact that many CRISPR studies provide insufficient data to check for consistency or assess calibration. Proper experimental procedures including signal calibration are critical to the assessment, design, and future development of CRISPR kinetics assays and CRISPR diagnostics. This review then presents guidelines for the calibration of fluorescence-based CRISPR assays. Fluorescence is the most common detection modality, and incorrect calibration is implicated in high-profile, gross errors in the field. Also presented is a review of enzymatic kinetic rates and reporter molecule degradation as the major factor limiting CRISPR assay sensitivity. Lastly, progress in, and criticism of, microfluidic applications of CRISPR assays is summarized.},
}
@article {pmid41178318,
year = {2025},
author = {Leung, CY and Wimmer, EA and Ahmed, HMM},
title = {Synthetic biology approaches to generate temperature-sensitive alleles for the Sterile Insect Technique.},
journal = {Insect science},
volume = {},
number = {},
pages = {},
doi = {10.1111/1744-7917.70186},
pmid = {41178318},
issn = {1744-7917},
support = {ResearchcooperationLowerSaxony-Israel//Nieders&#xe4;chsisches Ministerium f&#xfc;r Wissenschaft und Kultur/ ; Project D44003//International Atomic Energy Agency/ ; },
abstract = {The Sterile Insect Technique (SIT) is an environmentally friendly, sustainable pest control approach, which uses large-scale releases of sterile insects to suppress or eradicate target populations through infertile matings. The efficiency of SIT is enhanced by male-only releases requiring genetic sexing strains (GSSs) that are classically based on selectable recessive visible markers or temperature-sensitive lethal (tsl) mutations and a rescue by a wild-type allele translocated to the male-determining chromosome. The transfer of identified or designed temperature-sensitive alleles might allow the generation of neoclassical GSSs in additional SIT target species. By using precise genome-editing tools, such as CRISPR/Cas, the creation of specific mutations in target genes and the integration of a wild-type copy is feasible without the introduction of foreign DNA. This might ease regulation of neoclassical GSSs, since they are not considered transgenic. However, integration and expression of genes at male-determining loci or chromosomes is not reliably established. Therefore, additional strategies to link temperature-sensitive phenotypes to female development are required, which could be achieved by targeting genes involved in dosage compensation or sex determination. To create temperature-sensitive alleles, rational protein design using advanced modeling and prediction tools to evaluate and tailor the effect of mutations on protein stability and temperature sensitivity can be used. In addition, emerging synthetic biology strategies such as temperature-inducible N-degrons or temperature-sensitive inteins provide powerful tools to generate temperature sensitivity. Such approaches should enable conditional control over proteins causing female lethality or sex conversion and therefore promise straightforward generic approaches to generate GSSs for male-only production in SIT target species.},
}
@article {pmid41177343,
year = {2025},
author = {Rahimian, M and Panahi, B},
title = {Genomic insights into Erwinia amylovora prophages: Diversity, defense strategies, and phage-host coevolution.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {},
number = {},
pages = {105846},
doi = {10.1016/j.meegid.2025.105846},
pmid = {41177343},
issn = {1567-7257},
abstract = {Erwinia amylovora, the causative agent of fire blight in Rosaceae plants like apple and pear, is a major agricultural pathogen responsible for significant economic losses. This gram-negative phytopathogen has the potential to acquire antibiotic resistance and virulence genes, a situation that becomes more severe and restricts treatment options. Using an in silico approach, we analyzed 268 E. amylovora genomes and identified seven high-quality temperate prophages, all classified under Caudoviricetes, with average genome sizes of 44.2 kbp and 51 % GC content. These prophages exhibited unique genomic features, including tRNA genes (Ph-Ea644), anti-defense systems like ardc (Ph-Ea6-96), and regulatory/lysis genes (Ph-EaFC01). Comparative genomics and phylogenetic analyses grouped them into five clades, with Ph-Ea4-96, Ph-Ea3-97, and Ph-Ea2-97 being genetically identical. Functional annotation revealed streptomycin resistance genes and a CAZyme (GH23) in Ph-Ea7-3, virulence factors (e.g., alginate biosynthesis proteins), and six auxiliary metabolic genes (AMGs) linked to metabolic adaptation. Additionally, Ph-Ea644 encoded a cell wall-binding receptor protein. The prophages also carried defense systems (Gabija, CBASS) and 31 anti-CRISPR proteins (ACRs), suggesting evasion of host immunity. CRISPR-Cas analysis indicated fewer arrays and spacers in prophage-containing strains, underscoring CRISPR's role in lysogeny resistance. These findings highlight the genomic plasticity of E. amylovora prophages, their interactions with bacterial defenses, and their potential influence on pathogen evolution. This study enhances understanding of temperate phages in agricultural pathogens and underscores challenges in phage-based biocontrol strategies.},
}
@article {pmid41177179,
year = {2025},
author = {Bacci, L and Pollutri, D and Ripa, IJ and D'Andrea, M and Marchand, V and Motorin, Y and Hesse, AM and Couté, Y and Filipek, K and Penzo, M},
title = {Ribosomal protein L5 (RPL5/uL18) I60V mutation is associated to increased translation and modulates drug sensitivity in T-cell acute lymphoblastic leukemia cells.},
journal = {Biochemical pharmacology},
volume = {},
number = {},
pages = {117497},
doi = {10.1016/j.bcp.2025.117497},
pmid = {41177179},
issn = {1873-2968},
abstract = {Somatic mutations in ribosomal proteins (RPs), including RPL5, have been reported in approximately 10 % of pediatric patients with T-cell acute lymphoblastic leukemia (T-ALL). In cancer, the incorporation of mutant RPs into ribosomes often disrupts canonical ribosome function, thereby contributing to disease development. In this study, we aimed to characterize the effects of the RPL5-I60V mutation in the context of T-ALL, focusing on its impact on translation and cellular responses to a panel of compounds in vitro. Using CRISPR-Cas9, we generated a homozygous knock-in mutant in Jurkat cells and investigated its effects on ribosome biogenesis. We observed both quantitative and qualitative alterations in the production of the large ribosomal subunit. Ribosomes containing the mutant RPL5 protein exhibited intrinsically increased protein synthesis activity, which correlated with enhanced cellular proliferation. We then evaluated the response of these mutant cells to a panel of compounds targeting protein synthesis at various levels-including an MNK1 inhibitor, metformin, silvestrol, homoharringtonine, anisomycin, resveratrol, and hygromycin B-as well as cytarabine, a chemotherapeutic agent commonly used in T-ALL treatment. Our results showed that the RPL5-I60V mutation confers increased sensitivity to most of these compounds, with the exception of hygromycin B. This study advances our understanding of how oncoribosomes contribute to cancer pathogenesis and highlights the therapeutic potential of directly or indirectly targeting altered ribosomes, offering insights for the development of personalized treatment strategies.},
}
@article {pmid41176953,
year = {2025},
author = {Lankireddy, SV and Lekkala, S and Khadgi, A and Sripathi, VR and Janga, MR},
title = {Molecular biology of Cotton Leafroll Dwarf Virus (CLRDV) and potential application of CRISPR-Cas technology for developing virus-resistant cotton.},
journal = {Virology},
volume = {614},
number = {},
pages = {110730},
doi = {10.1016/j.virol.2025.110730},
pmid = {41176953},
issn = {1096-0341},
abstract = {Cotton leafroll dwarf virus (CLRDV) poses an increasing threat to global cotton production. Transmitted by the cotton aphid (Aphis gossypii) in a persistent, circulative manner, CLRDV exhibits a wide geographical distribution, with documented presence in South America, Africa, Asia, and the USA. Infection can result in either cotton blue disease (CBD) in South America or cotton leafroll dwarf disease (CLRDD) in the USA, both of which are associated with CLRDV. The considerable genetic diversity and frequent recombination events within CLRDV populations contribute to this symptom variability and complicate both diagnosis and management. While resistant cultivars have reduced disease impact in South America, these lines remain susceptible to emerging US strains, underscoring the urgent need for region-specific resistance breeding. Current molecular diagnostics rely on RT-PCR, but there is a need for rapid, field-deployable detection tools. Recent advances, such as CRISPR-Cas13a based SHERLOCK assays, offer sensitive and specific detection of CLRDV, with potential for on-site applications. Efficient screening techniques, supported by next-generation sequencing and transcriptomics, are essential for identifying novel resistance sources and elucidating virus-host interactions. CRISPR-based genome editing holds significant promise, as demonstrated in other crops. Targeted disruption of host susceptibility genes using CRISPR-Cas9, or direct degradation of viral genomes with RNA-targeting systems such as Cas12/Cas13, could offer durable, broad-spectrum resistance. By integrating molecular virology, high-throughput genomics, and precision gene editing, this review outlines a roadmap for translating these advances into sustainable, field-level solutions for CLRDV management and long-term cotton productivity.},
}
@article {pmid41176802,
year = {2025},
author = {Kalinina, NO and Spechenkova, NA and Taliansky, ME},
title = {Erratum to: Biotechnological Approaches to Plant Antiviral Resistance: CRISPR-Cas or RNA Interference?.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {10},
pages = {1450},
doi = {10.1134/S0006297925100013},
pmid = {41176802},
issn = {1608-3040},
}
@article {pmid41175344,
year = {2025},
author = {Shamloo, S and Schloßhauer, JL and Tiwari, S and Denise Fischer, K and Almolla, O and Ghebrechristos, Y and Kratzenberg, L and Bejoy, AM and Aifantis, I and Boccalatte, F and Wang, E and Imig, J},
title = {RNA binding of GAPDH controls transcript stability and protein translation in acute myeloid leukemia.},
journal = {RNA biology},
volume = {22},
number = {1},
pages = {1-23},
doi = {10.1080/15476286.2025.2580180},
pmid = {41175344},
issn = {1555-8584},
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/metabolism/pathology ; *Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism/genetics ; *Protein Biosynthesis ; *RNA, Messenger/genetics/metabolism ; RNA-Binding Proteins/metabolism/genetics ; 5' Untranslated Regions ; Cell Line, Tumor ; *RNA Stability ; Protein Binding ; Gene Expression Regulation, Leukemic ; Cell Proliferation ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) ; },
abstract = {Dysregulation of RNA binding proteins (RBPs) is a hallmark in cancerous cells. In acute myeloid leukaemia (AML) RBPs are key regulators of tumour proliferation. While classical RBPs have defined RNA binding domains, RNA recognition and function in AML by non-canonical RBPs (ncRBPs) remain unclear. Given the inherent complexity of targeting AML broadly, our goal was to uncover potential ncRBP candidates critical for AML survival using a CRISPR/Cas-based screening. We identified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a pro-proliferative factor in AML cells. Based on cross-linking and immunoprecipitation (CLIP), we are defining the global targetome, detecting novel RNA targets mainly located within 5'UTRs, including GAPDH, RPL13a, and PKM. The knockdown of GAPDH unveiled genetic pathways related to ribosome biogenesis, translation initiation, and regulation. Moreover, we demonstrated a stabilizing effect through GAPDH binding to target transcripts including its own mRNA. The present findings provide new insights on the RNA functions and characteristics of GAPDH in AML.},
}
@article {pmid40958477,
year = {2025},
author = {Tao, XL and Lei, YM and Zhou, XM and Chen, ZP and Ma, Y and Ma, PY and Song, DQ and Zhuo, Y},
title = {Allosteric Activation of Cas12a via PAM Topological Engineering for Direct and Rapid Detection of Nucleases.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {64},
number = {45},
pages = {e202515521},
doi = {10.1002/anie.202515521},
pmid = {40958477},
issn = {1521-3773},
support = {22374123//National Natural Science Foundation of China/ ; SWU-XJLJ202303//The Fundamental Research Funds for the Central Universities/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry ; Allosteric Regulation ; CRISPR-Cas Systems ; *DNA/chemistry/metabolism ; *Endodeoxyribonucleases/metabolism/chemistry ; Bacterial Proteins ; },
abstract = {A contemporary question in the intensely active field of CRISPR-Cas12a-based molecular diagnostics is how to simplify the multistep conversion process for detecting nonnucleic acid targets. Herein we describe an allosteric Y-shaped DNA structure for Cas12a activation via protospacer-adjacent motif (PAM) topological engineering (Y-COPE) to achieve straightforward and diverse nuclease monitoring. The newly designed topological structure of the Y-COPE is characterized by a split PAM embedded at the three-way junction and protospacers flanking both sides. This unique spatial configuration of the PAM effectively prevents Cas12a activation. Upon target cleavage, the released truncated fragments can dynamically correct the PAM, which promptly restores the dsDNA conformation for Cas12a activation and accomplishes signal output. Theoretical calculation results revealed that, compared with the canonical dsDNA activator, in the Y-COPE, there was a 1.8 Å increase in the center distance between Lys595 of Cas12a and the PAM, which led to a 24.2 kcal mol[-1] increase in binding free energy. This clearly revealed the underlying inhibition mechanism of the topological configuration of the PAM for Cas12a activation. This study advances the understanding of the dynamic response of Cas12a to topological PAM conformations and introduces the universal concept of CRISPR-based nonnucleic acid detection to benefit the next-generation molecular diagnostics.},
}
@article {pmid40888991,
year = {2025},
author = {Lin, HK and Dai, J and Pusztai, L},
title = {Integrating large-scale in vitro functional genomic screen and multi-omics data to identify novel breast cancer targets.},
journal = {Breast cancer research and treatment},
volume = {214},
number = {3},
pages = {319-327},
pmid = {40888991},
issn = {1573-7217},
support = {BCRF-22-133, SAC220225//Breast Cancer Research Foundation Investigator Award (BCRF-22-133), Susan Komen Leadership Grant (SAC220225)/ ; },
mesh = {Humans ; Female ; *Breast Neoplasms/genetics/drug therapy/pathology ; *Genomics/methods ; DNA Copy Number Variations ; Cell Line, Tumor ; *Biomarkers, Tumor/genetics ; Mutation ; Gene Expression Regulation, Neoplastic ; Gene Expression Profiling ; CRISPR-Cas Systems ; Transcriptome ; Molecular Targeted Therapy ; Multiomics ; },
abstract = {PURPOSE: Our goal is to leverage publicly available whole transcriptome and genome-wide CRISPR-Cas9 screen data to identify and prioritize novel breast cancer therapeutic targets.<br><br>METHODS: We used DepMap dependency scores&#x2009;>&#x2009;0.5 to identify genes that are potential therapeutic targets in 48 breast cancer cell lines. We removed genes that were pan-essential or were not expressed in TCGA breast cancer cohort. Genes were prioritized based on druggability using the Drug-Gene Interaction Database. Targets were defined separately for ER+, HER2+, and TNBC. A broader list of genes with dependency score&#x2009;>&#x2009;0.25 were used to assess the associations between dependency scores and mutations and copy number variations (CNV) to identify potential synthetic lethal relationships and to map survival critical genes into biological pathways.<br><br>RESULTS: 66, 53, and 29 genes were prioritized as targets in ER+, HER2+, and TNBC, respectively. These included known actionable targets and many novel targets. ER+ included FOXA1, GATA3, LDB1, TRPS1, NAMPT, WDR26, and ZNF217; HER2+ cancers included STX4, HECTD1, and TBL1XR1; and TNBC included GFPT1 and GPX4. Synthetic lethal associations revealed 5 and 19 significant associations between potential survival critical genes and mutations in HER2+&#x2009;and TNBC, respectively. For example, PIK3CA mutation increased dependency on NDUFS3 in HER2+&#x2009;cancers, and CNTRL mutation increased dependency on electron transport chain (ETC) genes in TNBC. 329, 747, and 622 CNVs showed synthetic lethal association in ER+, HER2+, and TNBC, respectively.<br><br>CONCLUSION: We provide a genome-wide drug target prioritization list for breast cancer derived from integrated large-scale omics data.},
}
@article {pmid40590508,
year = {2025},
author = {Mohammadzadeh, R and Shahbazi, S and Khodaei, N and Sabzi, S},
title = {Emerging Therapeutic Strategies to Combat Antimicrobial Resistance in the Post-Antibiotic Era.},
journal = {Journal of basic microbiology},
volume = {65},
number = {11},
pages = {e70070},
doi = {10.1002/jobm.70070},
pmid = {40590508},
issn = {1521-4028},
mesh = {Humans ; *Bacterial Infections/therapy/microbiology/drug therapy ; *Anti-Bacterial Agents/pharmacology/therapeutic use ; *Drug Resistance, Bacterial/drug effects ; *Bacteria/drug effects/pathogenicity ; Probiotics/therapeutic use ; Bacteriophages ; Antimicrobial Peptides/therapeutic use/pharmacology ; Bacteriocins/therapeutic use ; Animals ; CRISPR-Cas Systems ; },
abstract = {Antimicrobial resistance (AMR) is a serious global health issue. This review aims to explore alternative therapeutic strategies for combating AMR. The goal is to evaluate emerging treatments that target resistant pathogens through novel mechanisms, bypassing the limitations of traditional antibiotics. Recent researches highlight several promising alternatives, including antibodies, antimicrobial peptides, bacteriocins, bacteriophages, and probiotics (in the clinical trials) and synthetic antimicrobial peptides, anti-virulence strategies, genetically modified phages, antibacterial oligonucleotides, CRISPR-Cas9, and predatory bacteria (in the research stage). These therapies demonstrate potential to overcome AMR by targeting specific bacterial mechanisms, reducing toxicity, and evading resistance. Alternative therapies for AMR present significant promise, offering new avenues for treatment. Despite challenges in optimization and delivery, these therapies could revolutionize the way bacterial infections are treated. Continued research is crucial to address hurdles and ensure these therapies can be safely and effectively implemented in clinical settings, shaping the future of infection management.},
}
@article {pmid41174869,
year = {2025},
author = {Li, S and Zhang, B and Ma, P and Zhang, Y and Hu, Z and Wu, X and Chen, Q and Zhao, Y},
title = {The 13-lipoxygenase GmLOX6 is involved in JA biosynthesis and serves as a positive regulator of salt stress tolerance in soybean.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {3},
pages = {e70550},
pmid = {41174869},
issn = {1365-313X},
support = {2023ZD04036//Biological Breeding-National Science and Technology Major Project/ ; 2021YFD1201104-02-02//National Key Research and Development Program of China/ ; 2023YFD2300101//National Key Research and Development Program of China/ ; 31971899//National Natural Science Foundation of China/ ; 32272072//National Natural Science Foundation of China/ ; 32272093//National Natural Science Foundation of China/ ; U23A20192//National Natural Science Foundation of China/ ; },
mesh = {*Glycine max/genetics/physiology/enzymology/metabolism ; *Lipoxygenase/metabolism/genetics ; *Oxylipins/metabolism ; *Cyclopentanes/metabolism ; *Salt Tolerance/genetics/physiology ; *Plant Proteins/metabolism/genetics ; Salt Stress ; Gene Expression Regulation, Plant ; Reactive Oxygen Species/metabolism ; Plant Growth Regulators/metabolism ; Plants, Genetically Modified ; alpha-Linolenic Acid/metabolism ; CRISPR-Cas Systems ; },
abstract = {Salinity represents a major abiotic stressor that significantly impairs soybean growth and yield. Although jasmonic acid (JA) has been firmly established as a key regulator of plant defense against salt stress, the precise functions of lipoxygenase (LOX) genes responsible for initiating JA biosynthesis remain poorly defined. Here, a comprehensive genome-wide analysis of the soybean LOX gene family was performed, and a detailed functional characterization of GmLOX6 was carried out. Subcellular localization confirmed that GmLOX6 is targeted to chloroplasts, while enzymatic assays demonstrated that it acts as a 13-LOX enzyme with a strong preference for α-linolenic acid as substrate. To clarify its role under salt stress, we generated both overexpression and CRISPR/Cas9-mediated knockout lines of soybean. Phenotypic and molecular evaluations revealed that GmLOX6 facilitates JA production under salt stress, thereby contributing to enhanced JA accumulation. This elevation in JA levels was associated with improved salt tolerance through multiple physiological adaptations, including the activation of antioxidant enzymes for the detoxification of reactive oxygen species (ROS), enhanced Na[+] extrusion to preserve ionic balance, and reinforced membrane stability. Moreover, GmRWP-RK11 was identified as a transcriptional repressor of GmLOX6. Functional disruption of GmRWP-RK11 via CRISPR/Cas9 conferred greater salt tolerance, further supporting its negative regulatory role. Collectively, these findings uncover a novel regulatory axis in which GmLOX6-mediated JA biosynthesis enhances soybean resistance to salinity through modulation of ROS homeostasis and Na[+] transport. These insights provide an expanded understanding of the transcriptional and biochemical mechanisms underpinning JA-driven stress adaptation in soybean.},
}
@article {pmid41174319,
year = {2026},
author = {Fatmi, MQ and Nadeem, A and Abbasov, M and Sajjad, M},
title = {Computational Methods to Engineer Cas Proteins for Efficient Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2979},
number = {},
pages = {279-300},
pmid = {41174319},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; Molecular Dynamics Simulation ; *CRISPR-Cas Systems/genetics ; *Protein Engineering/methods ; *Computational Biology/methods ; Mutation ; *CRISPR-Associated Proteins/genetics/chemistry ; },
abstract = {The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) system has revolutionized genome editing through programmable, sequence-specific deoxyribonucleic acid (DNA) targeting. Yet, its broader application remains limited by off-target effects and context-dependent efficiency. To address these challenges, we present an integrated computational protocol with easy-to-do steps for researchers to guide the rational design of CRISPR/Cas variants with improved stability and specificity. The integrated workflow begins with coevolutionary coupling analysis to identify conserved and covarying residues critical for function. These residues are then evaluated for energetically favorable substitutions through mutant stability prediction, followed by network centrality analysis to evaluate the impact of mutations on intramolecular communication pathways, preserving key allosteric interactions. Finally, molecular dynamics (MD) simulations validate the structural integrity and dynamic behavior of the selected variants. Network analysis and molecular dynamics (MD) simulations are applied iteratively, allowing insights from MD to refine network-based evaluations and vice versa. This multiscale strategy offers a streamlined and systematic approach for engineering optimized Cas proteins for genome editing applications.},
}
@article {pmid41173880,
year = {2025},
author = {Binenbaum, J and Adamkova, V and Fryer, H and Xu, L and Gorringe, N and Włodzimierz, P and Burns, R and Papikian, A and Jacobsen, SE and Henderson, IR and Harris, CJ},
title = {CRISPR targeting of H3K4me3 activates gene expression and unlocks centromere-proximal crossover recombination in Arabidopsis.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9587},
pmid = {41173880},
issn = {2041-1723},
support = {ERC Starting Grant (TransPlantMemory)//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; EP/Z001749/1//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; ERC Advanced Grant (EvoPanCen)//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; EP/X025306/1//RCUK | Engineering and Physical Sciences Research Council (EPSRC)/ ; URF\R1\201016//Royal Society/ ; doctoral studentship//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/V003984/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; },
mesh = {*Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/genetics/metabolism ; *Histones/metabolism/genetics ; *Gene Expression Regulation, Plant ; *Centromere/genetics/metabolism ; *Crossing Over, Genetic ; *CRISPR-Cas Systems/genetics ; Histone-Lysine N-Methyltransferase/genetics/metabolism ; Disease Resistance/genetics ; Meiosis/genetics ; Plants, Genetically Modified ; },
abstract = {H3K4me3 is a fundamental and highly conserved chromatin mark across eukaryotes, playing a central role in many genome-related processes, including transcription, maintenance of cell identity, DNA damage repair, and meiotic recombination. However, identifying the causal function of H3K4me3 in these diverse pathways remains a challenge, and we lack the tools to manipulate it for agricultural benefit. Here we use the CRISPR-based SunTag system to direct H3K4me3 methyltransferases in the model plant, Arabidopsis thaliana. Targeting of SunTag-SDG2 activates the expression of the endogenous reporter gene, FWA. We show that SunTag-SDG2 can be employed to increase pathogen resistance by targeting the H3K4me3-dependent disease resistance gene, SNC1. Meiotic crossover recombination rates impose a limit on the speed with which new traits can be transferred to elite crop varieties. We demonstrate that targeting of SunTag-SDG2 to low recombining centromeric regions can significantly stimulate proximal crossover formation. Finally, we reveal that the effect is not specific to SDG2 and is likely dependent on the H3K4me3 mark itself, as the orthogonal mammalian-derived H3K4me3 methyltransferase, PRDM9, produces a similar effect on gene expression with reduced off-target potential. Overall, our study supports an instructive role for H3K4me3 in transcription and meiotic recombination and opens the door to precise modulation of important agricultural traits.},
}
@article {pmid41173850,
year = {2025},
author = {Brown, BC and Tokolyi, A and Morris, JA and Lappalainen, T and Knowles, DA},
title = {Large-scale causal discovery using interventional data sheds light on gene network structure in k562 cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9628},
pmid = {41173850},
issn = {2041-1723},
support = {K99HG012373//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; K99HG012792//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01AG057422//U.S. Department of Health &amp; Human Services | NIH | National Institute on Aging (U.S. National Institute on Aging)/ ; U01AG068880//U.S. Department of Health &amp; Human Services | NIH | National Institute on Aging (U.S. National Institute on Aging)/ ; R01MH106842//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; },
mesh = {Humans ; *Gene Regulatory Networks ; K562 Cells ; CRISPR-Cas Systems ; },
abstract = {Inference of directed biological networks is an important but notoriously challenging problem. The recent proliferation of large-scale CRISPR perturbation data provides a new opportunity to tackle this problem by leveraging the transcriptional response to the presence of a gene-targeting guide. Here, we introduce inverse sparse regression (inspre), an approach to learning causal networks that leverages large-scale intervention-response data. Applied to 788 genes from the genome-wide perturb-seq dataset, inspre discovers a network with small-world and scale-free properties. We integrate our network estimate with external data, finding relationships between gene eigencentrality and both measures of gene essentiality and gene expression heritability. Our analysis helps to elucidate the structure of networks that may underlie complex traits.},
}
@article {pmid39832721,
year = {2025},
author = {Mao, X and Xiong, J and Cai, M and Wang, C and He, Q and Wang, B and Chen, J and Xiao, Z and Wang, B and Han, S and Zhang, Y},
title = {SCARB1 links cholesterol metabolism-mediated ferroptosis inhibition to radioresistance in tumor cells.},
journal = {Journal of advanced research},
volume = {77},
number = {},
pages = {207-219},
doi = {10.1016/j.jare.2025.01.026},
pmid = {39832721},
issn = {2090-1224},
mesh = {*Ferroptosis/genetics ; Humans ; *Radiation Tolerance/genetics ; *Cholesterol/metabolism ; *Scavenger Receptors, Class B/metabolism/genetics ; Lipid Peroxidation ; Cell Line, Tumor ; Animals ; *Neoplasms/metabolism/radiotherapy/pathology/genetics ; Mice ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; },
abstract = {INTRODUCTION: Ferroptosis is an iron-dependent form of cell death triggered by the excessive accumulation of lipid peroxides. Understanding the regulatory mechanisms of ferroptosis and developing strategies to target this process hold significant clinical applications in tumor therapy.<br><br>OBJECTIVE: Our study aims to search for novel candidate genes involved in the regulation of ferroptosis and to investigate their mechanism of action in ferroptosis and tumor therapy.<br><br>METHODS: We employed a CRISPR-Cas9 library to perform a genome-wide screen under ferroptosis inducer treatment conditions, revealing Scavenger Receptor Class B Member 1(SCARB1) as a novel candidate gene involved in ferroptosis regulation. Subsequently, lipidomic analyses, metabolic interventions, and relevant cellular experimental analyses were performed to elucidate the role of SCARB1 in ferroptosis, lipid peroxidation, and tumor therapy.<br><br>RESULTS: Our study confirmed that SCARB1 significantly inhibits ferroptosis and lipid peroxidation induced by ferroptosis inducers. Mechanistically, SCARB1 inhibits ferroptosis through the regulation of cholesterol metabolism, and the upregulation of CoQ10 level is demonstrated to mediate the suppression of ferroptosis by SCARB1 after lipidomic analysis and metabolic intervention. Interestingly, SCARB1 exerts a tumor suppressive effect regarding tumor growth, migration and invasion, which is possibly independent of ferroptosis regulation. However, SCARB1 promotes radioresistance through the upregulation of cholesterol metabolism and inhibition of ferroptosis, while the combination of ferroptosis inducers can overcome radioresistance in tumor cells with high SCARB1 expression.<br><br>CONCLUSION: This study establishes a theoretical foundation for the regulation of ferroptosis by SCARB1 and highlights the potential of targeting lipid metabolism to overcome radioresistance in cancer therapy. The identification of SCARB1 as a key player in ferroptosis and its dual role in tumor suppression and radioresistance provides new avenues for therapeutic intervention in cancer treatment.},
}
@article {pmid41172978,
year = {2025},
author = {Sancho-Shimizu, V},
title = {VUS no more: Decoding inborn errors of immunity for clinical action.},
journal = {Cell},
volume = {188},
number = {22},
pages = {6105-6106},
doi = {10.1016/j.cell.2025.09.009},
pmid = {41172978},
issn = {1097-4172},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Phenotype ; },
abstract = {Saturation genome editing meets functional phenotyping to turn sequencing ambiguity into actionable diagnoses.},
}
@article {pmid41172903,
year = {2025},
author = {Zhang, J and Zhu, M and Yan, H and Qiao, J and Liu, Y},
title = {Split CRISPR/Cas systems: Pioneering solutions for molecular diagnostics challenges.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118177},
doi = {10.1016/j.bios.2025.118177},
pmid = {41172903},
issn = {1873-4235},
abstract = {Split CRISPR/Cas systems have recently emerged as revolutionary tools in molecular diagnostics, addressing the limitations of conventional CRISPR-based detection methods, such as low sensitivity and specificity for low-abundance targets and limited regulatory flexibility. This review highlights "split-activation" strategies that enhance analytical performance without requiring pre-amplification. It elaborates on two key approaches: split activator-mediated Cas systems for ultrasensitive RNA detection and split crRNA architectures for multiplex nucleic acid analysis. These innovations achieve significant improvements in sensitivity for femtomolar-level biomarkers while maintaining superior single-base discrimination. We examine their applications in clinical and on-site monitoring, analyze current challenges including background noise, multiplex capacity, and cost considerations. Future directions include developing customized Cas variants, nanomaterial-based amplification-free workflows, and integrated microfluidic platforms. This technology holds great promise for precision diagnostics, particularly in resource-limited settings.},
}
@article {pmid41171933,
year = {2025},
author = {Li, S and Vonesch, SC and Roy, KR and Tu, CS and Steudle, F and Nguyen, M and Jann, C and Steinmetz, LM},
title = {The editable landscape of the yeast genome reveals hotspots of structural variant formation.},
journal = {Science advances},
volume = {11},
number = {44},
pages = {eady9875},
pmid = {41171933},
issn = {2375-2548},
mesh = {*Genome, Fungal ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Saccharomyces cerevisiae/genetics ; *Genomic Structural Variation ; Whole Genome Sequencing ; INDEL Mutation ; },
abstract = {It is unclear how CRISPR editing outcomes vary across the genome and whether undesirable events such as structural variants (SVs) are predictable or preventable. To define a genome-wide map of editability, we performed whole-genome sequencing on 1875 budding yeast clones edited across 16 chromosomes by CRISPR-Cas9 and donor-templated repair. We found that unintended edits, including short indels and SVs, were enriched in specific genomic and sequence contexts. We developed a predictive model, SCORE (System for CRISPR Outcome and Risk Evaluation), which revealed 4.8% of the genome as SV prone, consisting of 562 SV hotspots. Donor repair-enhancing strategies suppressed SV formation in regions with moderate, but not high, predicted risk. Applying SCORE to the Sc2.0 synthetic yeast genome revealed a markedly altered SV landscape due to the removal of endogenous repetitive elements and the insertion of loxP sites. Our study provides the genome-scale map of SV hotspots after CRISPR editing and predictive and experimental tools to mitigate their formation.},
}
@article {pmid41171921,
year = {2025},
author = {Zhang, H and You, J and Zhou, H and Zhang, Z and Wu, H and Zhang, D and Pan, X and Zhang, W and Zhang, X and Rao, Z},
title = {The two-component nuclease-active KELShedu system confers broad antiphage activity via abortive infection.},
journal = {Science advances},
volume = {11},
number = {44},
pages = {eadv4747},
pmid = {41171921},
issn = {2375-2548},
mesh = {*Escherichia coli/virology/genetics/metabolism ; *Bacteriophages/physiology ; *Escherichia coli Proteins/metabolism/genetics ; *DNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Nucleotides/metabolism ; },
abstract = {Bacteriophages and bacteria engage in a continuous evolutionary arms race, driving the development of intricate bacterial defense systems such as CRISPR-Cas, BREX (Bacteriophage Exclusion), Gabija, and Shedu. Here, we characterize a two-component KELShedu system in Escherichia coli that confers resistance to phages via abortive infection. The KELShedu system comprises KELA, a double-stranded DNA-binding protein, and KELB, a metal ion-dependent nuclease harboring the DUF4263 domain. In addition, we find that physiological levels of nucleotide triphosphates (NTPs) inhibit the DNA cleavage activity of the KELShedu system, suggesting that KELShedu's activation depends on reduced intracellular NTP levels during phage invasion. Our research demonstrates that the KELShedu system responds to nucleotide depletion triggered by phage replication, leading to nonspecific degradation of cellular DNA and ultimately inducing abortive infection. These insights into the KELShedu system expand the repertoire of bacterial antiphage mechanisms and lay the groundwork for applications in microbial engineering and therapeutic development.},
}
@article {pmid41171707,
year = {2025},
author = {Vadakkethil, AA and Panda, S and Mitra, A and Dash, M and Baig, MJ and Angadi, UB and Kumar, D and Jaiswal, S and Asif Iquebal, M and Molla, KA},
title = {CRISPR-GATE: a one-stop repository and guide to computational resources for genome editing experimentation.},
journal = {Briefings in bioinformatics},
volume = {26},
number = {5},
pages = {},
pmid = {41171707},
issn = {1477-4054},
support = {//Indian Council of Agricultural Research/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Computational Biology/methods ; *Software ; *Databases, Genetic ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Internet ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein (CRISPR-Cas) has emerged and evolved as a revolutionary genome editing technology, transforming research across diverse biological disciplines. Over the past decade, this technology has unveiled numerous opportunities for precise genome manipulation. However, the processes of discovering Cas proteins, repurposing them as editing tools, selecting appropriate candidate tool from the CRISPR-toolbox, designing experiments, and analyzing data are often complex and require careful consideration. To support researchers at every stage of CRISPR experimentation, a wide array of web resources has been developed. In this article, we provide a comprehensive overview of standalone and web-based tools that assist in the identification of CRISPR-Cas systems and the design of guide RNAs (gRNAs). We also highlight tools for evaluating gRNA efficiency, predicting CRISPR-Cas9 mutation profiles, as well as tools for base editing and prime editing, and the analysis and visualization of experimental results. Additionally, we introduce CRISPR-Gateway for Accessing Tools and Resources (CRISPR-GATE), an all-inclusive web repository that consolidates publicly available tools for genome editing research. This repository offers a categorized and user-friendly interface, allowing researchers to quickly access relevant tools based on their specific needs. CRISPR-GATE aims to streamline the search for CRISPR resources, facilitating both education and accelerating innovation. The web repository can be accessed from https://crispr-gate.daasbioinfromaticsteam.in/.},
}
@article {pmid41171314,
year = {2025},
author = {Zaheer, A and AboQuella, NM and Wadan, AS and Saad, HA and Kumar, D and Panjwani, S and Rath, S and Ahmed, SI},
title = {CRISPR-based gene therapy for huntington's disease: current advances and future prospects.},
journal = {Neurogenetics},
volume = {26},
number = {1},
pages = {76},
pmid = {41171314},
issn = {1364-6753},
mesh = {*Huntington Disease/therapy/genetics ; Humans ; *Genetic Therapy/methods/trends ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Huntingtin Protein/genetics ; Disease Models, Animal ; },
abstract = {CRISPR-Cas9 technology offers transformative potential in treating Huntington's Disease (HD) by directly addressing its genetic root causes. This manuscript explores the pathophysiological mechanisms of HD, characterized by toxic mutant huntingtin (mHTT) protein resulting from expanded CAG repeats in the HTT gene, and the challenges posed by current therapeutic limitations. We comprehensively review the mechanisms of CRISPR-based therapeutic strategies, including excision of expanded repeats, allele-specific targeting, and epigenome editing, highlighting their efficacy in preclinical studies using animal models and human iPSCs. Delivery methods, such as viral and non-viral vectors, are analysed for their role in optimizing therapeutic outcomes while minimizing off-target effects and immune responses. Ethical and safety considerations, especially regarding precision and long-term impacts, are critically examined alongside emerging strategies to enhance specificity. With ongoing clinical trials and advancements in delivery systems, CRISPR technology represents a paradigm shift in addressing HD and broader neurodegenerative conditions. This review underscores the promise of gene editing in overcoming existing barriers and paving the way for transformative therapeutic approaches.},
}
@article {pmid41170849,
year = {2025},
author = {Yu, T and Xie, J and Huang, X and Huang, J and Bao, G and Yuan, W and Gao, C and Liu, C and Hu, J and Yang, W and Li, G},
title = {BaeR and H-NS control CRISPR-Cas-mediated immunity and virulence in Acinetobacter baumannii.},
journal = {mSystems},
volume = {},
number = {},
pages = {e0106725},
doi = {10.1128/msystems.01067-25},
pmid = {41170849},
issn = {2379-5077},
abstract = {Acinetobacter baumannii balances its remarkable ability to acquire antibiotic resistance genes via horizontal gene transfer (HGT) with the immune defense functions of its CRISPR-Cas system, forming a dynamic equilibrium governed by intricate transcriptional regulation. However, the regulatory mechanisms underlying the I-Fb CRISPR-Cas system in A. baumannii remain poorly understood. This study elucidated a multitiered regulatory axis mediated by BaeR and H-NS that coordinates immune defense and virulence expression in the I-Fb CRISPR-Cas system. Using DNA pull-down and electrophoretic mobility shift assay (EMSA), we demonstrated that H-NS directly binds AT-rich regions within the cas3 promoter, suppressing both interference activity and adaptive immunity of the I-Fb CRISPR-Cas system. Intriguingly, the two-component regulator BaeR controlled this suppression by positively regulating H-NS expression. The results revealed that Δcas3 mutants exhibited increased biofilm thickness, elevated the extracellular matrix component poly N-acetyl glucosamine (PNAG) production, upregulated pilus expression, and significantly enhanced epithelial cell adhesion. Strikingly, Δh-ns-cas3 and ΔbaeR-cas3 double-knockout strains showed no statistically significant differences in virulence phenotypes compared to the Δcas3 single mutants. These findings indicate CRISPR-Cas-mediated inhibition of biofilm formation is abolished upon cas3 deletion, thereby releasing the regulatory constraints imposed by BaeR and H-NS. This dysregulation leads to excessive biofilm and extracellular matrix component accumulation, ultimately amplifying bacterial colonization capacity and pathogenicity in host environments. This discovery reveals the dual regulatory roles of BaeR and H-NS in the A. baumannii I-Fb CRISPR-Cas system, mediating both immune defense and virulence modulation. These insights establish a theoretical foundation for novel antimicrobial strategies targeting CRISPR-Cas regulatory networks.IMPORTANCEA. baumannii, a leading cause of drug-resistant nosocomial infections, evolves antibiotic resistance through horizontal gene transfer (HGT) while employing CRISPR-Cas systems to limit foreign DNA invasion. This study reveals that the I-Fb CRISPR-Cas system, typically a defense mechanism, functions as a repressor of virulence traits in A. baumannii. We demonstrate that the transcriptional regulators H-NS and BaeR form a hierarchical axis suppressing Cas3 expression, thereby constraining biofilm formation and host adhesion. Strikingly, CRISPR-Cas deficiency enhances virulence, thickens biofilms, elevates PNAG production, and enhances epithelial colonization through escape from BaeR-/H-NS-mediated control. This work redefines CRISPR-Cas as a dual-function module balancing immune defense and pathogenicity, exposing the BaeR-H-NS-Cas3 axis as a druggable target for novel anti-infectives aimed at disrupting bacterial adaptive evolution.},
}
@article {pmid41072237,
year = {2026},
author = {Xin, M and Liu, J and Zhou, H and Bu, S and Hao, Z and Sun, H and Lu, J and Feng, X and Jiang, X and Wang, Q and Wan, J},
title = {An H1N1 virus biosensor based on enzyme activity-gated PER-CRISPR/Cas12a cascade signal amplification.},
journal = {Enzyme and microbial technology},
volume = {192},
number = {},
pages = {110759},
doi = {10.1016/j.enzmictec.2025.110759},
pmid = {41072237},
issn = {1879-0909},
mesh = {*Influenza A Virus, H1N1 Subtype/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; Influenza, Human/diagnosis/virology ; SARS-CoV-2/isolation &amp; purification/genetics ; RNA, Viral/analysis/genetics ; Nucleic Acid Amplification Techniques/methods ; DNA, Viral/analysis/genetics ; DNA, Single-Stranded ; Bacterial Proteins ; },
abstract = {The rapid and accurate detection of the H1N1 influenza virus is a key link in epidemic prevention and control. This study innovatively constructed a cascade signal amplification biosensor based on DNA polymerase activity regulation, aiming to achieve ultra-sensitive and highly specific detection of viral nucleic acids. This biosensor has the following significant advantages: (i) Molecular lock-key regulation mechanism: A functional DNA inhibitor is designed to form a complex with Taq DNA polymerase, and the target H1N1 RNA is specifically recognized to release enzyme activity inhibition, converting the target presence information into a PER reaction initiation signal. (ii) Cascade signal amplification system: The single-stranded DNA generated by PER activates Cas12a trans-cleavage activity, achieving a three-level signal amplification of enzyme activity activation → nucleic acid synthesis → CRISPR cleavage. The biosensor exhibits a linear detection range between 1 pM and 1 μM, with a detection limit of 25 fM. Moreover, the platform showed high versatility and could be readily adapted for the detection of other pathogens such as SARS-CoV-2 by simply modifying the nucleic acid sequences of the inhibitor and activator. This study not only provides a new tool for the screening of H1N1 influenza virus, but also offers a novel strategy for the development of next-generation molecular detection technologies suitable for point-of-care diagnostics, indicating considerable application potential.},
}
@article {pmid40785075,
year = {2025},
author = {Zhu, L and Lin, Y and Yang, G and He, G and Pan, Z and Yu, G and Yao, J and Li, M and Zhou, L and Jiang, D},
title = {A Rapid On-Site Visualization Detection System for Fusarium oxysporum f. sp. cubense Tropical Race 4 Utilizing RPA-CRISPR/Cas12a.},
journal = {Plant disease},
volume = {109},
number = {10},
pages = {2062-2069},
doi = {10.1094/PDIS-11-24-2512-SR},
pmid = {40785075},
issn = {0191-2917},
mesh = {*Fusarium/genetics/isolation &amp; purification ; *Plant Diseases/microbiology ; *Musa/microbiology ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Fusarium wilt of banana, a disease with devastating impacts on banana plants, is primarily caused by a pathogenic fungus called Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). To facilitate control and interrupt the spread of Fusarium wilt of banana, we developed a rapid on-site visualization system for detecting Foc TR4 based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a. Based on Foc TR4-specific sequences, primers, crRNA, and ssDNAs were designed. The detection system exhibited high specificity, with amplification signals observed exclusively in samples containing Foc TR4. Additionally, the system showed high sensitivity, with a limit of detection (LOD) of approximately 20 copies, and high efficiency, with detection results generated within 1 h from time of amplification. Notably, this method does not require large-scale instruments, making it a convenient and rapid approach. The detection system represents the first instance of on-site visualization of Foc TR4. This system can be employed for early detection of Foc TR4 and can provide a technical reference for the rapid detection and applications in the field of other pathogens.},
}
@article {pmid40715781,
year = {2025},
author = {Zhang, H and Li, YC and Pang, D and Xie, C and Zhang, T and Li, Y and Li, Y and Jiang, ZY and Bu, GL and Liu, MM and Chen, YR and Fei, HX and Lin, RB and Wu, PH and Du, WT and Zhao, GX and Luo, YL and Han, P and Zhong, Q and Sun, C and Zeng, MS},
title = {Desmocollin 2 is a dominant entry receptor for Epstein-Barr virus infection of epithelial cells.},
journal = {Nature microbiology},
volume = {10},
number = {11},
pages = {2768-2780},
pmid = {40715781},
issn = {2058-5276},
mesh = {Animals ; Humans ; *Epithelial Cells/virology/metabolism ; *Virus Internalization ; *Desmocollins/metabolism/genetics ; *Herpesvirus 4, Human/physiology ; *Epstein-Barr Virus Infections/virology/metabolism ; Cell Line ; *Receptors, Virus/metabolism/genetics ; Cricetinae ; CRISPR-Cas Systems ; Viral Envelope Proteins/metabolism ; Membrane Glycoproteins/metabolism ; Viral Proteins ; Molecular Chaperones ; },
abstract = {Epstein-Barr virus (EBV) can infect B cells and epithelial cells, and cause lymphomas and various epithelial malignancies. During epithelial cell infection, EBV employs a complex combination of viral glycoproteins and host receptors. However, the exact mechanism and whether a dominant receptor exists remain unclear. Here we identify desmocollin 2 (DSC2) as a dominant EBV entry receptor for epithelial cell infection using CRISPR-Cas9 screening. Knockout of DSC2 reduced EBV infection in both nasopharyngeal and gastric epithelial cell lines, and infection was rescued when DSC2 expression was restored. Expression of human DSC2 in non-EBV-susceptible hamster cell lines enabled susceptibility to EBV. Furthermore, we found that DSC2 directly binds to EBV glycoprotein H/glycoprotein L through its extracellular domain, particularly the preEC-EC2 regions, which could be targeted by polyclonal antibodies, therefore blocking EBV infection in primary epithelial cells. DSC2 enabled virus entry independent of Ephrin receptor A2. These findings could aid development of currently unavailable animal models and support development of targeted therapies.},
}
@article {pmid41170434,
year = {2025},
author = {Araújo, MRB and Dos Santos, LS and Prates, FD and Perini, HF and Silva, JS and Ramos, JN and Bokermann, S and Sacchi, CT and de Mattos Guaraldi, AL and Campos, KR and Cardoso, TDCS and Castro, DLC and Silva, MA and Sousa, M&#xc2;B and Vieira, VV and Santos, MBN and Camargo, CH and Andrade, BS and da Silva, MV and Sant'Anna, LO and Viana, MVC and Azevedo, V},
title = {Virulence and mutations analysis based on the whole genome of a Brazilian Corynebacterium diphtheriae strain isolated from a cutaneous infection.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1579154},
pmid = {41170434},
issn = {1664-302X},
abstract = {Corynebacterium diphtheriae is the main etiological agent of diphtheria, a potentially fatal disease whose most severe signs and symptoms result from the action of an exotoxin, the diphtheria toxin (DT). Although non-toxigenic C. diphtheriae strains have been associated with several diseases, including cutaneous infections and endocarditis, they are not monitored in many countries, and their mechanisms of virulence and antimicrobial resistance remain underexplored. Therefore, this study aimed to provide a comprehensive characterization -through genomic, in vitro, and in vivo analyses - of a non-toxigenic C. diphtheriae strain (46855) isolated from a leg lesion, highlighting its pathogenic potential and resistance profile. The isolate was assigned to a novel sequence type (ST-925) and was found to be resistant to tetracycline and rifampin. Multiple antimicrobial resistance genes were predicted in the genome, such as tet(33), rbpA, and rpoB2, in addition to mutations in the rpoB gene. A diverse set of virulence-associated genes related to adhesion, iron uptake systems, gene regulation, and post-translational modification was also identified. The isolate was able to form biofilm in vitro and exhibited strong virulence in Galleria mellonella larvae and A549 human pneumocyte cells. Finally, the structural analysis of the rpoB gene, carried out for the first time in this study, linked the observed mutations to rifampin resistance in C. diphtheriae. In summary, the data revealed that C. diphtheriae 46855, although non-toxigenic, harbors multiple genes associated with antimicrobial resistance and virulence, emphasizing the need for greater surveillance and functional studies on non-toxigenic strains.},
}
@article {pmid41170433,
year = {2025},
author = {Zhang, S and Chu, M and Sun, X},
title = {The arms race in bacteria-phage interaction: deciphering bacteria defense and phage anti-defense mechanisms through metagenomics.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1687307},
pmid = {41170433},
issn = {1664-302X},
abstract = {Bacteriophages are viruses that specifically infect bacteria and co-evolve with their hosts through mutual interactions. They represent one of the most significant drivers of microbial diversity, influencing its evolution, generation, and maintenance. To counter bacteriophage infection, bacteria have developed sophisticated immune systems, including both passive adaptations, such as inhibiting phage adsorption and preventing DNA entry, and active defense systems such as restriction-modification systems and CRISPR-Cas systems. The ongoing arms race between bacteriophages and bacteria has left distinct evolutionary signatures in their genomic sequences. Advances in large-scale genomic and metagenomic sequencing technologies, coupled with bioinformatics approaches, have greatly enhanced our understanding of bacteria-phage interaction mechanisms, driving progress in bacteriophage biology. This review systematically analyses the diverse immune strategies bacteria employ against phage infection, elucidates the coordination and interrelationships among different anti-phage mechanisms, and highlights potential directions for future research.},
}
@article {pmid41168338,
year = {2025},
author = {Elsayed, EM and Stukenberg, D and Meier, D and Schmeck, B and Becker, A},
title = {RECKLEEN is a lambda Red/CRISPR-Cas9 based single plasmid platform for enhanced genome editing in Klebsiella pneumoniae.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1509},
pmid = {41168338},
issn = {2399-3642},
support = {LOEWE/2/13/519/03/06.001(0002)/74//Hessisches Ministerium f&#xfc;r Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; },
mesh = {*Klebsiella pneumoniae/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Plasmids/genetics ; *Bacteriophage lambda/genetics ; *Genome, Bacterial ; },
abstract = {Klebsiella pneumoniae (Kp) has evolved as a major public health threat due to its multidrug-resistance (MDR) and hypervirulence. Current Kp genome-editing tools are constrained by cumbersome workflows, low flexibility, and limited scalability. Here, we present the RECKLEEN system -Recombineering/CRISPR-based KLebsiella Engineering for Efficient Nucleotide editing - as a single plasmid platform designed for precise genetic manipulation of Kp. RECKLEEN combines lambda Red recombineering with powerful CRISPR-Cas9-based targeted counterselection, achieving up to 99.998% killing efficiency. By implementing the near PAM-less SpG Cas9 variant in RECKLEEN, the compatible target sequence spectrum was significantly broadened. This approach enables deletions, point mutations, and DNA integrations, with efficiencies reaching 100% of the counter-selected clones. Simultaneous multi-target deletions were accomplished with up to 72% efficiency. To streamline the process, we developed a toolbox of eleven plasmids based on a modular cloning standard, enabling time- and resource-efficient assembly of editing constructs. This allows a 5-days workflow, from plasmid construction to the generation of strains with the desired genetic modification(s). The efficacy of RECKLEEN extends to various MDR Kp strains, such as ATCC 700721, ATCC BAA-1705, and ATCC 700603, demonstrating its broad applicability. RECKLEEN significantly enhances genome-editing capabilities for Kp, advancing research into its pathology and MDR mechanisms.},
}
@article {pmid41167881,
year = {2025},
author = {Meng, X and Yue, Y and Huang, M and Duan, Z and Liu, K and Wu, L},
title = {DNAzyme-CRISPR driven dual-mode biosensor with nanozyme signal amplification for on-site Pb[2+] detection.},
journal = {Analytica chimica acta},
volume = {1379},
number = {},
pages = {344711},
doi = {10.1016/j.aca.2025.344711},
pmid = {41167881},
issn = {1873-4324},
mesh = {*Lead/analysis ; *DNA, Catalytic/chemistry/metabolism ; *Biosensing Techniques/methods ; Electrochemical Techniques ; *CRISPR-Cas Systems ; Food Contamination/analysis ; Limit of Detection ; Colorimetry ; Cerium/chemistry ; },
abstract = {BACKGROUND: Lead ion (Pb[2+]) is a toxic heavy metal that poses severe threats to food safety. Traditional methods like inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) rely on bulky instrumentation, which are limited by high costs, complex sample preparation requirements, and inability to meet the demands for rapid on-site testing. Nanozyme-based biosensors have emerged as promising alternatives, yet single-mode sensors often suffer from matrix interference in complex food samples. Therefore, developing a rapid and reliable on-site method is critical for Pb[2+] detection.<br><br>RESULTS: To address this challenge, a CRISPR/Cas12a-driven dual-mode biosensor integrating Pt/CeO2 nanozyme-mediated peroxidase activity with GR-5 DNAzyme recognition was developed. The biosensor's design capitalizes on GR-5 DNAzyme for Pb[2+]-specific recognition, triggering CRISPR/Cas12a-mediated cleavage of electrochemical/colorimetric signal probe SH-ssDNA-Pt/CeO2 to generate dual signals. This innovative platform synergizes electrochemical precision and colorimetric simplicity for Pb[2+] detection in complex food matrices. The biosensor achieved an ultra-sensitive electrochemical response (linear range: 0.002-200&#xa0;nM; limit of detection: 0.14 pM) alongside a robust colorimetric readout (linear range: 0.5-2000&#xa0;nM; limit of detection: 0.47&#xa0;nM), representing significant sensitivity improvements over conventional single-mode sensors. Crucially, the intrinsic cross-verification mechanism between orthogonal signal modalities minimized false positives while ensuring >90.5&#xa0;% recovery in spiked corn, edible oil, beef and red wine samples with RSD <5&#xa0;%. These results highlight the potential of the dual-mode sensor as a practical, field deployable sensing platform for the detection of Pb[2+].<br><br>SIGNIFICANCE: This work innovatively integrates the specificity of CRISPR with the catalytic properties of nanozymes into a self-validating electrochemical/colorimetric dual-mode system. The detection method not only establishes a robust platform for highly sensitive, reliable and visualized detection of Pb[2+] in complex food matrices, but also pioneers a new paradigm for multi-modal biosensor design.},
}
@article {pmid41165985,
year = {2026},
author = {Bicknell, R and Koltunow, AMG},
title = {Pilosella: A Dicotyledonous Model for Studying Aposporous, Autonomous Apomixis.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2987},
number = {},
pages = {209-220},
pmid = {41165985},
issn = {1940-6029},
mesh = {*Apomixis/genetics ; Seeds/genetics/growth &amp; development ; *Asteraceae/genetics/physiology/growth &amp; development ; Ovule/genetics ; CRISPR-Cas Systems ; },
abstract = {Pilosella, a member of the Compositae, is a model system used to study the molecular genetics of aposporous apomixis. These plants are small, rapidly growing perennials that are easy to cultivate both in the greenhouse and in tissue culture. Apomixis in Pilosella occurs by apospory where mitotically derived embryo sacs arise adjacent to cells undergoing female gamete meiosis in the ovule. Seed initiation is autonomous, where both embryo and endosperm form without fertilization in the aposporous embryo sac. Apomixis is not fully penetrant in Pilosella. Instead, plants are facultatively apomictic, and apomixis can be easily scored through the simple decapitation of the immature capitulum bud. Natural sexual and facultatively apomictic forms are readily cross-compatible, facilitating comparative studies of inheritance and allele function. A wide range of experimental methods have been described for these plants, including histological techniques for studying the cytological aspects of apomixis, an efficient Agrobacterium-mediated transformation system, CRISPR/Cas9 mutagenesis, and mapping approaches that use deletion mutation and segregation in polyhaploid populations. Freely available online resources include a genome assembly, a molecular map based on cDNA markers and a transcriptome database. Collectively, these resources make Pilosella a highly tractable experimental system for studying the genetic control of native apomixis.},
}
@article {pmid41165983,
year = {2026},
author = {Radoeva, T and Rigola, D and Op den Camp, RHM and van Dijk, PJ and Underwood, CJ},
title = {Targeted Mutagenesis in Natural Apomicts.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2987},
number = {},
pages = {181-191},
pmid = {41165983},
issn = {1940-6029},
mesh = {*Apomixis/genetics ; CRISPR-Cas Systems ; *Mutagenesis ; *Taraxacum/genetics ; Parthenogenesis/genetics ; Chromosome Mapping ; Genes, Plant ; },
abstract = {Apomixis-clonal reproduction through seeds-is an alternative reproductive strategy that takes place in less than 0.1% of plant species and has evolved independently in diverse plant lineages. To date, the genetic basis of apomixis has been unraveled genetically in only a few genera. The identification of causal apomixis genes is technically challenging, as apomictic species are typically polyploid and the genetic loci associated with apomixis are often in low-recombination regions limiting conventional fine-mapping. In triploid apomictic dandelion (Taraxacum officinale), after conventional genetic mapping, deletion mapping, and complete apomixis loci haplotype assembly, we made use of targeted mutagenesis using CRISPR/Cas9 technology to identify the Taraxacum officinale PARTHENOGENESIS (ToPAR) gene that is responsible for embryogenesis in the absence of fertilization. Here, we report the methods used to clone the ToPAR gene by targeted mutagenesis and we expect that the general principles could be applied in other systems to identify novel apomixis genes.},
}
@article {pmid41165418,
year = {2025},
author = {Kumar, U and Dwivedi, D and Das, U},
title = {Advancements in CRISPR-Mediated Multiplex Genome Editing: Transforming Plant Breeding for Crop Improvement and Polygenic Trait Engineering.},
journal = {Biotechnology journal},
volume = {20},
number = {11},
pages = {e70148},
doi = {10.1002/biot.70148},
pmid = {41165418},
issn = {1860-7314},
support = {DBTHRDPMU/JRF/BET-24/I/2024-25/376//Department of Biotechnology/ ; 24J/01/00130//Council of Scientific and Industrial Research/ ; 3/1/3/BRET-2024/HRD (L1)//Indian Council of Medical Research/ ; AICE-JRF/SRF-KK04002406//Indian Council of Agricultural Research/ ; },
mesh = {*Gene Editing/methods ; *Plant Breeding/methods ; *Crops, Agricultural/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Multifactorial Inheritance/genetics ; Genome, Plant ; },
abstract = {With accelerating climate change and the urgent need to stack polygenic traits, multiplex CRISPR/Cas offers a scalable route to resilient crops-yet low editing efficiency and regeneration bottlenecks remain critical constraints. This review centers on multiplex strategies for polygenic trait engineering in plants, surveying compact nucleases (Cas9, Cas12, Cas13 and emerging ultra-compact variants), polycistronic gRNA platforms (tRNA-gRNA arrays, self-cleaving ribozymes, Csy4 processing), and delivery routes (Agrobacterium, biolistics, protoplast transfection, viral vectors). We highlight concrete outcomes-for example, targeted edits in PYL ABA-receptors increased rice grain yield by up to 31% in field tests-and applications from yield and disease resistance to abiotic-stress tolerance, nutrient biofortification and de novo domestication. Technical risks (off-targets, mosaicism, chromosomal rearrangements, transformability) are appraised alongside emerging fixes: compact/engineered nucleases, RNA-processing arrays, morphogenic regulators, and AI-driven sgRNA design integrated with multi-omics. By prioritizing multiplex approaches for polygenic trait stacking, the review argues that these tools are essential to accelerate precision breeding for climate-adapted agriculture.},
}
@article {pmid41163074,
year = {2025},
author = {Dashti, M and Mohammaddust Sarab, M and Shad, F and Dehnavi, S},
title = {CRISPR-mediated engineering of mesenchymal stromal/stem cells: a summary of recent progress in immunological applications for regenerative medicine and cancer therapy.},
journal = {Stem cell research &amp; therapy},
volume = {16},
number = {1},
pages = {592},
pmid = {41163074},
issn = {1757-6512},
mesh = {Humans ; *Mesenchymal Stem Cells/immunology/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Neoplasms/therapy/immunology ; *Regenerative Medicine/methods ; Gene Editing ; Animals ; *Mesenchymal Stem Cell Transplantation ; },
abstract = {Mesenchymal stromal/stem cells (MSCs) have introduced as a cornerstone of regenerative medicine, owing to their immunomodulatory properties and therapeutic potential in autoimmune and inflammatory disorders. Although, their clinical application is often restricted due to immune rejection and heterogeneity in immunoregulatory responses. The advent of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has revolutionized MSC engineering, enabling precise genetic modifications to enhance their immunological efficacy. This review explores how CRISPR-mediated editing of MSCs can mitigate immunogenicity, amplify anti-inflammatory functions, and repurpose MSCs for targeted immunotherapy. Key strategies include knockout of β2-microglobulin to evade T-cell recognition, augmentation of anti-inflammatory mediators like interleukin (IL)-10 and TNF-alpha stimulated gene/protein 6 (TSG-6), and disruption of pro-inflammatory pathways such as toll-like receptor 4 (TLR4)/NF-κB. In addition, CRISPR-engineered MSCs demonstrate promise in reshaping tumor microenvironments and combating bacterial infections through enhanced innate immunity. Despite challenges including off-target effects and delivery optimization, CRISPR-tailored MSCs represent a transformative approach to overcoming immunological barriers, paving the way for universal, off-the-shelf therapies in rheumatoid arthritis, cancer, and beyond.},
}
@article {pmid41147512,
year = {2025},
author = {Mirzaei, F and Mosaffa Jahromi, A and Molavi, H and Kabelitz, D and Kalantar, K and Meri, S},
title = {Targeting RNA-Binding proteins Roquin-1 and Regnase-1 could enhance CAR-iPSC-derived macrophage immunotherapy for solid tumors: a perspective and challenges.},
journal = {RNA biology},
volume = {22},
number = {1},
pages = {1-7},
doi = {10.1080/15476286.2025.2581385},
pmid = {41147512},
issn = {1555-8584},
mesh = {Humans ; *Ribonucleases/genetics/metabolism ; *Neoplasms/therapy/immunology/genetics ; *Macrophages/immunology/metabolism ; Tumor Microenvironment/immunology ; *RNA-Binding Proteins/genetics ; *Induced Pluripotent Stem Cells/cytology/metabolism/immunology ; Animals ; *Immunotherapy/methods ; Receptors, Chimeric Antigen/genetics/metabolism/immunology ; *Ubiquitin-Protein Ligases/genetics ; CRISPR-Cas Systems ; Gene Editing ; *Immunotherapy, Adoptive/methods ; Transcription Factors ; },
abstract = {Solid tumours present major treatment obstacles because of their immunosuppressive microenvironment and poor response to traditional chimeric antigen receptor (CAR)-based immunotherapies. Recent advances in cellular engineering have introduced CAR-macrophages derived from induced pluripotent stem cells (CAR-iMacs) as a promising approach to get around these obstacles. CAR-iMacs are designed to attack tumours, but their phenotypic plasticity can cause them to transform into M2-like macrophages in the tumour environment (TME), where they may instead suppress immune responses and promote tumour progression and metastasis. Roquin-1 and Regnase-1 are RNA-binding proteins that act as negative regulators of inflammatory genes that contribute to the phenotypic plasticity of macrophages. This perspective highlights a novel approach to augmenting anti-tumour responses of CAR-iMacs by simultaneously knocking out Roquin-1 and Regnase-1 via CRISPR-Cas9 gene editing. This approach drives a shift from an immunosuppressive M2-like state to an M1 state, promoting sustained pro-inflammatory signalling, boosting phagocytic and cytotoxic capabilities within the tumour microenvironment. Addressing a serious constraint in conventional adoptive cell therapies, this dual-targeting platform could provide a potent and scalable immunotherapeutic treatment for solid malignancies.},
}
@article {pmid41037400,
year = {2025},
author = {Combredet, C and Ansel, M and Brunet, T},
title = {A selection-based knockout approach for a choanoflagellate reveals regulation of multicellular development by Hippo signaling.},
journal = {Cell reports},
volume = {44},
number = {10},
pages = {116345},
doi = {10.1016/j.celrep.2025.116345},
pmid = {41037400},
issn = {2211-1247},
mesh = {*Choanoflagellata/genetics/growth &amp; development/metabolism ; *Signal Transduction ; *Gene Knockout Techniques/methods ; *Protein Serine-Threonine Kinases/metabolism/genetics ; Animals ; CRISPR-Cas Systems/genetics ; },
abstract = {Choanoflagellates, the closest living relatives of animals, provide crucial insights into animal origins. The multicellular choanoflagellate Salpingoeca rosetta can be genetically modified, but existing knockout (KO) pipelines are time consuming and have variable efficiency. Here, we present a fast and robust KO method for S. rosetta. We use CRISPR-Cas9 to inactivate target genes by interrupting, or fully replacing, their coding sequence with a selectable antibiotic resistance cassette. We inactivated three known S. rosetta multicellular developmental regulators (rosetteless, couscous, and jumble) and two homologs of Hippo pathway genes that control multicellular size in animals (warts and yorkie). Interestingly, warts-KO rosettes were consistently larger than their wild-type counterparts. RNA sequencing revealed that Warts and Yorkie regulated several extracellular matrix genes involved in multicellularity (including couscous), suggesting that Hippo signaling regulates multicellular size in choanoflagellates by modulating matrix secretion. We discuss the potential of our method to accelerate choanoflagellate functional genetics.},
}
@article {pmid41026602,
year = {2025},
author = {Kalchschmidt, J and Kanno, T and Park, S and Dubois, WD and Zhao, Y and Trzaskoma, P and Thomas, CJ and Staudt, LM and O'Shea, JJ and Jung, S and Casellas, R},
title = {Biphasic control of the B cell transcriptome by mTORC1 and GSK3.},
journal = {Cell reports},
volume = {44},
number = {10},
pages = {116361},
doi = {10.1016/j.celrep.2025.116361},
pmid = {41026602},
issn = {2211-1247},
mesh = {Humans ; *Mechanistic Target of Rapamycin Complex 1/metabolism/genetics ; *B-Lymphocytes/metabolism ; *Glycogen Synthase Kinase 3/metabolism/genetics ; *Transcriptome/genetics ; Lymphoma, B-Cell/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; },
abstract = {A central question in immune regulation is how cells coordinate transcriptional responses to environmental cues. It remains unclear whether transcriptional regulation is controlled by isolated mechanism or integrated regulatory programs. Here, we develop a high-sensitivity, genome-wide CRISPR-Cas9 screening platform with 47 transcriptional reporters in human B cell lymphoma, identifying 4,440 regulators and 17,638 regulatory interactions. To enable the exploration of these networks, we establish B-LEARN, an interactive portal for data visualization and discovery. Our results reveal a large number of shared regulators across our 47 screens that act as context-dependent activators or repressors. Globally, we uncover a biphasic regulatory architecture in which mTORC1 and GSK3 exert opposing control over the B cell transcriptome. Notably, mTOR inhibition broadly activates key B cell genes, an effect antagonized by GSK3. Thus, B cell transcription is governed by an integrated, pathway-driven circuit, offering new targets to modulate gene expression in lymphoma and autoimmune disease.},
}
@article {pmid40693307,
year = {2025},
author = {Xia, Y and Guo, R and Lu, T and Jiang, S and You, K and Xia, X and Du, K and Kang, X},
title = {PagHB7/PagABF4-PagEPFL9 Module Regulates Stomatal Density and Drought Tolerance in Poplar.},
journal = {Plant biotechnology journal},
volume = {23},
number = {11},
pages = {4857-4871},
doi = {10.1111/pbi.70273},
pmid = {40693307},
issn = {1467-7652},
support = {2024M760220//the China Postdoctoral Science Foundation/ ; 2021YFD2200105//the National Key R&amp;D Program of China during the 14th Five-year Plan Period/ ; },
mesh = {*Populus/genetics/physiology/metabolism ; *Plant Stomata/physiology/genetics ; Droughts ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; *Transcription Factors/metabolism/genetics ; Plants, Genetically Modified ; Stress, Physiological ; CRISPR-Cas Systems ; Drought Resistance ; },
abstract = {Epidermal patterning factor-like 9 (EPFL9) influences stomatal density and growth in poplar. There have been no reports on homeobox 7 (HB7) and ABRE binding factor 4 (ABF4) regulating stomatal density or drought tolerance by targeting EPFL9 in poplar. This study revealed that EPFL9 was specifically localised in guard cells in leaves and responded to drought stress. By constructing CRISPR/Cas9-mediated PagEPFL9 gene-edited lines, we found that epfl9 mutant plants showed significantly reduced stomatal density, inhibited growth and enhanced drought resistance. However, PagEPFL9 overexpression increased its drought stress sensitivity by increasing the stomatal density. PagHB7 was demonstrated to be an upstream regulator of PagEPFL9 by yeast one-hybrid screening library experiments, yeast one-hybrid experiments, electrophoretic mobility shift assay and dual luciferase reporter gene assay experiments. Yeast two-hybrid, bimolecular fluorescence complementation, split luciferase complementation assays, GST pull-down, electrophoretic mobility shift assay and dual luciferase reporter gene assay experiments further demonstrated that PagHB7 interacted with PagABF4 and that PagABF4 enhanced the inhibitory effect of PagHB7 on PagEPFL9. Knockout plants of PagHB7, a negative regulator of PagEPFL9, had a significantly increased stomatal density and reduced drought tolerance. Poplars overexpressing PagABF4 showed similar phenotypes to poplars knocking out PagEPFL9, with stomatal density significantly lower than that of WT, which may result in greater drought tolerance. Our study demonstrates that PagHB7 and PagABF4 interact with each other and regulate stomatal density by targeting PagEPFL9, thereby affecting drought resistance in poplar. This study provides new genetic resources for molecular design breeding of plant growth and drought tolerance.},
}
@article {pmid41162659,
year = {2025},
author = {Campbell, IW and Basta, DW and Zingl, FG and Sullivan, EJ and Doranga, S and Waldor, MK},
title = {Anoxia activates CRISPR-Cas immunity in the mouse intestine.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {41162659},
issn = {2058-5276},
support = {P30 DK034854/DK/NIDDK NIH HHS/United States ; R01 AI042347/AI/NIAID NIH HHS/United States ; Investigator program//Howard Hughes Medical Institute (HHMI)/ ; },
abstract = {The natural context in which CRISPR-Cas systems are active in Enterobacteriaceae has remained enigmatic. Here we find that the Citrobacter rodentium type I-E CRISPR-Cas system is activated by the oxygen-responsive transcriptional regulator Fnr in the anoxic environment of the mouse intestine. Since Fnr-dependent regulation is predicted in ~41% of Enterobacteriaceae cas3 orthologues, we propose that anoxic regulation of CRISPR-Cas immunity is an adaptation that protects Enterobacteriaceae against threats from foreign DNA within the intestinal microbiome.},
}
@article {pmid41162404,
year = {2025},
author = {Yan, X and Liu, B and Zhou, S and Fan, Y and Wei, S and Qiu, D and Xiang, H and Zhou, J and Mergny, JL and Monchaud, D and Ju, H and Zhou, J},
title = {A Chimeric Photo-Controllable CRISPR/Cas12a System for Universal and Fast Diagnostics.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c04782},
pmid = {41162404},
issn = {1520-6882},
abstract = {The potential of clustered regularly interspaced short palindromic repeats (CRISPR) and corresponding CRISPR-associated (Cas) protein systems (CRISPR/Cas) systems for biomedical applications is tremendous; however, precise control of their activity is essential to better harness this potential and, beyond this, to develop reliable diagnostic reagents. Herein, we report on such a strategy by controlling the CRISPR/Cas12a activity using a photo-controllable CRISPR RNA (crRNA). To this end, the 3' end of crRNA was conjugated to a G-quadruplex (G4) block through a photocleavable linker: upon photo irradiation, the G4 trigger is removed, thus allowing for the DNA target to access and hybridize with the crRNA, and thus be processed by the CRISPR/Cas12a system. The efficiency of this approach was demonstrated by the detection of human papillomavirus 16 DNA in 50 clinical samples: our one-pot strategy was found to be as efficient as the routinely implemented method (qPCR), with 95.7% sensitivity and 100% specificity, in addition to be faster (25 versus 60 min) and both simpler and less expensive (being implementable as lateral flow test strips). Collectively, this new and fully controllable CRISPR/Cas system holds great potential for next-generation clinical diagnostics.},
}
@article {pmid41162070,
year = {2025},
author = {Gautam, V and Jambagi, SR and Muthugounder, M},
title = {Genome editing of detoxification gene repertoires in insects using clustered regularly interspaced short palindromic repeats (CRISPR): A systematic review and meta-analysis.},
journal = {Pesticide biochemistry and physiology},
volume = {215},
number = {},
pages = {106687},
doi = {10.1016/j.pestbp.2025.106687},
pmid = {41162070},
issn = {1095-9939},
mesh = {Animals ; *Gene Editing ; *Insecta/genetics ; *Insecticide Resistance/genetics ; Insecticides/pharmacology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Inactivation, Metabolic/genetics ; },
abstract = {Decoding the molecular mechanisms underlying insect resistance to insecticides and host plant adaptation is essential for effective and sustainable Insecticide Resistance Management (IRM). Reverse genetic approaches targeting "target site resistance" can help control pests without harming pollinators and beneficial biocontrol agents. Understanding the role of mutations involved in xenobiotic resistance enables the judicious use of pesticides. CRISPR-based genome editing allows precise manipulation of detoxification genes, helping to decipher their roles in resistance development. This review provides a comprehensive overview of CRISPR-mediated genome editing in insect detoxification genes and their involvement in resistance mechanisms. In addition to synthesizing overall data trajectories, we present study-level effect sizes that highlight context-specific responses to gene editing, offering insights that can inform future experimental designs and functional validation studies.},
}
@article {pmid41161594,
year = {2025},
author = {Fu, H and Xu, W and Huang, M and Cong, Y},
title = {Molecular detection of Salmonella.},
journal = {Journal of food protection},
volume = {},
number = {},
pages = {100659},
doi = {10.1016/j.jfp.2025.100659},
pmid = {41161594},
issn = {1944-9097},
abstract = {The genus Salmonella consists of a group of globally significant foodborne pathogens that pose substantial public health risks. Traditional detection methods are inadequate for rapid diagnosis and effective epidemic surveillance due to limitations such as time-consuming procedures and insufficient sensitivity. In recent years, development of molecular techniques has driven innovations in Salmonella detection. Nucleic acid-based detection methods including polymerase chain reaction (PCR), real-time fluorescent quantitative PCR (qPCR), whole genome sequencing (WGS) and more emerge as crucial approaches for Salmonella detection due to their high sensitivity, specificity, and rapidity. Our review systematically summarized technological advancements in molecular detection of Salmonella, including specific genetic targets and drug resistance genes used for molecular detection, typing technologies, and emerging techniques such as CRISPR-Cas systems and microfluidic chips. This review comprehensively covers a wide array of molecular detection and characterization technologies, including conventional PCR, qPCR, multiplex PCR, digital PCR (dPCR), isothermal amplification techniques (such as loop-mediated isothermal amplification, recombinase polymerase amplification), genotyping methods (including pulsed-field gel electrophoresis, multilocus sequence typing etc.), WGS, melting curve analysis (MCA), and other emerging technologies. The review also discusses the balance between sensitivity and specificity in complex samples, challenges regarding the cost and accessibility of advanced technologies, as well as prospects for future development directions including portable point-of-care testing devices, automated detection equipment. Ongoing optimization of molecular detection technologies will provide critical support for the prevention and control of Salmonella infections.},
}
@article {pmid41161575,
year = {2025},
author = {Verma, R and Das, G and Manjunathachar, H and Muwel, N and Choudhary, R and Kumar, S and Nath, S and Gattani, A and Gupta, V and Sharma, RK and Ajith, Y},
title = {CRISPR-Cas systems: Pioneering next-generation diagnostic tools for parasitic diseases.},
journal = {Molecular and biochemical parasitology},
volume = {},
number = {},
pages = {111708},
doi = {10.1016/j.molbiopara.2025.111708},
pmid = {41161575},
issn = {1872-9428},
abstract = {Parasitic diseases pose significant threats to both human and veterinary health, causing morbidity, mortality, and economic losses. Effective diagnostics are critical, yet conventional methods such as microscopy, serology, and polymerase chain reaction (PCR) are limited by low sensitivity, cross-reactivity, or dependence on costly equipment and skilled personnel. Isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), have improved point-of-care (POC) applications but remain limited by nonspecific amplification and reduced sensitivity for low-copy targets. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems have emerged as transformative tools in molecular diagnostics, offering high sensitivity, specificity, rapidity, and cost-effectiveness. This review presents an overview of CRISPR-Cas systems, their historical development, classification (Class 1 and Class 2, Types I-VI), molecular mechanisms, and diagnostic potential in parasitic diseases, with illustrative examples from studies published between 2017 and May 2025. Despite significant progress, CRISPR-based diagnostics face challenges such as off-target activity, dependence on nucleic acid amplification, and complex sample preparation. Future directions focus on amplification-free detection, multiplexed assay development, and integration with nanotechnology, microfluidics, smartphone-based devices, and artificial intelligence. CRISPR-Cas technologies thus represent a promising frontier in next-generation diagnostics for parasitic disease surveillance, control, and personalized healthcare in both human and veterinary health.},
}
@article {pmid41161091,
year = {2025},
author = {Hong, SH and Kim, I and Lee, G and Kim, EH and Bae, E and Suh, JY},
title = {Structural and mechanistic investigation of the anti-CRISPR protein AcrIE5 using NMR spectroscopy and AlphaFold modeling.},
journal = {Biochemical and biophysical research communications},
volume = {789},
number = {},
pages = {152854},
doi = {10.1016/j.bbrc.2025.152854},
pmid = {41161091},
issn = {1090-2104},
abstract = {The CRISPR-Cas system employs RNA-guided endonucleases to protect bacteria and archaea from invading bacteriophages and plasmids. In response, bacteriophages have evolved anti-CRISPR proteins that inhibit diverse types of the CRISPR-Cas system. AcrIE5 was discovered from the mobile genetic elements of Pseudomonas aeruginosa, and potently inhibits the type I-E CRISPR-Cas system of P. aeruginosa. Here, we determined the solution structure of AcrIE5 using NMR spectroscopy, which adopts a novel αβ fold comprising three α-helices and two β-strands. AcrIE5 harbors a mobile loop between the β-strands that is conserved among homologs encoded by MGEs infecting Pseudomonas species, but truncated in homologs from MGEs of other bacteria. AlphaFold correctly reproduced the experimental structure of AcrIE5 and predicted its binding at a cleft formed by Cas8e, Cas7e, and Cas5e within the P. aeruginosa type I-E Cascade. The mobile loop and α-helices of AcrIE5 mediated key interactions with Cas8e at the PAM recognition site, as well as with adjacent Cas7e and Cas5e. AcrIE5 did not bind individual subunits of Cascade with high affinity, suggesting that it recognizes a composite interface of the functional Cascade assembly. Taken together, our findings suggest that AcrIE5 may compete with DNA binding to the PAM recognition site of type I-E Cascade, similar to AcrIE3 and AcrIE4, and also highlight a potential functional role of the conserved mobile loop in host-specific anti-CRISPR activity.},
}
@article {pmid41160700,
year = {2025},
author = {Elliott, SD and Ready, PJ and Wrinn, CM and Ma, Q and Edward, M and Niescier, RF and Escobar, I and Sun, J and Ganga, AK and McAtee, CK and Atiş, &#x130;S and Koleske, AJ and Bordey, A and Breslow, DK},
title = {A CRISPR activation screen reveals a cilia disassembly pathway mutated in focal cortical dysplasia.},
journal = {Science advances},
volume = {11},
number = {44},
pages = {eaeb7238},
pmid = {41160700},
issn = {2375-2548},
mesh = {*Cilia/metabolism/genetics/pathology ; Humans ; *Mutation ; *Malformations of Cortical Development/genetics/metabolism/pathology ; Cytoskeletal Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; rhoA GTP-Binding Protein/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Armadillo Domain Proteins/genetics/metabolism ; Animals ; Signal Transduction ; Focal Cortical Dysplasia ; },
abstract = {Defective assembly of primary cilia causes ciliopathies, but cilia disassembly and its role in disease remain poorly understood. From a genome-wide CRISPR activation (CRISPRa) screen for negative regulators of ciliary function, we find here that the F2R G protein-coupled receptor, sterile alpha and TIR motif-containing 1 (SARM1) hydrolase, ryanodine receptors, peri-centrosomal calcium signaling, and RhoA form a functional pathway that is necessary and sufficient for cilia disassembly. Highlighting the significance of this pathway, several components are somatically mutated in focal cortical dysplasia (FCD), a neurological disorder characterized by intractable epilepsy. Supporting the functional impact of these variants, patient-derived SARM1 and RhoA mutations potentiate cilia loss, and a RhoA variant impairs cortical development. Conversely, SARM1 inhibition restores cilia in cells with FCD-associated alterations. Together, our work identifies a pathway for cilia disassembly, implicates aberrant pathway activation as a feature of FCD-associated mutations, and illustrates the potential of CRISPRa screening to provide insight into diseases caused by somatic mutations.},
}
@article {pmid40991409,
year = {2025},
author = {Smith, SL and Iwamoto, Y and Manimaran, A and Drubin, DG},
title = {Harnessing fusion of genome-edited human stem cells to rapidly screen for novel protein functions in vivo.},
journal = {Molecular biology of the cell},
volume = {36},
number = {11},
pages = {ar141},
doi = {10.1091/mbc.E25-06-0301},
pmid = {40991409},
issn = {1939-4586},
mesh = {Humans ; *Gene Editing/methods ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Endocytosis/genetics ; Cell Fusion/methods ; Actin Cytoskeleton/metabolism ; CRISPR-Cas Systems/genetics ; Clathrin/metabolism ; Lysosomes/metabolism ; },
abstract = {Genome editing has enabled the integration of fluorescent protein coding sequences into genomes, resulting in expression of in-frame fusion proteins under the control of their natural gene regulatory sequences. While this technique overcomes the well-documented artifacts associated with gene overexpression for biological processes sensitive to altered protein stoichiometry, such as clathrin-mediated endocytosis (CME), editing genomes of metazoan cells incurs a significant time cost compared with simpler organisms, such as yeast. Editing two or more genes to express multiple fluorescent fusion proteins in a single cell line has proven to be a powerful strategy for uncovering spatial dynamic, and therefore functional, relationships among different proteins, but it can take many months to edit each gene within the same cell line. Here, by utilizing cell fusions, we quickly generated cells expressing pairwise permutations of fluorescent fusion proteins in genome-edited human cells to reveal previously undetected protein-organelle interactions. We fused human induced pluripotent stem cells (hiPSCs) that express in-frame fusions of CME and actin cytoskeleton proteins with hiPSCs that express fluorescently tagged organelle markers, uncovering novel interactions between CME proteins, branched actin filament networks, and lysosomes.},
}
@article {pmid40963020,
year = {2025},
author = {Chauhan, VP and Sharp, PA and Langer, R},
title = {Engineered prime editors with minimal genomic errors.},
journal = {Nature},
volume = {646},
number = {8087},
pages = {1254-1260},
pmid = {40963020},
issn = {1476-4687},
mesh = {*Gene Editing/methods ; *INDEL Mutation/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Deoxyribonuclease I/metabolism/genetics ; Humans ; *Genome/genetics ; DNA/genetics/metabolism/chemistry ; *Genomics ; },
abstract = {Prime editors make programmed genome modifications by writing new sequences into extensions of nicked DNA 3' ends[1]. These edited 3' new strands must displace competing 5' strands to install edits, yet a bias towards retaining the competing 5' strands hinders efficiency and can cause indel errors[2]. Here we discover that nicked end degradation, consistent with competing 5' strand destabilization, can be promoted by Cas9-nickase mutations that relax nick positioning. We exploit this mechanism to engineer efficient prime editors with strikingly low indel errors. Combining this error-suppressing strategy with the latest efficiency-boosting architecture, we design a next-generation prime editor (vPE). Compared with previous editors, vPE features comparable efficiency yet up to 60-fold lower indel errors, enabling edit:indel ratios as high as 543:1.},
}
@article {pmid41160667,
year = {2025},
author = {Du, X and Goh, PK and Ma, C and Coughlan, E and Greatorex, S and Porter, LH and Russ, B and Cummins, KD and Sek, K and Slaney, CY and Scott, AM and Oliaro, J and Neeson, PJ and Risbridger, GP and Taylor, RA and Trapani, JA and Turner, SJ and Darcy, PK and Wiede, F and Tiganis, T},
title = {Targeting PTPN2 enhances human CAR T cell efficacy and the development of long-term memory in mouse xenograft models.},
journal = {Science translational medicine},
volume = {17},
number = {822},
pages = {eadk0627},
doi = {10.1126/scitranslmed.adk0627},
pmid = {41160667},
issn = {1946-6242},
mesh = {Animals ; Humans ; *Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism/antagonists &amp; inhibitors ; *Xenograft Model Antitumor Assays ; Mice ; *Immunologic Memory ; *Receptors, Chimeric Antigen/metabolism/immunology ; Immunotherapy, Adoptive ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; },
abstract = {Chimeric antigen receptor (CAR) T cells have been ineffective against solid tumors, where the hostile tumor microenvironment limits CAR T cell function and persistence. Protein tyrosine phosphatase N2 (PTPN2) attenuates T cell receptor and cytokine signaling to maintain T cell tolerance. Here, we used CRISPR-Cas9 gene editing or an inhibitor to target PTPN2 in human CAR T cells specific for the Lewis Y (LeY) neoantigen, which is expressed in most epithelial tumors. Targeting PTPN2 increased CAR and cytokine signaling, including interferon signaling, and enhanced the antigen-induced expansion, activation, and cytotoxicity of anti-LeY CAR T cells in vitro and in vivo. The deletion of PTPN2 in CAR T cells repressed the growth of human tumor and patient-derived xenografts in mice, when compared with unedited CAR T cells, and prolonged mouse survival. The administration of inhibitor also enhanced the ability of α-LeY CAR T cells to repress tumor growth. Cellular indexing of transcriptomes and epitopes by sequencing analysis of splenic PTPN2-deficient CD8[+] CAR T cells in tumor-bearing mice revealed that PTPN2 deficiency favored the generation of CD45RA[+] CAR T cells expressing markers of long-lived stem cell memory (SCM) CAR T cells. Flow cytometric analysis reaffirmed that the deletion or inhibition of PTPN2 promoted the intratumoral accumulation of SCM CD8[+] CAR T cells and the overall persistence of CD8[+] CAR T cells. These data support the use of gene editing or small-molecule inhibitors targeting PTPN2 in human CAR T cells to treat solid tumors.},
}
@article {pmid41160625,
year = {2025},
author = {Bish, LM and Fuss, JL and Panaccione, DG},
title = {Gene editing of the thioester reductase step in the biosynthesis of lysergic acid amides.},
journal = {PloS one},
volume = {20},
number = {10},
pages = {e0334651},
doi = {10.1371/journal.pone.0334651},
pmid = {41160625},
issn = {1932-6203},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Lysergic Acid/analogs &amp; derivatives/metabolism ; *Amides/metabolism ; *Oxidoreductases/genetics/metabolism ; Peptide Synthases/genetics/metabolism ; Fungal Proteins/genetics/metabolism ; },
abstract = {Ergot alkaloids derived from lysergic acid are important in agriculture, as food and feed contaminants, and in medicine, as the foundation of several pharmaceuticals. The fungus Metarhizium brunneum makes several lysergic acid amides, with lysergic acid α-hydroxyethylamide (LAH) being produced in by far the highest concentration. The multifunctional enzyme lysergyl peptide synthetase 3 (Lps3) has multiple domains that play important roles in lysergic acid amide synthesis. We hypothesized a role for the reductase domain of Lps3 in liberating LAH from an enzyme-bound precursor and tested this hypothesis with CRISPR/Cas9-based gene editing experiments. We transformed M. brunneum with a Cas9/single guide RNA complex and a donor DNA that replaced the tyrosine at the active site of the reductase domain of Lps3 with a phenylalanine. Sanger sequencing of edited and wild-type genes demonstrated successful editing of the reductase domain without non-target mutations in Lps3. High performance liquid chromatography of the edited strain showed a significant reduction of LAH and accumulation of the precursor lysergic acid. The phenotype was similar when the edited allele of lpsC was in a wild-type background or in backgrounds with late pathway genes easO or easP knocked out, except no LAH was detectable when the edit was in the easO knockout background. The data demonstrate that the reductase domain plays a key role or roles in formation of LAH. The abundant lysergic acid accumulating in the mutants, as opposed to later pathway intermediates in LAH biosynthesis (such as lysergyl-alanine), indicated severe debilitation of Lps3. The data indicate a requirement for the reductase domain of Lps3 in synthesis of lysergic acid amides and demonstrate the feasibility of the CRISPR/Cas9-based approach for editing genes in Metarhizium species.},
}
@article {pmid41160062,
year = {2025},
author = {Yilmaz Çolak, &#xc7;},
title = {Harnessing CRISPR technology for the diagnosis of Bordetella pertussis: advances and implications.},
journal = {Future microbiology},
volume = {},
number = {},
pages = {1-8},
doi = {10.1080/17460913.2025.2581522},
pmid = {41160062},
issn = {1746-0921},
abstract = {Following the discovery of the prokaryotic adaptive immune system known as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins, this technology has revolutionized biotechnology as a multifaceted genome-editing tool with a wide range of applications. CRISPR technology has not only provided novel treatment options, especially for genetic diseases, but also transformed molecular diagnostic platforms. The specific, sensitive, and adaptable nature of the CRISPR-Cas systems has led to the development of innovative solutions for the detection of diseases, including viral and bacterial infections. This review provides an overview of the CRISPR-Cas systems and mainly focuses on the application of CRISPR-based assays for the detection of Bordetella pertussis, which is the main causative agent of a highly infectious disease, whooping cough. The review emphasizes the need for novel diagnostic tools for B. pertussis, along with highlighting some future perspectives, since its diagnosis can be challenging due to nonspecific early symptoms and interference from closely related Bordetella species. In this regard, CRISPR-based diagnostic platforms can offer a promising avenue for rapid and accurate detection of B. pertussis, helping the management of whooping cough.},
}
@article {pmid41159723,
year = {2025},
author = {Duan, Z and Yang, R and Lai, T and Jiang, W and Zhang, J and Chen, B and Liao, L},
title = {Development of a CRISPR/Cas9-induced gene editing system for Pseudoalteromonas fuliginea and its applications in functional genomics.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0177125},
doi = {10.1128/aem.01771-25},
pmid = {41159723},
issn = {1098-5336},
abstract = {Pseudoalteromonas has been used as a model system to study cold adaptation and is of widespread interest in biotechnology and ecology. To explore its physiological responses to extreme cold, uncover functional genes, and clarify their ecological roles, efficient genetic tools are essential. However, existing genetic manipulation methods in Pseudoalteromonas rely on traditional homology-based recombination, which is laborious and time-consuming in this bacterial system. Consequently, improving editing efficiency is crucial for advancing both basic research and applied potential. Here, we established a CRISPR/Cas9 system in Pseudoalteromonas and carried out an extensive investigation of the Type II CRISPR/Cas9 platform for gene editing in Pseudoalteromonas fuliginea, a representative species thriving in the frigid polar oceans. To validate the feasibility of the CRISPR/Cas system in P. fuliginea, multiple genes were selected as targets, and the gene editing effects were confirmed through phenotypic changes or gene expression. We have successfully achieved both gene knockouts and insertions in P. fuliginea, encompassing the deletion of genes such as fliJ, indA, and genes encoding Pf sRNAs, as well as the in vivo insertion of 3×FLAG and the gfp gene. The average CRISPR/Cas9 gene editing efficiency in P. fuliginea exceeded 70%. In summary, we developed an efficient CRISPR/Cas9-based editing system in P. fuliginea, which can be utilized to accelerate the development of Pseudoalteromonas as a model system for addressing fundamental questions related to extreme environmental adaptation and to fulfill its potential biotechnological applications.IMPORTANCEPseudoalteromonas fuliginea is a marine bacterium with great potential for ecological and biotechnological research, yet its genetic manipulation has long been a technical challenge. In this study, we developed a gene editing system based on CRISPR technology that enables efficient and precise genome modification in this organism. Using this system, we successfully deleted, inserted, and tagged multiple genes, including regulatory and non-coding elements, with high success rates. Notably, several of these genes are linked to key traits such as motility and stress response, which contribute to microbial adaptation in polar environments. This tool allows researchers to directly test gene function and study microbial adaptation in cold marine environments. The ability to perform reliable genetic edits in P. fuliginea opens new possibilities for its use as a model organism and will support future advances in microbial ecology, environmental microbiology, and marine biotechnology.},
}
@article {pmid41157107,
year = {2025},
author = {Shpiliukova, K and Kachanov, A and Brezgin, S and Chulanov, V and Ivanov, A and Kostyushev, D and Kostyusheva, A},
title = {m[6]A RNA Modification: Technologies Behind Future Anti-Cancer Therapy.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {20},
pages = {},
doi = {10.3390/molecules30204091},
pmid = {41157107},
issn = {1420-3049},
support = {075-15-2025-519//Ministry of Science and Higher Education of the Russian Federation (Federal scientific and technical program for the development of genetic technologies for 2019-2030/ ; },
mesh = {Humans ; *Adenosine/analogs &amp; derivatives/metabolism/genetics ; *Neoplasms/genetics/drug therapy/therapy/metabolism ; Methylation ; Animals ; Epigenesis, Genetic ; *Antineoplastic Agents/pharmacology/therapeutic use ; *RNA/genetics/metabolism ; *RNA Processing, Post-Transcriptional ; RNA Methylation ; },
abstract = {N6-methyladenosine (m[6]A) modifications are among the most prevalent epigenetic marks in eukaryotic RNAs, regulating both coding and non-coding RNAs and playing a pivotal role in RNA metabolism. Given their widespread influence, m[6]A modifications are deeply implicated in the pathogenesis of various cancers, including highly aggressive malignancies such as lung cancer, melanoma, and liver cancer. Dysregulation of m[6]A dynamics-marked by an imbalance in methylation and demethylation-can drive tumor progression, enhance metastatic potential, increase aggressiveness, and promote drug resistance, while also exerting context-dependent tumor-suppressive effects. Given this dual role, precise modulation of m[6]A levels and the activity of its regulatory enzymes (writers, erasers, and readers) represent a promising therapeutic avenue. In this review, we highlight recent advances in targeting m[6]A machinery, including small-molecule inhibitors, antisense oligonucleotides, and CRISPR/Cas-based editing tools, capable of both writing and erasing m[6]A marks and altering m[6]A methylation sites per se. By evaluating these strategies, we aim to identify the most effective approaches for restoring physiological m[6]A homeostasis or for strategically manipulating the m[6]A machinery for therapeutic benefit.},
}
@article {pmid41156655,
year = {2025},
author = {Peláez Sánchez, RG and González Restrepo, J and Pineda, S and Cuartas-López, AM and Martínez Garro, JM and Torres-Castro, M and Urrego, R and López-Rojas, LE and Salazar Florez, JE and Monroy, FP},
title = {Bioinformatic Identification of CRISPR-Cas Systems in Leptospira Genus: An Update on Their Distribution Across 77 Species.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {10},
pages = {},
doi = {10.3390/pathogens14101044},
pmid = {41156655},
issn = {2076-0817},
mesh = {*CRISPR-Cas Systems/genetics ; *Leptospira/genetics/classification ; *Computational Biology/methods ; Genome, Bacterial ; Bacteriophages/genetics ; Leptospirosis/microbiology ; },
abstract = {UNLABELLED: Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the Leptospira genus. Genome editing in Leptospira has been difficult to perform. Currently, the functionality of the CRISPR-Cas system has been demonstrated in species such as Leptospira interrogans. However, the different CRISPR-Cas systems present in most of the 77 species are unknown. Therefore, the objective of this study was to identify these arrays across the genomes of all described Leptospira species using bioinformatics tools.<br><br>METHODS: a bioinformatics workflow was followed: genomes were downloaded from the NCBI database; Cas protein detection was carried out using the CRISPR-CasFinder and RAST web servers; functional analyses of Cas proteins were performed with InterProScan, ProtParam, Swiss Model, Alphafold3, Swiss PDB Viewer, and Pymol; conservation pattern detection was conducted using MEGA12, and Seqlogos; spacer identification was carried out with the Actinobacteriophages database and BLAST version 1.4.0; and bacteriophage detection was performed using PHASTER, and PHASTEST.<br><br>RESULTS: Cas proteins were detected in 36 out of the 77 species of the Leptospira species, including Cas1 to Cas9 and Cas12. These proteins were classified into Class 1 and Class 2 systems, corresponding to types I, II, and V. Direct repeats and spacers were detected in 19 species, with the direct repeats exhibiting two conserved nucleotide motifs. Analysis of spacer sequences revealed 323 distinct bacteriophages. Additionally, three intact bacteriophages were detected in the genomes of four Leptospira species. Notably, two saprophytic species have complete CRISPR-Cas systems.<br><br>CONCLUSIONS: The presence of Cas proteins, direct repeats, and spacer sequences with homology to bacteriophage genomes provides evidence for a functional CRISPR-Cas system in at least 19 species.},
}
@article {pmid41155971,
year = {2025},
author = {Serrano, DR and Juste, F and Anaya, BJ and Ramirez, BI and Sánchez-Guirales, SA and Quispillo, JM and Hernandez, EM and Simon, JA and Trallero, JM and Serrano, C and Rawat, S and Lalatsa, A},
title = {Exosome-Based Drug Delivery: A Next-Generation Platform for Cancer, Infection, Neurological and Immunological Diseases, Gene Therapy and Regenerative Medicine.},
journal = {Pharmaceutics},
volume = {17},
number = {10},
pages = {},
doi = {10.3390/pharmaceutics17101336},
pmid = {41155971},
issn = {1999-4923},
support = {PID2024-156769OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; Innovation in Pharmacology, Nanotechnology, and personalized medicine by 3D printing//Universidad Complutense de Madrid/ ; },
abstract = {Exosomes, naturally derived extracellular vesicles, have emerged as powerful bio-nanocarriers in precision medicine. Their endogenous origin, biocompatibility, and ability to encapsulate and deliver diverse therapeutic payloads position them as transformative tools in drug delivery, gene therapy, and regenerative medicine. This review presents a comprehensive analysis of exosome-based therapeutics across multiple biomedical domains, including cancer, neurological and infectious diseases, immune modulation, and tissue repair. Exosomes derived from stem cells, immune cells, or engineered lines can be loaded with small molecules, RNA, or CRISPR-Cas systems, offering highly specific and low-immunogenic alternatives to viral vectors or synthetic nanoparticles. We explore endogenous and exogenous loading strategies, surface functionalization techniques for targeted delivery, and innovations that allow exosomes to traverse physiological barriers such as the blood-brain barrier. Furthermore, exosomes demonstrate immunomodulatory and regenerative properties in autoimmune and degenerative conditions, with promising roles in skin rejuvenation and cosmeceuticals. Despite their potential, challenges remain in large-scale production, cargo loading efficiency, and regulatory translation. Recent clinical trials and industry efforts underscore the accelerating momentum in this field. Exosomes represent a promising platform in precision medicine, though further standardization and validation are required before widespread clinical use. This review offers critical insights into current technologies, therapeutic mechanisms, and future directions to unlock the full translational potential of exosomes in clinical practice.},
}
@article {pmid41155374,
year = {2025},
author = {Honjo, A and Yako, H and Miyamoto, Y and Yagi, M and Yamamoto, M and Nishi, A and Sakagami, H and Yamauchi, J},
title = {Knocking Down FRMD4A, a Factor Associated with the Brain Development Disorder and a Risk Factor for Alzheimer's Disease, Using RNA-Targeting CRISPR/Cas13 Reveals Its Role in Cell Morphogenesis.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
doi = {10.3390/ijms262010083},
pmid = {41155374},
issn = {1422-0067},
mesh = {*Alzheimer Disease/genetics/metabolism/pathology ; Animals ; *CRISPR-Cas Systems ; Neurons/metabolism/cytology ; Mice ; *Morphogenesis/genetics ; *Cytoskeletal Proteins/genetics/metabolism ; *Membrane Proteins/genetics/metabolism ; Gene Knockdown Techniques ; Humans ; *Brain/metabolism ; Cell Line ; },
abstract = {Genetic truncation or mutation of the gene encoding band 4.1, ezrin, radixin, and moesin (FERM) domain protein containing 4A (FRMD4A) is associated with brain developmental diseases, including microcephaly with global developmental delay. It has also been identified as a risk factor for Alzheimer's disease. By analogy with other FERM domain-containing proteins, FRMD4A is believed to regulate cell morphogenesis and/or cell polarization in central nervous system (CNS) cells; however, it remains unclear whether and how dysfunction of FRMD4A and/or its closely homologous protein FRMD4B causes abnormal morphogenesis in neuronal cells. Here, we describe for the first time the roles of FRMD4A and FRMD4B in process elongation in neuronal cells. Knockdown of Frmd4a or Frmd4b using specific RNA-targeting clustered regularly interspaced short palindromic repeat (CRISPR) and Cas13-fitted gRNAs led to decreased process elongation in primary cortical neurons. Similar decreases in neuronal marker expression were observed in the N1E-115 cell line, a model of neuronal differentiation. Furthermore, hesperetin, an aglycone of the citrus flavonoid hesperidin known to promote neuroprotective signaling, recovered the decreased process elongation induced by the knockdown of Frmd4a or Frm4b. Hesperetin also stimulated phosphorylation of mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPKs/ERKs), which could help promote neuronal processes. These results suggest that FRMD4A and FRMD4B regulate process elongation through a possible signaling pathway linked to the sustained phosphorylation of MAPKs/ERKs. Crucially, this study reveals that, at the molecular and cellular levels, hesperetin can restore normal phenotypes when FRMD4A protein or FRMD4B protein is impaired.},
}
@article {pmid41155368,
year = {2025},
author = {Hossain, MM and Sultana, F and Mostafa, M and Khan, I and Tran, LP and Mostofa, MG},
title = {Reinforced Defenses: R-Genes, PTI, and ETI in Modern Wheat Breeding for Blast Resistance.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
doi = {10.3390/ijms262010078},
pmid = {41155368},
issn = {1422-0067},
mesh = {*Triticum/genetics/microbiology/immunology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; *Plant Breeding ; *Plant Immunity/genetics ; *Genes, Plant ; Magnaporthe/pathogenicity ; Host-Pathogen Interactions/genetics/immunology ; },
abstract = {Wheat blast, caused by Magnaporthe oryzae pathotype Triticum (MoT), poses a major threat to wheat (Triticum aestivum) cultivation, particularly in South America and Bangladesh. The rapid evolution and spread of the pathogen necessitate the development of durable and broad-spectrum resistance in wheat cultivars. This review summarizes current insights into the multi-layered defense mechanisms of wheat, encompassing resistance (R) genes, pattern-triggered immunity (PTI), and effector-triggered immunity (ETI) against MoT. The R-genes provide race-specific resistance through ETI, while both ETI and PTI are required to form integral layers of the plant immune system that synergistically reinforce host defense network. Recent advances in genomics, transcriptomics, and molecular breeding have facilitated the discovery and deployment of key R-genes and signaling components involved in PTI and ETI pathways. Integrating these immune strategies through gene pyramiding, marker-assisted selection (MAS), and genome editing offers a promising route towards enhanced and durable resistance in hosts. Harnessing and optimizing these multilayered immune systems will be pivotal to securing wheat productivity amid the growing threat of wheat blast.},
}
@article {pmid41155240,
year = {2025},
author = {Carbone, F},
title = {Special Issue: Latest Research on Plant Genomics and Genome Editing.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
doi = {10.3390/ijms26209946},
pmid = {41155240},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; *Genome, Plant ; *Genomics/methods ; *Plants/genetics ; CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; },
abstract = {Over the past ten years, plant science has undergone a remarkable transformation driven by the convergence of next-generation sequencing, increasingly sophisticated bioinformatics tools, and the rise of targeted genome editing platforms [...].},
}
@article {pmid41154642,
year = {2025},
author = {Zhang, H and Yang, Y and Yang, T and Cao, P and Yu, C and Liang, L and Liu, R and Chen, Z},
title = {Engineering a High-Fidelity MAD7 Variant with Enhanced Specificity for Precision Genome Editing via CcdB-Based Bacterial Screening.},
journal = {Biomolecules},
volume = {15},
number = {10},
pages = {},
doi = {10.3390/biom15101413},
pmid = {41154642},
issn = {2218-273X},
support = {2023YFC3402300//National Key R&amp;D Program of China/ ; 22208044//National Natural Science Foundation of China/ ; 22278058//National Natural Science Foundation of China/ ; XLYC2203075//"Xingliao Talent Plan"project/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; 2025JH2/101330156//Natural Science Foundation of Liaoning Province/ ; 2023JJ12SN030//Science and Technology Innovation Foundation of Dalian/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; DUT25LAB105//Fundamental Research Funds for the Central Universities/ ; DUT25RC(3)019//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Bacterial Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endonucleases/genetics/metabolism ; Mutation ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) nucleases enable precise genome editing, but off-target cleavage remains a critical challenge. Here, we report the development of MAD7_HF, a high-fidelity variant of the MAD7 nuclease engineered through a bacterial screening system leveraging the DNA gyrase-targeting toxic gene ccdB. This system couples survival to efficient on-target cleavage and minimal off-target activity, mimicking the transient action required for high-precision editing. Through iterative selection and sequencing validation, we identified MAD7_HF, harboring three substitutions (R187C, S350T, K1019N) that enhanced discrimination between on- and off-target sites. In Escherichia coli assays, MAD7_HF exhibited a >20-fold reduction in off-target cleavage across multiple mismatch contexts while maintaining on-target efficiency comparable to wild-type MAD7. Structural modeling revealed that these mutations stabilize the guide RNA-DNA hybrid at on-target sites and weaken interactions with mismatched sequences. This work establishes a high-throughput bacterial screening strategy that allows the identification of Cas12a variants with improved specificity at a given target site, providing a useful framework for future efforts to develop precision genome-editing tools.},
}
@article {pmid41153450,
year = {2025},
author = {Nardon, E and Azzalini, E and Paladin, D and Boscarino, D and Bonin, S},
title = {CRISPR/Cas Tools for the Detection of Borrelia sensu lato in Human Samples.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
doi = {10.3390/genes16101233},
pmid = {41153450},
issn = {2073-4425},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Lyme Disease/diagnosis/microbiology/genetics ; *Borrelia burgdorferi Group/genetics/isolation &amp; purification ; DNA, Bacterial/genetics ; Real-Time Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Bacterial Outer Membrane Proteins/genetics ; Antigens, Surface ; Bacterial Vaccines ; Lipoproteins ; },
abstract = {BACKGROUND/OBJECTIVES: Lyme disease diagnosis remains challenging due to the limitations of current methods. While PCR-based assays are widely used, their sensitivity can be affected by sample type and the inhibition of host DNA. This study aimed to evaluate the feasibility and sensitivity of a CRISPR/Cas12-based detection system for Borrelia burgdorferi sensu lato, comparing its performance with real-time PCR.<br><br>METHODS: DNA from three Borrelia genospecies (B. burgdorferi, B. garinii, and B. afzelii) was amplified targeting the OspA gene. Detection was performed using a Cas12/crRNA system with a fluorescent ssDNA reporter. Sensitivity assays were conducted on serial dilutions of Borrelia DNA, with and without human genomic DNA, and results were compared with qPCR.<br><br>RESULTS: Direct detection of Borrelia DNA without amplification was not feasible. However, when combined with PCR, the Cas12/crRNA system reliably detected as few as 5 genome copies per reaction. End-point PCR extended to 60 cycles improved detection robustness for B. garinii and B. afzelii, although sensitivity decreased in the presence of human genomic DNA.<br><br>CONCLUSIONS: The Cas12/crRNA-based system offers a sensitive and accessible alternative to qPCR, especially in settings lacking real-time PCR instrumentation. Future developments may include integration with isothermal amplification and microfluidic platforms to enhance direct detection capabilities.},
}
@article {pmid41153439,
year = {2025},
author = {Stunf Pukl, S},
title = {Genetic Therapy of Fuchs Endothelial Corneal Dystrophy: Where Are We? A Review.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
doi = {10.3390/genes16101222},
pmid = {41153439},
issn = {2073-4425},
mesh = {*Fuchs' Endothelial Dystrophy/therapy/genetics ; Humans ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Collagen Type VIII/genetics ; Mutation ; Transcription Factor 4/genetics ; Animals ; Gene Editing ; },
abstract = {OBJECTIVES: The incidence of Fuchs endothelial corneal dystrophy (FECD) is growing, and with it, the unmet need for a corneal transplant. Among alternative treatment modalities, only genetic therapy represents a causal therapy.<br><br>METHODS: Following the SNARA protocol, the PubMed and ClinicalTrials databases were searched using the keywords Fuchs endothelial corneal dystrophy, FECD, genetic therapy, and CRISPR-Cas9.<br><br>RESULTS: FECD is polyfactorial disease and mutations or polymorphisms in at least 15 different genes were connected to the disease. For the early-onset form of the disease, exclusive connection to mutations in COL8A2 was confirmed, while for the late-onset form, the most characteristic mutation is the expansion of the CTG18.1 triplet in the TCF4 gene, making these two possible targets. While the CRISPR-Cas9 approach represents the mainstay of genetic therapy development recently, the application of this method to FECD contains several obstacles, studied in preclinical settings. DT-168 and the Ad-Cas9-Col8a2gRNA molecules were developed for FECD treatment and preclinically tested, and phase I and II clinical studies for DT-168 are also already being performed.<br><br>CONCLUSIONS: The review of the literature proved that genetic therapy for FECD is at the level of preclinical research and that there are several specific challenges connected to the target genetic mutation as well as the delivery of possible treatment and duration of the effect. Further studies in the field might bring solutions in the future for alternative treatments for this common corneal disease.},
}
@article {pmid41153385,
year = {2025},
author = {Boti, MA and Diamantopoulos, MA and Scorilas, A},
title = {RNA-Targeting Techniques: A Comparative Analysis of Modern Approaches for RNA Manipulation in Cancer Research and Therapeutics.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
doi = {10.3390/genes16101168},
pmid = {41153385},
issn = {2073-4425},
mesh = {Humans ; *Neoplasms/genetics/therapy ; RNA Interference ; CRISPR-Cas Systems ; Oligonucleotides, Antisense/therapeutic use/genetics ; Animals ; *RNA/genetics ; },
abstract = {RNA-targeting techniques have emerged as powerful tools in cancer research and therapeutics, offering precise and programmable control over gene expression at the post-transcriptional level. Once viewed as passive intermediates in the central dogma, RNA molecules are now recognized as dynamic regulators of cellular function, capable of influencing transcription, translation, and epigenetic regulation. Advances in high-throughput sequencing technologies, transcriptomics, and structural RNA biology have uncovered a diverse landscape of coding and non-coding RNAs involved in oncogenesis, drug resistance, and tumor progression. In response, several RNA-targeting strategies have been developed to modulate these transcripts, including antisense oligonucleotides (ASOs), RNA interference (RNAi), CRISPR-Cas13 systems, small molecules, and aptamers. This review provides a comparative analysis of these technologies, highlighting their molecular mechanisms, therapeutic potential, and current limitations. Emphasis is placed on the translational progress of RNA-targeting agents, including recent FDA approvals and ongoing clinical trials for cancer indications. Through a critical comparison of these strategies, this review underscores the growing significance of RNA-targeting technologies as a foundation for next-generation cancer therapeutics and precision oncology.},
}
@article {pmid41152294,
year = {2025},
author = {Nemoto, A and Imaizumi, K and Miya, F and Hiroi, Y and Yamada, M and Ideno, H and Saitoh, S and Kosaki, K and Okuno, H and Okano, H},
title = {Rescue of imprinted genes by epigenome editing in human cellular models of Prader-Willi syndrome.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9442},
pmid = {41152294},
issn = {2041-1723},
mesh = {*Prader-Willi Syndrome/genetics/therapy ; Humans ; *Genomic Imprinting/genetics ; Induced Pluripotent Stem Cells/metabolism ; *Gene Editing/methods ; DNA Methylation/genetics ; CRISPR-Cas Systems ; *Epigenome/genetics ; Hypothalamus/metabolism ; Organoids/metabolism ; Chromosomes, Human, Pair 15/genetics ; Epigenesis, Genetic ; Epigenome Editing ; },
abstract = {Prader-Willi syndrome (PWS) is a genomic imprinting disorder caused by the loss of function of the paternal chromosome 15q11-13, resulting in a spectrum of symptoms associated with hypothalamic dysfunction. PWS patients lack the expression of paternally expressed genes (PEGs) in the 15q11-13 locus but possess an epigenetically silenced set of these genes in the maternal allele. Thus, activation of these silenced genes can serve as a therapeutic target for PWS. Here, we leverage CRISPR-based epigenome editing system to modulate the DNA methylation status of the PWS imprinting control region (PWS-ICR) in induced pluripotent stem cells (iPSCs) derived from PWS patients. Successful demethylation in the PWS-ICR restores the PEG expression from the maternal allele and reorganizes the methylation patterns in other PWS-associated imprinted regions beyond the PWS-ICR. Remarkably, these corrected epigenomic patterns and PEG expression are maintained following the differentiation of these cells into hypothalamic organoids. Finally, the single-cell transcriptomic analysis of epigenome-edited organoids demonstrates a partial restoration of the transcriptomic dysregulation observed in PWS. This study highlights the utility of epigenome editing technology as a therapeutic approach in addressing PWS and potentially other imprinting disorders.},
}
@article {pmid41152284,
year = {2025},
author = {Mishal, R and Meléndez-Zajgla, J and Rueda-Zarazúa, B and Labra-Barrios, ML and Castañón-Sánchez, CA and Uribe Carvajal, S and Padierna-Mota, L and Hernández-Hernández, JM and Leon-Avila, G and Pérez Rangel, A and Hernández-Martínez, E and Angeles-Morales, EB and Albalawi, IK and Luna-Arias, JP},
title = {RNA-seq analysis of wild-type and mutated TBPL1 gene in breast cancer cells lines through CRISPR/Cas9 approach reveals novel molecular signatures.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {37578},
pmid = {41152284},
issn = {2045-2322},
support = {Ph.D. fellowship 465228//Consejo Nacional de Humanidades, Ciencia y Tecnologia, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; PhD. fellowship 752614//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; },
mesh = {Humans ; *Breast Neoplasms/genetics/pathology ; *CRISPR-Cas Systems ; Female ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *TATA-Box Binding Protein/genetics/metabolism ; *Mutation ; RNA-Seq ; Transcriptome ; Cell Proliferation/genetics ; Gene Expression Profiling ; Cell Movement/genetics ; },
abstract = {Breast cancer is the leading cause of death among women globally. Several genes have been found to be transcriptionally dysregulated in cancer, according to recent studies. TATA-box binding protein (TBP) and its two paralogs, TBPL1 and TBPL2, play roles in human transcription. The TBPL1 gene is implicated in colorectal carcinomas by suppressing the expression of miR-18a. However, its function in breast cancer remains undisclosed. TBPL1 is distantly related to TBP and possesses a 40% similarity with TBP's core domain. In the present study, we explored the potential role of the TBPL1 gene in transcriptome regulation by knocking out the TBPL1 gene through the CRISPR/Cas9 method. Following the knockout of the TBPL1 gene, we examined the gene transcription patterns and compared them to wild-type cell lines. We observed disparate signatures of upregulated and downregulated genes in wild-type and mutated conditions. Healthy breast MCF-12F, and T47D, SKBR3, and MDA-MB-231 breast cancer cell lines were assessed, as these cancer cells exhibit overexpression of the TBPL1 gene. Next-generation sequencing data revealed distinct marker genes regulated by the TBPL1 gene and their potential involvement in cell migration, proliferation, anti-apoptosis, and metastasis. Additionally, we also discovered novel lncRNAs implicated in the transcriptome analysis of the TBPL1 knocked-out gene. Our investigation indicated that this gene might affect varied stages of breast cancer cell lines' cellular properties, such as cell duplication, morphology, and growth. It might also contribute to tumor formation in more aggressive cell lines like MDA-MB-231 in vivo.},
}
@article {pmid41152217,
year = {2025},
author = {Song, Z and Guo, J and Fan, Z and Huang, S and Li, G and Zhao, Z and Chen, B and Huang, S and Zheng, W and Wei, Y and Chen, Y and Huang, X and Liu, J and Wu, L and Wang, X},
title = {Noncanonical target-strand cytosine base editing via engineered Un1Cas12f1 platform.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9499},
pmid = {41152217},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Cytosine/metabolism ; Mice ; Male ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; Protein Engineering ; },
abstract = {CRISPR/Cas-derived base editors harness various deaminase or glycosylase activities to target bases within non-target strand (NTS) of the R-loop, catalyzing base conversions independent of double-strand break formation. To develop miniature BEs compatible with therapeutic viral vectors, we explore the compact Cas12f system. Through computational modeling and mutagenesis, we establish a highly active enUn1Cas12f1 protein, and subsequently construct the derivative cytosine BE (CBE). Remarkably, the engineered CBE exhibits an unexpected activity to also edit the target strand (TS), indicating its substantially expanded editable space. We refine this activity via a focused alanine scan, establishing a nickase-CBE that preferentially install TS edits (TSminiCBE). Further engineering with a non-specific DNA binding domain yields an optimized TS-editing BE that enables in vivo base edits in mice (male). Overall, through extensive engineering of the Cas12f platform and repurposing its intrinsic dynamics, our work establishes a strand-selectable miniature CBE toolkit with strong potential for diverse applications.},
}
@article {pmid41151616,
year = {2025},
author = {Terhalle, E and Rademacher, J},
title = {[Nontuberculous Mycobacteria: Diagnostic Challenges and Individualized Therapeutic Approaches].},
journal = {Deutsche medizinische Wochenschrift (1946)},
volume = {150},
number = {22},
pages = {1360-1366},
doi = {10.1055/a-2502-6525},
pmid = {41151616},
issn = {1439-4413},
mesh = {Humans ; *Mycobacterium Infections, Nontuberculous/diagnosis/drug therapy/epidemiology/therapy ; *Nontuberculous Mycobacteria/isolation &amp; purification ; Anti-Bacterial Agents/therapeutic use ; Precision Medicine ; },
abstract = {Non-tuberculous mycobacteria (NTM) are increasingly recognized as clinically relevant pathogens, particularly in countries with a low tuberculosis incidence. Recent data from Denmark demonstrate a continuous annual rise in NTM-related pulmonary disease (NTM-PD) of 4.6% over 3 decades, with more than half of the isolates associated with true disease. Structural lung diseases such as bronchiectasis, prior tuberculosis, and chronic pulmonary conditions are major risk factors, alongside immunodeficiencies and immunosuppressive therapies. The diagnosis of NTM-PD requires a combination of clinical symptoms, radiological findings, and the microbiological confirmation. Novel diagnostic tools, such as anti-GPL IgA serology and a CRISPR-Cas-based cfDNA assay, show promise for differentiating colonization from disease and monitoring treatment response, but the sputum culture remains essential for species identification and drug susceptibility testing. Treatment is complex and species-specific, with macrolides forming the backbone of most regimens. Refractory cases, particularly those involving Mycobacterium abscessus, pose therapeutic challenges and often require multidisciplinary management. Inhaled liposomal amikacin (ALIS) has shown benefit in refractory MAC disease. Clinical decision-making must balance efficacy, tolerability, and long-term adherence, highlighting the need for individualized treatment strategies and regular monitoring. This review outlines current evidence and practical recommendations for clinicians managing NTM-PD.},
}
@article {pmid41149352,
year = {2025},
author = {Qin, Y and Xie, J and Zhen, S},
title = {CRISPR-Cas12a and DNA Tetrahedron Assemblies Amplified Fluorescence Anisotropy for the Sensitive Detection of Hepatitis B Virus DNA.},
journal = {Biosensors},
volume = {15},
number = {10},
pages = {},
pmid = {41149352},
issn = {2079-6374},
support = {22322409//National Natural Science Foundation of China/ ; 21974109//National Natural Science Foundation of China/ ; SWU-KF25011//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Hepatitis B virus/genetics ; *DNA, Viral/analysis ; *Fluorescence Polarization/methods ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques ; Limit of Detection ; Hepatitis B ; },
abstract = {Fluorescence anisotropy (FA) has been widely used for analyzing biomolecules due to its high throughput, homogeneous detection, and strong resistance to photobleaching. However, the traditional FA method suffers from low sensitivity when the target molecules are small and rotate rapidly, often producing insignificant changes in the FA value. In this study, by combining double signal amplification through the trans-cleavage of CRISPR-Cas12a and DNA tetrahedron assemblies with a large molecular size, a new, fast, simple and highly sensitive FA method was constructed to achieve the quantitative detection of hepatitis B virus DNA (HBV-DNA). The experimental results showed that the linear range of this method was 0.5-9 nmol/L, and the detection limit (LOD = 3σ/k) was 48 pmol/L. In addition, the method demonstrated excellent selectivity and anti-interference, and it was successfully applied to detect HBV-DNA in human serum, indicating that this method has the potential for clinical diagnosis.},
}
@article {pmid41149312,
year = {2025},
author = {Zhou, Z and Cho, IH and Kadam, US},
title = {CRISPR-Cas-Based Diagnostics in Biomedicine: Principles, Applications, and Future Trajectories.},
journal = {Biosensors},
volume = {15},
number = {10},
pages = {},
pmid = {41149312},
issn = {2079-6374},
mesh = {*CRISPR-Cas Systems ; Humans ; *Biosensing Techniques ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems, originally identified as prokaryotic adaptive immune mechanisms, have rapidly evolved into powerful tools for molecular diagnostics. Leveraging their precise nucleic acid targeting capabilities, CRISPR diagnostics offer rapid, sensitive, and specific detection solutions for a wide array of targets. This review delves into the fundamental principles of various Cas proteins (e.g., Cas9, Cas12a, Cas13a) and their distinct mechanisms of action (cis- and trans-cleavage). It highlights the diverse applications spanning infectious disease surveillance, cancer biomarker detection, and genetic disorder screening, emphasizing key advantages such as speed, high sensitivity, specificity, portability, and cost-effectiveness, particularly for point-of-care (POC) testing in resource-limited settings. The report also addresses current challenges, including sensitivity limitations without pre-amplification, specificity issues, and complex sample preparation, while exploring promising future trajectories like the integration of artificial intelligence (AI) and the development of universal diagnostic platforms to enhance clinical translation.},
}
@article {pmid41149303,
year = {2025},
author = {Zhu, Z and Ma, H and Yao, H and Yuan, Y and Miao, X and Su, S},
title = {CRISPR-Enhanced Colorimetric Aptasensor for Adenosine Triphosphate Detection Based on MoS2-Based Nanozymes.},
journal = {Biosensors},
volume = {15},
number = {10},
pages = {},
pmid = {41149303},
issn = {2079-6374},
support = {SYW2025037//the Science and Technology Program of Suzhou/ ; 2024GRFX045//the Visiting Scholar Project for Higher Vocational Colleges in Jiangsu Province/ ; TC2024JCYL23//the Science and Technology Program of Taicang/ ; 2023JXKYTD01//the Innovation Team Funds of Suzhou Chien-shiung Institute of Technology/ ; },
mesh = {Colorimetry/methods ; *Adenosine Triphosphate/analysis ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide ; Molybdenum/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Disulfides/chemistry ; CRISPR-Cas Systems ; },
abstract = {As the direct energy source in organisms, accurate and simple detection of adenosine triphosphate (ATP) is of great significance. Herein, a colorimetric aptasensor for ATP determination was designed by integrating the CRISPR/Cas12a system with an aptamer, and with Prussian blue nanocube and gold nanoparticle co-functionalized MoS2 (MoS2-PBNCs-AuNPs) nanozymes. As expected, the introduced CRISPR/Cas12a system and aptamer could efficiently amplify the detection signal and improve the specific recognition ability, respectively. Meanwhile, the catalytic activity of the MoS2-PBNCs-AuNPs nanozymes can be regulated with the concentration of ATP. The high-affinity binding of ATP to the aptamer competitively inhibited aptamer-crRNA hybridization, causing fewer Cas12 proteins to be activated. As a result, the uncleaved single-stranded DNA (ssDNA) adsorbed onto the surface of nanozymes to effectively enhance their catalytic oxidation capability toward 3,3',5,5'-tetramethylbenzidine (TMB). According to this phenomenon, this CRISPR-enhanced colorimetric aptasensor can detect down to 0.14 μM ATP with high selectivity, reproducibility, and stability. In addition, acceptable recoveries and low relative standard deviations of the aptasensor for ATP determination suggest that it is promising for application in early detection of clinical-related diseases.},
}
@article {pmid41148810,
year = {2025},
author = {Liu, H and Zhang, P},
title = {Advances in β-Thalassemia Gene Therapy: CRISPR/Cas Systems and Delivery Innovations.},
journal = {Cells},
volume = {14},
number = {20},
pages = {},
pmid = {41148810},
issn = {2073-4409},
mesh = {*beta-Thalassemia/therapy/genetics ; Humans ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Animals ; *Gene Transfer Techniques ; beta-Globins/genetics ; },
abstract = {β-thalassemia is an inherited blood disorder caused by mutations in the β-globin (HBB) gene, leading to reduced or absent β-globin production, resulting in chronic anemia. While current therapies, including blood transfusions and hematopoietic stem cell transplantation, offer symptomatic relief, they are limited by complications and their limited accessibility. CRISPR-based gene editing technologies provide new therapeutic avenues by enabling the precise correction of HBB mutations or the reactivation of fetal hemoglobin (HbF) through the targeting of regulatory elements such as BCL11A. These approaches have shown promising preclinical and clinical outcomes. However, efficient and safe delivery remains a major challenge. Viral vectors offer high efficiency but raise concerns about immunogenicity and insertional mutagenesis, whereas non-viral systems such as lipid nanoparticles and engineered exosomes offer lower toxicity and modularity but face targeting limitations. This review highlights recent progress in CRISPR-based therapies for β-thalassemia and emerging delivery strategies to enhance clinical translation.},
}
@article {pmid41134871,
year = {2025},
author = {Charlier, J and Sherkatghanad, Z and Makarenkov, V},
title = {Similarity-based transfer learning with deep learning networks for accurate CRISPR-Cas9 off-target prediction.},
journal = {PLoS computational biology},
volume = {21},
number = {10},
pages = {e1013606},
doi = {10.1371/journal.pcbi.1013606},
pmid = {41134871},
issn = {1553-7358},
mesh = {*Deep Learning ; *CRISPR-Cas Systems/genetics ; Neural Networks, Computer ; Computational Biology/methods ; *Gene Editing/methods ; Humans ; Machine Learning ; },
abstract = {Transfer learning has emerged as a powerful tool for enhancing predictive accuracy in complex tasks, particularly in scenarios where data is limited or imbalanced. This study explores the use of similarity-based pre-evaluation as a methodology to identify optimal source datasets for transfer learning, addressing the dual challenge of efficient source-target dataset pairing and off-target prediction in CRISPR-Cas9, while existing transfer learning applications in the field of gene editing often lack a principled method for source dataset selection. We use cosine, Euclidean, and Manhattan distances to evaluate similarity between the source and target datasets used in our transfer learning experiments. Four deep learning network architectures, i.e. Multilayer Perceptron (MLP), Convolutional Neural Networks (CNNs), Feedforward Neural Networks (FNNs), and Recurrent Neural Networks (RNNs), and two traditional machine learning models, i.e. Logistic Regression (LR) and Random Forest (RF), were tested and compared in our simulations. The results suggest that similarity scores are reliable indicators for pre-selecting source datasets in CRISPR-Cas9 transfer learning experiments, with cosine distance proving to be a more effective dataset comparison metric than either Euclidean or Manhattan distances. An RNN-GRU, a 5-layer FNN, and two MLP variants provided the best overall prediction results in our simulations. By integrating similarity-based source pre-selection with machine learning outcomes, we propose a dual-layered framework that not only streamlines the transfer learning process but also significantly improves off-target prediction accuracy. The code and data used in this study are freely available at: https://github.com/dagrate/transferlearning_offtargets.},
}
@article {pmid41117290,
year = {2025},
author = {Ye, T and Xue, M and Xu, Y and Fan, M and Yuan, M and Yu, J and Cao, H and Hao, L and Wu, X and Yin, F and Xu, F},
title = {Inverted Tetrahedral DNA Reporters Enable Label-Free Ratiometric CRISPR Electrochemical Aptasensing of Kanamycin.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {43},
pages = {27694-27702},
doi = {10.1021/acs.jafc.5c06928},
pmid = {41117290},
issn = {1520-5118},
mesh = {*Electrochemical Techniques/methods/instrumentation ; *Kanamycin/analysis ; *Biosensing Techniques/methods/instrumentation ; *DNA/chemistry/genetics ; Milk/chemistry ; CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry/genetics ; Animals ; *Anti-Bacterial Agents/analysis ; Limit of Detection ; Food Contamination/analysis ; },
abstract = {Integrating CRISPR technology with electrochemical sensing has promising potential in point-of-care testing applications. However, inappropriate immobilization of the reporter on the heterogeneous surface leads to a poor trans-cleavage efficiency. Additionally, the accuracy and reliability of electrochemical sensing still face challenges. Herein, an inverted tetrahedral DNA reporter was developed for electrochemical CRISPR aptasensing. Thiol-modified single-strand oligonucleotides were self-assembled on the edge of tetrahedral DNA nanostructures (TDNs) as a scaffold, enabling an inverted immobilization of DNA tetrahedra via the Au-S bond. The trans-cleavage activity of CRISPR/Cas12a on the single-stranded oligonucleotides resulted in the TDNs dissociating from the electrode surface. The recovery of electron transfer of potassium ferricyanide on the electrode enhances the electrochemical response, while the signal of adsorbed methylene blue on the skeleton of TDNs decreases, enabling a ratiometric signal output. As a proof of concept, the proposed inverted tetrahedral DNA reporters were employed to develop a label-free ratiometric electrochemical aptasensing method for kanamycin detection. Under the optimal conditions, as low as 0.35 pM kanamycin was detected in 50 min with a 4 orders of magnitude dynamic range from 1 pM to 10 nM. Furthermore, the practical application ability of the proposed method for kanamycin detection in a spiked milk sample was also demonstrated. This work offers a new perspective for electrochemical CRISPR sensing development.},
}
@article {pmid41052905,
year = {2025},
author = {Sun, Y and Hong, Z and Wang, W and Zhang, H and Ren, X and He, X and Kan, T and Fan, Y and Wang, C and Cao, Y and Zhang, H},
title = {Establishment of an efficient and versatile genome editing platform for L. ruthenicum.},
journal = {Plant physiology},
volume = {199},
number = {2},
pages = {},
doi = {10.1093/plphys/kiaf486},
pmid = {41052905},
issn = {1532-2548},
support = {2022BBF01001-03//Key Research &amp; Development Program of Ningxia Hui Autonomous Region/ ; 32170547//National Natural Science Foundation of China/ ; 2024AAC03123//Natural Science Foundation of Ningxia Hui Autonomous Region/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Genome, Plant ; Promoter Regions, Genetic ; },
abstract = {Black goji berry (Lycium ruthenicum Murr.) is a valuable functional food and traditional medicinal plant owing to its rich content of anthocyanins, trace minerals, vitamins, and polysaccharides. However, limited genetic manipulation tools have hindered functional genomic studies and trait improvement in this species. In this study, we optimized the genetic transformation system for L. ruthenicum, achieving a remarkably high transformation efficiency of 95.4%. Based on this system, we developed a clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated gene knockout approach, using the Arabidopsis U6 (AtU6) promoter to drive sgRNA expression and the cauliflower mosaic virus 35S (35S) promoter to drive Cas9 expression, achieving editing efficiencies of 68.8% at the phytoene desaturase (LrPDS) locus and 96.0% at the betaine aldehyde dehydrogenase (LrBADH2) locus. Furthermore, we established an adenine base editing (ABE) system using the ribosomal protein subunit 5A (RPS5A) promoter to drive tRNA adenine deaminase-8e (TadA-8e-nSpCas9) cassette expression, achieving an editing efficiency of 72.2% at the LrPDS locus. To broaden protospacer adjacent motif (PAM) compatibility, we introduced the PAM-relaxed variant SpRY, enabling successful A-to-G editing at an acetolactate synthase (LrALS) target site containing a noncanonical NAN PAM, with an efficiency of 5.3%. Additionally, we developed a multiplex ABE system based on the tRNA-processing strategy, which enabled simultaneous editing at 2 independent loci with an efficiency of 33.3%. Our study establishes a robust genome editing toolkit for L. ruthenicum, offering valuable tools for functional gene analysis and molecular breeding in this economically important species.},
}
@article {pmid39565542,
year = {2025},
author = {Zhou, L and Li, X and Ji, Z and Zhou, C and Yang, L and Li, Y and Fu, C and Gu, L and Zhang, S and Gao, J and Yue, P and Yu, H},
title = {Generation of Ext1 Gene-Edited Mice Model Via Dual sgRNAs/Cas9 System and Phenotypic Analyses.},
journal = {Molecular biotechnology},
volume = {67},
number = {11},
pages = {4233-4246},
pmid = {39565542},
issn = {1559-0305},
support = {32160147//National Natural Science Foundation of China/ ; YCSW2020229//Youth and Middle-aged Scientific and Technological Innovation Leading Talents Program of the Corps/ ; 2020GXNSFAA297097//Natural Science Foundation of Guangxi Zhuang Autonomous Region/ ; },
mesh = {Animals ; *N-Acetylglucosaminyltransferases/genetics/metabolism ; Exostosin 1 ; Mice ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Disease Models, Animal ; Phenotype ; Male ; Female ; *Exostoses, Multiple Hereditary/genetics/pathology ; Frameshift Mutation ; Exons ; Humans ; },
abstract = {Hereditary multiple exostoses (HME) is an autosomal dominant skeletal disease. Genetic linkage analyses have identified that mutations in the exostosin glycosyltransferase (EXT)1 and EXT2 genes are linked to HME pathogenesis, with EXT1 mutation being the most frequent. The aim of this study was to generate a mice model with Ext1 gene editing to simulate human EXT1 mutation and investigate the genetic pathogenicity of Ext1 through phenotypic analyses. We designed a pair of dual sgRNAs targeting exon 1 of the mice Ext1 gene for precise deletion of a 46 bp DNA fragment, resulting in frameshift mutation of the Ext1 gene. The designed dual sgRNAs and Cas9 proteins were injected into mice zygotes cytoplasm. A total of 14 mice were obtained via embryo transfer, among which two genotypic chimera mice had a deletion of the 46 bp DNA fragment in exon 1 of the Ext1 gene. By hybridization and breeding, we successfully generated heterozygous mice with edited Ext1 gene (Ext[+/-]). Off-target effect analysis did not reveal off-target mutations in Ext[+/-] mice caused by the two sgRNAs used. Compared to wild-type mice, Ext[+/-] mice exhibited lower body weights. X-ray imaging showed hyperplastic bone near caudal vertebrae only in male Ext[+/-] mice, with computed tomography values approximately at 200 HU for hyperplastic bone between ribs and spine regions. Furthermore, immunohistochemical analysis revealed fewer articular chondrocytes expressing EXT1 in edited mice compared to wild-type ones. Pathological section analysis demonstrated no structural or morphological abnormalities in heart, liver, lung, or kidney tissues from Ext[+/-] mice. In conclusion, we successfully generated an accurate DNA deletion model for studying Ext1 using dual sgRNAs/Cas9 systems. In conclusion, we successfully generated precise DNA deletions in the Ext1 mice model using the dual sgRNAs/Cas9 system. In conclusion, we observed significant phenotypic changes in Ext[+/-] mice, particularly bone hyperplasia in male individuals; however, no exostosis was detected in the gene-edited mice. The introduction of a frameshift mutation into the Ext1 gene through CRISPR/Cas9 technology resulted in novel phenotypic alterations, highlighting the genetic pathogenicity of Ext1. Therefore, our Ext[+/-] mice serve as a valuable model for further biomedical investigations related to the Ext1 gene.},
}
@article {pmid41148687,
year = {2025},
author = {Nass, NM and Zaher, KA},
title = {Beyond the Resistome: Molecular Insights, Emerging Therapies, and Environmental Drivers of Antibiotic Resistance.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {14},
number = {10},
pages = {},
doi = {10.3390/antibiotics14100995},
pmid = {41148687},
issn = {2079-6382},
abstract = {Antibiotic resistance remains one of the most formidable challenges to modern medicine, threatening to outpace therapeutic innovation and undermine decades of clinical progress. While resistance was once viewed narrowly as a clinical phenomenon, it is now understood as the outcome of complex ecological and molecular interactions that span soil, water, agriculture, animals, and humans. Environmental reservoirs act as silent incubators of resistance genes, with horizontal gene transfer and stress-induced mutagenesis fueling their evolution and dissemination. At the molecular level, advances in genomics, structural biology, and systems microbiology have revealed intricate networks involving plasmid-mediated resistance, efflux pump regulation, integron dynamics, and CRISPR-Cas interactions, providing new insights into the adaptability of pathogens. Simultaneously, the environmental dimensions of resistance, from wastewater treatment plants and aquaculture to airborne dissemination, highlight the urgency of adopting a One Health framework. Yet, alongside this growing threat, novel therapeutic avenues are emerging. Innovative β-lactamase inhibitors, bacteriophage-based therapies, engineered lysins, antimicrobial peptides, and CRISPR-driven antimicrobials are redefining what constitutes an "antibiotic" in the twenty-first century. Furthermore, artificial intelligence and machine learning now accelerate drug discovery and resistance prediction, raising the possibility of precision-guided antimicrobial stewardship. This review synthesizes molecular insights, environmental drivers, and therapeutic innovations to present a comprehensive landscape of antibiotic resistance. By bridging ecological microbiology, molecular biology, and translational medicine, it outlines a roadmap for surveillance, prevention, and drug development while emphasizing the need for integrative policies to safeguard global health.},
}
@article {pmid41148514,
year = {2025},
author = {Tripathi, A and Vishwakarma, K and Tripathi, S and Jadaun, JS and Nayak, AK},
title = {Utilization of MADS-Box genes for agricultural advancement: current insights and future prospects.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {20},
pmid = {41148514},
issn = {1573-4978},
mesh = {*MADS Domain Proteins/genetics/metabolism ; Gene Expression Regulation, Plant/genetics ; Plant Proteins/genetics/metabolism ; *Crops, Agricultural/genetics ; CRISPR-Cas Systems ; Oryza/genetics ; *Agriculture/methods ; Solanum lycopersicum/genetics ; Phylogeny ; Plants, Genetically Modified/genetics ; Gene Editing/methods ; Genes, Plant ; },
abstract = {MADS-box genes constitute a highly conserved family of transcription factors integral to the regulation of a diverse array of plant developmental processes, encompassing floral organ specification, fruit maturation, root architecture and adaptation to abiotic stresses. These transcription factors encode proteins containing the distinctive MADS (MCM1, AGAMOUS, DEFICIENS, and SRF) domain, which mediates DNA binding and orchestrates interaction with co-regulators, thereby enabling the precise transcriptional control of developmental gene networks. Functional characterization through transgenic approaches including overexpression, knockdown, and CRISPR/Cas9-based mutagenesis-has revealed the capacity of MADS-box gene manipulation to modulate key agronomic traits, such as yield potential, as well as resilience to salinity, drought, and temperature fluctuations. In rice, targeted editing of OsMADS18 using CRISPR/Cas9 generated a substantial quantitative variation in tiller and panicle number, demonstrating the direct contribution of MADS-box gene function to biomass and yield performance. Similarly, CRISPR/Cas9-mediated disruption of the RIPENING INHIBITOR (RIN) gene in tomato (Solanum lycopersicum) underscored its central role in regulating fruit ripening, linking MADS-box gene activity to postharvest quality and development. Phylogenomic studies reveal strong conservation of MADS-box gene lineages in monocot grasses, as evidenced by clustered short internal branches, whereas eudicots, particularly Solanaceae present well-differentiated subclades, reflecting lineage-specific diversification events. Notably, network analysis highlight the high connectivity and central regulatory position of many MADS-box proteins, underlining their roles as master integrators of developmental and environmental signalling involved in both floral and vegetative transitions. A mechanistic understanding of these regulatory circuits offers translational opportunities to engineer crops with improved performance and resilience, reinforcing the pivotal role of MADS-box genes in crop improvement.},
}
@article {pmid41148377,
year = {2025},
author = {Baars, J and Kurm, V and Scholten, B and Griekspoor, Y and Lavrijssen, B and Steens, JA and Smulders, MJM and van Peer, A},
title = {On site discrimination between two closely related commercial strains of oyster mushroom using a loop-mediated isothermal amplification (LAMP) test.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {17},
pmid = {41148377},
issn = {1573-4978},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Pleurotus/genetics/classification ; DNA, Fungal/genetics ; Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Protection of the intellectual property (IP) rights on new crop varieties is important as it allows the breeding company or entity that produced the variety to earn back (part of) the investment. Infringement on the IP rights of mushroom varieties is not uncommon. In order to combat infringement of the IP rights on two strains of Oyster mushroom (SPOPPO and ALLERPO) it is important to be able to readily recognize and discriminate the two strains in commercial practice. This article describes the development of tools for the on-site identification of two closely related sporeless strains of Oyster mushroom.<br><br>METHODS AND RESULTS: To develop a reliable method of discriminating between SPOPPO and ALLERPO, we used either the LAMP technique or a modification of that technique. It allows for fast (within 30&#xa0;min) identification of the commercially used strains SPOPPO and ALLERPO with high specificity. Fast on-site answers on strain identity can be important when experiencing unexpected strain behavior or when strains are of suspect origin. Both strains are discriminated from sporulating strains by a LAMP reaction on the intact version of the msh4 gene; sporeless strains contain a msh4 gene with a large insert that renders the associated protein inactive.<br><br>CONCLUSIONS: SPOPPO and ALLERPO are distinguished from each other and other commercially used Pleurotus ostreatus strains by LAMP reactions that target genomic regions with strain specific recombinations. To our knowledge, this is the first time LAMP reactions have been developed to discriminate between Pleurotus ostreatus strains.},
}
@article {pmid41145060,
year = {2025},
author = {Monteiro Belo Dos Santos, S and Van Tricht, C and Lammertyn, J and Spasic, D},
title = {Zoonotic disease detection at the point-of-care: the best of RPA and CRISPR-Cas.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118139},
doi = {10.1016/j.bios.2025.118139},
pmid = {41145060},
issn = {1873-4235},
abstract = {Biosensors are increasingly crucial in detecting biomarkers for emerging zoonotic diseases at the point-of-care (POC). This imminence was recently highlighted by the deficient response during the SARS-CoV-2 pandemic. While polymerase chain reaction (PCR) is the common nucleic acid (NA) testing method for zoonotic diseases in laboratory settings, it is impractical for the POC settings due to the equipment-related cost, lack of portability and user-friendliness. Recent advances in NA amplification introduced isothermal methods, such as recombinase polymerase amplification (RPA), which is known for its low temperature (37-42 °C), short incubation time (5-20 min) and suitability for integration in miniaturized, portable, low-cost, highly sensitive diagnostic platforms. However, RPA susceptibility to false positive results steered to its combination with CRISPR-Cas12/13, leading to the rise of SHERLOCK and DETECTR. This review first explores RPA-CRISPR-Cas bioassay development as either two- or one-step. This is followed by a discussion on the integration of canonical RPA, or its combination with CRISPR-Cas, into different diagnostic platforms towards NA amplification at the POC (e.g., mobile laboratories, centrifugal, or pump-free platforms). Finally, the advantages, limitations, and outlook for POC-based diagnostics of zoonotic diseases with RPA(-CRISPR-Cas) are discussed, highlighting the need for innovative technologies to address global health challenges. While promising, many of these approaches still require further research to achieve streamlined, single-step reactions and seamless integration into diagnostic platforms. Moreover, despite two decades of RPA(-CRISPR-Cas) development, technology readiness is limited, still missing validated platforms, integrated sample preparation, and AI-powered results analysis enabling real time epidemiological monitoring.},
}
@article {pmid41144208,
year = {2026},
author = {Shaw, S and Sateriale, A and Pawlowic, MC and Vinayak, S and Brooks, CF and Byerly, JH and Striepen, B},
title = {Genetic Manipulation of Cryptosporidium parvum.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2978},
number = {},
pages = {173-185},
pmid = {41144208},
issn = {1940-6029},
mesh = {*Cryptosporidium parvum/genetics ; Animals ; Mice ; CRISPR-Cas Systems ; *Cryptosporidiosis/parasitology ; Gene Editing/methods ; Transfection/methods ; Humans ; },
abstract = {Cryptosporidium parvum can be reliably genetically manipulated using CRISPR/Cas9 driven homologous repair coupled to in vivo propagation in immunodeficient mice. This chapter will guide through foundational procedures for excystation, transfection, infection, collection, and purification of transgenic Cryptosporidium parvum. The genetic tools for Cryptosporidium research were expanded significantly over the last 5 years. While we cannot cover all advances in detail, we will highlight novel selection markers, conditional mutagenesis strategies, and genetic crosses.},
}
@article {pmid41141466,
year = {2025},
author = {Mohiuddin, M},
title = {Monitoring and Assessment of Circulating Tumor DNA in Cancers Using Ultrarapid Sensitivity as an Innovative Practice.},
journal = {Health science reports},
volume = {8},
number = {10},
pages = {e71409},
pmid = {41141466},
issn = {2398-8835},
abstract = {BACKGROUND: Liquid biopsy with circulating tumor DNA (ctDNA) has rapidly emerged as a new paradigm for assessing tumor burden, genetic heterogeneity, and therapeutic response in a real-time, noninvasive manner. However, ctDNA is often visually low (sometimes < 0.1% of the total circulating cell-free DNA), creating a significant challenge for reliable detection (especially for early-stage disease and minimal residual disease).<br><br>DISCUSSION: New technologies for structural variant (SV)-based ctDNA assays, nanomaterial-based electrochemical sensors, magnetic nano-electrode platforms, and fragment-enriched library preparation have improved sensitivity to attomolar concentrations and less in some populations. In some cancers, ctDNA may provide early evidence of recurrence (i.e., >&#x2009;1 year) before being clinically evident using traditional metrics. These technologies allow for unprecedented opportunities and sensitivity for early detection, monitoring of treatment response, and early detection of molecular recurrence. Nevertheless, a barrier remains for widespread clinical application owing to pre-analytical technique variability, analytical platform variability, cost, and the necessity of large-scale, prospective trials.<br><br>CONCLUSION: This study will analyze new innovative technology-based ultrasensitive ctDNA assay detection and future research and clinical applications for breast, colorectal, lung, lymphoid, and gastroesophageal cancers, and studies assessing ctDNA for monitoring treatment. Prospects for ctDNA detection utilizing multiplexed CRISPR-Cas ctDNA assays, microfluidic point-of-care (POC) devices, and AI-based error suppression methods may be the next horizon for ctDNA liquid biopsy technology.},
}
@article {pmid41141388,
year = {2026},
author = {Wang, T and Yu, M and Liu, P and Song, Z and Li, C and Yang, J and Liu, N},
title = {In vivo gene therapy: A strategy for mutations, degenerations, and tumors.},
journal = {Genes &amp; diseases},
volume = {13},
number = {1},
pages = {101808},
pmid = {41141388},
issn = {2352-3042},
abstract = {Gene mutations, organ function degeneration, and carcinogenesis are the primary threats to human health. Gene therapy, which involves the addition, deletion, regulation, and editing of genes, as well as the development of genetic vaccines, can potentially cure genetic mutation disorders, degenerative diseases, and cancers. Ex vivo gene therapy has recently been used to treat monogenetic mutation diseases of the hematopoietic system and cancers. However, in vivo gene therapy remains inapplicable. The primary elements of in vivo gene therapy include deoxyribonucleic acid (DNA) nucleases (e.g., zinc finger nucleases, transcription activator-like effector nucleases), CRISPR-Cas system, base editors, prime editors, and delivery vectors (e.g., viral and non-viral vehicles). According to the development of DNA nucleases and delivery vectors, in vivo gene therapy can be made available for future clinical use. The current review summarizes the development of DNA nucleases and delivery vectors for in vivo gene therapy, emphasizing recent progress.},
}
@article {pmid41139369,
year = {2025},
author = {Wei, H and Li, D and Xie, K and Lou, S and Dong, G and Guo, F and Lian, G and Pan, X and Zeng, Z and Han, N and Gao, Z and Bian, H},
title = {Creation of new rice germplasm with cross-resistance to auxin herbicides picloram and dicamba by genome editing of OsAFB4.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {11},
pages = {282},
pmid = {41139369},
issn = {1432-2242},
support = {LGN21C130006//Natural Science Foundation of Zhejiang Province/ ; 32201834//Innovative Research Group Project of the National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics/drug effects/growth &amp; development ; *Herbicide Resistance/genetics ; *Gene Editing ; *Picloram/pharmacology ; *Herbicides/pharmacology ; *Dicamba/pharmacology ; Indoleacetic Acids ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; Plant Breeding ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; },
abstract = {Genome Editing of OsAFB4 in rice cultivar Jiahe212 confers cross-resistance to synthetic auxin herbicides picloram and dicamba without reducing grain yield, providing a novel germplasm for herbicide-resistant rice breeding. Synthetic auxin herbicides (SAHs) are widely employed in global agriculture owing to low toxicity and high efficiency in weed management. Mutations in OsAFB4, an AUXIN SIGNALING F-BOX gene, led to specific resistance to SAH picloram in rice. However, potential application of OsAFB4 modification for resistance to other SAHs and its effect on agronomic traits in rice remain uncharacterized. In this study, we employed CRISPR/Cas9-mediated genome editing to generate OsAFB4 mutations in the elite commercial rice cultivar Jiahe212 (JH212). Hydroponic-culture experiments showed that Osafb4 mutant lines exhibited significant resistance to both SAHs: picloram and dicamba. Promoter activity assays using a pOsAFB4:GUS reporter line revealed that SAH treatments induced significant upregulation of GUS expression specifically in shoot apices. Under picloram or dicamba treatment, expressions of OsIAA1, OsIAA3, OsIAA9 and OsIAA20 were significantly upregulated in wild-type plants, while remarkably suppressed in Osafb4 mutants, revealing a critical role for OsAFB4 in regulating auxin-responsive IAA genes during OsAFB4-mediated sensitivity to SAH. Selected for field trials, the Cas9-free Osafb4-13 mutant line exhibited robust resistance to both picloram and dicamba without reducing grain yield compared to untreated controls. This study provides a new cross-herbicide-resistant rice germplasm without reducing grain yield by targeted editing of OsAFB4.},
}
@article {pmid41104823,
year = {2025},
author = {Li, X and Ma, J and Luo, Y and Zhu, D and Wang, L and Su, S},
title = {Enhancing Performance in Electrochemical Early Diagnosis of African Swine Fever Based on CRISPR-Responsive DNA Nanoflowers.},
journal = {Analytical chemistry},
volume = {97},
number = {42},
pages = {23401-23408},
doi = {10.1021/acs.analchem.5c04188},
pmid = {41104823},
issn = {1520-6882},
mesh = {*African Swine Fever Virus/genetics/isolation &amp; purification ; *African Swine Fever/diagnosis ; Animals ; *Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Swine ; *Nanostructures/chemistry ; *DNA, Viral/analysis/genetics ; Early Diagnosis ; Limit of Detection ; Nucleic Acid Amplification Techniques ; *DNA/chemistry ; },
abstract = {Accurate, ultrasensitive, and fast detection of the African swine fever virus (ASFV) can efficiently prevent its spread and reduce the losses. Herein, an electrochemical biosensor was designed for high-performance detection of ASFV DNA by coupling with CRISPR technology and signal amplification technology. Porous DNA nanoflowers (DNFs) were prepared by rolling circle amplification (RCA), which was preconjugated Cas12a-crRNA complex to improve detection sensitivity, shorten detection time, and simplify detection steps. In the presence of ASFV DNA, the trans-cleavage activity of Cas12a was activated, degrading DNFs into DNA fragments and causing a significant electrochemical signal change. Combined with the CRISPR-Cas12a system, the detection limit of the DNF-amplified biosensor (3.57 aM) is 3 orders of magnitude lower than that of the conventional RCA-amplified biosensor (2.90 fM). Moreover, the designed electrochemical biosensor showed excellent reproducibility, storage stability, and practical analysis ability, suggesting that it has a promising application in the early diagnosis of African swine fever (ASF).},
}
@article {pmid41084819,
year = {2025},
author = {Gao, Z and Yang, X and Ren, X and Ma, H and Wu, D and Du, Y and Fan, Q and Ma, Q and Wei, Q},
title = {Ultrasensitive Detection of m[6] A-Modified RNA Using CRISPR/Cas12a-Integrated Iontronic Biosensor with Hydrophobized Nanochannels: Toward Early Cancer Diagnosis by Machine Learning.},
journal = {Analytical chemistry},
volume = {97},
number = {42},
pages = {23609-23621},
doi = {10.1021/acs.analchem.5c05377},
pmid = {41084819},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Machine Learning ; *Adenosine/analogs &amp; derivatives/analysis/chemistry ; *CRISPR-Cas Systems ; *Early Detection of Cancer/methods ; *RNA, Long Noncoding/genetics/analysis ; Hydrophobic and Hydrophilic Interactions ; *Neoplasms/diagnosis ; Limit of Detection ; Aluminum Oxide/chemistry ; },
abstract = {N[6] -methyladenosine (m[6] A), the most prevalent internal modification in eukaryotic RNAs, has emerged as a focal point of intensive research in recent years owing to its pivotal regulatory roles in carcinogenesis, progression, and metastasis. However, conventional methods for site-specific detection of m[6] A modifications are plagued by operational complexity, pose challenges for quantitative assessment of methylation levels, and exhibit elevated false-positive rates, severely limiting their utility in clinical and mechanistic studies. In this study, we engineered an ultrasensitive iontronic biosensor leveraging a hydrophobized anodic aluminum oxide (AAO) nanochannel platform, synergistically integrating the precise target recognition capability of the CRISPR/Cas12a system with the efficient signal amplification of the clamped hybridization chain reaction (CHCR). This integration enables ultrasensitive and specific detection of m[6] A-modified RNA with a low detection limit of 32 aM. Validation experiments targeting MALAT1 and HOTAIR lncRNAs demonstrated that the sensor achieves exceptional specificity in qualitative analysis of m[6] A modifications. Furthermore, combinatorial detection of these two lncRNAs enables robust discrimination between cancer patients and healthy individuals. Through in-depth mining of latent data patterns via machine learning, the random forest (RF) model yielded a cancer diagnostic accuracy of 96.7%. This study establishes a novel and potent paradigm for early cancer diagnosis, with far-reaching implications for epitranscriptomic research and clinical translation.},
}
@article {pmid41081763,
year = {2025},
author = {Wang, X and Feng, S and Chen, H and Zhou, B and Fan, T and Qin, Y and Zhao, L and Jiang, Y and Chen, Y},
title = {Development of an Aptamer/CRISPR-Cas12a-Based Dual-Modal Biosensor for Fusobacterium nucleatum Detection in Non-Invasive Colorectal Cancer Screening.},
journal = {Analytical chemistry},
volume = {97},
number = {42},
pages = {23360-23369},
doi = {10.1021/acs.analchem.5c04132},
pmid = {41081763},
issn = {1520-6882},
mesh = {*Fusobacterium nucleatum/isolation &amp; purification ; *Colorectal Neoplasms/diagnosis/microbiology ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry ; *Early Detection of Cancer/methods ; Feces/microbiology ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Colorectal cancer (CRC) is the third most common cancer and leading cause of cancer-related deaths worldwide. However, current CRC screening methods are complex, invasive, and tend to exhibit low sensitivity. Recent evidence has highlighted gut microbiota dysbiosis, especially elevated Fusobacterium nucleatum levels, as a promising biomarker for CRC. In this study, a sensitive and specific detection platform was developed for F. nucleatum by combining a highly specific aptamer with rolling circle amplification (RCA) and the CRISPR/Cas12a technology. The aptamer enables specific target recognition, while RCA amplifies the target signal, and the Cas12a-mediated cleavage of a fluorescence-quenching substrate generates a quantifiable fluorescence or grayscale signal. Using a microplate reader, this assay achieved a limit of detection (LOD) of 3.68 CFU/mL; furthermore, by incorporating smartphone-assisted ImageJ grayscale analysis, it elevated the LOD to 4.30 CFU/mL, thereby enabling a dual-mode output along with on-site applicability. Additionally, the strong correlation between the two signals allowed for mutual validation. Upon application to clinical fecal samples, the developed method sensitively distinguished CRC patients from healthy controls, and its results correlated with the quantitative polymerase chain reaction results. This triple-synergistic platform, integrating aptamer specificity, RCA amplification, and CRISPR/Cas12a sensitivity, enables the noninvasive, ultrasensitive detection of F. nucleatum, supporting early CRC screening, prognosis monitoring, and microbiome-targeted therapy. Moreover, this approach overcomes the challenges of detecting low-abundance bacteria in early stage CRC and advances the precision of microbiome-based diagnostics for CRC.},
}
@article {pmid40905611,
year = {2025},
author = {Hu, Y and Yan, H and Zhang, Y and Yu, Q and Xue, T and Zhang, X and Zeng, Q and Yang, H and Xia, X and Xu, Y and Deng, R and Li, J},
title = {In-Field Molecular Diagnostics of Plant Pathogens Using Bioluminescent CRISPR-Guided Caspase Assay.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {64},
number = {44},
pages = {e202508870},
doi = {10.1002/anie.202508870},
pmid = {40905611},
issn = {1521-3773},
support = {2023YFB3208302//National Key Research and Development Program of China/ ; //New Cornerstone Investigator Program/ ; 2025NSFTD0001//Sichuan Science and Technology/ ; 22522408//National Natural Science Foundation of China/ ; 22074100//National Natural Science Foundation of China/ ; },
mesh = {*Plant Diseases/microbiology ; *Caspases/metabolism ; *Luminescent Measurements/methods ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {In-field molecular diagnostics of plant pathogens are critical for crop disease management and precision agriculture, but tools are still lacking. Herein, we present a bioluminescent molecular diagnostic assay capable of detecting viable pathogens directly in minimally processed plant samples, enabling rapid and precise in-field crop disease diagnosis. The assay, called bioluminescent craspase diagnostics (BioCrastics), leverages newly discovered RNA-activated protease of CRISPR (Craspase) with enzymatic luminescence to generate a cascaded amplification, thus bypasses nucleic acid purification and amplification while achieving sub-nanogram sensitivity for fungal pathogens. Using wheat stripe rust as a proof of concept, we demonstrate direct pathogen detection in crude leaf homogenates within 40 min, early identification of infections 6 days prior to symptom emergence. Notably, the assay, via targeting pathogenic RNAs, specifically quantifies viable fungi, overcoming false positives from dead pathogens-a limitation of PCR-based methods that impairs disease risk assessment. Featuring simplified sample processing, portable detection, and species-specific accuracy, BioCrastics establishes a field-deployable tool that bridges the gap between laboratory-level precision and on-farm diagnostic needs for crop disease management.},
}
@article {pmid41138779,
year = {2025},
author = {Afresham, S and Khan, MK and Mughal, MAS and Mehmood, MS and Ali, S and Bashir, M and Abbas, Z and Azeem, A and Ahmed, W and Imran, M and Abbas, RZ and Sindhu, ZU and Sajid, MS},
title = {Recent Advancements in the Diagnosis of Parasitic Diseases.},
journal = {Molecular and biochemical parasitology},
volume = {},
number = {},
pages = {111706},
doi = {10.1016/j.molbiopara.2025.111706},
pmid = {41138779},
issn = {1872-9428},
abstract = {Parasitic infections present a significant health risk to the public, affecting millions of people, particularly in underdeveloped and developing countries. In developing countries, these infections are also responsible for causing significant economic challenges due to elevated healthcare expenditure. Accurate diagnosis and effective treatment methods are essentially required to combat this global issue. For decades, traditional diagnostic methods such as microscopy, serological testing, histopathology, and culturing have been used for the diagnosis of these parasitic infections. While these methods can be effective and helpful in many ways, they often consume a lot of time, require an elevated level of expertise, and have limited applications particularly in endemic regions having issues like poor infrastructure and limited access to healthcare facilities. This review aims to highlight the urgent need for a revolution to replace these conventional techniques with more affordable, quick, and field-adjustable tools such as rapid diagnostic tests (RDTs) and molecular methods and provides a comprehensive picture of advanced diagnostic tools used in the identification of parasites. With the advancements in science and technology, molecular methods such as Polymerase chain reaction, Next generation sequencing, and isothermal loop-mediated amplification have remarkably enhanced the sensitivity and accuracy of parasite detection and identification. The range of these diagnostic methods has further extended by advanced serological methods, imaging techniques, and immunological methods. Moreover, the innovations in nanotechnology, CRISPR-Cas methods, and multi-omics techniques for identification of parasite DNA, antigens, metabolites, and host responses are invaluable for diagnostic accuracy, comprehensive understanding of parasite biology, and for the discovery of new therapeutic targets and diagnostic biomarkers. However, further research and developments are required for an effective and long-lasting impact of these advancements.},
}
@article {pmid41138228,
year = {2025},
author = {Hodge, CA and Donegan, NP and Armstrong, DA and Hayden, MS and Howell, AL},
title = {Enhanced cleavage of genomic CCR5 using CASX2[Max].},
journal = {RNA biology},
volume = {22},
number = {1},
pages = {1-18},
doi = {10.1080/15476286.2025.2577449},
pmid = {41138228},
issn = {1555-8584},
mesh = {Humans ; *CRISPR-Cas Systems ; *Receptors, CCR5/genetics/metabolism/chemistry ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. CasX2 (PlmCas12e) belongs to the class II CRISPR system derived from Planctomycetes, a non-pathogenic bacterium present in aquatic and terrestrial soils and offers several advantages as a potential therapeutic CRISPR system over Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9). These advantages include its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and the absence of pre-existing immunity in humans. We compared the cleavage efficiency and double-stranded break repair characteristics between CasX2 and CasX2[Max], a recently generated CasX2 variant with three amino acid substitutions, for targeting CCR5, a gene that encodes the CCR5 receptor important for HIV-1 infection. Two single guide RNAs (sgRNAs) were designed that flank the 32 bases deleted in the natural CCR5 ∆32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2[Max] using sgRNAs with 20 nt and 23 nt spacer lengths, enabled cleavage of genomic CCR5. Structural modelling indicated that two of the CasX2[Max] amino acid substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2[Max] in cellular gene excision. In sum, CasX2[Max] consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.},
}
@article {pmid41137488,
year = {2025},
author = {Kang, YW and Park, HH},
title = {The anti-CRISPR protein AcrIE8.1 inhibits the type I-E CRISPR-Cas system by directly binding to the Cascade subunit Cas11.},
journal = {FEBS letters},
volume = {},
number = {},
pages = {},
doi = {10.1002/1873-3468.70201},
pmid = {41137488},
issn = {1873-3468},
support = {2024//Chung-Ang University/ ; RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; },
abstract = {CRISPR-Cas systems provide adaptive immunity to bacteria by recognizing and destroying foreign genetic elements. The type I-E CRISPR-Cas system utilizes a multi-subunit Cascade complex to detect target DNA and recruit the Cas3 nuclease for degradation. To overcome this defense, bacteriophages have evolved anti-CRISPR (Acr) proteins that inhibit various steps of the CRISPR interference pathway. Here, we determined the crystal structure of AcrIE8.1, an uncharacterized Acr, revealing it binds to Cas11, a Cascade subunit, to disrupt function. AcrIE8.1 has a compact fold with a defined Cas11-binding interface, suggesting a unique inhibitory mechanism among AcrIE proteins. These findings highlight Cas11 as a critical target for Acr-mediated immune evasion. Impact statement Through a combination of structural and biochemical analyses, we demonstrate that AcrIE8.1 directly binds to the Cas11 subunit of the Cascade complex to inhibit the CRISPR-Cas system. This represents a novel inhibitory strategy not previously observed among AcrIE proteins.},
}
@article {pmid41137484,
year = {2025},
author = {Lee, SY and Park, HH},
title = {Investigating the molecular mechanisms underlying the anti-CRISPR function of AcrIIA13b protein.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.70304},
pmid = {41137484},
issn = {1742-4658},
support = {RS-2025-02316334//NRF-Korea/ ; },
abstract = {The CRISPR-Cas systems of adaptive immunity in bacteria and archaea provide resistance against phages and other mobile genetic elements. Counteractive anti-CRISPR (Acr) proteins in phages and archaeal viruses impede these CRISPR-Cas systems. Although CRISPR-Cas systems have revolutionized genome editing, potential off-target events remain a safety concern. Hence, a thorough comprehension of the structural and molecular basis of diverse Acrs is imperative to unravel the fundamental mechanisms governing CRISPR-Cas regulation. Here, we present the structure of AcrIIA13b from Staphylococcus haemolyticus and analyze its structural and functional features to reveal the molecular basis underlying the inhibition of Cas9 by AcrIIA13b. Our structural analysis shows that AcrIIA13b eliminates the cleavage activity of Staphylococcus aureus Cas9 (SauCas9) by blocking the PAM-binding region of Cas9 so that Cas9 cannot recognize the target DNA. In addition, we demonstrate that the 15 amino acid residues at the N terminus of AcrIIA13b, which were revealed to be important for its dimerization, are critical for its inhibitory activity against Cas9. Our findings shed light on the molecular basis of AcrIIA13b-mediated CRISPR-Cas inhibition and provide valuable insights into the arms race between bacteria and phages.},
}
@article {pmid41136894,
year = {2025},
author = {Liu, H and Zhu, G and Chen, L and Ye, H and Zhang, Y and Han, G},
title = {Machine learning prediction of bacterial optimal growth temperature from protein domain signatures reveals thermoadaptation mechanisms.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {957},
pmid = {41136894},
issn = {1471-2164},
support = {32172769//National Natural Science Foundation of China/ ; },
abstract = {UNLABELLED: Cultivating the vast majority of uncultured microbes requires knowledge of their physiological preferences, particularly optimal growth temperature (OGT). We present a machine learning approach that utilizes protein domain frequencies from bacterial genomes to predict OGT across a wide continuous range (1–83 °C). Our Random Forest model, trained on a dataset of 1,498 genomes, achieved high predictive accuracy (R[2]=0.853 on test data, 82.4% of predictions within a ± 10 °C error margin), substantially advancing current capabilities and offering a practical tool to guide cultivation experiments. Analysis of the model identified key protein domain signatures associated with thermal adaptation. The enrichment of domains related to polyamine metabolism, the tRNA methyltransferase family, and CRISPR-Cas systems was positively correlated with higher OGTs, providing genomic evidence for their roles in thermotolerance. Conversely, domains involved in redox homeostasis, transport, and nucleic acid binding were more abundant at lower temperatures. These findings not only facilitate targeted cultivation efforts but also deepen our understanding of the molecular strategies bacteria employ to thrive across diverse thermal niches.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-12162-z.},
}
@article {pmid40618724,
year = {2025},
author = {Botkin, JR and Curtin, SJ},
title = {CRISPR-Cas9 Mutagenesis and Gene Overexpression to Enhance Resistance to Ascochyta medicaginicola in Medicago truncatula.},
journal = {Molecular plant-microbe interactions : MPMI},
volume = {38},
number = {5},
pages = {751-761},
doi = {10.1094/MPMI-05-25-0053-R},
pmid = {40618724},
issn = {0894-0282},
mesh = {*Medicago truncatula/microbiology/genetics/immunology ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology/immunology/genetics ; *Ascomycota/physiology ; *Disease Resistance/genetics ; Mutagenesis ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; },
abstract = {Alfalfa (Medicago sativa), the most widely cultivated forage legume globally, is vulnerable to Ascochyta medicaginicola, the fungus causing spring black stem and leaf spot (SBS) disease, which significantly reduces yield. SBS disease also affects Medicago truncatula, a diploid model legume with extensive genetic resources, including susceptible and resistant accessions. Using comparative genomics, four candidate genes for disease resistance were identified, MtTCAR1, MtPHO2A, MtCPR1-like, and MtPAM16. CRISPR/Cas9 mutagenesis was applied to generate independent mutant plants in the R108 accession, and disease resistance was evaluated by a detached leaf qPCR-based pathogen assay. MtCPR1-like mutant plants exhibited a 34% reduction in pathogen biomass, along with variable constitutive expression of pathogenesis-related genes. Additionally, a fifth candidate gene, MtKCS12, identified through transcriptomic analysis, was overexpressed in transformed plants, resulting in a 71.4 to 80.9% reduction in pathogen biomass compared with wild-type segregants. This study validates gene editing and transgenic approaches for improving SBS disease resistance in M. truncatula, and future research will focus on applying these strategies to enhance resistance in economically important alfalfa. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.},
}
@article {pmid41136797,
year = {2025},
author = {Basarali, MK and Daemi, A and Tahiraga, RG and Özbolat, G and Hooshiar, MH and Shirazi, MSR and Döğüş, Y},
title = {Artificial intelligence-driven epigenetic CRISPR therapeutics: a structured multi-domain meta-analysis of therapeutic efficacy, off-target prediction, and gRNA optimization.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {223},
pmid = {41136797},
issn = {1438-7948},
mesh = {*Artificial Intelligence ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Epigenesis, Genetic ; *Genetic Therapy/methods ; },
abstract = {CRISPR-based epigenetic editing enables reversible regulation of gene expression without permanent DNA modification. The integration of artificial intelligence (AI) enhances guide RNA (gRNA) design, off-target prediction, and delivery optimization. We conducted a systematic review and meta-analysis (2015-2025) in accordance with PRISMA 2020 guidelines to evaluate the impact of AI on the precision, safety, and therapeutic efficacy of epigenetic CRISPR tools. From 540 screened records, 58 studies met inclusion criteria, of which 41 provided extractable quantitative data for meta-analysis and 17 contributed to qualitative synthesis. Random-effects models, subgroup analyses, and bias assessments were applied. Pooled analyses demonstrated strong positive effects across three domains: therapeutic efficacy (SMD = 1.67), gRNA optimization (SMD = 1.44), and off-target prediction (AUC = 0.79). Publication bias was minimal, and subgroup analyses indicated the strongest impact in therapeutic applications. Deep learning models were consistently associated with higher effect sizes. Qualitative synthesis revealed trends in interpretable AI, omics integration, and delivery innovations, underscoring AI's role in safer and more precise CRISPR editing. Overall, AI significantly improves the precision and therapeutic performance of CRISPR-based epigenetic tools, with the strongest effects observed in therapeutic efficacy, supporting their potential for personalized gene editing.},
}
@article {pmid41136394,
year = {2025},
author = {Chow, JT and Desjardins, A and Lee, DKC and Grigore, IA and Lee, L and Fu, NJ and Chau, S and Lee, BY and Gabra, MM and Salmena, L},
title = {A microRNA CRISPR screen reveals microRNA-483-3p as an apoptotic regulator in prostate cancer cells.},
journal = {Cell death &amp; disease},
volume = {16},
number = {1},
pages = {752},
pmid = {41136394},
issn = {2041-4889},
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Prostatic Neoplasms/genetics/pathology/metabolism ; Male ; *Apoptosis/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Survival/genetics ; },
abstract = {The development of traditional protein-targeted cancer therapies is a slow and arduous process, often taking years or even decades. In contrast, RNA-based therapies targeting crucial microRNA (miRNA) offer a faster alternative due to the sequence-specific nature of miRNA inhibitor binding. This, combined with the capacity of individual miRNA to influence multiple cellular pathways, makes these small RNA attractive targets for cancer therapy. While miRNA are known to be dysregulated in prostate cancer (PCa), identifying their individual contributions to disease progression and the identification of therapeutically actionable miRNA targets in PCa has been challenging due to limited profiling and lack of screening tools. To address this need, we developed miRKOv2, a miRNA-only CRISPR knockout library enabling systematic, genome-wide loss-of-function screens to identify miRNA essential for PCa cell survival. Our screens uncovered 70 potential essential miRNA candidates, with miR-483 demonstrating the most significant impact on PCa cell viability. Functional characterization revealed that miR-483 disruption potentiated apoptosis in PCa cell lines. Mechanistically, we uncovered a novel regulatory axis wherein miR-483-3p directly modulates a BCLAF1/PUMA/BAK1 apoptotic signaling network, highlighting its critical role in maintaining PCa cell survival. Our findings provide novel insights into the complex regulatory role of miRNA in PCa progression and offer a potential therapeutic strategy for targeting miRNA-mediated pathways in metastatic disease.},
}
@article {pmid41135510,
year = {2025},
author = {Yang, Q and Sun, Y and Sun, L and Chi, T and Chen, Z},
title = {Cryo-EM structure of the RfxCas13d-crRNA-off-target-RNA complex.},
journal = {Structure (London, England : 1993)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.str.2025.09.010},
pmid = {41135510},
issn = {1878-4186},
abstract = {The CRISPR-Cas system is crucial for the adaptive immune response of prokaryotes and has been widely applied for genetic engineering. Cas13d, a type VI-D CRISPR-Cas effector, functions as RNA-guided ribonuclease and has been engineered for programmable RNA editing, which is a commonly used, active, and well-characterized small type VI editor. Here, we determined cryoelectron microscopy (cryo-EM) structures of Ruminococcus flavefaciens Cas13d in a RfxCas13d-crRNA-off-target-RNA ternary complex and RfxCas13d-crRNA binary complex at 3.10 and 3.13 Å resolution. The ternary complex consists of RfxCas13d, crRNA, and a captured short off-target ssRNA at a complex state of binding proximal mismatched RNA. RfxCas13d undergoes conformational changes with or without the off-target RNA, but the catalytic sites remain unchanged. Mg[2+] aids in stabilizing the crRNA repeat region structure, which may be crucial for RNA binding. This discovery provides the foundation for developing RfxCas13d as a mature tool and offers a framework for advancing transcriptome engineering.},
}
@article {pmid41134772,
year = {2025},
author = {Xu, X and Zhu, L and Xu, X and Chen, J and Du, X and Zhu, L and Yu, S and Qian, L and Jiang, X and Zhou, L and Dong, Y and Wang, Y and Huang, Y and Wang, Y},
title = {TSSKL is essential for sperm mitochondrial morphogenesis and male fertility in moths.},
journal = {PLoS genetics},
volume = {21},
number = {10},
pages = {e1011914},
pmid = {41134772},
issn = {1553-7404},
mesh = {Animals ; Male ; *Spermatozoa/metabolism ; *Moths/genetics ; *Mitochondria/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Fertility/genetics ; Bombyx/genetics ; Female ; Infertility, Male/genetics ; Morphogenesis/genetics ; Testis/metabolism ; CRISPR-Cas Systems ; },
abstract = {Sperm deliver male genomic DNA to the ovum, playing a pivotal role in sexual reproduction across the animal kingdom. The molecular regulation of sperm morphogenesis has consequently become a focal point of genetic research, with dual implications for both reproductive medicine and sustainable agriculture. Here, we characterize the functional role of the testis-specific serine/threonine protein kinase-like (TSSKL) gene in the model lepidopteran insect Bombyx mori and the globally destructive crop pest Plutella xylostella. RNA-seq and qPCR analyses revealed TSSKL's testis-specific expression pattern. Using CRISPR/Cas9-mediated mutagenesis, we demonstrate that TSSKL knockout induces complete male sterility, while female fertility remains unaffected compared to wild-type. Fluorescence microscopy and ultrastructural analyses revealed that TSSKL deletion leads to severe morphological defects in both eupyrene and apyrene sperm, accompanied by impaired mitochondrial dynamics and aberrant autophagy. Comparative transcriptome and functional analyses linked these phenotypes to dysregulated energy metabolism pathways. Crucially, this sterility phenotype is conserved in P. xylostella, recapitulating the findings in B. mori. Our study demonstrates that TSSKL is crucial for male fertility, coordinating both structural and metabolic aspects of sperm development. Beyond advancing fundamental knowledge of insect reproductive biology, this work also identifies TSSKL as an ideal target for lepidopteran pest control through precision sterility induction.},
}
@article {pmid41133737,
year = {2025},
author = {Devine, R and Noble, K and Wilkinson, B and Hutchings, M},
title = {Microbe Profile: Streptomyces formicae KY5: an ANT-ibiotic factory.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {10},
pages = {},
pmid = {41133737},
issn = {1465-2080},
mesh = {*Streptomyces/genetics/metabolism/isolation &amp; purification/classification ; *Ants/microbiology ; Multigene Family ; Anti-Bacterial Agents/biosynthesis ; Animals ; Antifungal Agents/metabolism/pharmacology ; Genome, Bacterial ; Secondary Metabolism/genetics ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Streptomyces formicae KY5 was isolated from a Tetraponera penzigi plant-ant nest. It is primarily known for its production of the formicamycins, antibiotics with potent activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus, and additionally produces an antifungal compound that inhibits multi-drug-resistant fungal pathogens including Lomentospora prolificans. S. formicae is genetically tractable using CRISPR-Cas9 gene editing, allowing for detailed analysis of the formicamycin biosynthetic gene cluster. AntiSMASH analysis predicts the genome to encode at least 45 secondary metabolite biosynthetic gene clusters, many of which appear to encode novel compounds. Current research efforts are focussing on characterising the regulation of secondary metabolism at a global level in order to switch on pathways that are not typically expressed under standard laboratory conditions with the aim of identifying novel antimicrobials.},
}
@article {pmid41132368,
year = {2025},
author = {Morais, C and Costa, SS and Hanke, D and Santos, A and Krüger-Haker, H and Pomba, C and Feßler, AT and Schwarz, S and Couto, I},
title = {Genomic analysis of the Staphylococcus pseudintermedius mobilome associated with antimicrobial resistance.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1640322},
pmid = {41132368},
issn = {1664-302X},
abstract = {The increasing antimicrobial resistance (AMR) in Staphylococcus pseudintermedius causing skin and soft-tissue infections (SSTIs) in companion animals is a public health concern. The aim of this study was to verify if mobile genetic elements (MGEs), in particular plasmids, are related to the carriage of AMR genes among circulating and clinically relevant S. pseudintermedius. In total, 56 S. pseudintermedius, representing predominant and emerging clonal lineages associated with SSTIs in dogs and cats collected in Lisbon (Portugal), were subjected to plasmid DNA extraction and digestion with EcoRI and XbaI. Each unique restriction pattern was assigned to a plasmid profile. A subset of 17 strains was further selected for hybrid whole genome sequencing (WGS) on Oxford Nanopore MinION and Illumina MiSeq platforms. Thirty-one of the 56 S. pseudintermedius strains carried one or more plasmid(s), mostly of small or medium sizes, corresponding to eight plasmid profiles. Two of the identified plasmids carried AMR determinants; plasmid pSP-G3C4, isolated from ST71 strains, carried the tetracycline resistance gene tet(K) and plasmid pSP5912, isolated from a ST2061 strain, harbored the qacG biocide resistance gene. Other AMR determinants were detected as part of MGEs integrated into the bacterial chromosomal DNA, namely Tn552, Tn552-like, Tn553, Tn916, Tn5405-like, Tn5801, Tn5801-like GI6287 and pRE25-like elements. In addition, a new chromosomal cassette, carrying fusC, was identified in a ST1183 strain. The 12 methicillin-resistant S. pseudintermedius studied carried staphylococcal cassette chromosome mec (SCCmec) type III (n = 5), SCCmec type IVg (n = 3), SCCmec NA45 (n = 1), ΨSCCmec 57395 (n = 1), the recently described cassettes SCCmec 7017-61515 (n = 1), or SCCmec type V(T)SL/154 (n = 1). Most strains carried intact prophages without AMR determinants. Intact restriction-modification systems were detected in 12 out of the 17 strains and CRISPR/Cas in five strains, four of which were methicillin-susceptible. The results of this study suggest that the AMR content in S. pseudintermedius is mainly related to MGEs integrated into the chromosomal DNA rather than located on plasmids. These results provide important insights that may lead to a better understanding of multidrug resistance in S. pseudintermedius towards improved SSTIs treatment in companion animals.},
}
@article {pmid41129856,
year = {2025},
author = {Zhou, J and Ren, XM and Gao, J and Wu, RP and Chen, L and Li, Z},
title = {Amplification-free detection of mycoplasma pneumoniae via CRISPR-Cas12a and deep learning-optimized crRNAs on a lateral flow platform.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {268},
number = {},
pages = {117196},
doi = {10.1016/j.jpba.2025.117196},
pmid = {41129856},
issn = {1873-264X},
abstract = {Accurate and rapid diagnosis of Mycoplasma pneumoniae infection is essential for reducing its significant health burden. An amplification-free CRISPR-Cas12a-mediated detection platform has been developed, incorporating a deep learning-optimized crRNA library (CCDLCL) targeting conserved regions of the MP P1 gene. The system enables visual readout via lateral flow strips, supporting its potential as a point-of-care testing (POCT) nucleic acid testing strategy. Through computational design and screening, 16 highly active crRNAs were identified from an initial set of over 50 candidates. Combinatorial use of these crRNAs demonstrated synergistic enhancement of fluorescence signal intensity and reaction kinetics. Compared to single-crRNA assays, the multiplexed crRNA library improved sensitivity by 16.8-fold, achieving a limit of detection (LOD) of 0.15 pM, and reduced time to signal saturation by 30 %. When deployed on lateral flow strips, the assay exhibited a tenfold increase in visual detection sensitivity, with a LOD of 100 pM. Clinical evaluations confirmed high specificity-showing no cross-reactivity with SARS-CoV-2, hepatitis B virus (HBV), or human genomic DNA-and over 95 % agreement with standard clinical results without target pre-amplification, delivering outcomes within 45 min. This study establishes a deep learning-facilitated crRNA design framework and a novel crRNA library-based detection system, offering a feasible approach for POCT nucleic acid testing in resource-limited settings and paving the way for streamlined clinical translation of CRISPR-Cas diagnostics.},
}
@article {pmid41125583,
year = {2025},
author = {Yang, J and Li, H and Li, M and Song, R and Shen, T and Wang, G and Xu, D and Hao, M and Jia, A and Rehman, SU and Hua, L and Liang, Y and Chi, C and Lan, C and Deng, XW and Dubcovsky, J and Song, B and Wang, X and Chen, S},
title = {Genome-assisted identification of wheat leaf rust resistance gene Lr.ace-4A/Lr30.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9339},
pmid = {41125583},
issn = {2041-1723},
mesh = {*Triticum/genetics/microbiology/immunology ; *Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Plant Leaves/microbiology/genetics ; Basidiomycota ; Genome, Plant ; *Genes, Plant ; CRISPR-Cas Systems ; Puccinia ; },
abstract = {Leaf rust is a devastating disease of wheat. Growing rust-resistant wheat varieties is the best strategy to mitigate this threat. Here, we generate a 10.51-gigabase chromosome-scale assembly of the durum wheat landrace PI 192051. Using mutagenesis and transcriptome sequencing, we identify the leaf rust resistance gene Lr.ace-4A within a recombination-sparse region of PI 192051 and demonstrate that Lr.ace-4A is identical to the previously designated Lr30 gene in hexaploid wheat. Lr.ace-4A/Lr30 encodes a non-canonical coiled-coil nucleotide-binding leucine-rich repeat receptor, featuring tandem nucleotide-binding domains. This gene is both necessary and sufficient to confer resistance to leaf rust, as demonstrated by CRISPR/Cas9-induced mutations and transgenic complementation. Lr.ace-4A provides near-immunity resistance in durum wheat, though its effectiveness is diminished in hexaploid wheat. Two amino acid polymorphisms differentiate the resistant and susceptible Lr.ace-4A haplotypes, with transgenic plants carrying either susceptible variant showing susceptibility. The cloning of Lr.ace-4A will accelerate its deployment in wheat breeding programs.},
}
@article {pmid41125159,
year = {2025},
author = {Zhou, C and Zhu, S and Luo, C and Wang, W and Fan, H and Gao, Y and Xu, X and Wang, Q and You, Y and Xie, T},
title = {From IscB to Cas9: Engineering and advances in the next generation of miniature gene editing tools.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108743},
doi = {10.1016/j.biotechadv.2025.108743},
pmid = {41125159},
issn = {1873-1899},
abstract = {The CRISPR-Cas system, distinguished by its inherent modularity and broad programmability, has catalyzed a paradigm shift in genome engineering due to its unprecedented accuracy, specificity, and on-target efficiency, now serving as the cornerstone of modern genome manipulation. The efficient delivery of gene editing tools remains a major technical hurdle to clinical application, primarily due to the lack of compact editors. The recent identification of the transposon-associated nuclease IscB as an evolutionary ancestor of Cas9 has provided important insights into the molecular evolution of the CRISPR-Cas9 system. Notably, IscB is a highly compact nuclease, approximately one-third the size of Cas9, capable of precise nucleic acid cleavage in eukaryotic cells under the guidance of ωRNA. These features make it a promising candidate for the development of next-generation miniaturized genome editors. However, natural IscB exhibits limited editing performance in eukaryotic systems. This review first outlines the biochemical function of the transposon IscB and briefly traces the evolutionary origin of the Cas9 system. It then describes and compares the structural characteristics and cleavage mechanisms of OgeuIscB and Cas9. Subsequent sections summarize various engineering strategies for current IscB systems, including the development of base editors and recent advances in their application. Finally, the limitations of existing systems are discussed, and potential directions for future optimization are proposed, aiming to provide new insights and facilitate the advancement of IscB-based miniaturized editors.},
}
@article {pmid41124242,
year = {2025},
author = {Lu, X and Zhu, Y and Wei, C and Cheng, L and Goodier, KD and Kong, J and Gao, X and Yu, D and Liu, X and Long, Y and Lin, J and Ma, J and Su, Y and Mao, HQ},
title = {A multistep platform identifies spleen-tropic lipid nanoparticles for in vivo T cell-targeted delivery of gene-editing proteins.},
journal = {Science advances},
volume = {11},
number = {43},
pages = {eady5579},
pmid = {41124242},
issn = {2375-2548},
mesh = {Animals ; *Gene Editing/methods ; *Spleen/metabolism/cytology ; Mice ; *T-Lymphocytes/metabolism/immunology ; *Nanoparticles/chemistry ; *Lipids/chemistry ; *Gene Transfer Techniques ; Humans ; Receptors, CCR5/genetics ; CRISPR-Cas Systems ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are a promising nonviral delivery system for gene-editing proteins, but optimal formulations remain underexplored. Unlike messenger RNA-based approaches, ribonucleoprotein delivery enables immediate genome editing without relying on endogenous translation. However, intracellular delivery remains a major challenge due to protein size, charge variability, and susceptibility to denaturation and degradation. Here, we present a multistep screening platform to optimize LNP formulations for gene-editing protein delivery, focusing on in vivo T cell targeting. Through in vitro screening of a composition library, we identified top-performing candidates. In vivo screening in Ai9 mice revealed a spleen-tropic LNP formulation that preferentially targets T cells, enabling efficient gene editing in vivo. Using this LNP formulation, we achieved targeted knockout of CCR5 and PD-1 in splenic T cells, supporting potential applications in HIV resistance and cancer immunotherapy. Furthermore, a machine learning-guided mechanistic study revealed key design principles for LNP-based protein delivery, highlighting unexplored opportunities for in vivo genome-editing therapies.},
}
@article {pmid41123979,
year = {2025},
author = {Wang, S and Wei, Z and Feng, Y and Gan, Z and Yu, L and Cheng, H and Xiao, Y},
title = {Rationally Trapped Polycrystalline Perovskite in Lanthanide MOF Cages for Ammonia-Mediated Nucleic Acid Intelligent Visualization.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {},
number = {},
pages = {e07214},
doi = {10.1002/smll.202507214},
pmid = {41123979},
issn = {1613-6829},
support = {82273895//National Natural Science Foundation of China/ ; 82304440//National Natural Science Foundation of China/ ; 82073811//National Natural Science Foundation of China/ ; 2024M763727//China Postdoctoral Science Foundation/ ; },
abstract = {Perovskites nanoparticles (PNPs), promising materials in fluorescence biosensing, have has their practical applications stymied by poor stability in polar solvents. Integrating PNPs into metal-organic frameworks (MOFs) offers a solution by enhancing their compatibility with various environments. Lanthanide MOFs (Ln-MOFs) are particularly advantageous due to their customizable structure, enhanced stability, and intrinsic fluorescence. Herein, the mechanism by which hybrid materials achieve a balance between physical and fluorescence properties is elucidated. Through theoretical calculations, 2,2'-bipyridine-5,5'-dicarboxylic acid is selected as the ligand to sensitize Eu[3+] and facilitate Pb[2+] chemisorption. In addition, 4-bromobutyric acid is not only employed to create hierarchical Eu-MOFs and optimize the framework for in situ growth of polycrystalline perovskites but also utilized to generate zwitterionic ligands through an SN2 reaction with MOF cages restricted n-octylamine, ensuring the stable dispersion of hybrid materials in ethyl acetate (polar solvent). The prepared PNPs@Ln-MOF exhibits significantly enhanced fluorescence lifetime (50-fold) and stability in polar solvents. Besides, the highly sensitive fluorescence color shift of PNPs@Ln-MOF in response to ammonia offers a generalizable strategy for ammonia-mediated biosensing device. Supported by CRISPR/Cas technology, this device allows for precise on-site nucleic acid assay (LOD = 200 fM), pioneering advanced applications of perovskite-based hybrid materials in biosensing.},
}
@article {pmid41065718,
year = {2025},
author = {Zeng, B and Sheng, A and Zhang, X and Wang, X and Bao, Y and Huang, Y and Huang, Y and Shan, L and Xu, X and Qin, Y and Yang, Y and Deng, Y and Tian, L and Wang, J and Ma, L},
title = {CRISPR/Cas12a Integrated with a Microfluidic System Enhanced Analysis of Programmed Cell Death Ligand 1 Expression in Circulating Tumor Cells from Non-Small Cell Lung Cancer Patients.},
journal = {ACS sensors},
volume = {10},
number = {10},
pages = {7388-7402},
doi = {10.1021/acssensors.5c01152},
pmid = {41065718},
issn = {2379-3694},
mesh = {Humans ; *Carcinoma, Non-Small-Cell Lung/blood/pathology/metabolism ; *Neoplastic Cells, Circulating/metabolism/pathology ; *B7-H1 Antigen/genetics/metabolism/blood ; *Lung Neoplasms/blood/pathology/metabolism ; *CRISPR-Cas Systems ; *Lab-On-A-Chip Devices ; *Microfluidic Analytical Techniques/methods ; Cell Line, Tumor ; },
abstract = {The detection of programmed cell death ligand 1 (PD-L1) positive circulating tumor cells (CTCs) in peripheral blood has significant clinical value for predicting and evaluating the efficacy of immunotherapy in patients with non-small cell lung cancer (NSCLC). However, traditional methods remain limited by low sensitivity and the precise quantification remains a challenge. A dual-mode microfluidic analysis chip was constructed here that included clustered regularly interspaced short palindromic repeats/Cas12a quantification and immunofluorescence visualization. Quantification of the PD-L1 protein on the surface of CTCs (20 to 10[7] cell/mL) was achieved selectively and sensitively by amplifying the nucleic acid target to generate a strong fluorescent signal, even with very low levels of target cells. The system effectively detected PD-L1[+] CTCs expression in peripheral blood samples from patients with NSCLC and monitored the efficacy of PD-1/PD-L1 targeted immune checkpoint inhibitors in real time. It exhibited excellent performance for clinical applications in monitoring the prognosis in patients with NSCLC.},
}
@article {pmid41123685,
year = {2025},
author = {Yang, S and Liu, Y and Zhang, J and Xu, J and Li, T and Huang, H and Zhu, Z and Li, M and Wang, H and Yang, C},
title = {An integrated lab-in-a-tube platform for point-of-care detection of blaKPC in urinary tract infections.},
journal = {Mikrochimica acta},
volume = {192},
number = {11},
pages = {748},
pmid = {41123685},
issn = {1436-5073},
support = {82472374//National Natural Science Foundation of China/ ; YG2024LC02//Cross-Research Fund of Biomedical Engineering of Shanghai Jiaotong University/ ; 2024ZY008//The Clinical and Excellence Program at the Institute of Molecular Medicine, Shanghai Jiao Tong University School of Medicine/ ; },
mesh = {Humans ; *Urinary Tract Infections/microbiology/diagnosis/urine ; *Klebsiella pneumoniae/enzymology/genetics/drug effects/isolation &amp; purification ; *beta-Lactamases/genetics/urine ; *Bacterial Proteins/genetics/urine ; *Point-of-Care Systems ; Klebsiella Infections/urine/diagnosis/microbiology ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; },
abstract = {Carbapenem-resistant Klebsiella pneumoniae (CRKP), predominantly mediated by the blaKPC carbapenemase gene, poses a critical therapeutic challenge for urinary tract infections (UTIs). To enable rapid identification at the point of need, we engineered TubeCARE (tube-integrated platform for carbapenem antimicrobial resistance evaluation), an integrated, disposable lab-in-a-tube system for direct blaKPC detection from urine samples. This self-contained system uniquely combines urine processing, nucleic acid extraction, recombinase polymerase amplification (RPA), and CRISPR/Cas12a reaction within a single sealed unit, offering dual-modality outputs: smartphone-based real-time fluorescence or naked-eye lateral flow strip interpretation. The full workflow delivers "sample-in, result-out" in 40 min, eliminating external multi-step nucleic acid extraction and reducing contamination risks. Both detection modes demonstrated laboratory-grade sensitivity (1 CFU/mL) with 100% specificity against non-target carbapenemase genes. Clinical validation using 24 urine samples (20 blaKPC-positive, 4 negative) showed 100% concordance with quantitative PCR. In a representative UTI case, TubeCARE enabled 40-min bedside blaKPC detection; early implementation would circumvent 72-h diagnostic delays, facilitating timely carbapenem avoidance and preventing bloodstream complications. Featuring integrated lab-in-a-tube operation, cost-effective manufacturing, user-friendly workflow, and laboratory-grade accuracy, TubeCARE provides actionable antimicrobial resistance surveillance in diverse clinical and resource-limited settings.},
}
@article {pmid40938646,
year = {2025},
author = {Lee, JH and Lee, E-S and Kyung, SM and Xiang, X-R and Park, H-E and Shin, M-K and Yoo, HS},
title = {Functional analysis of the intracellular survival of Mycobacterium avium subsp. paratuberculosis in THP-1 cells using CRISPR interference.},
journal = {Journal of bacteriology},
volume = {207},
number = {10},
pages = {e0024425},
doi = {10.1128/jb.00244-25},
pmid = {40938646},
issn = {1098-5530},
support = {//National Institute of Wildlife Disease Control and Prevention/ ; },
mesh = {Humans ; *Mycobacterium avium subsp. paratuberculosis/genetics/physiology/pathogenicity ; *Macrophages/microbiology ; THP-1 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Virulence/genetics ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; *Microbial Viability ; CRISPR-Cas Systems ; Paratuberculosis/microbiology ; },
abstract = {UNLABELLED: Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne's disease in ruminants and a potential zoonotic agent linked with Crohn's disease in humans. Despite the possible risk to public health, few studies have focused on the virulence of MAP against human macrophages. Therefore, a functional analysis of mycobacterial genes associated with virulence, especially the intracellular survival of MAP, was performed after infection of MAP CRISPR interference (CRISPRi) mutants in the human THP-1 macrophages. MAP mutants were targeted to four genes (mdh, pknG, MAP1981c, and icl). The optimal concentration of anhydrotetracycline (ATc) was determined to be 5 µg/mL by measuring the survival of the cells and the downregulation of gene expression levels in the cells up to Day 3. The clump formation and intracellular survival of MAP were investigated using transmission electron microscopy and the colony-forming units, respectively. The clump formation of MAP mutants induced by CRISPRi was decreased in THP-1 macrophages at 24 and 72 h post-infection. The survival rates of the MAP mutants significantly decreased with increasing ATc concentration and time course of infection in MAP-mdhKD, MAP1981cKD, and MAP-iclKD. Conversely, the survival rate of THP-1 macrophages increased with increasing ATc concentration. Our results suggest that these genes might be closely related to MAP virulence along with intracellular survival in THP-1 macrophages. These data can provide novel insights into the utilization of CRISPRi in further research on MAP virulence by exploring intracellular survival using mycobacterial genes related to the virulence of MAP during host infection.<br><br>IMPORTANCE: Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP) is a worldwide issue in the dairy industry and has a possible connection to Crohn's disease (CD) in humans. Despite its potential contribution to the etiology of CD, there have been few studies focusing on the virulence of MAP against human macrophages. In the current study, we investigated MAP virulence along with intracellular survival in human THP-1 macrophages using functional analysis of MAP CRISPR interference (CRISPRi) mutants at the knockdown of genes associated with mycobacterial virulence. The identified potential genes represent novel candidate classes that could be necessary for MAP virulence by exploring intracellular survival during host infection and could provide novel insights for future studies on the utilization of CRISPRi.},
}
@article {pmid40919935,
year = {2025},
author = {Alberts, ME and Kurtz, MP and Müh, U and Bernardi, JP and Bollinger, KW and Dobrila, HA and Duncan, L and Laster, HM and Orea, AJ and Pannullo, AG and Rivera-Rosado, JG and Torres, FV and Ellermeier, CD and Weiss, DS},
title = {Analysis of essential genes in Clostridioides difficile by CRISPRi and Tn-seq.},
journal = {Journal of bacteriology},
volume = {207},
number = {10},
pages = {e0022025},
doi = {10.1128/jb.00220-25},
pmid = {40919935},
issn = {1098-5530},
support = {DBI-1852070//National Science Foundation/ ; R01 AI155492/AI/NIAID NIH HHS/United States ; R21 AI159071/AI/NIAID NIH HHS/United States ; },
mesh = {*Clostridioides difficile/genetics/metabolism ; *Genes, Essential ; *DNA Transposable Elements ; Mutagenesis, Insertional ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Essential genes are interesting in their own right and as potential antibiotic targets. To date, only one report has identified essential genes on a genome-wide scale in Clostridioides difficile, a problematic pathogen for which treatment options are limited. That foundational study used large-scale transposon mutagenesis to identify 404 protein-encoding genes as likely to be essential for vegetative growth of the epidemic strain R20291. Here, we revisit the essential genes of strain R20291 using a combination of CRISPR interference (CRISPRi) and transposon insertion site sequencing (Tn-seq). First, we targeted 181 of the 404 putatively essential genes with CRISPRi. We confirmed essentiality for >90% of the targeted genes and observed morphological defects for >80% of them. Second, we conducted a new Tn-seq analysis, which identified 346 genes as essential, of which 283 are in common with the previous report and might be considered a provisional essential gene set that minimizes false positives. We compare the list of essential genes to those of other bacteria, especially Bacillus subtilis, highlighting some noteworthy differences. Finally, we used fusions to red fluorescent protein (RFP) to identify 18 putative new cell division proteins, 3 of which are conserved in Bacillota but of largely unknown function. Collectively, our findings provide new tools and insights that advance our understanding of C. difficile.IMPORTANCEClostridioides difficile is an opportunistic pathogen for which better antibiotics are sorely needed. Most antibiotics target pathways that are essential for viability. Here, we use saturation transposon mutagenesis and gene silencing with CRISPR interference to identify and characterize genes required for growth on laboratory media. Comparison to the model organism Bacillus subtilis revealed many similarities and a few striking differences that warrant further study and may include opportunities for developing antibiotics that kill C. difficile without decimating the healthy microbiota needed to keep C. difficile in check.},
}
@article {pmid40897994,
year = {2025},
author = {Kumar, N},
title = {Genome Editing for Fertility: Unlocking the Promise of CRISPR/Cas9 in Addressing Male Infertility - A Narrative Review.},
journal = {Reproductive sciences (Thousand Oaks, Calif.)},
volume = {32},
number = {10},
pages = {3221-3239},
pmid = {40897994},
issn = {1933-7205},
mesh = {*Gene Editing/methods ; Humans ; Male ; *CRISPR-Cas Systems ; *Infertility, Male/genetics/therapy ; Animals ; *Fertility/genetics ; *Genetic Therapy/methods ; },
abstract = {Male infertility remains a significant global reproductive health challenge, frequently attributed to genetic mutations impairing spermatogenesis and sperm function. This narrative review aims to explore the genetic and molecular underpinnings of male infertility and evaluate the emerging role of Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein 9 (CRISPR/Cas9) genome editing as a diagnostic and therapeutic tool, while addressing its associated ethical, technical, and safety considerations. A Comprehensive literature search was conducted across PubMed, Scopus, Web of Science databases, covering studies published between September 1992 and April 2025. Keywords included "male infertility," "genetic causes of male infertility," "genome editing," "CRISPR/Cas9 and male infertility," "genome editing in male reproduction," "ethical concerns of CRISPR," and "future fertility treatments." Eligible studies focused on genetic correction strategies, spermatogonial stem cell applications, off-target effects, mosaicism, and ethical implications of gene editing. The review synthesizes current knowledge on genetic and epigenetic etiologies of male infertility. It discusses the therapeutic potential of CRISPR/Cas9 in correcting these defects and restoring fertility in preclinical models. Critical challenges, including off-target gene editing, germline mosaicism, long-term safety, and ethical debates surrounding human germline modification, were examined. The review also considers future advancements in genome editing and artificial sperm development. CRISPR/Cas9 represents a transformative platform in reproductive medicine with promising implications for treatment of genetically linked male infertility. However, its clinical translation demands rigorous validation, transparent ethical deliberation, and robust regulatory frameworks. Future innovations combining genome editing, regenerative biology, and precision diagnostics may revolutionize fertility care, but must proceed with caution to ensure safety, efficacy, and ethical integrity.},
}
@article {pmid40782836,
year = {2025},
author = {Agboola, OE and Agboola, SS and Odeghe, OB and Olaiya, OE and Ayinla, ZA and Akinsanya, PO and Ilesanmi, OS and Ibrahim, TK and Adegbuyi, TA and Kolawole, OA and Omotuyi, IO and Oyinloye, BE},
title = {Computational genome engineering through AI-CRISPR-precision medicine integration in modern therapeutics.},
journal = {Annales pharmaceutiques francaises},
volume = {83},
number = {6},
pages = {1073-1085},
doi = {10.1016/j.pharma.2025.08.001},
pmid = {40782836},
issn = {2772-803X},
mesh = {Humans ; *Precision Medicine/methods ; *Gene Editing/methods ; *Artificial Intelligence ; Drug Discovery ; *CRISPR-Cas Systems ; *Genetic Engineering/methods ; Machine Learning ; },
abstract = {The convergence of precision medicine strategies, CRISPR gene editing technologies, and artificial intelligence (AI) is causing a revolutionary change in the pharmaceutical industry in recent times. Latest trends and future directions of these integrated technologies in pharmaceutical science and molecular biology are presented in the present exhaustive review. With more than 250 gene-editing clinical trials being tracked internationally as of February 2025, the recent clinical successes point toward the therapeutic potency of CRISPR-based therapeutics. In parallel, AI-based drug discovery platforms are recording fantastic hit rates; compared to conventional industry benchmarks, AI-emerging drugs reflect 80-90% Phase I trial success rates. Therapeutic development paradigms are being transformed by the intersection of machine learning algorithms, multi-omics technologies, and precision medicine paradigms. The review provides insights into the revolutionary potential of these converging approaches in addressing unmet medical requirements and optimizing therapeutic benefits through syntheses of existing evidence from clinical trials, regulatory matters, and technological innovations.},
}
@article {pmid41123679,
year = {2025},
author = {Rocha, DC and Omoregbee, MO and Contiliani, DF and Mandlik, R and Li, G and Mascoveto, J and Coleman, G and Culver, JN and Leal, DR and de Souza, AA and Qi, Y},
title = {Transgene-free genome editing in citrus and poplar trees using positive and negative selection markers.},
journal = {Plant cell reports},
volume = {44},
number = {11},
pages = {244},
pmid = {41123679},
issn = {1432-203X},
support = {IOS-2132693//Division of Integrative Organismal Systems/ ; 2224203//Division of Integrative Organismal Systems/ ; IOS-2428015//Division of Integrative Organismal Systems/ ; 2021-67013-34554//National Institute of Food and Agriculture/ ; 2020-33522-32274//National Institute of Food and Agriculture/ ; 2024-33522-42755//National Institute of Food and Agriculture/ ; 2020-70029- 33161//National Institute of Food and Agriculture/ ; DE-SC0023011//Department of Energy/ ; 2023/09068-9//FAPESP/ ; },
mesh = {*Populus/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Citrus/genetics ; Transgenes/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant ; Genetic Markers ; Herbicide Resistance/genetics ; },
abstract = {Transgene-free genome editing of the gene of interest in citrus and poplar has been achieved by co-editing the ALS gene via transient transgene expression of an efficient cytosine base editor. CRISPR-Cas genome editing systems have been widely used in plants. However, such genome-edited plants are nearly always transgenic in the first generation when Agrobacterium-mediated transformation is used. Transgene-free genome-edited plants are valuable for genetic analysis and breeding as well as simplifying regulatory approval. It can be challenging to generate transgene-free genome-edited plants in vegetatively propagated or perennial plants. To advance transgene-free genome editing in citrus and poplar, we investigated a co-editing strategy using an efficient cytosine base editor (CBE) to edit the ALS gene to confer herbicide resistance combined with transient transgene expression and potential mobile RNA-based movement of CBE transcripts to neighboring, non-transgenic cells. An FCY-UPP based cytotoxin system was used to select non-transgenic plants that survive after culturing on 5-FC containing medium. While the editing efficiency is higher in poplar than in citrus, our results show that the CBE-based co-editing strategy works in both citrus and poplar, albeit with low efficiency for biallelic edits. Unexpectedly, the addition of the TLS mobile RNA sequence reduced genome editing efficiency in both transgenic and non-transgenic plants. Although a small fraction of escaping plants is detected in both positive and negative selection processes, our data demonstrate a promising approach for generating transgene-free base-edited plants.},
}
@article {pmid41123065,
year = {2025},
author = {Sohail, M and Ma, S and Mushtaq, B and Haider, MU and Li, B and Zhang, X and Huang, H},
title = {Fabricating Four-Element Doped Carbon Dots-Based Fluorescent Ratiometric Reporter Platform for CRISPR/Cas-Driven Precise Sensing of Nucleic Acids.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c03228},
pmid = {41123065},
issn = {1520-6882},
abstract = {Conventional CRISPR/Cas sensing platforms exhibit poor efficiency concerning reporter-based demerits, including their interference-labile nature, photobleaching, low robustness due to a single output signal, and probe-concentration dependence. Herein, a carbon dots (CDs)-based dual-emissive fluorescent ratiometric CRISPR/Cas reporter platform was fabricated for biosensing and other analytical applications to bottleneck the demerits of conventional reporters, integrating the benefits of a ratiometric strategy and four-element doped carbon dots (4D CDs) as a transducer. Briefly, doping enhances the optical and physicochemical traits of CDs and minimizes the effect of the interfering species. A series of state-of-the-art N, P, S, and Cu codoped CDs (4D CDs) were synthesized using the hydrothermal approach and statistical tools, such as Box-Behnken design, analysis of variance, and others, enhancing photophysical traits, surface features, and sensitivity of CDs. The red-emissive CDs were prepared by using the same procedure but different precursors. The optimum 4D CDs (blue-emissive) and red-emissive CDs were used to unleash the principle of the fluorescent ratiometric CRISPR/Cas reporter system for diverse applications. Finally, the designed 4D CDs-based CRISPR/Cas biosensor was applied for nucleic acid monitoring, such as the COVID-19 nucleic acid. This project disclosed the controlled-doping principle to synthesize 4D CDs and unleashed the mechanism of ratiometric dual-emissive CRISPR/Cas-powered reporters for precise sensing applications. We anticipate the implementation of this technology in commercial analytical, biosensing, point-of-care, and other applications.},
}
@article {pmid41122064,
year = {2025},
author = {Balobaid, A and Waterworth, WM and Vila Nova, SF and Causier, B and Sharma, V and Park, MR and Pandey, MK and West, CE},
title = {Arabidopsis thaliana FANCONI ANAEMIA I (FANCI) has roles in the repair of interstrand crosslinks and CRISPR-Cas9 induced DNA double strand breaks.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {2},
pages = {e70533},
doi = {10.1111/tpj.70533},
pmid = {41122064},
issn = {1365-313X},
support = {//King Saud University/ ; BB/S002081/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/Y010426/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/metabolism/genetics ; *DNA Repair/genetics ; *DNA Breaks, Double-Stranded ; CRISPR-Cas Systems/genetics ; Mitomycin/pharmacology ; Cross-Linking Reagents ; Mutation ; },
abstract = {DNA repair is crucial for genome stability, in particular for plants which are exposed to high levels of damage arising from UV irradiation, soil pollutants and reactive oxygen species. Damage that affects both strands of the DNA duplex is harder to repair due to both the lack of a template strand and the potential for physical separation of fragmented chromosomes. As such, DNA double-strand breaks (DSBs) and interstrand DNA crosslinks (ICL) are particularly cytotoxic forms of damage. Here we report the functions of FANCONI ANAEMIA I (FANCI), an Arabidopsis thaliana homologue of the mammalian ICL repair protein. We show that in plant cells, as in mammals, FANCI forms a nuclear localised complex with FANCD2. Genetic analysis of plants lacking FANCI displays significant hypersensitivity to the DNA crosslinking reagent mitomycin C. Furthermore, mutation of FANCI in combination with mutations in a second ICL repair factor, METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81), results in increased levels of programmed cell death compared to the corresponding single mutants, revealing roles in maintaining plant genome stability. Sequence analysis of mutational repair of CRISPR-Cas9-induced DSBs revealed that FANCI promotes single nucleotide insertions and reduces longer deletions. This pattern of mutations may reflect roles for FA proteins in replication-coupled repair of a subset of DSBs. Taken together, this analysis finds evidence for multiple roles for FANCI in the maintenance of plant genome stability.},
}
@article {pmid41121375,
year = {2025},
author = {Amieva, R and Rico-San Román, L and Pastor-Fernández, I and Hemphill, A and Boubaker, G and Collantes-Fernández, E and Ortega-Mora, LM and Horcajo, P},
title = {Loss of NcBPK1 impairs bradyzoite differentiation and enhances virulence in Neospora caninum.},
journal = {Parasites &amp; vectors},
volume = {18},
number = {1},
pages = {422},
pmid = {41121375},
issn = {1756-3305},
support = {PRE2020-092101//Spanish Ministry of Science, Innovation and Universities/ ; },
mesh = {*Neospora/pathogenicity/genetics/growth &amp; development ; Animals ; Virulence ; Mice ; *Coccidiosis/parasitology/veterinary ; *Protozoan Proteins/genetics/metabolism ; Female ; Cattle ; Macrophages/parasitology ; Pregnancy ; Virulence Factors/genetics ; Cattle Diseases/parasitology ; CRISPR-Cas Systems ; Host-Parasite Interactions ; Disease Models, Animal ; Gene Deletion ; },
abstract = {BACKGROUND: Neospora caninum is an apicomplexan parasite responsible for bovine neosporosis, a disease that leads to substantial economic losses in cattle due to abortion and reduced productivity. The pathogenesis of N. caninum is shaped by complex host-parasite interactions, and virulence is known to vary between strains. BPK1 (Bradyzoite pseudokinase 1), a pseudokinase previously identified as a potential virulence factor in Toxoplasma gondii, has not yet been functionally characterized in N. caninum.<br><br>METHODS: To investigate the role of NcBPK1 in parasite virulence, a knockout strain (Nc&#x394;BPK1) was generated using CRISPR/Cas9 genome editing. The virulence of the mutant was evaluated in a pregnant mouse model by assessing neonatal survival and parasite burden in dam tissues. In vitro assays were conducted to examine parasite replication in bovine macrophages and to analyze the expression of stage-specific genes.<br><br>RESULTS: Deletion of NcBpk1 resulted in enhanced parasite virulence in vivo, as shown by a decrease in neonatal survival and higher parasite loads in maternal brain tissue. The Nc&#x394;BPK1 mutant also displayed enhanced replication in bovine macrophages and reduced expression of bradyzoite-specific genes, suggesting a defect in stage conversion.<br><br>CONCLUSIONS: These findings indicate that NcBPK1 is crucial for regulating the balance between acute replication and chronic persistence. Its absence promotes rapid tachyzoite proliferation and worsens disease outcomes. This study sheds light on the molecular mechanisms underlying N. caninum virulence. Further research is needed to elucidate the signaling pathways and protein interactions involving NcBPK1.},
}
@article {pmid41121349,
year = {2025},
author = {Safarzadeh Kozani, P and Safarzadeh Kozani, P},
title = {Preventing secondary primary malignancies (SPMs) in CAR-T cell therapy through site-specific transgene integration into genomic safe harbors (GSHs).},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {1155},
pmid = {41121349},
issn = {1479-5876},
mesh = {Humans ; *Transgenes/genetics ; *Neoplasms, Second Primary/prevention &amp; control/genetics ; *Receptors, Chimeric Antigen/metabolism ; Animals ; *Immunotherapy, Adoptive/adverse effects ; Gene Editing ; *Genomics ; CRISPR-Cas Systems ; },
abstract = {Chimeric antigen receptor (CAR)-T cell therapy has revolutionized oncology by achieving durable remissions in refractory hematologic malignancies. However, emerging reports link this therapy to second primary malignancies, including CAR+ lymphomas and leukemias, driven by insertional mutagenesis from semi-random viral vector integration near oncogenes or tumor suppressor loci. These rare but serious complications underscore the dual challenge of eradicating primary tumors while mitigating delayed genotoxic risks. Conventional CAR-T cell manufacturing, reliant on gamma-retroviral or lentiviral vectors, introduces genomic instability through integration into fragile sites or transcriptionally active regions. CRISPR/Cas9-mediated genome editing further amplifies risks via off-target double-strand breaks and chromosomal rearrangements. This review evaluates genomic safe harbors (GSHs)-such as AAVS1, TRAC, CCR5, ROSA26 and CLYBL-as loci validated for stable, high-level CAR transgene expression without oncogenic disruption. GSHs meet stringent criteria: distal from cancer-related genes, resistant to epigenetic silencing, and transcriptionally permissive. Preclinical studies demonstrate that site-directed CAR integration into GSHs preserves antitumor efficacy while eliminating malignant transformation risks. Challenges persist in optimizing homology-directed repair efficiency, mitigating residual dsDNA toxicity, and standardizing regulatory frameworks for long-term genomic surveillance. Emerging technologies-base/prime editing, hybrid nucleases, and rigorous monitoring-promise enhanced precision and safety. By reconciling therapeutic innovation with genomic integrity, GSH-engineered CAR-T cells herald a paradigm shift toward precision immunotherapies, offering curative potential while preempting secondary oncogenesis. Collaborative efforts to refine manufacturing, harmonize global standards, and prioritize patient-specific risk stratification will be critical to advancing this transformative approach.},
}
@article {pmid41121307,
year = {2025},
author = {Li, C and Peng, W and Zhong, Z and Zhang, C and Wang, X and Qin, R and Lei, Q and Lv, J and Liu, F and Zhao, Y and Lv, Z and Li, C and Yang, S and Zhang, H and Tao, Z and Sun, C},
title = {CRISPR/Cas9 library screening reveals that STK19 has synergistic antitumor effects when combined with cisplatin on tongue squamous cell carcinoma.},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {1142},
pmid = {41121307},
issn = {1479-5876},
support = {82360568, 81960543//Natural Science Foundation of China/ ; 82160452//Natural Science Foundation of China/ ; 202401AY070001-137//Applied Basic Research Foundation of Yunnan Province/ ; 202301AY070001-247//Yunnan Fundamental Research Projects/ ; 2023YJKTB01//Basic Research Program of the First People's Hospital of Qujing/ ; 202305AF150091//Yunnan Province Academician Expert Workstation/ ; },
mesh = {*Cisplatin/pharmacology/therapeutic use ; *Tongue Neoplasms/drug therapy/genetics/pathology ; Humans ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Animals ; Drug Synergism ; *Carcinoma, Squamous Cell/drug therapy/genetics/pathology ; *Protein Serine-Threonine Kinases/metabolism/genetics ; Xenograft Model Antitumor Assays ; *Antineoplastic Agents/pharmacology/therapeutic use ; DNA Damage/drug effects ; Mice ; Mice, Nude ; Cell Proliferation/drug effects ; },
abstract = {BACKGROUND: Tongue squamous cell carcinoma (TSCC) is a common oral cancer that has a high propensity for recurrence and metastasis. Therefore, TSCC has a 50% 5-year survival rate. Platinum-based chemotherapy is an effective treatment for squamous cell carcinoma, however, chemotherapy resistance remains a major issue. Therefore, innovative and effective drug combinations are needed to improve TSCC patient prognosis.<br><br>METHODS: In this study, we conducted an in vitro CRISPR/Cas9 library screen using two TSCC cell lines (Tscca and Cal27) to identify specific genes that, when inhibited, synergize with cisplatin to effectively suppress tumor growth.<br><br>RESULTS: We identified STK19 as a potential drug target. Inhibition of STK19 enhances the response of TSCC to cisplatin. Through genetic and pharmacological methods, it has been demonstrated that reducing STK19 activity enhances cisplatin-induced DNA damage. The mechanism involves the depletion of MGMT with STK19 inhibition, leading to conditional lethality and synergistic reduction of tumors in vivo when combined with cisplatin. Overall, in this study, unbiased genetic testing was used to successfully identify synthetic lethal drug combinations for TSCC.<br><br>CONCLUSION: STK19 was identified as a promising target that could enhance the killing effects of cisplatin on tongue squamous carcinoma cells, offering a novel therapeutic option for individuals who are insensitive to conventional treatment methods.},
}
@article {pmid41121296,
year = {2025},
author = {Shi, X and Lu, S and Tang, Q and Zhao, X},
title = {Targeted modification of cis-elements in the CUL3 gene to restore exon 9 inclusion for treating Gordon syndrome.},
journal = {Human genomics},
volume = {19},
number = {1},
pages = {119},
pmid = {41121296},
issn = {1479-7364},
support = {ZR2022MH300//the Shandong Province Natural Science Foundation/ ; 82271540//the National Natural Science Foundation of China/ ; QDFY+X2024131//the Natural Science Foundation of the Affiliated Hospital of Qingdao University/ ; },
mesh = {*Cullin Proteins/genetics ; Humans ; *Exons/genetics ; RNA Splice Sites/genetics ; Introns/genetics ; RNA Splicing/genetics ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {BACKGROUND: The weak splice acceptor site (AS) of exon 9 underlies almost all pathogenic variants of Cullin3 (CUL3) causing exon 9 skipping in Gordon syndrome, emphasizing the need for splicing-targeted therapeutic strategies. This study explored universal therapeutic targets to modulate AS and investigated their potential and mechanisms for restoring normal splicing.<br><br>RESULTS: Through bioinformatic prediction, minigene assays, EMSA, CRISPR/Cas9-mediated construction of mutant cell lines and RIP, three rescue sites in the polypyrimidine (Py) tract of intron 8 were identified, including A(-9)T, A(-10)T and AA(-9, -10)TT, with AA(-9, -10)TT most effectively promoting exon inclusion by extending the Py-tract to increase U2AF2 binding. Additionally, previous candidate target A18G was confirmed to rescue exon 9 skipping by weakening hnRNP A1 splicing inhibition in endogenous cell models.<br><br>CONCLUSIONS: Our findings highlight the therapeutic potential of AA(-9, -10)TT and A18G in CUL3-related Gordon syndrome, suggesting the targeted modification of cis-elements could be an ideal and universal strategy to develop treatments for splicing-related diseases.},
}
@article {pmid41120304,
year = {2025},
author = {Wang, Z and Liu, F and Chen, N and Wu, J and Li, X and Fang, M and Yan, M and Zhang, J and Deng, B and Wang, L and Wang, X and Liu, M and Zeng, D and Zou, Z and Wang, B and Songyang, Z and He, B and Liu, Q},
title = {Chromatin looping-based CRISPR screen identifies TLK2 as chromatin loop formation regulator in cancer stemness plasticity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9288},
pmid = {41120304},
issn = {2041-1723},
support = {82321003//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82341020//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82473123//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82173367//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82403653//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82302929//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82303009//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2022A1515010915//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; },
mesh = {Humans ; *Chromatin/metabolism/genetics ; Animals ; *Neoplastic Stem Cells/metabolism/pathology ; Female ; Mice ; Kruppel-Like Factor 4 ; *Breast Neoplasms/genetics/pathology/metabolism ; Cell Line, Tumor ; *Protein Serine-Threonine Kinases/metabolism/genetics ; CCCTC-Binding Factor/metabolism/genetics ; Cell Plasticity/genetics ; Kruppel-Like Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Cell Cycle Proteins/metabolism ; Chromosomal Proteins, Non-Histone/metabolism ; Lung Neoplasms/secondary/genetics ; },
abstract = {Targeting cancer cell plasticity through chromatin organization is an emerging research area, yet the molecular mechanisms that govern chromatin loop formation remain unclear. Here, we develop a CRISPR screen based on our engineered live-cell CTCF-cohesin contact reporters to identify regulators of chromatin loops. Our findings reveal that tousled-like kinase 2 (TLK2) functions as a key regulator of chromatin loop formation during the cancer stemness transition. Mechanistically, TLK2 phosphorylates DYNLL1, enhancing its interaction with CTCF to promote CTCF-cohesin hub formation at the KLF4 locus. Suppressing TLK2 impairs cancer stemness plasticity, sensitizes cancer cells to cytotoxic stress in vitro, and reduces lung metastases and enhances immunotherapy response in breast cancer mouse models. Clinically, elevated TLK2 expression correlates with poor prognosis in breast cancer patients. Collectively, these findings identify TLK2 as a potential therapeutic target for mitigating cancer stemness plasticity, highlighting chromatin loop-targeting therapy as a promising strategy to eradicate cancer stem cells.},
}
@article {pmid41118579,
year = {2025},
author = {Hanai, Y and Hilario, PLL and Shiraishi, Y and Yoshida, N and Murakami, S and Shimizu, Y and Kano, N and Kojima, M and Murai, K and Kawai, T and Okamura, K},
title = {The knock-in atlas: a web resource for targeted protein trap by CRISPR/Cas9 in human and mouse cell lines.},
journal = {Nucleic acids research},
volume = {53},
number = {19},
pages = {},
doi = {10.1093/nar/gkaf1050},
pmid = {41118579},
issn = {1362-4962},
support = {//NAIST for the Creation of Innovation in Science and Technology/ ; 24KJ1692//JSPS Research Fellowship/ ; 25KJ1828//NAIST Granite Program/ ; 17K20145//JSPS Found for the Promotion of Joint International Research/ ; //Academic Assistant Grant by Office for Gender Equality at NAIST/ ; //Takeda Science Foundation/ ; 20H03468//KAKENHI/ ; 23K14546//JSPS/ ; },
mesh = {Humans ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Gene Knock-In Techniques/methods ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; Internet ; Cell Line ; RNA-Binding Proteins/genetics ; HeLa Cells ; *Databases, Genetic ; },
abstract = {Various cell engineering techniques have been developed by leveraging the CRISPR-Cas9 technology, but large-scale resources for targeted gene knock-in are still limited. Here we introduce the Knock-in Atlas, a web resource for gene tagging by fluorescent proteins by inserting artificial exons in target gene introns. To produce knock-in cells efficiently and reproducibly, we carefully chose and catalogued guide RNAs (gRNAs) for targeting genes in the human and mouse genomes by taking the gRNA efficacy scores and protein structures around the insertion sites into account. As of August 2025, we have characterized knock-in cell lines for 350 proteins, with a focus on RNA binding proteins, by flow cytometry and confocal microscopy. The transfection and flow cytometry protocols were optimized for several cell lines including HEK293T, eHAP1, HeLa, THP-1, Neuro2a, mouse embryonic fibroblast (MEF) and mouse embryonic stem cell (mESC). A website has been launched to organize the results of initial characterization including flow cytometry data after transfection, confocal microscopy, and western blot results for the genes for which knock-in HEK293T cell lines were already made. The site also provides a database to organize the information of pre-designed gRNAs for the human and mouse genomes. <https://rnabio.naist.jp/atlas/>.},
}
@article {pmid41118576,
year = {2025},
author = {Shu, WJ and Ma, Z and Jia, L and Guo, B and Tian, X and He, C and Wang, F},
title = {MiR-ON-CRISPR: a microRNA-activated CRISPR-dCas9 system for precise gene therapy in living cells and mouse models of sepsis.},
journal = {Nucleic acids research},
volume = {53},
number = {19},
pages = {},
doi = {10.1093/nar/gkaf1037},
pmid = {41118576},
issn = {1362-4962},
support = {32271512//National Natural Science Foundation of China/ ; 82572281//National Natural Science Foundation of China/ ; 32201199//National Natural Science Foundation of China/ ; 2023-JC-ZD-43//Natural Science Basic Research Program of Shaanxi/ ; 2025JC-YBQN-1129//Natural Science Basic Research Program of Shaanxi/ ; 2022JC-56//Natural Science Basic Research Program of Shaanxi/ ; 2024SF-YBXM-138//Key Research and Development Program of Shaanxi/ ; 0959202513035//Young Talent Fund of Xi'an Association for Science and Technology/ ; 24YXYJ0031//Xi'an Science and Technology Research Program/ ; },
mesh = {Animals ; *MicroRNAs/genetics/metabolism ; *Sepsis/therapy/genetics/pathology ; *CRISPR-Cas Systems ; Mice ; Disease Models, Animal ; *Genetic Therapy/methods ; Humans ; Gene Editing/methods ; Endoplasmic Reticulum Stress/genetics ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; NF-E2-Related Factor 2/genetics/metabolism ; },
abstract = {The CRISPR-dCas9 technology is a powerful tool for manipulating the expression of target genes in a variety of biomedical applications. Nevertheless, it is imperative that the activity of the CRISPR-dCas9 system be tightly controlled to improve its safety and applicability. In this study, we successfully designed a microRNA-activated CRISPR-dCas9 system, termed miR-ON-CRISPR, in which the core components (dCas9 and sgRNA) are both regulated by endogenous miRNA. Our findings demonstrated that the miR-ON-CRISPR system can regulate firefly luciferase reporter gene expression to faithfully visualize miRNA activity and image the differentiation status of neural cells. Moreover, the miR-ON-CRISPR was designed as an AND/OR gate system, thereby enabling the simultaneous detection of two distinct miRNAs. Furthermore, the system was adapted to achieve cell type-specific killing by activating the exogenous DTA genes or endogenous BAX genes. Finally, in mouse models of sepsis, the miR-ON-CRISPR system was shown to alleviate the sepsis-induced liver injury as well as the associated oxidative stress damage and endoplasmic reticulum stress via activating the nuclear erythroid 2-related factor 2 gene. In conclusion, this proof-of-concept study demonstrates the feasibility of the miR-ON-CRISPR system for cell type-specific control of CRISPR-dCas9 activity and its therapeutic applications in the treatment of genetic diseases.},
}
@article {pmid41118570,
year = {2025},
author = {Braithwaite, J and Cannon, C and Chalmers, R and Edwards, H},
title = {Single-colony resolution of CRISPR-Cas adaptation in E. coli reveals altered spacer-source bias during solid-phase growth.},
journal = {Nucleic acids research},
volume = {53},
number = {19},
pages = {},
doi = {10.1093/nar/gkaf1044},
pmid = {41118570},
issn = {1362-4962},
support = {RPG-2020-079//Leverhulme Trust/ ; //University of Nottingham/ ; },
mesh = {*Escherichia coli/genetics/growth &amp; development ; *CRISPR-Cas Systems ; Plasmids/genetics ; Escherichia coli Proteins/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; Lac Operon ; Adaptation, Physiological/genetics ; *DNA, Intergenic/genetics ; },
abstract = {CRISPR-Cas systems provide adaptive immunity by integrating short DNA fragments from mobile genetic elements into host arrays. While the core biochemical mechanism of adaptation is well defined, its modulation by physiological contexts is less well understood. Here, we present a visual papillation assay that enables single-colony detection of CRISPR-Cas adaptation in Escherichia coli. Spacer acquisition restores the reading frame of a disrupted lacZ gene, forming blue papillae on lactose X-gal plates. The assay is semi-quantitative, highly sensitive, capable of detecting single events among 109 cells, and responds predictably to Cas1-Cas2 expression levels. Spacer mapping revealed a major shift in source bias: in liquid culture, 64% of spacers were plasmid-derived, but on solid medium this dropped to ∼9%. Adjusting inducer concentration to match liquid conditions did not restore plasmid bias, indicating a physiological basis linked to colony growth. Accounting for the molar excess of chromosomal DNA, the 9% plasmid share reflects near-neutral DNA source sampling rather than plasmid overrepresentation. These findings suggest that the spatial and metabolic structure of colonies strongly shapes the adaptation landscape. The assay provides a scalable platform for dissecting condition-specific features of CRISPR-Cas adaptation, including spacer origin, sequence features, and growth context.},
}
@article {pmid41118394,
year = {2025},
author = {Gouin, Y and Wilcockson, A and Chan, AM and Suttle, CA and Zhong, KX},
title = {The genome of Gallaecimonas pentaromativorans strain 10A, isolated from a Pacific oyster, sheds light on an environmentally widespread genus with remarkable metabolic potential.},
journal = {PloS one},
volume = {20},
number = {10},
pages = {e0334406},
pmid = {41118394},
issn = {1932-6203},
mesh = {Animals ; *Genome, Bacterial ; Phylogeny ; *Ostreidae/microbiology ; },
abstract = {Bacteria in the genus Gallaecimonas are known for their ability to breakdown complex hydrocarbons, making them of particular ecological and biotechnological significance. However, few species have been isolated to date, and their ecological distribution has yet to be examined. Here, we report a novel strain of G. pentaromativorans, designated as strain 10A, which was isolated from a Pacific oyster (Magallana gigas, a.k.a. Crassostrea gigas) collected from a farm experiencing a mass mortality event in British Columbia (BC), Canada. Gallaecimonas pentaromativorans strain 10A is a rod-shaped, motile bacterium and has a circular genome of 4,322,156 bp encoding 3,928 protein-coding sequences (CDS). Phylogenetic analysis showed that strain 10A is closely related to members of G. pentaromativorans. Like other Gallaecimonas members, strain 10A is predicted to harbor specific pathways involved in degrading xenobiotic compounds including polycyclic aromatic hydrocarbons (PAHs), producing biosurfactants, and assimilating nitrate and sulfate; however, it is uniquely equipped with an additional 166 genes belonging to 147 protein families, including a putative higB-higA that likely contributes to enhanced stress response. Strain 10A also possesses Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated (Cas) system (CRISPR-Cas), prevalent in Gallaecimonas (detected in three out of four species), implying a potential defense mechanism against exogenous mobile genetic elements such as plasmids and viruses. We also mined publicly available databases to establish the widespread distribution of bacteria in the genus Gallaecimonas in seawater, sediments, and freshwater across latitude, suggesting its versatility and importance to environmental processes. Ultimately, this study demonstrates that the genome of G. pentaromativorans strain 10A, isolated from a Pacific oyster, may encode a suite of putative functions, including xenobiotic breakdown, biosurfactant production, and CRISPR-Cas defense. This plasticity and breadth in metabolic function help to explain the cosmopolitan distribution of members of this genus.},
}
@article {pmid41068553,
year = {2025},
author = {Wang, YM and Xu, T and Duan, JQ and Zhao, L and You, D and Lai, SY and Qing, Y and Ge, LP and Liu, ZH and Sun, J and Zeng, X and Xu, ZW and Zhu, L},
title = {An Integrated One-Tube RPA-CRISPR/Cas13d Assay Coupled with Lateral Flow for Rapid PRRSV-1 Detection.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {42},
pages = {27080-27088},
doi = {10.1021/acs.jafc.5c07919},
pmid = {41068553},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; Animals ; *Porcine respiratory and reproductive syndrome virus/genetics/isolation &amp; purification ; Swine ; *Porcine Reproductive and Respiratory Syndrome/virology/diagnosis ; RNA, Viral/genetics ; },
abstract = {Rapid and scalable diagnostic technologies are essential for controlling infectious diseases. We present STEP (Single-Tube Extraction-free Platform for CRISPR/Cas13d detection), a streamlined, equipment-minimal CRISPR-based platform enabling rapid, sensitive, and accurate viral RNA detection. STEP integrates isothermal RPA amplification with CRISPR-based nucleic acid cleavage, providing multimodal readouts including lateral flow strips, in-tube fluorescence, and fluorescence quantification. Lab-free extraction reagents and lyophilized formulations enhance user friendliness of STEP and stability for point-of-care testing (POCT), reduce cost, and eliminate cold-chain requirements. Optimization of baseline time and the ability to operate at both ambient and body temperatures minimize temporal and equipment constraints. Clinical evaluation showed 100% sensitivity and specificity versus RT-qPCR, delivering a sample-to-answer workflow within 35 min. STEP provides a robust platform for decentralized infectious disease diagnostics and rapid public health response, combining speed, user friendliness, and minimal instrumentation requirements.},
}
@article {pmid41015034,
year = {2025},
author = {Fontana, L and Martinucci, P and Amistadi, S and Felix, T and Mombled, M and Tachtsidi, A and Corre, G and Chalumeau, A and Hardouin, G and Martin, J and Romano, O and Amendola, M and Antoniou, P and Miccio, A},
title = {Multiplex base editing of BCL11A regulatory elements to treat sickle cell disease.},
journal = {Cell reports. Medicine},
volume = {6},
number = {10},
pages = {102376},
doi = {10.1016/j.xcrm.2025.102376},
pmid = {41015034},
issn = {2666-3791},
mesh = {*Anemia, Sickle Cell/genetics/therapy ; *Gene Editing/methods ; Humans ; Fetal Hemoglobin/metabolism/genetics ; Animals ; *Repressor Proteins/genetics ; CRISPR-Cas Systems/genetics ; Mice ; Hematopoietic Stem Cells/metabolism ; *Regulatory Sequences, Nucleic Acid/genetics ; DNA Breaks, Double-Stranded ; *Carrier Proteins/genetics/metabolism ; },
abstract = {Sickle cell disease (SCD) is a genetic anemia caused by the production of an abnormal adult hemoglobin. Elevated levels of fetal hemoglobin (HbF) in adulthood reduce disease severity. A promising therapy involves the treatment of hematopoietic stem/progenitor cells (HSPCs) with CRISPR-Cas9 to downregulate the HbF repressor BCL11A via generation of double-strand breaks (DSBs) in the +58-kb enhancer. To improve safety and HbF induction, we use base editors to target both the +58-kb and +55-kb enhancers without generating DSBs. We dissect key DNA motifs recognized by transcriptional activators and identify critical nucleotides. Multiplex base editing efficiently disrupts these sites, reactivating HbF to levels exceeding those achieved with CRISPR-Cas9-induced editing, while minimizing DSBs and genomic rearrangements. Base editing is effective in long-term repopulating HSPCs and results in robust HbF reactivation in vivo. These findings demonstrate that multiplex base editing of BCL11A enhancers is a safe, efficient, and durable strategy to treat SCD.},
}
@article {pmid40993398,
year = {2025},
author = {Datlinger, P and Pankevich, EV and Arnold, CD and Pranckevicius, N and Lin, J and Romanovskaia, D and Schaefer, M and Piras, F and Orts, AC and Nemc, A and Biesaga, PN and Chan, M and Neuwirth, T and Artemov, AV and Li, W and Ladstätter, S and Krausgruber, T and Bock, C},
title = {Systematic discovery of CRISPR-boosted CAR T cell immunotherapies.},
journal = {Nature},
volume = {646},
number = {8086},
pages = {963-972},
pmid = {40993398},
issn = {1476-4687},
mesh = {Humans ; Animals ; Mice ; *Receptors, Chimeric Antigen/immunology/genetics/metabolism ; *T-Lymphocytes/immunology/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Immunotherapy, Adoptive/methods ; Female ; Gene Editing ; Gene Knockout Techniques ; rho GTP-Binding Proteins/genetics/deficiency ; Xenograft Model Antitumor Assays ; Male ; Leukemia/therapy/immunology/genetics ; *Immunotherapy/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success in treating blood cancers, but CAR T cell dysfunction remains a common cause of treatment failure[1]. Here we present CELLFIE, a CRISPR screening platform for enhancing CAR T cells across multiple clinical objectives. We performed genome-wide screens in human primary CAR T cells, with readouts capturing key aspects of T cell biology, including proliferation, target cell recognition, activation, apoptosis and fratricide, and exhaustion. Screening hits were prioritized using a new in vivo CROP-seq[2] method in a xenograft model of human leukaemia, establishing several gene knockouts that boost CAR T cell efficacy. Most notably, we discovered that RHOG knockout is a potent and unexpected CAR T cell enhancer, both individually and together with FAS knockout, which was validated across multiple in vivo models, CAR designs and sample donors, and in patient-derived cells. Demonstrating the versatility of the CELLFIE platform, we also conducted combinatorial CRISPR screens to identify synergistic gene pairs and saturation base-editing screens to characterize RHOG variants. In summary, we discovered, validated and biologically characterized CRISPR-boosted CAR T cells that outperform standard CAR T cells in widely used benchmarks, establishing a foundational resource for optimizing cell-based immunotherapies.},
}
@article {pmid40993381,
year = {2025},
author = {Knudsen, NH and Escobar, G and Korell, F and Kienka, T and Nobrega, C and Anderson, S and Cheng, AY and Zschummel, M and Armstrong, A and Bouffard, A and Kann, MC and Goncalves, S and Pope, HW and Pezeshki, M and Rojas, A and Suermondt, JSMT and Phillips, M and Berger, TR and Park, S and Salas-Benito, D and Darnell, EP and Birocchi, F and Leick, MB and Larson, RC and Doench, JG and Sen, D and Yates, KB and Manguso, RT and Maus, MV},
title = {In vivo CRISPR screens identify modifiers of CAR T cell function in myeloma.},
journal = {Nature},
volume = {646},
number = {8086},
pages = {953-962},
pmid = {40993381},
issn = {1476-4687},
mesh = {*Multiple Myeloma/therapy/immunology/genetics/pathology ; Animals ; Humans ; Mice ; *Receptors, Chimeric Antigen/immunology/metabolism/genetics ; *T-Lymphocytes/immunology/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; Female ; *Immunotherapy, Adoptive ; Male ; Cell Proliferation ; Cell Line, Tumor ; B-Cell Maturation Antigen/immunology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Chimeric antigen receptor (CAR) T cells are highly effective in haematological malignancies[1]. However, progressive loss of CAR T cells contributes to relapse in many patients[2-4]. Here we performed in vivo loss-of-function CRISPR screens in CAR T cells targeting B cell maturation antigen to investigate genes that influence CAR T cell persistence and function in a human multiple myeloma model. We tracked the expansion and persistence of CRISPR library-edited T cells in vitro and at early and late time points in vivo to track the performance of gene-modified CAR T cells from manufacturing to survival in tumours. The screens revealed context-specific regulators of CAR T cell expansion and persistence. Ablation of RASA2 and SOCS1 enhanced T cell expansion in vitro, whereas loss of PTPN2, ZC3H12A and RC3H1 conferred early growth advantages to CAR T cells in vivo. Notably, we identified cyclin-dependent kinase inhibitor 1B (encoded by CDKN1B), a cell cycle regulator, as the most important factor limiting CAR T cell fitness at late time points in vivo. CDKN1B ablation increased CAR T cell proliferation and effector function, significantly enhancing tumour clearance and overall survival. Our findings reveal differing effects of gene perturbation on CAR T cells over time and in different environments, highlight CDKN1B as a promising target to generate highly effective CAR T cells for multiple myeloma and underscore the potential of in vivo screening for identifying genes to enhance CAR T cell efficacy.},
}
@article {pmid40993380,
year = {2025},
author = {Mittler, E and Tse, AL and Tran, PT and Florez, C and Janer, J and Varnaite, R and Kasikci, E and Mv, VK and Loomis, M and Christ, W and Cazares, E and Bakken, RR and Martin, CK and Zeng, X and Raymond, JL and Shahsavani, M and Khanal, S and Wilkinson, ER and Oktavia, RM and Slough, MM and Haslwanter, D and Han, J and Berrigan, J and Rosendal, E and Kielian, M and Manicassamy, B and Överby, AK and Falk, A and Barba-Spaeth, G and Rey, FA and Klingström, J and Gavathiotis, E and Herbert, AS and Chandran, K and Gredmark-Russ, S},
title = {LRP8 is a receptor for tick-borne encephalitis virus.},
journal = {Nature},
volume = {646},
number = {8086},
pages = {945-952},
pmid = {40993380},
issn = {1476-4687},
mesh = {Animals ; Mice ; Humans ; *Encephalitis Viruses, Tick-Borne/physiology/metabolism/pathogenicity ; Reelin Protein ; *LDL-Receptor Related Proteins/metabolism/genetics ; *Encephalitis, Tick-Borne/virology/prevention &amp; control/metabolism ; *Receptors, Virus/metabolism/genetics ; Female ; Cell Line ; Virus Internalization ; Viral Envelope Proteins/metabolism ; Male ; Neurons/virology/metabolism ; Brain/metabolism/virology ; Virus Attachment ; HEK293 Cells ; CRISPR-Cas Systems/genetics ; },
abstract = {Tick-borne encephalitis virus (TBEV) causes tick-borne encephalitis (TBE), a severe and sometimes life-threatening disease characterized by viral invasion of the central nervous system with symptoms of neuroinflammation[1,2]. As with other orthoflaviviruses-enveloped, arthropod-borne RNA viruses-host factors required for TBEV entry remain poorly defined. Here we used a genome-scale CRISPR-Cas9-based screen to identify LRP8, an apolipoprotein E and reelin receptor with high expression in the brain, as a TBEV receptor. LRP8 downregulation reduced TBEV infection in human cells, and its overexpression enhanced infection. LRP8 bound directly to the TBEV E glycoprotein and mediated viral attachment and internalization into cells. An LRP8-based soluble decoy blocked infection of human cell lines and neuronal cells and protected mice from lethal TBEV challenge. LRP8's role as a TBEV receptor has implications for TBEV neuropathogenesis and the development of antiviral countermeasures.},
}
@article {pmid40963013,
year = {2025},
author = {Tamura, S and Nelson, AD and Spratt, PWE and Hamada, EC and Zhou, X and Kyoung, H and Li, Z and Arnould, C and Barskyi, V and Krupkin, B and Young, K and Zhao, J and Holden, SS and Sahagun, A and Keeshen, CM and Lu, C and Ben-Shalom, R and Taloma, SE and Schamiloglu, S and Li, YC and Min, L and Jenkins, PM and Pan, JQ and Paz, JT and Sanders, SJ and Matharu, N and Ahituv, N and Bender, KJ},
title = {CRISPR activation for SCN2A-related neurodevelopmental disorders.},
journal = {Nature},
volume = {646},
number = {8086},
pages = {983-991},
pmid = {40963013},
issn = {1476-4687},
mesh = {Animals ; Mice ; *NAV1.2 Voltage-Gated Sodium Channel/genetics/deficiency ; Humans ; *Neurodevelopmental Disorders/genetics/therapy/physiopathology ; Haploinsufficiency/genetics ; Dependovirus/genetics ; Male ; Female ; *CRISPR-Cas Systems/genetics ; Seizures/genetics/therapy/chemically induced ; Pyramidal Cells/metabolism/pathology ; Gene Knock-In Techniques ; Phenotype ; Synapses/metabolism ; Disease Models, Animal ; Proof of Concept Study ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Genetic Therapy/methods ; },
abstract = {Most neurodevelopmental disorders with single gene diagnoses act via haploinsufficiency, in which only one of the two gene copies is functional[1]. SCN2A haploinsufficiency is one of the most frequent causes of neurodevelopmental disorder, often presenting with autism spectrum disorder, intellectual disability and, in a subset of children, refractory epilepsy[2]. Here, using SCN2A haploinsufficiency as a proof-of-concept, we show that upregulation of the existing functional gene copy through CRISPR activation (CRISPRa) can rescue neurological-associated phenotypes in Scn2a haploinsufficient mice. We first show that restoring Scn2a expression in adolescent heterozygous Scn2a conditional knock-in mice rescues electrophysiological deficits associated with Scn2a haploinsufficiency (Scn2a[+/-]). Next, using an adeno-associated virus CRISPRa-based treatment in adolescent mice, we show that we can correct intrinsic and synaptic deficits in neocortical pyramidal cells, a major cell type that contributes to neurodevelopmental disorders and seizure aetiology in SCN2A haploinsufficiency. Furthermore, we find that systemic delivery of CRISPRa protects Scn2a[+/-] mice against chemoconvulsant-induced seizures. Finally, we also show that adeno-associated virus CRISPRa treatment rescues excitability in SCN2A haploinsufficient human stem-cell-derived neurons. Our results showcase the potential of this therapeutic approach to rescue SCN2A haploinsufficiency and demonstrates that rescue even at adolescent stages can ameliorate neurodevelopmental phenotypes.},
}
@article {pmid40919940,
year = {2025},
author = {Le, Y and Liu, X and Zhou, S and Wu, P and Zhang, M and Sun, J and Ni, J and Wang, H},
title = {A thermostable Cas9-based genome editing system for thermophilic acetogenic bacterium Thermoanaerobacter kivui.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {10},
pages = {e0117025},
doi = {10.1128/aem.01170-25},
pmid = {40919940},
issn = {1098-5336},
support = {32470130//National Natural Science Foundation of China/ ; BK20231326//Natural Science Foundation of Jiangsu Province/ ; 2020YFA0906800//National Key Research and Development Program of China/ ; M2022-10//State Key Laboratory of Microbial Technology Open Projects Fund/ ; },
mesh = {*Thermoanaerobacter/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Ethanol/metabolism ; Alcohol Dehydrogenase/genetics/metabolism ; Metabolic Engineering ; Genome, Bacterial ; *CRISPR-Associated Protein 9/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Hot Temperature ; },
abstract = {Thermoanaerobacter kivui is a thermophilic acetogenic bacterium capable of thriving at elevated temperatures up to 66°C. It metabolizes carbohydrates such as glucose, mannose, and fructose and can also grow lithotrophically utilizing hydrogen (H2) and carbon dioxide (CO2) or carbon monoxide (CO), with acetate serving as its main product. A simple and efficient genome editing system for T. kivui would not only facilitate the understanding of the physiological function of enzymes involved in energy and carbon metabolism but also enable metabolic engineering. To address this issue, we developed a thermostable Cas9-based genome editing system for targeted gene knockout and gene integration into the T. kivui genome. Gene knockout assays were conducted on the adh gene, responsible for encoding alcohol dehydrogenase, and the ldh gene, encoding lactate dehydrogenase. Furthermore, the adhE gene from Thermoanaerobacter ethanolicus, which encodes a bifunctional aldehyde/alcohol dehydrogenase enzyme, was successfully integrated into the T. kivui genome. As a result, the engineered strain was able to produce ethanol. Following a liquid culturing period with kanamycin sulfate for about 72 hours, the efficiency of gene editing was enhanced, resulting in a ratio of mutants out of all colonies obtained of 90%. The results confirm the validity and efficiency of the thermostable Cas9-based genome editing system in T. kivui for gene editing.IMPORTANCEThermophilic acetogenic microorganisms represent an emerging metabolic engineering platform for the production of various biochemicals from hydrogen and carbon dioxide, or synthesis gas, under conditions of high-temperature fermentation. Gas fermentation has gained significant research interest due to its excellent thermodynamics, economic feasibility, and multisubstrate utilization. However, a major obstacle to the use of thermophilic acetogenic microorganisms as metabolic engineering platforms is the scarcity of genetic tools. This study demonstrates a proof of concept for a thermostable Cas9-based genome editing of the thermophilic acetogenic bacterium T. kivui. The system is an important expansion to the genetic toolbox of T. kivui, enabling a better understanding of key enzyme functions and the construction of cell factories for the biotechnological conversion of carbon dioxide and organic substrates into value-added products.},
}
@article {pmid40891847,
year = {2025},
author = {Li, L and Luo, K and Zhang, S and Wang, X and Wang, S and Liu, X and Zang, S and Liu, Y and Zhou, C and Luo, C},
title = {A three-plasmid-containing CRISPR-Cas9 platform to engineer Bacillus velezensis 916 as an efficient biocontrol agent.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {10},
pages = {e0138925},
doi = {10.1128/aem.01389-25},
pmid = {40891847},
issn = {1098-5336},
mesh = {*Bacillus/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Plasmids/genetics ; *Biological Control Agents ; Plant Diseases/prevention &amp; control/microbiology ; Lipopeptides/genetics ; Bacterial Proteins/genetics ; Peptide Synthases/genetics ; Oryza/microbiology ; },
abstract = {Bacillus velezensis (Bv) is a widely used biocontrol agent against plant diseases, mainly because its genome contains numerous non-ribosomal peptide synthetases (NRPS) gene clusters for the synthesis of various cyclic lipopeptides (CLPs). The domesticated strain Bv916, capable of co-producing four CLPs, has been successfully applied for green control of rice sheath blight and angular leaf spot. To enhance Bv916's biological control efficacy while maintaining environmental safety, it is essential to establish a food-grade gene editing platform in Bv916. Here, a three-plasmid CRISPR-Cas9 platform for Bv916 was constructed using the thermosensitive origin pET194ts, constitutive P43 promoters for Cas9, the specific promoter Psrf for single guide RNAs (sgRNAs), and three resistance gene expression cassettes. By replacing the native promoters of ComX and RecA in Bv916 with the strong promoters P43 and PrepU, respectively, this platform achieved a single-gene editing efficiency of 96%, while the simultaneous dual-gene editing efficiency reached 61%, with each round completed within five business days. Furthermore, this gene editing platform is used to replace promoters of four NRPS gene clusters (loc, srf, bl, and fen) in Bv916 with strong constitutive promoters (PB, PA, P43, and PrepU), generating the derivative BvLSBF. Compared to Bv916, BvLSBF showed 6.8-fold, 5.9-fold, 10.9-fold, and 6.2-fold increases in locillomycin, surfactin, bacillomycin L, and fengycin, respectively. Its antagonistic activity against plant pathogens was also significantly enhanced. This system enables further development of Bv916 as a cell factory and integration of multiple biocontrol factors, offering significant potential for sustainable agriculture.IMPORTANCEIn this study, a food-grade three-plasmid CRISPR-Cas9 platform for Bv916 was established by incorporating the optimized BvCas9 under the constitutive promoter P43, single guide RNAs (sgRNAs), and homologous recombination fragments into three thermosensitive shuttle vectors. This gene editing system was used to achieve gene insertion, deletion, and replacement in Bv916, particularly by editing four non-ribosomal peptide synthetase (NRPS) gene clusters. This resulted in increased production of four cyclic lipopeptides and significantly enhanced antibacterial and antifungal activity.},
}
@article {pmid41118255,
year = {2025},
author = {Ong, CJN and Elesho, OE and Bramwell, BB and Cabuhat, KSP and Bacalzo, GD and Nuevo, JJM and Fortaleza, JAG},
title = {Staphylococcus aureus: Antimicrobial resistance, quorum sensing, and antibiofilm approaches.},
journal = {European journal of microbiology &amp; immunology},
volume = {},
number = {},
pages = {},
doi = {10.1556/1886.2025.00050},
pmid = {41118255},
issn = {2062-509X},
abstract = {Staphylococcus aureus is a clinically important bacterial pathogen causing infections from superficial skin lesions to life-threatening systemic diseases. The emergence of methicillin-resistant S. aureus (MRSA) has compounded the global health burden, particularly in low- and middle-income countries, as its quorum-sensing (QS) mediated mechanisms contribute to its persistence, resistance, and evasion from host immune responses and antimicrobial treatments. Thus, these features compromise the effectiveness of conventional antibiotics, urging the need for alternative therapeutic approaches. To resolve these issues, several non-antibiotic antibiofilm approaches have been developed. Bacteriophages and phage-derived enzymes show promising specificity in lysing bacterial cells and disrupting biofilms. Antimicrobial peptides (AMPs), with their broad-spectrum activity, destabilize bacterial membranes and modulate immune responses. Monoclonal antibodies can neutralize toxins or inhibit adhesion molecules within biofilms. Phytochemicals have demonstrated activity against QS pathways and efflux pumps. Metal ion chelators like deferiprone interfere with iron acquisition, which is essential for biofilm stability. Nanoparticles (NPs), ranging from metallic and polymeric to lipid-based and cyclodextrin-based systems, enhance drug delivery and biofilm penetration. CRISPR-Cas systems provide precise genome editing to target resistance genes and virulence factors. Rhamnolipids disrupt biofilm matrix integrity, while enzymes such as dispersin B degrade extracellular polymeric substances. Photodynamic and laser therapies offer localized disruption of biofilm structures through oxidative stress. Collectively, this review offers a transformative complementary approach to traditional antibiotics, enhancing treatment efficacy while potentially reducing the emergence of resistance. Continued research on delivery systems, safety profiles, and synergistic combinations will be pivotal for their clinical translation against S. aureus infections.},
}
@article {pmid41117937,
year = {2025},
author = {Menge, S and Segura, I and Hartmann, M and Decker, L and Kiran, S and Danzer, KM and Iben, S and Harbauer, AB and Oeckl, P and Freischmidt, A},
title = {Comparing loss of individual fragile X proteins suggests strong links to cellular senescence and aging.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {82},
number = {1},
pages = {358},
pmid = {41117937},
issn = {1420-9071},
mesh = {Humans ; *Cellular Senescence/genetics ; *Fragile X Mental Retardation Protein/genetics/metabolism ; *Aging/genetics/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Autophagy ; Cell Line, Tumor ; Proteomics/methods ; Mitochondria/metabolism ; Fragile X Syndrome/metabolism/genetics/pathology ; Neurodegenerative Diseases/metabolism/genetics/pathology ; },
abstract = {Members of the fragile X protein (FXP) family (FMR1, FXR1 and FXR2) are differentially expressed in most types of cancer and major neurodegenerative diseases. While increased expression of FXR1 in cancer has been linked to senescence evasion and consequently tumor initiation and progression, decreased expression of FXPs in neurodegeneration may contribute to pathogenic protein aggregation and death of vulnerable neurons. However, due the causal role in fragile x syndrome, most data are available about loss of FMR1 in neurons while functions of FXR1 and especially FXR2 remain largely unexplored. To address this knowledge gap, and to directly compare functions of the FXPs, we used proteomics of CRISPR/Cas9 edited HAP1 cells carrying knockouts of the individual FXPs for identification of cellular mechanisms associated with these proteins. Further exploration of proteomic findings suggests roles of the FXPs in ribosome biogenesis, autophagy and mitochondrial health linked to organismal aging, and cellular senescence. Validation of FXP induced defects relevant for neurodegenerative diseases in neuroblastoma cell line SH-SY5Y upon FXP knockdown revealed high cell type specificity of individual FXP functions. Overall, we provide a comprehensive overview and comparison of cellular mechanisms related to the individual FXPs, as well as starting points for further studying this protein family in respective cell types of FXP associated diseases, and in aging in general.},
}
@article {pmid41117602,
year = {2025},
author = {Keshry, SS and Nayak, U and Mamidi, P and Mohanty, S and Ghorai, U and Swain, RK and Chattopadhyay, S},
title = {CRISPR-Induced Mutations of mk2b and mk3 Host Proteins Enhance Chikungunya Virus Susceptibility and Modulate Host Immune Responses in Zebrafish.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {20},
pages = {e71112},
doi = {10.1096/fj.202501236RR},
pmid = {41117602},
issn = {1530-6860},
support = {113-2334-6704/2K23/1//CSIR | Human Resource Development Centre, Council of Scientific And Industrial Research (HRDC)/ ; IF180156//Department of Science and Technology, Ministry of Science and Technology, India (DST)/ ; BT/PR42322/TRM/120/525/2021//Department of Biotechnology, Ministry of Science and Technology, India (DBT)/ ; },
mesh = {Animals ; *Zebrafish/genetics/virology/immunology ; *Chikungunya virus/immunology ; *Chikungunya Fever/immunology/genetics/virology ; *Protein Serine-Threonine Kinases/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; *Intracellular Signaling Peptides and Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Host factors are essential at every stage of the viral life cycle and therefore represent attractive and potentially effective targets for the development of antiviral therapeutics. This study highlights the crucial roles of host factors, specifically mitogen-activated protein kinase 2 (mk2) and mitogen-activated protein kinase 3 (mk3), both of which are stress-stimulated serine/threonine kinases. The roles of mk2 and mk3 were investigated by generating single (mk2b-/- and mk3-/-) and double knockouts (mk2b-/-mk3-/-) in a zebrafish model using the CRISPR-Cas9 technique, followed by chikungunya virus (CHIKV) infection. All knockout lines exhibited significantly higher CHIKV titers and severe phenotypes compared to the WT control, with mk3[-]/[-] showing the greatest susceptibility. After CHIKV infection, expression levels of TNF-α changed across all knockout models. Notably, mk2b[-]/[-] and mk2b[-]/[-]mk3[-]/[-] double knockout larvae exhibited reduced TNF-α expression, suggesting that higher levels of TNF-α may be associated with viral clearance via the p38-MK2-TNF-α signaling axis. In contrast, mk3-/- zebrafish exhibited increased vulnerability to CHIKV through alternative, yet unidentified, pathways. Furthermore, an increase in viral titer corresponded with an enhanced host immune response, as indicated by significantly higher expression levels of ifnɸ1 and rsad2 in all knockout groups. In conclusion, this study confirms that the mk2b and mk3 host proteins are essential in controlling CHIKV infection at the organism level. These findings might have implications towards designing strategies for future antiviral therapeutics. Furthermore, the knockout model of mk2b and mk3 in zebrafish could serve as a valuable tool for studying their roles in other viral infections.},
}
@article {pmid41116722,
year = {2025},
author = {Lin, Y and Jiang, D and Dong, X and Li, Y and Wu, X and Li, R and Li, F and Sun, D and Yu, Y},
title = {A label-free orthogonal dual-channel CRISPR-Cas platform for simultaneous detection of Mycobacterium tuberculosis and respiratory syncytial virus.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc05269f},
pmid = {41116722},
issn = {1364-548X},
abstract = {A label-free, orthogonal dual-channel CRISPR-Cas platform is developed for the simultaneous detection of diverse pathogens. Cas12a is reported by a G4-PPIX complex, and Cas13 by a split Broccoli-DFHBI aptamer, which ensure exclusive channel specificity and eliminate optical cross-talk. Clinical tests on 50 samples show 100% concordance with PCR.},
}
@article {pmid41115973,
year = {2025},
author = {Mladenov, E and Kallies, M and Stuschke, M and Gkika, E and Iliakis, G},
title = {CRISPR/Cas9 generated DSB clusters mimic complex lesions induced by high-LET radiation and shift repair from c-NHEJ to mutagenic repair pathways.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {36480},
pmid = {41115973},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Humans ; *DNA Breaks, Double-Stranded/radiation effects ; *DNA End-Joining Repair/radiation effects/genetics ; *Linear Energy Transfer ; Hypoxanthine Phosphoribosyltransferase/genetics ; Animals ; *DNA Repair ; Cell Line ; Poly (ADP-Ribose) Polymerase-1/genetics/metabolism ; Mutagenesis ; },
abstract = {DNA double-strand break (DSB) clusters are a hallmark of high-linear energy transfer (high-LET) radiation and are associated with pronounced biological effects, including reduced cell survival and elevated genomic instability. Our previous work in Chinese hamster cells, engineered with variably designed clusters of I-SceI recognition sites, integrated at multiple genomic locations, revealed that DSB clusters suppress classical non-homologous end-joining (c-NHEJ) and induce chromosomal translocations that ultimately increase cell lethality. Here, we extend this line of investigation to human cell lines and generate DSB clusters using alternative approaches that do not require prior genetic manipulation of the test cell lines. We employ CRISPR/Cas9-technology to generate DSB clusters of specific design at a selected genomic locus and examine their consequences on locus integrity. We target Exon 3 of the human HPRT (hHPRT) gene and introduce single DSBs or DSB clusters of varying numbers and inter-DSB distances. Alterations at the locus reflecting hHPRT gene inactivation, are quantified as mutations causing resistance to 6-thioguanine (6TG). Our results show that DSB clusters are markedly more potent inducers of mutations than single DSBs and that DSBs spaced within ~ 600 base pairs synergize in mutation induction. Mechanistic analyses using small-molecule inhibitors and engineered gene knockout cell lines reveal that the increased mutagenicity of clustered DSBs is primarily mediated by DNA end resection and PARP1-dependent alternative end-joining (alt-EJ) pathways. These findings reinforce the biological relevance of DSB clusters as a severe form of complex DNA damage and provide mechanistic insights into high-LET radiation-induced increased cell killing and genomic instability.},
}
@article {pmid41114873,
year = {2025},
author = {Su, CW and Tsai, LC and Hsu, YC and Lu, YS and Lee, JC and Linacre, A and Hsieh, HM},
title = {Saliva identification by RT-LAMP integrated with CRISPR-Cas and LFA.},
journal = {Forensic science, medicine, and pathology},
volume = {},
number = {},
pages = {},
pmid = {41114873},
issn = {1556-2891},
support = {MOST 110-2320-B-015-001 and MOST 111-2320-B-015-001-MY2//National Science and Technology Council/ ; },
abstract = {Saliva is a frequently encountered body fluid at crime scenes, however currently there are no definite means to rapidly identify a body fluid as being saliva. In this study, a novel detection method for saliva using a modified Loop-mediated Isothermal Amplification (LAMP) integrated with CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein) and LFA (Lateral Flow Assay) was developed to detect the expression of a saliva-specific gene: follicular dendric cell secreted protein (FDCSP). To determine the specificity of the assay, RNA from saliva plus other commonly encountered body fluids was tested (peripheral blood, semen, vaginal fluid, and menstrual blood): positive results were only observed from RNA extracted from known saliva samples and RNA from all the other body fluids exhibited a negative result. To assess the reproducibility, triplicates were used from one saliva sample, and the assay was performed on three different days: positive results were observed from all triplicates. The limit of detection was 2[-6] (0.3906 ng RNA) or 2[-7] (0.1953 ng RNA). This preliminary study for the identification of saliva requires no complex equipment and is easy to perform, offering an alternative means for body fluid identification.},
}
@article {pmid41113410,
year = {2025},
author = {Feng, L and Huang, Y and Zhao, R and Zhang, K and Yang, W},
title = {[Effect of Different Caenorhabditis elegans U6 Promoters on the Efficiency of CRISPR/Cas9-Mediated Gene Editing].},
journal = {Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition},
volume = {56},
number = {4},
pages = {1038-1044},
pmid = {41113410},
issn = {1672-173X},
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Promoter Regions, Genetic/genetics ; *RNA, Small Nuclear/genetics ; Plasmids/genetics ; Caenorhabditis elegans Proteins/genetics ; },
abstract = {OBJECTIVE: To investigate the effects of Caenorhabditis elegans (C. elegans) endogenous U6 promoters on dpy-10 gene editing efficiency.<br><br>METHODS: We screened endogenous U6 small nuclear RNA (snRNA) genes of C. elegans from the WormBase database and constructed 14 editing plasmids targeting dpy-10 by replacing the U6 r07e5.16 promoter in the pSX524 plasmid (Peft-3::cas9::tbb-2 terminator::U6 r07e5.16::dpy-10 sgRNA) through molecular cloning. Gene editing was performed in wild-type C. elegans using a standardized microinjection protocol. Gene editing efficiency and the high-efficiency gene editing index were quantified based on the screening of dpy-10 mutant phenotypes in the F1 progeny.<br><br>RESULTS: A total of 15 U6 snRNA genes (r07e5.16, f35c11.9, t20d3.13, k09b11.15, k09b11.16, w05b2.8, c28a5.7, f54c8.9, k09b11.11, k09b11.12, k09b11.14, t20d3.12, f54c8.8, f54c8.10, and k09b11.13) were identified from the WormBase database. Based on the editing efficiency and high-efficiency gene editing index, the activity of these promoters was evaluated, and 4 U6 promoters (w05b2.8, c28a5.7, f54c8.9, and k09b11.11) were found to have significantly enhanced gene editing success rates, outperforming other promoters, including U6 r07e5.16 and U6 k09b11.12 , which are commonly used in the C. elegans research community. Notably, the gRNA[F+E] scaffold did not show superior editing efficiency over the gRNA scaffold when paired with the optimal U6w05b2.8 promoter.<br><br>CONCLUSION: In this study, U6 promoters that significantly improve gene editing efficiency in C. elegans are identified and the critical role of promoter optimization in CRISPR-Cas9 systems is highlighted. These findings provide a valuable foundation for improving genome editing strategies and offer new ideas for optimizing the CRISPR technology applied in nematode research.},
}
@article {pmid41112309,
year = {2025},
author = {Cruz-Cárdenas, JA and López-Arredondo, A and Cázares-Preciado, JA and Rodríguez-Gonzalez, M and Palomares, LA and Brunck, MEG},
title = {Development of CRISPR/Cas9-mediated CD16b[-/-] and CD32a[-/-] promyelocytic cell lines to study FcγR signaling in human neutrophils.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1633609},
pmid = {41112309},
issn = {1664-3224},
mesh = {Humans ; *Receptors, IgG/genetics/metabolism/immunology ; *Neutrophils/immunology/metabolism ; *CRISPR-Cas Systems ; *Signal Transduction/immunology ; HL-60 Cells ; Phagocytosis ; GPI-Linked Proteins/genetics ; Reactive Oxygen Species/metabolism ; Cytokines/metabolism ; },
abstract = {INTRODUCTION: Neutrophils use Fc gamma receptors (FcγRs) to recognize IgG-opsonized pathogens, triggering antimicrobial functions including phagocytosis, ROS production, and cytokine release. CD16b, the most abundant FcγR on neutrophils, plays a key role in initiating these responses, while CD32a is another abundant FcγR on neutrophils that contributes to modulating immune functions. CD16b lacks an intracellular domain and its signaling mechanisms remain unclear. The prevalence of the CD16b-deficient phenotype on donor neutrophils is estimated at <1% of the global population, which complicates its study. To address this, we employed CRISPR/Cas9 to generate HL-60-derived neutrophil-like cells deficient for CD16b or CD32a, that facilitate investigation of their respective roles in neutrophil biology.<br><br>METHODS: We disrupted the FCGR3B or FCGR2A genes using CRISPR/Cas9 in the HL-60 cell line and differentiated clones into neutrophil-like cells using 1.3% DMSO. Functional assays were performed, including phagocytosis, ROS production, SYK phosphorylation, and cytokine responses.<br><br>RESULTS AND DISCUSSION: Both CD16b[-/-] and CD32a[-/-] HL-60-derived clones maintained neutrophilic differentiation and phagocytic capacity but displayed impaired Fc&#x3b3;R-mediated ROS production and SYK phosphorylation, with more pronounced defects in CD16b[-/-] cells. Cytokine production was altered in both lines, with CD16b[-/-] cells producing less IL-6 and IL-1&#x3b2;, and CD32a[-/-] cells producing less TNF-&#x3b1; and IL-10. This model provides new insights into the distinct roles of CD16b and CD32a in neutrophil activation and immune responses.},
}
@article {pmid41110011,
year = {2025},
author = {De La Mora, CL and Havey, MJ and Krysan, PJ},
title = {Efficient production of gene-edited onion (Allium cepa) plants using biolistic delivery of cas9 RNPs and transient expression constructs.},
journal = {Plant cell reports},
volume = {44},
number = {11},
pages = {243},
pmid = {41110011},
issn = {1432-203X},
support = {1844304//Division of Integrative Organismal Systems/ ; },
mesh = {*Onions/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Biolistics/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; Plant Proteins/genetics/metabolism ; },
abstract = {Delivery of Cas9/sgRNA RNPs and DNA vectors designed to transiently express morphogenic regulatory genes or an antibiotic resistance gene enabled efficient recovery of gene edited onion lines. We developed a protocol for efficiently producing gene edited onion plants that does not depend on stable transformation. The process makes use of transient gene expression to enrich for gene editing among plants regenerated from immature embryos bombarded with ribonucleoprotein (RNP) complexes composed of CRISPR-associated protein 9 (Cas9) and single guide RNAs (sgRNAs). We used the Allium cepa Downy Mildew Resistant 6 (AcDMR6) gene as the target gene for our studies and produced a total of 47 onion plants with edited AcDMR6 alleles, including 13 homozygous plants, 12 biallelic plants, 7 heterozygous plants, and 15 chimeric plants. The most effective strategy for producing gene edited onion plants involved co-delivering plasmids encoding a hygromycin-resistance protein and plant developmental regulator genes along with the Cas9/sgRNA RNPs followed by transient Hygromycin selection for 48 h. Using this approach, up to 12% of the regenerated onion plants carried edited alleles of AcDMR6. By comparison, no editing was observed among the 146 plants regenerated from explants transfected with the Cas9/sgRNA RNPs alone. The strategy we describe here for using transient gene expression to enrich for gene editing in onion could potentially be extended to other crop species as well.},
}
@article {pmid41071600,
year = {2025},
author = {Zhang, W and Feng, ZY and Feng, Z and Lian, R and Liu, Z and Zhang, J},
title = {A CRISPR-Customizable Copper-Coordinated DNA Nanoplatform Potentiates Cuproptosis Through Circadian and Metabolic Pathway Manipulation.},
journal = {ACS nano},
volume = {19},
number = {41},
pages = {36701-36717},
doi = {10.1021/acsnano.5c12641},
pmid = {41071600},
issn = {1936-086X},
mesh = {*Copper/chemistry/pharmacology ; Humans ; Animals ; *DNA/chemistry/pharmacology ; *Circadian Rhythm/drug effects ; Mice ; Metabolic Networks and Pathways/drug effects ; Apoptosis/drug effects ; *Antineoplastic Agents/pharmacology/chemistry ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; ARNTL Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Despite the promise of cuproptosis in antitumor therapy, developing strategies to enhance its therapeutic efficacy within the tumor microenvironment remains a challenge. Inspired by the chronotherapy that manipulate circadian rhythms to enhance drug effectiveness, herein we report for a CRISPR-customized copper-DNA nanoplatform (Cu-RNP) that synergistically induces multimodal cell death, including potentiated cuproptosis, by manipulating circadian and metabolic pathways. Cu-RNP integrates coordination-driven self-assembly of Cu[2+]-DNA nanospheres with Cas13d/crRNA ribonucleoproteins targeting BMAL1. Upon cellular internalization, the acidic and reducing endo/lysosomal environment triggers Cu-RNP disassembly, releasing RNP to silence BMAL1 and disrupt circadian oscillations, leading to WEE1 downregulation and p21 upregulation, thereby inducing apoptosis. Simultaneously, liberated Cu[2+] generates cytotoxic hydroxyl radicals for chemodynamic therapy (CDT) and concurrently depletes GSH, promoting mitochondrial copper overload for cuproptosis. Importantly, we demonstrate that silencing BMAL1 disrupts circadian rhythms, inhibits glycolysis, enhances mitochondrial respiration, and redirects metabolic flux to the TCA cycle, thereby amplifying the cell's vulnerability to copper-induced cuproptosis. In vitro and in vivo results demonstrate that Cu-RNP sensitizes cancer cells to cuproptosis and elicit strong antitumor response through the synergistic combination of cuproptosis, CDT, apoptosis, and circadian-metabolic modulation. This study demonstrates a mechanistic link between BMAL1-regulated circadian rhythms and cuproptosis sensitivity, suggesting a potential treatment strategy for multimodal, cuproptosis-potentiating cancer therapies.},
}
@article {pmid41024711,
year = {2025},
author = {Sanchez, A and Zhou, C and Tulaiha, R and Ramirez, F and Wang, L and Zhang, X},
title = {CRISPR Screen Identifies BAP1 as a Deubiquitinase Regulating SPIN4 Stability.},
journal = {Biochemistry},
volume = {64},
number = {20},
pages = {4318-4326},
pmid = {41024711},
issn = {1520-4995},
support = {R35 GM146979/GM/NIGMS NIH HHS/United States ; R35 GM154945/GM/NIGMS NIH HHS/United States ; T32 GM149439/GM/NIGMS NIH HHS/United States ; },
mesh = {*Ubiquitin Thiolesterase/metabolism/genetics ; Humans ; *Tumor Suppressor Proteins/metabolism/genetics ; *CRISPR-Cas Systems ; *Cell Cycle Proteins/metabolism/genetics ; Ubiquitination ; HEK293 Cells ; Protein Stability ; Ubiquitin-Protein Ligases/metabolism ; Proteolysis ; Microtubule-Associated Proteins ; Phosphoproteins ; },
abstract = {Protein homeostasis is tightly controlled by the coordinated actions of E3 ubiquitin ligases and deubiquitinases (DUBs). We previously identified Spindlin-4 (SPIN4), a histone H3K4me3 reader, as a degradation substrate of DCAF16. In this study, we confirmed this degradation pathway using an E3 ligase-focused CRISPR-Cas9 knockout screen. Furthermore, through a DUB-focused CRISPR-Cas9 knockout screen and biochemical analyses, we demonstrated that the deubiquitinase BAP1 interacts with and stabilizes SPIN4 via its deubiquitination activity. Inhibition or loss of BAP1 reduces SPIN4 levels, highlighting its critical role in maintaining SPIN4 homeostasis. Proteomics and interactome analyses further support this regulatory axis. These findings reveal a dynamic balance controlling SPIN4 stability, with potential implications for epigenetic regulation and disease processes.},
}
@article {pmid41109516,
year = {2025},
author = {Pandya, K and Kumar, D},
title = {CRISPR/Cas Genome Editing for Neurodegenerative Diseases: Mechanisms, Therapeutic Advances, and Clinical Prospects.},
journal = {Ageing research reviews},
volume = {},
number = {},
pages = {102922},
doi = {10.1016/j.arr.2025.102922},
pmid = {41109516},
issn = {1872-9649},
abstract = {Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD) are major public health challenges. Current treatments are only symptomatic and do not address the underlying pathogenic genetic mechanisms. The development of the CRISPR/Cas genome editing tool has increased possibilities for targeted repair of pathological mutations. CRISPR/Cas9, Cas12, and Cas13 systems enable targeted editing and transcriptome modulation in various preclinical models. CRISPR/Cas9 disruption of mutant APP and Tau genes in AD models has restored normal genetic function. This technique reduces toxic protein aggregation. Results in neurodegenerative phenotype in these models are ameliorated. Correction of CAG nucleotide repeats in HD, and reduction of alpha-synuclein expression in PD. Advancements in engineered Cas variants with enhanced specificity, such as SpCas9-HF1 and prime editors, with innovative delivery strategies including adeno-associated virus (AAVs) and nanoparticle-based systems, have improved genome editing. However, challenges remain, including off-target effects, mosaicism, and delivery across the BBB, and long-term safety. Ethical consideration focuses on somatic versus germline editing, equitable access, and regulatory oversight. While somatic editing shows acceptance in treating neurological disorders. Germline interventions face strict regulations due to potential multigeneration impacts. Collectively, these technologies are the vanguard of precision molecular medicine, advancing from symptom management towards potentially curative gene therapies for neurological disorders.},
}
@article {pmid41109509,
year = {2025},
author = {Gujjar, RS and Sherin, S and Batra, S and Mahto, A and Goswami, SK and Kumar, R and Tiwari, RK and Singh, A and Kumari, M},
title = {Emerging trends in the development of efficient CAS nucleases for meticulous gene editing in plants.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {},
number = {},
pages = {112820},
doi = {10.1016/j.plantsci.2025.112820},
pmid = {41109509},
issn = {1873-2259},
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease) is a formidable and precise gene-editing system, invented as a natural immune system of bacterial defense. The system relies on a guide RNA/CrRNA (Crispr RNA) sequence, specifically designed to bind to the target nucleic acid, enabling the Cas nuclease to act like a molecular scissor and cleave the target sequence. Recent research has substantially advanced the development, optimization, and application of Cas nucleases, particularly Cas9, Cas12, and their engineered orthologs to enhance the fidelity, flexibility, and proficiency of specific edits at the target site. This review focuses on the latest advancements in the research on Cas nucleases such as Cas9, Cas12a, Cas12b, CasΦ, Cas13, and Cas14, along with their mode of function and the exclusive features of each nuclease. Further, we have elaborated on the emergence of redesigned Cas9 orthologs, such as dCas9 (deactivated Cas9) and nCas9 (Cas9 nickase). The modified Cas9 orthologs have empowered the CRISPR-based editing with sophisticated techniques such as base editing and prime editing, which are capable of providing preferred edits with more precision and specificity. These advanced genome editing approaches have been favorably employed to improve the desirable agronomic traits such as stress tolerance, herbicide resistance, and yield stability in a wide range of crops, including rice, maize, wheat, tomato, and cotton. KEY MESSAGE: Our manuscript offers the recent advancement in the research on Cas nucleases, and their modified orthologs to perform CRISPR-based gene edits with enhanced more precision and specificity.},
}
@article {pmid41108631,
year = {2025},
author = {Harzli, I and Mostafa, K and Mutlu, VA and Shamsi, IH and Kavas, M},
title = {Exploring Novel Microbial Approaches for Enhancing Crop Resilience to Abiotic Stress: Mechanisms and Applications.},
journal = {Physiologia plantarum},
volume = {177},
number = {5},
pages = {e70582},
doi = {10.1111/ppl.70582},
pmid = {41108631},
issn = {1399-3054},
support = {121O463//T&#xfc;bitak/ ; },
mesh = {*Crops, Agricultural/microbiology/physiology ; *Stress, Physiological ; Mycorrhizae/physiology ; Gene Editing ; Plant Breeding ; },
abstract = {The current state of agriculture heavily relies on chemical fertilizers and pesticides, which can negatively impact plant nutritional quality, plant health, and productivity. Additionally, abiotic stresses pose significant challenges to global agricultural productivity, threatening food security and crop sustainability. Therefore, developing and implementing sustainable alternatives to chemical fertilizers and pesticides is crucial to enhance agricultural productivity and resilience. Recent research highlights the potential of microorganisms, such as plant growth-promoting rhizobacteria (PGPR), mycorrhizal fungi, and endophytes, as sustainable solutions to improve plant resilience under abiotic stress conditions. However, challenges including scalability, ecological impacts, and the need for standardized application methods persist. This review explores novel microbial approaches to improving crop resilience against abiotic stress, focusing on how microorganisms interact with plants to mitigate stress impacts. Key mechanisms include the production of stress-alleviating compounds, enhanced nutrient uptake, and modulation of plant stress response pathways. We also examine advanced strategies in plant breeding, emphasizing CRISPR/Cas-mediated genome editing technologies as powerful tools for elucidating plant-microbe interactions. A thorough understanding of these interactions is essential for effectively applying genome editing to enhance the functional capacities of plants or associated microbes, ultimately improving key agronomic traits. This review provides a comprehensive overview of these innovative microbial approaches and their practical applications in sustainable agriculture, offering insights into future research directions, such as developing novel microbial strains and optimizing field applications.},
}
@article {pmid41108328,
year = {2025},
author = {Liu, C and Yao, D and Jiang, Y and Huo, C and Zhang, J and Yi, S and Yang, W and Han, Y and Liu, Y and Wang, X},
title = {Electroporation-based CRISPR/Cas9 Gene Editing in Haliotis Discus Hannai.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {27},
number = {6},
pages = {148},
pmid = {41108328},
issn = {1436-2236},
support = {ZR2023QC107//Natural Science Foundation of Shandong Province/ ; ZR2022QC233//Natural Science Foundation of Shandong Province/ ; 2022LZGC015//Key Research and Development Program of Shandong Province/ ; SDAIT-14-01//Modern Agricultural Industry Technology System of Shandong Province/ ; tstp20240518//Special Funds for the Taishan Scholar Project of Shandong Province/ ; 202203//Special Supporting Funds for Leading Talents above Provincial Level in Yantai City/ ; 2023HD//Aquaculture Seed Industry Promotion Project of Yantai City/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Electroporation/methods ; *Myostatin/genetics ; *Gastropoda/genetics/embryology ; Aquaculture ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Abalone, a marine mollusk with significant economic and ecological value, plays a crucial role in sustainable aquaculture. The development and application of CRISPR-Cas9 gene-editing technology have opened up a new path for improving breeding efficiency. CRISPR/Cas9-mediated gene editing has been achieved in abalones via microinjection. In this study, a gene encoding myostatin MSTN in H. discus hannai; was selected as target for conducting the CRISPR-Cas9 gene editing experiment in combination with an electroporation delivery system. Our results showed that all three sgRNAs effectively targeted and cleaved the target segment, with sgRNA1 and sgRNA2 exhibiting high in vitro activity. After electroporation, the effects of transfection on embryonic development of fertilized eggs were observed and statistically analyzed. 12.7 ± 5.4% of the fertilized eggs were damaged and deformed after electroporation. Twenty-four hours after electroporation, surviving larvae were collected for DNA extraction and sequencing. Two potential mutations within the target region of MSTN were identified by sequencing. These results provide a reference for the improvement and development of CRISPR-mediated gene editing methods in marine mollusks such as abalones.},
}
@article {pmid41107544,
year = {2025},
author = {Whyms, C and Zhao, Y and Addo-Yobo, D and He, H and Whittington, AC and Trasanidou, D and Salazar, CRP and Staals, RHJ and Li, H},
title = {The twist-and-squeeze activation of CARF-fused adenosine deaminase by cyclic oligoadenylates.},
journal = {The EMBO journal},
volume = {},
number = {},
pages = {},
pmid = {41107544},
issn = {1460-2075},
support = {R35 GM152081/GM/NIGMS NIH HHS/United States ; },
abstract = {The recently identified CARF (CRISPR-associated Rossman-fold) family of proteins play a critical role in prokaryotic defense, mediating cOA (cyclic oligoadenylate)-stimulated ancillary immune responses in the type III CRISPR-Cas systems. Whereas most previously characterized CARF proteins contain nucleic acids or protein degradation effectors, a subset of the family, including the CARF-fused adenosine deaminase (ADA) (Cad1), has recently been shown to convert ATP to ITP. The enzymatic mechanism and the activation process of Cad1, however, remain incompletely understood. Here we present biochemical and structural analyses of a ring nuclease Cad1, revealing its substrate binding specificity and a sequential activation process by cOAs. Despite an overall structural similarity to canonical ADA enzymes, the ADA domain of Cad1 possesses unique structural features that confer a specificity for ATP. Supported by mutational analysis, our structural work demonstrates an allosteric link between the cOA-binding CARF and the ADA domain through a protein network within the hexameric enzyme assembly. Binding of a cA4 molecule to paired CARF domains induces a twisting of the linked ADA domains around one another, which remodels their active sites and alters interactions with neighboring ADA domains, thereby driving a sequential conformational activation mechanism.},
}
@article {pmid41005541,
year = {2025},
author = {Kristof, A and Karunakaran, K and Ferry, Y and Mizote, P and Allen, C and Briggs, S and Blazeck, J},
title = {A next-generation platform for highly optimized CRISPR-mediated transcriptional repression.},
journal = {Journal of biotechnology},
volume = {408},
number = {},
pages = {192-200},
doi = {10.1016/j.jbiotec.2025.09.012},
pmid = {41005541},
issn = {1873-4863},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; HEK293 Cells ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockdown Techniques ; Protein Engineering/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Repressor Proteins/genetics/metabolism ; Nuclear Localization Signals/genetics ; },
abstract = {CRISPR interference (CRISPRi), the fusion of transcriptional repressor domains to nuclease-inactive Cas9, is a powerful genetic tool enabling site-specific suppression of gene expression. However, its performance remains inconsistent across cell lines, gene targets, or single guide RNAs (sgRNAs) employed. This study described the development process of a novel, highly optimized CRISPRi repressor for mammalian gene regulation through a multi-pronged protein engineering approach: (1) truncating established domains, (2) characterizing candidate domains, (3) creating combinatorial domain fusion libraries, and (4) optimizing NLS configuration. First, by evaluating several truncations of MeCP2, a well-established repressor, we see that the ultra-compact NCoR/SMRT interaction domain (NID) significantly enhances CRISPRi gene knockdown performance, exceeding levels observed with canonical MeCP2 subdomains by an average of ∼40 %. Incorporating this optimized MeCP2 NID truncation with a diverse panel of authenticated repressor domains, we next assemble and screen combinatorial multi-domain libraries, discovering four new repressor fusions. Upon follow-up nuclear localization signal (NLS) configuration analysis, we see that affixing one carboxy-terminal NLS enhances gene knockdown efficiency of the repressors by an average of ∼50 %. Through rigorous validation of NLS-tagged repressor fusions across several cell lines, multiple sgRNA targets, and genome-wide dropout screens, we establish that our strongest system, dCas9-ZIM3-NID-MXD1-NLS, achieves superior gene silencing capabilities over alternative CRISPRi platforms. In addition to developing dCas9-ZIM3-NID-MXD1-NLS, a uniquely potent transcriptional repressor, we envision that the multi-domain engineering approach utilized in this study will be valuable framework enabling future strides in CRISPR platform development.},
}
@article {pmid40962145,
year = {2025},
author = {Gao, Y and Zhou, Y and Wang, L and Zhang, N and Qin, W and Meng, W and Zhou, C},
title = {Synergistic strategy for high-yield 2,3-butanediol and acetoin production in Bacillus licheniformis MW03 based on metabolic engineering.},
journal = {Journal of biotechnology},
volume = {408},
number = {},
pages = {232-243},
doi = {10.1016/j.jbiotec.2025.09.006},
pmid = {40962145},
issn = {1873-4863},
mesh = {*Acetoin/metabolism/analysis ; *Butylene Glycols/metabolism ; *Bacillus licheniformis/genetics/metabolism ; *Metabolic Engineering/methods ; Bacterial Proteins/genetics/metabolism ; Fermentation ; Alcohol Oxidoreductases/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Gene Editing ; },
abstract = {Bacillus licheniformis is an efficient platform for 2,3-butanediol (2,3-BD) and acetoin production due to its rapid glucose utilization rate and adaptability to industrial fermentation conditions. Here, we isolated the B. licheniformis strain MW03 with high yield of acetoin and 2,3-BD, which carried genetic mutations in acoR and budC, respectively encoding an acetoin dehydrogenase regulator and meso-2,3-BD dehydrogenase. To further confirm the physiological effects on acetoin and 2,3 BD biosynthesis, gene editing was performed using the CRISPR-Cas9 system, followed by phenotypic screening and genotype validation. The knockout of acoR and budC increased the acetoin maximum titer by 21.2 % and 49.2 %, respectively. Moreover, the optical purity of D-(-)-2,3-BD reached 92.7 % following the knockout of budC. Heterologous expression of acoR from B. licheniformis 2709 in both the wild type and acoR knockout mutant strongly inhibited acetoin accumulation compared to native acoR, which emphasized the regulatory role of AcoR in acetoin accumulation. Conversely, complementation of budC restored the synthesis of meso-2,3-BD synthesis, emphasizing its importance in this process. Overexpression of alsD in the acoR mutant increased the 2,3-BD titer by 61.9 % to 121.97 g/L, while the productivity reached 2.03 g/L·h. Finally, co-expression of bdhA and gldA increased 2,3-BD production by 25.6 %. This study elucidated the dual regulatory roles of acoR and budC in acetoin and 2,3-BD metabolism, establishing a "knockout-overexpression" synergic strategy, which offers theoretical support and practical guidance for further strain optimization.},
}
@article {pmid41106392,
year = {2025},
author = {Yang, M and Liu, S and Chen, G and Liu, X and Sun, D and Zhang, J and Wang, Y and Chen, S and Tian, R and Hu, Z},
title = {Structural and functional bases of F. rodentium Cas9 provide insights into CRISPR-Cas protein engineering.},
journal = {Cell genomics},
volume = {},
number = {},
pages = {101039},
doi = {10.1016/j.xgen.2025.101039},
pmid = {41106392},
issn = {2666-979X},
abstract = {The Faecalibaculum rodentium (Fr) CRISPR-Cas9 system exhibits enhanced gene-editing precision and efficiency compared to SpCas9, with distinctive advantages in targeting the TATA box in eukaryotic promoters. However, the underlying molecular mechanisms remained unexplored. Here, we present cryo-electron microscopy structures of the FrCas9-single guide RNA (sgRNA)-DNA complex in both the R-loop expansion and pre-catalytic states, shedding light on its specialized recognition of the 5'-NRTA-3' protospacer adjacent motif (PAM) and the unusual overwinding of the sgRNA-DNA heteroduplex. Our investigations into the structure and extensive mutational analyses reveal that the phosphate lock loop plays a pivotal role in finely adjusting FrCas9's off-target sensitivity and catalytic efficiency. Remarkably, targeted residue substitutions in the phosphate lock loop and the PAM-distal region were found to synergistically enhance both the editing precision and efficiency of FrCas9. These findings advance our understanding of Cas9's accuracy and potency mechanisms while providing a molecular foundation for the rational design and development of next-generation CRISPR technologies.},
}
@article {pmid41106357,
year = {2025},
author = {Gehrke, F and Puchta, H},
title = {CRISPR meets AI-based robotics: Advancing sustainable agriculture.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5785-5787},
doi = {10.1016/j.cell.2025.09.011},
pmid = {41106357},
issn = {1097-4172},
mesh = {*Artificial Intelligence ; *Robotics/methods ; Gene Editing/methods ; *Crops, Agricultural/genetics ; *Agriculture/methods ; *CRISPR-Cas Systems ; },
abstract = {In this issue of Cell, Xu and colleagues develop an approach integrating genome editing, artificial intelligence, and robotics to enhance crop improvement. By reconfiguring reproductive traits for automated pollination in crops such as tomatoes and soybeans, their approach accelerates hybrid seed production and yields crops with better stress tolerance, flavor, and resilience, supporting sustainable agriculture and crop diversity.},
}
@article {pmid41104521,
year = {2025},
author = {Braunreiter, K and Kempton, A and Mejia-Guerra, MK and Murray, A and Baine, S and Adegboye, K and Haile, A and Kumar Ahuja, SJ and Fedoce, A and Liu, C and Burch, P and Kabadi, AM},
title = {Characterization of a humanized mouse model of Duchenne muscular dystrophy to support the development of genetic medicines.},
journal = {Disease models &amp; mechanisms},
volume = {18},
number = {10},
pages = {},
doi = {10.1242/dmm.052182},
pmid = {41104521},
issn = {1754-8411},
support = {//Sarepta Therapeutics/ ; },
mesh = {Animals ; *Muscular Dystrophy, Duchenne/therapy/genetics/physiopathology/pathology/blood ; Disease Models, Animal ; Humans ; Dystrophin/metabolism/genetics ; Muscle, Skeletal/pathology/physiopathology ; Mice, Inbred mdx ; *Genetic Therapy ; Mice ; Male ; Diaphragm/pathology ; Biomarkers/blood/metabolism ; Gene Editing ; Muscle Fibers, Skeletal/pathology/metabolism ; CRISPR-Cas Systems/genetics ; Myocardium/pathology/metabolism ; Fibrosis ; },
abstract = {Duchenne muscular dystrophy (DMD) is a rare, progressive neuromuscular disease resulting from DMD variants, leading to loss of functional dystrophin. To evaluate human-targeted genetic medicines for functional dystrophin restoration, humanized genetic models containing the full human locus are required. This study characterized the hDMDΔ52/mdx mouse model previously reported by Pickar-Oliver and colleagues. Genomic characterization confirmed complete DMD duplication with identical exon 52 deletion junctions on both copies. Histological analysis showed increased diaphragm fibrosis and skeletal muscle central nuclei in hDMDΔ52/mdx mice versus hDMD/mdx controls. hDMDΔ52/mdx mice demonstrated reduced tibialis anterior specific force, decreased skeletal muscle fiber diameter, decreased resistance to eccentric contraction-induced damage and cardiac defects. Multiple serum biomarkers of disease were identified. Using a CRISPR/Cas9 gene-editing strategy to restore human functional dystrophin protein expression, detectable dystrophin expression in the heart and skeletal muscle and increased resistance to injury in the tibialis anterior muscle were observed. In summary, hDMDΔ52/mdx mice display multiple physiological and functional deficits associated with DMD pathology, which can be restored by human-targeted therapy, confirming the suitability of this model for developing human-targeted genetic medicines.},
}
@article {pmid41104129,
year = {2025},
author = {Pan, L and Wei, L and Luo, S and Ren, B and Li, M and Liang, L and Li, X and Wei, G},
title = {Klebsiella pneumoniae detection by a light-controlled one-pot RPA-CRISPR/Cas12a method.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1669860},
pmid = {41104129},
issn = {2235-2988},
mesh = {*Klebsiella pneumoniae/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; Humans ; *Klebsiella Infections/diagnosis/microbiology ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Ultraviolet Rays ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Klebsiella pneumoniae (KP) is a significant pathogenic bacterium responsible for severe infections in hospitals. However, existing traditional detection techniques, such as culture and PCR, are relatively inefficient. Therefore, this study aims to establish a rapid and convenient method for detecting KP.<br><br>METHODS: This study developed a single-tube detection method combining recombinant polymerase amplification (RPA) and light-controlled CRISPR/Cas12a. RPA primers were designed and screened for the rcsA gene of KP to effectively amplify the target. A light-controlled CRISPR/Cas12a system was created using crRNA modified with a photocleavable group (NPOM). The two systems were integrated into a single tube. Following RPA amplification, UV light-controlled release of crRNA inhibition activates CRISPR-mediated target recognition and Cas12a trans-cleavage, detecting fluorescent signals (FD) in conjunction with UV analysis.<br><br>RESULTS: The light-controlled RPA-CRISPR/Cas12a detection platform developed in this study uses a 15 &#x3bc;L reaction system. By optimizing key parameters such as RPA amplification time (20&#xa0;min), primer concentration (400 nM), UV light activation time (30 s), and crRNA/Cas12a concentration (300 nM), the platform achieves optimal detection efficiency. The platform has a fluorescence detection limit of 4.072&#xd7;10[2] copies/reaction and can specifically identify KP in seven common clinical strains. Clinical sample validation demonstrated that the method yields results fully consistent with PCR detection (30/30 agreement rate of 100%), showcasing excellent detection performance and clinical application potential.<br><br>CONCLUSION: We have successfully developed a light-controlled RPA-CRISPR/Cas12a detection system capable of rapidly and highly sensitively detecting KP. This system demonstrates significant advantages in terms of detection speed (completed in as little as 50 minutes), sensitivity (as low as 4.072&#xd7;10[2] copies/reaction), and ease of use, providing an efficient and reliable solution for clinical pathogen detection.},
}
@article {pmid41039096,
year = {2025},
author = {Johnston, M and Dissanayake-Perera, S and Collins, JJ and Stevens, MM and Dincer, C},
title = {Convergence of nanotechnology and CRISPR-based diagnostics.},
journal = {Nature nanotechnology},
volume = {20},
number = {10},
pages = {1365-1373},
pmid = {41039096},
issn = {1748-3395},
support = {13GW0493//Bundesministerium f&#xfc;r Bildung und Forschung (Federal Ministry of Education and Research)/ ; CiET2021\94//Royal Academy of Engineering/ ; },
mesh = {*Nanotechnology/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing ; },
abstract = {In addition to its broad application in genome engineering and therapeutics, clustered regularly interspaced short palindromic repeats (CRISPR) technology provides field-deployable methods for the highly sensitive and selective detection of nucleic acids. From a diagnostic perspective, CRISPR-based assays hold clear clinical potential for identifying a range of both infectious and non-communicable diseases. In this Perspective we evaluate recent nanotechnologies and nanomaterials that have been engineered to interface with CRISPR systems on a nanoscale level to realize the full potential of this versatile diagnostic tool. We assess biomolecules such as enzymes and oligonucleotides, some of the more commonly used synthetic nanoparticles and detection platforms that integrate nanotechnologies in new and innovative ways. We discuss current trends and look ahead to future challenges and opportunities, including non-nucleic acid target detection, pre-amplification-free detection of nucleic acids, the development of wearable devices and integration with artificial intelligence workflows.},
}
@article {pmid40975295,
year = {2026},
author = {Zheng, X and Xu, H and Huang, Y and Liu, X and Zhu, S and Liu, H and Gao, S},
title = {Development of an RT-RAA-CRISPR-Cas12a assay for rapid, sensitive and visual detection of Tilapia Lake Virus (TiLV).},
journal = {Journal of virological methods},
volume = {339},
number = {},
pages = {115266},
doi = {10.1016/j.jviromet.2025.115266},
pmid = {40975295},
issn = {1879-0984},
mesh = {Animals ; *Fish Diseases/virology/diagnosis ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Tilapia/virology ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; Aquaculture ; RNA, Viral/genetics ; DNA, Single-Stranded/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {In this study，we developed a new, highly efficient, and sequence-specific method for detecting Tilapia Lake Virus (TiLV) based on the clustered regularly interspaced short palindromic repeats (CRISPR) - CRISPR-associated protein 12a (Cas12a) system. TiLV is a highly contagious virus that has caused significant damage to the global aquaculture industry. Specific primers, CRISPR RNA (crRNA), and single-stranded DNA (ssDNA) reporters were designed to detect TiLV genome segment 3, with the ssDNA reporters modified at the 5' and 3' ends with fluorophore and quencher groups, respectively. The assay showed no cross-reactivity with other bacterial and viral pathogens in fish. The detection limit was 9.10 copies per reaction for recombinant plasmid standards and 91.82 fg/μL for TiLV RNA, demonstrating high sensitivity. The reverse transcription recombinase aided amplification (RT-RAA) coupled CRISPR/Cas12a method showed 100 % concordance with the standard fluorescence method, indicating its accuracy and suitability for clinical testing. This study innovatively combined the RT-RAA technique with the CRISPR/Cas12a reaction system, offering a new diagnostic method for TiLV that is fast, portable, highly specific, and sensitive. This enables on-site rapid screening for TiLV, ensuring aquaculture safety and the secure circulation of aquatic animal products.},
}
@article {pmid40935117,
year = {2026},
author = {Terada, T and Fujii, S and Yamanishi, N and Kajihara, R and Watanabe, T and Ezaki, R and Horiuchi, H and Matsuzaki, M},
title = {Potential of recombinant avian adeno-associated virus as a viral vector for CRISPR/Cas9 delivery to avian cells.},
journal = {Journal of virological methods},
volume = {339},
number = {},
pages = {115263},
doi = {10.1016/j.jviromet.2025.115263},
pmid = {40935117},
issn = {1879-0984},
mesh = {Animals ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; Chickens/genetics ; *Gene Editing/methods ; *Dependovirus/genetics ; Cell Line ; Green Fluorescent Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Fibroblasts/virology ; Staphylococcus aureus/genetics/enzymology ; },
abstract = {While genome editing has been established in chickens, where cultured primordial germ cell (PGC) systems are available, the implementation of genome editing remains a major challenge in many other birds due to the lack of robust PGC culture methods. Therefore, the development of reliable and efficient tools can significantly accelerate precision genome modification in avian species. Here, we evaluated the applicability of recombinant avian adeno-associated virus (rA3V) as a delivery vector for a CRISPR/Cas9 construct in avian cells using Staphylococcus aureus-derived Cas9 (SaCas9) and single-guide RNA (sgRNA). Infection with rA3V particles carrying an EGFP expression cassette (rA3V-EGFP) successfully induced EGFP expression in chicken fibroblasts (DF-1) cells, with approximately 80 % EGFP-positive cells at the maximum multiplicity of infection (MOI = 10,000). In plasmid-based transfection experiments, sgRNAs targeting the chicken tyrosinase locus and SaCas9 exhibited DNA cleavage activity in DF-1 cells. Furthermore, infection with rA3V particles encoding these CRISPR components successfully introduced indel mutations into the tyrosinase gene in DF-1 cells, with a calculated indel frequency of approximately 5.4 % at MOI = 40,000 without drug selection. Although EGFP expression was observed in quail fibrosarcoma cells, the percentage of EGFP-positive cells was much lower than that in DF-1 cells. In addition, in vivo infection with rA3V-EGFP of the chicken blastoderm failed to induce EGFP expression in germline cells, even at the highest applicable viral dose. In summary, rA3V can be used as a genome-editing vector in birds, although further investigation of its infectivity and tropism is necessary to expand its applicability to diverse avian species.},
}
@article {pmid40915071,
year = {2026},
author = {Li, Z and Jiao, Y and Tang, J and Dong, X and Thomas, R and Xie, B and Li, Y and Jacques, P},
title = {The pH-responsive regulator PlPacC and GATA transcription factor PlAreB are involved in the regulation of the biosynthesis of the antifungal lipopeptaibols leucinostatins in Purpureocillium lilacinum.},
journal = {Microbiological research},
volume = {302},
number = {},
pages = {128324},
doi = {10.1016/j.micres.2025.128324},
pmid = {40915071},
issn = {1618-0623},
mesh = {Hydrogen-Ion Concentration ; *Transcription Factors/genetics/metabolism ; *Antifungal Agents/metabolism ; *Fungal Proteins/genetics/metabolism ; *Hypocreales/metabolism/genetics/growth &amp; development ; Gene Expression Regulation, Fungal ; Nitrogen/metabolism ; Culture Media/chemistry ; CRISPR-Cas Systems ; },
abstract = {The biocontrol fungus Purpureocillium lilacinum PLBJ-1 produces leucinostatins, a class of non-ribosomal peptides (NRPs) with broad-spectrum antimicrobial activities. However, the molecular mechanisms underlying the optimization of culture conditions for leucinostatin production remain unexplored. Previous research showed that PLBJ-1 synthesizes leucinostatins more effectively in hand-made Potato Dextrose Broth (PDB-M) than in commercially available PDB (PDB-C). Elementary analysis of these two media indicated that the difference in leucinostatin yield was correlated with variations in pH dynamics and nitrogen content. Subsequent experiments under different initial pH and nitrogen levels confirmed that an alkaline environment and reduced nitrogen availability could enhance leucinostatin production. To investigate the regulators involved, CRISPR-Cas9-mediated gene disruptions were performed on the pH-responsive transcription factor PlPacC and the nitrogen regulator PlAreB. The disruption of either PlPacC or PlAreB resulted in a marked reduction in biomass and sporulation in P. lilacinum PLBJ-1. Specifically, PlPacC disruption impaired environmental pH regulation and significantly decreased leucinostatin production. In contrast, PlAreB disruption led to an increased leucinostatin yield. Overall, these findings demonstrate that environmental pH and nitrogen availability are the critical factors governing leucinostatin biosynthesis, acting through two key transcriptional regulators, PlPacC and PlAreB. This study lays a molecular foundation for future large-scale optimization of leucinostatin fermentation.},
}
@article {pmid40789509,
year = {2025},
author = {Tsai, HY and Chen, MH and Yun, J and Lai, LA and Valentine, JF and Bronner, MP and Brentnall, TA and Pan, S and Chen, R},
title = {Restricting metabolic plasticity enhances stress adaptation through the modulation of PDH and HIF1A in TRAP1-depleted colon cancer.},
journal = {Cancer letters},
volume = {632},
number = {},
pages = {217977},
doi = {10.1016/j.canlet.2025.217977},
pmid = {40789509},
issn = {1872-7980},
support = {P30 DK056338/DK/NIDDK NIH HHS/United States ; R01 CA211892/CA/NCI NIH HHS/United States ; R01 CA276173/CA/NCI NIH HHS/United States ; },
mesh = {*Colonic Neoplasms/metabolism/genetics/pathology ; Humans ; *Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; Glycolysis ; Glucose Transporter Type 1/genetics/metabolism ; Pyruvate Dehydrogenase Acetyl-Transferring Kinase ; Cell Line, Tumor ; Oxidative Phosphorylation/drug effects ; HCT116 Cells ; Reactive Oxygen Species/metabolism ; Gene Expression Regulation, Neoplastic ; Monocarboxylic Acid Transporters/genetics/metabolism ; Adaptation, Physiological ; CRISPR-Cas Systems ; HSP90 Heat-Shock Proteins ; Symporters ; },
abstract = {Metabolic plasticity allows cancer cells to survive under adverse conditions. To investigate the role of mitochondrial chaperone tumor necrosis factor receptor-associated protein 1 (TRAP1) in this process, we used CRISPR/Cas9 mediated genetic deletion to knock out (KO) TRAP1 in colon cancer cells. Depletion of TRAP1 triggered a series of events: induced metabolic reprogramming, increased glycolytic flux, downregulation of mitochondrial complex I, and elevated ROS generation. TRAP1-deficient cells showed tolerance to Oxidative Phosphorylation (OXPHOS) inhibitors and exhibited a higher extracellular acidification rate (ECAR). Additionally, TRAP1 depletion activated hypoxia response elements (HREs) and upregulated HIF1A target genes such as GLUT1 and MCT1. Furthermore, pyruvate dehydrogenase kinases 1 (PDK1) was upregulated in KO cells, leading to the inactivation of the tricarboxylic acid (TCA) cycle enzyme, pyruvate dehydrogenase (PDH). This metabolic shift towards glycolytic metabolism resulted in increased glycolytic metabolism, elevated lactic acid production, and higher glucose consumption, making TRAP1-depleted cancer cells more dependent on this altered metabolism for survival. Treatment with DCA, a PDK inhibitor, restored PDH activity, exacerbated oxidative stress, and increased cell death in KO cells. Our study here sheds light on how TRAP1 depletion affects metabolic plasticity, driving colon cancer cells to adapt to metabolic and oxidative stress. These findings highlight TRAP1 as a promising therapeutic target for manipulating metabolic plasticity and overcoming drug resistance in cancer therapy.},
}
@article {pmid41103153,
year = {2025},
author = {Chen, L and Yun, M and Chen, B and Xie, S and Liu, W and Wang, M and Yan, J and Cai, J and Yang, S and Peng, Q and Xie, D and Lin, Y and Jiang, B},
title = {Loss of CsCLV2 function causes dwarfism and determinates growth in cucumber.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70525},
doi = {10.1111/tpj.70525},
pmid = {41103153},
issn = {1365-313X},
support = {32202477//National Natural Science Foundation of China/ ; },
mesh = {*Cucumis sativus/genetics/growth &amp; development/metabolism ; *Plant Proteins/genetics/metabolism/physiology ; *Dwarfism/genetics ; Phenotype ; Meristem/growth &amp; development/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Cucumber (Cucumis sativus L.) is a globally important vegetable crop. Ideal plant architecture optimizes spatial utilization, enhances economic coefficient, and facilitates mechanized cultivation. In this study, we identified a dwarf mutant, csdw3, exhibiting reduced plant height, shortened internodes, and fewer internodes. Genetic analysis showed that this dwarf phenotype is controlled by a single recessive gene. Fine-mapping localized the causal locus to an 80 kb region on chromosome 1, where we discovered a 102 bp deletion in CsCLV2, a gene encoding a leucine-rich repeat receptor-like protein homologous to Arabidopsis CLAVATA2. CRISPR-Cas9-generated loss-of-function mutants recapitulated the dwarf phenotype, confirming the role of CsCLV2 in plant height regulation. Histological examination revealed that CsCLV2 disruption causes premature termination of shoot apical meristem (SAM) development, reducing both internode number and length. Protein interaction assays further demonstrated that CsCLV2 associates with receptor-like kinase CsCIK1 (CLAVATA3 INSENSITIVE RECEPTOR KINASES 1), indicating their cooperative function in the CLV-WUS signaling pathway to maintain meristem activity. Our findings uncover a regulator of plant height in cucumber and provide valuable genetic resources for breeding ideotypes optimized for yield and cultivation efficiency.},
}
@article {pmid41102733,
year = {2025},
author = {Marchenko, N and Nesbitt, NM and Alexandrova, E and Reisz, JA and D'Alessandro, A and Suh, J and Uryasev, S and Pennacchia, L and Bahou, WF},
title = {Biliverdin reductase B as a new target in breast cancer.},
journal = {Breast cancer research : BCR},
volume = {27},
number = {1},
pages = {179},
pmid = {41102733},
issn = {1465-542X},
support = {NCI CA284999/CA/NCI NIH HHS/United States ; P30CA046934/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Breast Neoplasms/genetics/pathology/metabolism/drug therapy ; Female ; Animals ; Cell Line, Tumor ; Mice ; *Oxidoreductases Acting on CH-CH Group Donors/genetics/metabolism/antagonists &amp; inhibitors ; Cell Proliferation ; Oxidation-Reduction ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Enhanced metabolic and mitochondrial activity inherent in actively proliferating cancer cells is associated with intracellular redox imbalance that impacts cellular viability. To restore redox homeostasis cancer cells evolve to activate redox protective mechanisms. This differential activation of redox defense pathways compared to normal cells provides a therapeutic window for novel targeted therapies in cancer. Although heme metabolism emerges as a crucial regulator of redox homeostasis and iron metabolism in cancer cells with frequent alteration in breast cancer, it remains largely unexplored, and no targeted translational approaches have been developed. Heme-regulated redox homeostasis is coordinately maintained through biosynthetic and degradation pathways. As a byproduct of TCA cycle, cytotoxic heme is initially derivatized by heme oxygenases and progressively metabolized to the potent antioxidant bilirubin by two non-redundant biliverdin reductases, BLVRA and BLVRB. BLVRB overexpression has been observed in breast cancers, although its function in breast cancer pathogenesis remains unknown.<br><br>METHODS: CRISPR/Cas9 deletion of BLVRB in multiple breast cancer cell lines demonstrated its profound effect on intracellular redox state and cell proliferation in vitro and in xenograft models. Integrated proteomic, metabolomic, and lipidomic studies identified and validated BLVRB-mediated adaptive metabolic responses required for breast cancer cell cytoprotection.<br><br>RESULTS: We have established BLVRB as a requisite component of the pro-survival redox defense mechanism in breast cancer cells. Targeted deletion of BLVRB induces reductive stress, leading to alterations in endoplasmic reticulum proteostasis and lipid composition. These defects impact plasma membrane functionality and endosomal recycling of multiple oncogenic receptors, such as HER2 and transferrin receptors.<br><br>CONCLUSIONS: These data collectively identify BLVRB as a novel metabolic target in breast cancer, distinct from other redox-regulating pathways. This study, along with our recent progress in developing novel specific BLVRB inhibitors, offers a unique translational opportunity for targeted therapies in personalized breast cancer medicine.},
}
@article {pmid41102523,
year = {2025},
author = {Savage, N and Danis, E and Chokshi, CR and Custers, S and Shaikh, MV and Miletic, P and Venugopal, C and Brown, KR and Vibhakar, R and Moffat, J and Singh, SK},
title = {CRISPR screen reveals SOX2 as a critical regulator of CD133 and cellular stress response in glioblastoma.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {36228},
pmid = {41102523},
issn = {2045-2322},
support = {1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; },
mesh = {*AC133 Antigen/metabolism/genetics ; *SOXB1 Transcription Factors/genetics/metabolism ; *Glioblastoma/genetics/metabolism/pathology ; Humans ; *CRISPR-Cas Systems ; Neoplastic Stem Cells/metabolism/pathology ; Cell Line, Tumor ; *Brain Neoplasms/genetics/metabolism/pathology ; Gene Expression Regulation, Neoplastic ; *Stress, Physiological ; Animals ; Mice ; },
abstract = {Glioblastoma (GBM) remains a formidable challenge in clinical settings due to limited treatments available. The surface protein CD133 marks glioblastoma stem cells (GSCs), cells capable of overcoming therapeutic pressures and correlate with more aggressiveness tumor phenotypes. In this study, we employed a CRISPR-Cas9 functional screen to deconvolute CD133 dynamics in tumors. This led us to establish that SOX2 is a key player in controlling the PROM1 gene, which in turn influences how cells react to stress factors, including those induced by chemoradiation treatment. The discoveries in this study shed light on the complex web of mechanisms that control the survival and resistance of GSCs, offering promising new avenues for targeting and potentially overcoming therapy resistance.},
}
@article {pmid41101942,
year = {2025},
author = {Lim, SL and Chin, CH and Chiou, YJ and Hsu, MT and Chiang, PW and Chen, HJ and Tu, YC and Tzou, WS and Tang, SL},
title = {Unveiling Unusual Ecofunctional Traits of Endozoicomonas Species Through Comprehensive Comparative Genomics.},
journal = {Environmental microbiology},
volume = {27},
number = {10},
pages = {e70191},
doi = {10.1111/1462-2920.70191},
pmid = {41101942},
issn = {1462-2920},
support = {AS-IA-109-L05//Academia Sinica/ ; },
mesh = {*Genome, Bacterial ; Genomics ; Phylogeny ; Prophages/genetics ; Bacterial Proteins/genetics ; *Gammaproteobacteria/genetics ; Quorum Sensing ; },
abstract = {Endozoicomonas is an omnipresent marine bacterial genus, associated with various marine organisms, that contributes to host health, nutrient cycling and disease resistance. Nonetheless, its genomic features remain poorly characterised due to a paucity of high-quality genomes. In this study, we sequenced 5 novel Endozoicomonas strains and re-sequenced 1 known strain to improve genomic resolution. By integrating these 6 high-quality genomes with 31 qualified published genomes, our pan-genomic analysis revealed variation in genetic traits among clades. Notably, Endozoicomonas lacks quorum-sensing capabilities, suggesting resistance to quorum quenching mechanisms. It also lacks the capacity to synthesise and transport vitamin B12, indicating that it does not supply this nutrient to holobionts. Remarkably, Endozoicomonas genomes encode 92 identified giant proteins (15-65 kbp). These proteins cluster into three major groups associated with antimicrobial peptide synthesis, exotoxin production and cell adhesion. Additionally, we found that Endozoicomonas has acquired prophages from diverse sources via infection or other types of gene transfer. Notably, CRISPR-Cas sequences suggest evolutionary trajectories independent of both prophage acquisition and phylogenetic lineage, implying potential geographic influences or environmental pressures. This study provides new insights into the genomic diversity of Endozoicomonas and its genetic adaptations to diverse hosts.},
}
@article {pmid41100915,
year = {2025},
author = {Kim, I and Suh, JY},
title = {Old and new tactics of CRISPR-centric competition between bacteria and bacteriophages.},
journal = {Current opinion in structural biology},
volume = {95},
number = {},
pages = {103168},
doi = {10.1016/j.sbi.2025.103168},
pmid = {41100915},
issn = {1879-033X},
abstract = {The CRISPR-Cas system provides adaptive immunity for prokaryotes against mobile genetic elements (MGEs) such as bacteriophages and plasmids. As a countermeasure, MGEs have evolved various anti-CRISPR (Acr) mechanisms that neutralize the CRISPR-mediated immunity. Canonical Acr proteins block target binding of Cas proteins in a stoichiometric or enzymatic manner. New findings reveal that Acr also disintegrates functional Cas complexes, induces promiscuous target binding, and mimics Cas proteins and crRNA with defective mutations. Here, we summarize a broad repertoire of structural and functional mechanisms underlying CRISPR-centric competition, highlighting recent discoveries of molecular machinery that modulates CRISPR function.},
}
@article {pmid41100703,
year = {2025},
author = {Liao, X and Li, Y and Wu, Y and Wen, L and Jing, M and Chen, B and Li, X and Shang, X},
title = {TEMC-Cas: Accurate Cas Protein Classification via Combined Contrastive Learning and Protein Language Models.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00631},
pmid = {41100703},
issn = {2161-5063},
abstract = {The accurate classification of Cas proteins is crucial for understanding CRISPR-Cas systems and developing genome-editing tools. Here, we present TEMC-Cas, a deep learning framework for accurate classification of Cas proteins that combines a finely tuned ESM protein language model with contrastive learning. Unlike traditional methods that rely on sequence similarity (e.g., BLAST, HMMs) or structural prediction, TEMC-Cas leverages evolutionary-scale modeling to capture distant homology while employing contrastive learning to distinguish closely related subtypes. The framework incorporates LoRA for efficient parameter adaptation and addresses class imbalance through weighted loss functions. TEMC-Cas achieves superior performance in classifying the Cas1-Cas13 families and 17 Cas12 subtypes, demonstrating particular strength in identifying remote homology. This approach provides a robust tool for the discovery of the CRISPR system and expands the toolbox for genome engineering applications. TEMC-Cas is now freely accessible at https://github.com/Xingyu-Liao/TEMC-Cas.},
}
@article {pmid41099996,
year = {2025},
author = {Shafi, Z and Shahid, M and Ilyas, T and Singh, K},
title = {Next-generation perspectives on microbially synthesized siderophores: molecular engineering, multi-omics insights, and applications for smart climate-resilient crops.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {10},
pages = {393},
pmid = {41099996},
issn = {1573-0972},
mesh = {*Siderophores/biosynthesis/genetics/metabolism ; *Crops, Agricultural/growth &amp; development/microbiology ; Iron/metabolism ; *Bacteria/metabolism/genetics ; Metabolomics ; Gene Editing ; Genomics ; Proteomics ; CRISPR-Cas Systems ; Multiomics ; },
abstract = {Siderophores, low-molecular-weight iron-chelating compounds synthesized by microbes, play a crucial role in iron (Fe) acquisition under Fe-limited conditions. In recent years, their significance in sustainable agriculture has gained increasing attention due to their multifaceted roles in plant growth promotion, stress alleviation, and disease suppression. This review presents next-generation insights into the biosynthesis, regulation, and applications of microbial siderophores, with a focus on advanced molecular and omics-based approaches. Innovations in synthetic biology and CRISPR/Cas-mediated genome editing have enabled precise manipulation of siderophore biosynthetic gene clusters, enhancing their production and functionality. Multi-omics platforms-genomics, transcriptomics, proteomics, and metabolomics-have revealed complex regulatory networks, unveiling cryptic pathways and inter-microbial variability in siderophore synthesis. Furthermore, the use of siderophore-producing plant growth-promoting rhizobacteria (PGPR) has shown promise in improving nutrient uptake, inducing systemic resistance, and mitigating abiotic stresses in crops. The integration of nano-formulations and encapsulation technologies has enhanced the stability and field efficacy of siderophore-based bioinoculants. This review also explores emerging strategies for developing microbial consortia and smart delivery systems to meet the challenges of climate-resilient agriculture. By bridging molecular insights with field-level applications, this article underscores the potential of siderophores as eco-friendly tools for next-generation sustainable farming practices.},
}
@article {pmid41097718,
year = {2025},
author = {Lye, SH and Polycarp, N and Durojaye, TJ and Tollefsbol, TO},
title = {Functional Heterogeneity and Context-Dependent Roles of LncRNAs in Breast Cancer.},
journal = {Cancers},
volume = {17},
number = {19},
pages = {},
pmid = {41097718},
issn = {2072-6694},
support = {R01CA178441/CA/NCI NIH HHS/United States ; },
abstract = {As with other non-coding RNAs (ncRNAs), the aberrant expression of long non-coding RNAs (lncRNAs) can be associated with different forms of cancers, including breast cancer (BC). Various lncRNAs may either promote or suppress cell proliferation, metastasis, and other related cancer signaling pathways by interacting with other cellular machinery, thus affecting the expression of BC-related genes. However, lncRNAs are characterized by features that are unlike protein-coding genes, which pose unique challenges when it comes to their study and utility. They are highly diverse and may display contradictory functions depending on factors like the BC subtype, isoform diversity, epigenetic regulation, subcellular localization, interactions with various molecular partners, and the tumor microenvironment (TME), which contributes to the intratumoral heterogeneity and phenotypic plasticity. While lncRNAs have potential clinical utility, their functional heterogeneity coupled with a current paucity of knowledge of their functions present challenges for clinical translation. Strategies to address this heterogeneity include improving classification systems, employing CRISPR/Cas tools for functional studies, utilizing single-cell and spatial sequencing technologies, and prioritizing robust targets for therapeutic development. A comprehensive understanding of the lncRNA functional heterogeneity and context-dependent behavior is crucial for advancing BC research and precision medicine. This review discusses the sources of lncRNA heterogeneity, their implications in BC biology, and approaches to resolve knowledge gaps in order to harness lncRNAs for clinical applications.},
}
@article {pmid41027589,
year = {2025},
author = {Jiang, W and Georgiadis, I and Fumagalli, T and Wang, S and Vasileiou, C and Dahlin, J and Borodina, I},
title = {In Vivo DNA Assembly in Yarrowia lipolytica.},
journal = {ACS synthetic biology},
volume = {14},
number = {10},
pages = {4116-4121},
doi = {10.1021/acssynbio.5c00296},
pmid = {41027589},
issn = {2161-5063},
mesh = {*Yarrowia/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Rad52 DNA Repair and Recombination Protein/genetics/metabolism ; Saccharomyces cerevisiae/genetics ; Homologous Recombination/genetics ; *DNA/genetics/metabolism ; Gene Editing/methods ; Green Fluorescent Proteins/genetics/metabolism ; },
abstract = {The oleaginous yeast Yarrowia lipolytica is an important platform organism for biotechnology applications. In this study, we established an in vivo DNA assembly system leveraging CRISPR-Cas9 for efficient genomic integration of multiple DNA fragments into the genome of Y. lipolytica. Using the green fluorescent protein mNeonGreen as a model, we demonstrated 53% correct assembly of three DNA fragments with homology arms as short as 50 bp. The system was further validated by constructing 2-3 step biosynthetic pathways for pigments betaxanthin and betanin. To improve the homologous recombination efficiency of Y. lipolytica, we expressed S. cerevisiae RAD52 (ScRAD52) or a Cas9-hBrex27 fusion protein. While ScRAD52 expression impaired growth, the cas9-hBrex27 fusion enhanced integration efficiency, particularly for multifragment pathway assemblies. The in vivo assembly method simplifies pathway construction and gene overexpression in Y. lipolytica.},
}
@article {pmid41021780,
year = {2025},
author = {Golla, SA and Abo-Hashesh, M and Gupta, D and Liu, Y and Mahadevan, R},
title = {Model-Based Optimization of a qCRISPRi Circuit for Dynamic Control of Metabolic Pathways.},
journal = {ACS synthetic biology},
volume = {14},
number = {10},
pages = {3890-3898},
doi = {10.1021/acssynbio.5c00095},
pmid = {41021780},
issn = {2161-5063},
mesh = {Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Metabolic Networks and Pathways/genetics ; Quorum Sensing/genetics ; Repressor Proteins/genetics/metabolism ; Trans-Activators/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; Gene Expression Regulation, Bacterial ; },
abstract = {Metabolic engineering enables sustainable chemical production but often imposes metabolic burdens that reduce cellular viability and productivity. Dynamic control strategies, such as quorum sensing (QS)-based circuits, can mitigate these effects by autonomously regulating gene expression in response to cell density. In this study, we investigated a QS-regulated CRISPR interference (qCRISPRi) circuit for the dynamic control of metabolic pathways, focusing on the role of leaky expression and regulator stringency. Using a combination of mathematical modeling and experiments, we evaluated how promoter leakiness and LuxR stringency influence key switching characteristics including maximum gene expression, switching density, fold repression, and transition time. Our results show that high leaky expression of dCas9 reduces switching density and represses GFP prematurely, whereas a high-stringency LuxR variant enhances switching precision by reducing leakiness and enabling sharper transitions. These model predictions were validated experimentally in E. coli, confirming that LuxR stringency improves dynamic circuit performance. Together, this work provides a quantitative framework for optimizing QS-based regulatory systems and offers generalizable design insights for implementing dynamic control in metabolic engineering.},
}
@article {pmid40992138,
year = {2026},
author = {Xiao, G and Shi, H and Lin, Q and Li, S and He, J and Zhang, G},
title = {A rapid CRISPR-Cas12a/T7EI integrated workflow for high-throughput screening of homozygous mutant cell lines.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {267},
number = {},
pages = {117152},
doi = {10.1016/j.jpba.2025.117152},
pmid = {40992138},
issn = {1873-264X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *High-Throughput Screening Assays/methods ; Gene Editing/methods ; *Mutation/genetics ; Homozygote ; Workflow ; THP-1 Cells ; Cell Line ; },
abstract = {Efficient screening for homozygous mutant cell lines, particularly those resulting from low-efficiency CRISPR-Cas9 editing, remains challenging. Here, we developed HomoSelect-CT, an integrated workflow combining CRISPR-Cas12a nucleic acid detection with T7 Endonuclease I (T7EI) genotyping, designed to streamline the screening process for homozygous mutant cell lines. This method requires no specialized instrumentation, enhancing accessibility and efficiency. We validated HomoSelect-CT by successfully identifying homozygous mutants in CRISPR-Cas9-edited THP-1 cells, which was confirmed by Sanger sequencing and Western blot (WB). These findings demonstrate that HomoSelect-CT is a robust and efficient alternative for the rapid isolation of genome-edited cell lines. The entire screening workflow, from monoclonal cultures to confirmed homozygous mutants, is completed in under 4 h, requiring only standard PCR equipment and routine reagents. Thus, HomoSelect-CT represents a significant advancement in CRISPR screening methodology, offering remarkable simplicity and enabling high-throughput screening that is particularly suitable for mutants arising from low-efficiency editing events.},
}
@article {pmid40829585,
year = {2025},
author = {Xu, C and Niu, X and Sun, H and Yan, H and Tang, W and Ke, A},
title = {Conversion of IscB and Cas9 into RNA-guided RNA editors.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5847-5861.e11},
doi = {10.1016/j.cell.2025.07.032},
pmid = {40829585},
issn = {1097-4172},
mesh = {Humans ; *RNA Editing ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; Adenosine Deaminase/metabolism/genetics ; RNA, Messenger/metabolism/genetics ; Gene Editing/methods ; RNA-Binding Proteins/metabolism/genetics ; DNA, Single-Stranded/metabolism ; Trans-Splicing ; },
abstract = {RNA-guided RNA editing represents an attractive alternative to DNA editing. However, the prevailing tool, CRISPR-Cas13, has collateral RNA cleavage activity that causes undesirable cytotoxicity in human cells. Here, we report an ultracompact RNA-editing platform engineered from IscB, which has comparable or higher activity than Cas13 but without cytotoxicity concerns. We show that IscB, the evolutionary ancestor of Cas9, has an intrinsic affinity for complementary single-stranded (ss)DNA and RNA. This activity becomes dominant when its double-stranded DNA binding activity is switched off through the deletion of its target-adjacent motif domain. The resulting R-IscB is comparable to or better than Cas13, can efficiently alter splicing outcomes in human cells, and can further mediate trans-splicing to correct any mutation at the mRNA level. R-IscB also drives efficient A-to-I editing on mRNA when fused to adenosine deaminase acting on RNA 2 (ADAR2) and mediates cleavage-based mRNA knockdown upon HNH engineering. Finally, we show that the same approach converts some Cas9s to RNA-targeting tools.},
}
@article {pmid40774253,
year = {2025},
author = {Zhou, Z and Zhu, S and Hong, Y and Jin, G and Ma, R and Lin, F and Zhang, Y and Lee, HY and Liu, N},
title = {Composite transposons with bivalent histone marks function as RNA-dependent enhancers in cell fate regulation.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5878-5894.e18},
doi = {10.1016/j.cell.2025.07.014},
pmid = {40774253},
issn = {1097-4172},
mesh = {Humans ; *DNA Transposable Elements/genetics ; Erythropoiesis/genetics ; *Histone Code ; CRISPR-Cas Systems ; *Enhancer Elements, Genetic ; *Histones/metabolism ; Animals ; *RNA/metabolism ; Myelopoiesis/genetics ; },
abstract = {Discrete genomic units can recombine into composite transposons that transcribe and transpose as single units, but their regulation and function are not fully understood. We report that composite transposons harbor bivalent histone marks, with activating and repressive marks in distinct regions. Genome-wide CRISPR-Cas9 screening, using a reporter driven by the hominid-specific composite transposon SVA (SINE [short interspersed nuclear element]-VNTR [variable number of tandem repeats]-Alu) in human cells, identified diverse genes that modify bivalent histone marks to regulate SVA transcription. SVA transcripts are critical for SVA's cis-regulatory function in selectively contacting and activating long-range gene expression. Remarkably, a subset of bivalent SVAs is activated during erythropoiesis to boost multiple erythroid gene expression, and knocking down these SVAs leads to deficient erythropoiesis. The RNA-dependent cis-regulatory function of SVA activates genes for myelopoiesis and can contribute to aging-associated myeloid-biased hematopoiesis. These results reveal that the cis-regulatory functions of composite transposons are bivalently regulated to control cell fate transitions in development and aging.},
}
@article {pmid40769155,
year = {2025},
author = {Park, JC and Uhm, H and Kim, YW and Oh, YE and Lee, JH and Yang, J and Kim, K and Bae, S},
title = {AI-generated MLH1 small binder improves prime editing efficiency.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5831-5846.e21},
doi = {10.1016/j.cell.2025.07.010},
pmid = {40769155},
issn = {1097-4172},
mesh = {Humans ; *Gene Editing/methods ; Animals ; *MutL Protein Homolog 1/metabolism/genetics/chemistry ; HeLa Cells ; Mice ; Mismatch Repair Endonuclease PMS2/metabolism ; CRISPR-Cas Systems ; DNA Mismatch Repair ; Protein Binding ; RNA, Guide, CRISPR-Cas Systems/metabolism ; },
abstract = {The prime editing (PE) system consists of a Cas9 nickase fused to a reverse transcriptase, which introduces precise edits into the target genomic region guided by a PE guide RNA. However, PE efficiency is limited by mismatch repair. To overcome this limitation, transient expression of a dominant-negative MLH1 (MLH1dn) has been used to inhibit key components of mismatch repair. Here, we designed a de novo MLH1 small binder (MLH1-SB) that binds to the dimeric interface of MLH1 and PMS2 using RFdiffusion and AlphaFold 3. The compact size of MLH1-SB enabled its integration into existing PE architectures via 2A systems, creating a PE-SB platform. The PE7-SB2 system significantly improved PE efficiency, achieving an 18.8-fold increase over PEmax and a 2.5-fold increase over PE7 in HeLa cells, as well as a 3.4-fold increase over PE7 in mice. This study highlights the potential of generative AI in advancing genome editing technology.},
}
@article {pmid40706591,
year = {2025},
author = {Marzook, NB and Song, OR and Baumgärtel, L and Bernitz, N and Mkandawire, TT and Watson, LC and Nunes, V and Warchal, S and MacRae, JI and Howell, M and Sateriale, A},
title = {The essential host genome for Cryptosporidium survival exposes metabolic dependencies that can be leveraged for treatment.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5947-5961.e15},
doi = {10.1016/j.cell.2025.07.001},
pmid = {40706591},
issn = {1097-4172},
mesh = {Animals ; *Cryptosporidiosis/parasitology/drug therapy/genetics/metabolism ; Humans ; *Cryptosporidium/genetics/metabolism/drug effects ; Glutathione/metabolism ; CRISPR-Cas Systems ; Mice ; Epithelial Cells/metabolism/parasitology ; Farnesyl-Diphosphate Farnesyltransferase/antagonists &amp; inhibitors/metabolism ; Cholesterol/biosynthesis ; Host-Parasite Interactions/genetics ; },
abstract = {Cryptosporidium is a leading cause of diarrheal disease, yet little is known regarding the infection cell biology of this intracellular intestinal parasite. To this end, we implemented an arrayed genome-wide CRISPR-Cas9 knockout screen to microscopically analyze multiple phenotypic features of a Cryptosporidium infection following individual host gene ablation. We discovered parasite survival within the host epithelial cell hinges on squalene, an intermediate metabolite in the host cholesterol biosynthesis pathway. A buildup of squalene within intestinal epithelial cells creates a reducing environment, making more reduced glutathione available for parasite uptake. Remarkably, the Cryptosporidium parasite has lost the ability to synthesize glutathione and has become dependent on this host import. This dependency can be leveraged for treatment with the abandoned drug lapaquistat, an inhibitor of host squalene synthase that shifts the redox environment, blocking Cryptosporidium growth in vitro and in vivo.},
}
@article {pmid40685751,
year = {2025},
author = {Qin, W and Lin, SJ and Zhang, Y and Huang, K and Petree, C and Boyd, K and Varshney, P and Varshney, GK},
title = {Rationally Designed TadA-Derived Cytosine Editors Enable Context-Independent Zebrafish Genome Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {39},
pages = {e09800},
doi = {10.1002/advs.202509800},
pmid = {40685751},
issn = {2198-3844},
support = {R24 OD034438/OD/NIH HHS/United States ; R24OD034438/RI/ORIP NIH HHS/United States ; /NH/NIH HHS/United States ; R24OD034438/RI/ORIP NIH HHS/United States ; },
mesh = {Animals ; *Zebrafish/genetics ; *Gene Editing/methods ; *Cytosine/metabolism ; CRISPR-Cas Systems/genetics ; Genome/genetics ; },
abstract = {CRISPR base editors are crucial for precise genome manipulation. Existing APOBEC-based cytosine base editors (CBEs), while powerful, exhibit indels and sequence context limitations, and editing CC and GC motifs is challenging and inefficient. To address these challenges, existing tRNA adenine deaminase (TadA)-derived CBEs are evaluated in zebrafish, and a series of zTadCBE variants is developed that demonstrate high editing efficiency, minimized off-target effects, and an expanded targeting range compared to existing tools. The approach integrates beneficial mutations from TadA-based adenine base editors (ABEs) with SpRYCas9n-enhanced protospacer-adjacent motif (PAM) compatibility. The expanded window zTadCBE variants enable the targeting of cytosines at a broader range of nucleotide positions relative to the PAM sequence, further enhancing the versatility of this tool. Using zTadCBEs, four zebrafish disease models affecting the auditory, nervous, metabolic, and muscular systems are generated directly in the F0 generation-models that cannot be efficiently produced using earlier CBE tools. Together, zTadCBE variants provide a robust and flexible toolkit for efficient and precise C-to-T base editing in zebrafish, facilitating rapid in vivo functional assessment of genetic variants.},
}
@article {pmid40683408,
year = {2025},
author = {Zhou, J and Zhou, C and Jiang, G and Wang, Y and Pan, L and Jia, X and Tu, X and Li, W and Wang, C},
title = {Engineering an Escherichia coli with performance-enhanced switch utilizing CRISPR-Cas9 system as living quorum quencher for biofilm formation inhibition.},
journal = {Environmental research},
volume = {285},
number = {Pt 2},
pages = {122383},
doi = {10.1016/j.envres.2025.122383},
pmid = {40683408},
issn = {1096-0953},
mesh = {*Biofilms/growth &amp; development ; *Escherichia coli/genetics/physiology ; *Quorum Sensing/genetics ; *CRISPR-Cas Systems ; Genetic Engineering ; },
abstract = {Quorum quenching (QQ) of signal molecules plays a critical role in disrupting bacterial communication, thereby suppressing biofilm formation. However, the wild-type QQ bacteria lacks the regulatory capacity to modulate gene expression levels. In this study, the QQ gene aiiO and reporter gene GFP were chromosomally integrated into Escherichia coli BW25113 using the clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system. The performance-enhanced switch in the engineering bacteria (EB) allowed it to express aiiO weakly without the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) and express aiiO strongly with IPTG, and 1.00 mM IPTG induction enhanced EB's QQ activity by 2.34-fold. In activated sludge systems, the inoculation of EB reduced biofilm formation by 18.56 % versus controls after 168 h, with the performance-enhanced switch enhancing inhibition to 24.72 %. EB reduced biofilm thickness by 22.96 %, total microbial biomass by 57.68 %, and significantly decreased extracellular polymeric substances secretion and adhesion strength of the biofilm (maximum reductions: 29.88 % and 34.31 %, respectively) across all sampling points versus controls. 1.00 mM IPTG addition sustainedly intensified these biofilm-inhibitory effects by EB, demonstrating the genetic switch's persistent functionality under environmentally relevant conditions. Furthermore, the genetically modified strain exhibited minimal environmental impact according to standardized assessments. Therefore, this study successfully constructed an implementable strategy for engineering bacteria-mediated biofilm control, with demonstrated applicability in complex environmental systems.},
}
@article {pmid41097540,
year = {2025},
author = {Chen, C and Wu, T and Liu, J and Gao, J},
title = {Threat and Control of tet(X)-Mediated Tigecycline-Resistant Acinetobacter sp. Bacteria.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {19},
pages = {},
doi = {10.3390/foods14193374},
pmid = {41097540},
issn = {2304-8158},
support = {32402890//National Natural Science Foundation of China/ ; 2023M732993//China Postdoctoral Science Foundation/ ; },
abstract = {Tigecycline is regarded as one of the last-resort antibiotics against multidrug-resistant (MDR) Acinetobacter sp. bacteria. Recently, the tigecycline-resistant Acinetobacter sp. isolates mediated by tet(X) genes have emerged as a class of global pathogens for humans and food-producing animals. However, the genetic diversities and treatment options were not systematically discussed in the era of One Health. In this review, we provide a detailed illustration of the evolution route, distribution characteristics, horizontal transmission, and rapid detection of tet(X) genes in diverse Acinetobacter species. We also detail the application of chemical drugs, plant extracts, phages, antimicrobial peptides (AMPs), and CRISPR-Cas technologies for controlling tet(X)-positive Acinetobacter sp. pathogens. Despite excellent activities, the antibacterial spectrum and application safety need further evaluation and resolution. It is noted that deep learning is a promising approach to identify more potent antimicrobial compounds.},
}
@article {pmid41097079,
year = {2025},
author = {Wu, KC and Chang, YH and Chiang, RY and Ding, DC},
title = {CAP-LAMP2b-Modified Stem Cells' Extracellular Vesicles Hybrid with CRISPR-Cas9 Targeting ADAMTS4 to Reverse IL-1β-Induced Aggrecan Loss in Chondrocytes.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199812},
pmid = {41097079},
issn = {1422-0067},
support = {TCRD 113-065//Hualien Tzu Chi Hospital/ ; },
mesh = {*Extracellular Vesicles/metabolism ; Humans ; *Chondrocytes/metabolism/cytology ; *ADAMTS4 Protein/genetics/metabolism ; *CRISPR-Cas Systems ; *Mesenchymal Stem Cells/metabolism/cytology ; *Aggrecans/metabolism ; *Interleukin-1beta/metabolism ; Cell Differentiation ; Cells, Cultured ; Gene Editing ; },
abstract = {Extracellular vesicles (EVs) from mesenchymal stem cells hold therapeutic promise for inflammatory and degenerative diseases; however, limited delivery and targeting capabilities hinder their clinical use. In this study, we sought to enhance the anti-inflammatory and chondroprotective effects of EVs through CAP-LAMP2b (chondrocyte affinity peptide fused to an EV membrane protein) engineering and ADAMTS4 gene editing hybrid vesicle formation. Human umbilical cord MSCs (hUCMSCs) were characterized via morphology, immunophenotyping, and trilineage differentiation. EVs from control and CAP-LAMP2b-transfected hUCMSCs were fused with liposomes carrying CRISPR-Cas9 ADAMTS4 gRNA. DiI-labeled EV uptake was assessed via fluorescence imaging. CAP-LAMP2b was expressed in hUCMSCs and their EVs. EVs exhibited the expected size (~120 nm), morphology, and exosomal markers (CD9, CD63, CD81, HSP70). CAP-modified hybrid EVs significantly enhanced chondrocyte uptake compared to control EVs and liposomes. IL-1β increased ADAMTS4 expression, whereas CAP-LAMP2b-ADAMTS4 EVs, particularly clone SG3, reversed these effects by reducing ADAMTS4 and restoring aggrecan. Western blotting confirmed suppressed ADAMTS4 and elevated aggrecan protein. CAP-LAMP2b-ADAMTS4 EVs, therefore, showed superior uptake and therapeutic efficacy in inflamed chondrocytes, attenuating inflammatory gene expression and preserving matrix integrity. These results support engineered EVs as a promising cell-free approach for cartilage repair and osteoarthritis treatment.},
}
@article {pmid41097026,
year = {2025},
author = {Jeong, SK and Park, JR and Kim, EG and Kim, KM},
title = {Development of Resistance to Damping-Off in Rice, Oryza sativa L., Using CRISPR/Cas9.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199761},
pmid = {41097026},
issn = {1422-0067},
mesh = {*Oryza/genetics/microbiology/growth &amp; development ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Diseases/genetics/microbiology ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; Rhizoctonia/pathogenicity ; Plant Proteins/genetics ; },
abstract = {Damping-off disease hinders rice seedling growth and reduces yield. Current control methods, such as seed or soil sterilization, rely on chemicals that cause environmental pollution and promote pathogen resistance. As a sustainable alternative, we targeted the damping-off resistance-related gene OsDGTq1 using CRISPR/Cas9. Field experiments first verified OsDGTq1's significance in resistance. The CRISPR/Cas9 system, delivered via Agrobacterium-mediated transformation, was used to edit OsDGTq1 in rice cultivar Ilmi. Lesions from major damping-off pathogens, Rhizoctonia solani and Pythium graminicola, were observed on G0 plants. All 37 regenerated plants contained T-DNA insertions. Among them, edits generated by sgRNA1-1, sgRNA1-2, and sgRNA1-3 resulted in the insertion of two thymine bases as target mutations. Edited lines were assigned names and evaluated for agronomic traits, seed-setting rates, and pathogen responses. Several lines with edited target genes showed distinct disease responses and altered gene expression compared to Ilmi, likely due to CRISPR/Cas9-induced sequence changes. Further studies in subsequent generations are needed to confirm the stability of these edits and their association with resistance. These results confirm that genome editing of OsDGTq1 alters resistance to damping-off. The approach demonstrates that gene-editing technology can accelerate rice breeding, offering an environmentally friendly strategy to develop resistant varieties. Such varieties can reduce chemical inputs, prevent pollution, and minimize seedling loss, ultimately enhancing food self-sufficiency and stabilizing rice supply.},
}
@article {pmid41097019,
year = {2025},
author = {Kapitonova, MA and Shabalina, AV and Dedkov, VG and Dolgova, AS},
title = {CRISPR-Cas12a-Based Isothermal Detection of Mammarenavirus machupoense Virus: Optimization and Evaluation of Multiplex Capability.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199754},
pmid = {41097019},
issn = {1422-0067},
support = {Ensuring chemical and biological safety of the Russian Federation//state program/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Endodeoxyribonucleases/genetics ; *CRISPR-Associated Proteins/genetics ; *Molecular Diagnostic Techniques/methods ; *Arenaviridae/genetics/isolation &amp; purification ; Animals ; Bacterial Proteins ; },
abstract = {Bolivian hemorrhagic fever (BHF) is a zoonotic disease caused by Mammarenavirus machupoense (MACV) featuring severe neurological and hemorrhagic symptoms and a high mortality rate. BHF is usually diagnosed by serological tests or real-time polymerase chain reaction (RT-PCR); these methods are often inaccessible in endemic regions due to a lack of laboratory infrastructure, creating a demand for sensitive and rapid equipment-free alternatives. Here, we present an isothermal method for MACV nucleic acid detection based on the Cas12a-based DETECTR system combined with recombinase polymerase amplification (RPA) in a single tube: the RT-RPA/DETECTR assay. We demonstrate the possibility of using more than one primer set for the simultaneous detection of MACV genetic variants containing multiple point mutations. The method was optimized and tested using specially developed virus-like armored particles containing the target sequence. The multiplex RT-RPA/DETECTR method achieved a limit of detection of approximately 5 × 10[4] copies/ mL (80 aM) of armored particles. The method was validated using clinical samples spiked with virus-like particles. The assay proved to be selective and reliable in detecting certain nucleotide substitutions simultaneously.},
}
@article {pmid41096922,
year = {2025},
author = {Xu, J and Pan, M and Zhu, Y and Wang, P and Jiang, L and Xu, D and Wang, X and Chen, L and Guo, W and Yang, H and Cao, D},
title = {CRISPR/Cas9-Mediated Targeted Mutagenesis of GmAS1/2 Genes Alters Leaf Shape in Soybean.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199657},
pmid = {41096922},
issn = {1422-0067},
support = {YBXM2529//National Nanfan Research Institute of CAAS/ ; },
mesh = {*Glycine max/genetics/anatomy &amp; histology/growth &amp; development ; *CRISPR-Cas Systems ; *Plant Leaves/genetics/anatomy &amp; histology/growth &amp; development ; Gene Expression Regulation, Plant ; Phenotype ; *Plant Proteins/genetics/metabolism ; *Mutagenesis ; *Genes, Plant ; Gene Editing ; Transcription Factors/genetics ; Gene Expression Profiling ; Mutation ; },
abstract = {ASYMMETRIC LEAVES1 (AS1) and AS2 play essential roles in regulating leaf development in plants. However, their functional roles in soybean remain poorly understood. Here, we identified two members of the soybean AS1 gene family, GmAS1a and GmAS1c, which exhibit high expression levels in stem and leaf tissues. Using the CRISPR/Cas9 system, we targeted four GmAS1 and three GmAS2 genes, generating mutant lines with distinct leaf development phenotypes, including wrinkling (refers to fine lines and creases on the leaf surface, like aged skin texture), curling (describes the inward or outward rolling of leaf edges, deviating from the typical flat shape), and narrow. We found that functional redundancy exists among the four GmAS1 genes in soybean. GmAS1 and GmAS2 cooperatively regulate leaf curling, leaf crinkling phenotypes, and leaf width in soybean, with functional redundancy also observed between these two genes. Transcriptome sequencing analysis of w3 mutant (as1b as1c as1d as2a as2b as2c) identified 1801 differentially expressed genes (DEGs), including 192 transcription factors (TFs). Gene ontology enrichment analysis revealed significant enrichment of DEGs in pathways associated with plant hormone biosynthesis and signal transduction. A detailed examination of the DEGs showed several genes involved in the development of leaf lateral organs, such as KNOX (SHOOT MERISTEMLESS (STM), KNAT1, KNAT2, and KNAT6), LOB (LBD25, LBD30), and ARP5, were down-regulated in w3/WT (wild-type) comparison. CRISPR/Cas9-mediated targeted mutagenesis of the GmAS1/2 genes significantly impairs leaf development and polarity establishment in soybean, providing valuable germplasm resources and a theoretical framework for future studies on leaf morphogenesis.},
}
@article {pmid41096782,
year = {2025},
author = {Huang, C and Liu, M and Kok, J},
title = {Chromosomal and Plasmid-Based CRISPRi Platforms for Conditional Gene Silencing in Lactococcus lactis.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199516},
pmid = {41096782},
issn = {1422-0067},
support = {201706350298//China Scholarship Council/ ; 201505990303//China Scholarship Council/ ; },
mesh = {*Lactococcus lactis/genetics ; *Plasmids/genetics ; *CRISPR-Cas Systems ; *Gene Silencing ; Streptococcus pyogenes/genetics ; Bacterial Proteins/genetics ; Gene Expression Regulation, Bacterial ; *Chromosomes, Bacterial/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Promoter Regions, Genetic ; },
abstract = {Inducible CRISPR interference (CRISPRi) systems were established in Lactococcus lactis using both plasmid and chromosomal approaches. Expression of nuclease-deficient Cas9 (dCas9) from Streptococcus pyogenes was placed under the control of the nisin-inducible promoter PnisA, while sgRNAs were transcribed from the constitutive Pusp45 promoter. To monitor expression, dCas9 was fused with superfolder GFP. Plasmid-based constructs successfully repressed a luciferase reporter gene and silenced the gene of the major autolysin, AcmA, leading to the expected morphological phenotype. However, plasmid systems showed leaky expression, producing mutant phenotypes even without induction. Chromosomal integration of dCas9 reduced its expression level by approximately 20-fold compared with plasmid-based expression, thereby preventing leaky activity and ensuring tight regulation. This chromosome-based (cbCRISPRi) platform enabled controlled repression of the essential gene ybeY, which resulted in severe growth defects. Restoration of wild-type phenotypes was achieved by introducing a synonymous codon substitution in the sgRNA target region. Transcriptome analysis of ybeY-silenced cells revealed downregulation of ribosomal protein genes and widespread effects on membrane-associated proteins, ATP synthase subunits, and various transporters. These inducible CRISPRi platforms provide robust and tunable tools for functional genomics in L. lactis, particularly for studying essential genes that cannot be deleted.},
}
@article {pmid41096720,
year = {2025},
author = {Peng, H and Li, J and Sun, K and Tang, H and Huang, W and Li, X and Wang, S and Ding, K and Han, Z and Li, Z and Xu, L and Wang, K},
title = {Advances and Applications of Plant Base Editing Technologies.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199452},
pmid = {41096720},
issn = {1422-0067},
support = {32472182//National Natural Science Foundation of China/ ; 2024ZD0407704//Biological Breeding-National Science and Technology Major Project/ ; 2022BBF02039//Science and Technology Department of Ningxia in China/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; Genome, Plant ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Base editing represents a major breakthrough in the field of genome editing in recent years. By fusing deaminases with the CRISPR/Cas system, it enables precise single-base modifications of DNA. This review systematically summarizes the development of base editing technologies, including cytosine base editors (CBEs), adenine base editors (ABEs), and glycosylase base editors (GBEs), with a particular focus on their applications in crop improvement as well as future trends and prospects. We highlight advances in the creation of novel germplasm with enhanced stress resistance and desirable agronomic traits through base editing in rice, wheat, maize, potato, and other crops, particularly for improving herbicide resistance, disease resistance, and grain quality. Furthermore, we analyze factors that influence base editing efficiency, noting that challenges remain, such as PAM sequence constraints, limited base conversion types, off-target effects, narrow editing windows, and efficiency variation. Future efforts should aim to optimize deaminase activity, expand PAM compatibility, and develop versatile tools to facilitate the broad application of base editing in molecular breeding. This review provides a timely reference for researchers and breeders, offering theoretical guidance and practical insights into harnessing base editing for crop genetic improvement.},
}
@article {pmid41096693,
year = {2025},
author = {He, J and Shi, N and Yao, H and Li, J and Wang, Y and Zhang, J},
title = {Genome Editing in the Chicken: From PGC-Mediated Germline Transmission to Advanced Applications.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199426},
pmid = {41096693},
issn = {1422-0067},
support = {2023ZD04053//the Biological Breeding-National Science and Technology Major Project/ ; 2025YFHZ0042//the Natural Science Foundation of Sichuan Province/ ; SCU2025D003//the fundamental research funds for the central universitie/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Chickens/genetics ; *Germ Cells/metabolism ; CRISPR-Cas Systems ; },
abstract = {Avian genome editing has historically lagged behind mammalian research. This disparity is primarily due to a unique reproductive biology that precludes standard techniques like pronuclear injection. A pivotal breakthrough, however, came from the development of efficient in vitro culture systems for primordial germ cells (PGCs). This has established the chicken as a tractable and powerful model for genetic engineering. Our review chronicles the technological evolution this has enabled, from early untargeted methods to the precision of modern CRISPR-based systems. We then analyze the broad applications of these tools, which are now used to engineer disease resistance, enhance agricultural traits, and develop novel platforms such as surrogate hosts and oviduct bioreactors. Collectively, these advances have established PGC-based genome editing as a robust and versatile platform. Looking forward, emerging precision editors and the expansion of these techniques to other avian species are poised to drive the next wave of innovation in poultry science and biotechnology.},
}
@article {pmid41096689,
year = {2025},
author = {Haldrup, SB and McClements, ME and Cehajic-Kapetanovic, J and Corydon, TJ and MacLaren, RE},
title = {Gene Therapy Strategies for the Treatment of Bestrophinopathies.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199421},
pmid = {41096689},
issn = {1422-0067},
support = {NA//Aarhus University/ ; NA//Fight for Sight Denmark/ ; NA//Synoptik Fonden/ ; NA//Maskinfabrikant Jochum Jensen og hustru Mette Marie Jensen, f. Poulsens Mindelegat (fond)/ ; NA//A.P. M&#xf8;ller Foundation/ ; NA//Mette Warburgs Fond/ ; NA//APT Holding/ ; 00038189//VELUX Foundation/ ; 24OC0088426//Novo Nordisk Foundation/ ; NA//NIHR Oxford Biomedical Research Centre/ ; 304408/Z/23/Z//Wellcome Discovery Award/ ; },
mesh = {Humans ; *Genetic Therapy/methods ; *Bestrophins/genetics/metabolism ; *Retinal Diseases/therapy/genetics ; *Eye Diseases, Hereditary/therapy/genetics ; Gene Editing/methods ; Mutation ; Animals ; CRISPR-Cas Systems ; Chloride Channels/genetics ; },
abstract = {The BEST1 gene encodes a transmembrane protein in the retinal pigment epithelium (RPE) in the eye, that functions as a calcium-dependent chloride channel (CaCC). Pathogenic variants in BEST1 are the underlying cause for bestrophinopathies, a group of inherited retinal disorders that vary in their pattern of inheritance, clinical appearance, and underlying molecular disease mechanisms. Currently, there are no treatments available for any of the bestrophinopathies, and gene therapy represents an attractive strategy due to the accessibility of the eye and slow disease progression. While gene augmentation may be effective for a subset of bestrophinopathies, others require allele-specific silencing or correction of the disease-causing variant to reconstitute expression of the BEST1 protein. This review aims to give an overview of the clinical diversity of bestrophinopathies and proposes the molecular disease mechanism of the pathogenic BEST1 variant as an important parameter for the choice of treatment strategy. Furthermore, we discuss the potential of different mutation-specific and mutation-independent CRISPR/Cas9-based gene editing strategies as a future treatment approach for bestrophinopathies.},
}
@article {pmid41096677,
year = {2025},
author = {Șerban, M and Toader, C and Covache-Busuioc, RA},
title = {CRISPR and Artificial Intelligence in Neuroregeneration: Closed-Loop Strategies for Precision Medicine, Spinal Cord Repair, and Adaptive Neuro-Oncology.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199409},
pmid = {41096677},
issn = {1422-0067},
mesh = {Humans ; *Artificial Intelligence ; *Precision Medicine/methods ; Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Spinal Cord Regeneration ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Nerve Regeneration/genetics ; },
abstract = {Repairing the central nervous system (CNS) remains one of the most difficult obstacles to overcome in translational neurosciences. This is due to intrinsic growth inhibitors, extracellular matrix issues, the glial scar-form barrier, chronic neuroinflammation, and epigenetic silencing. The purpose of this review is to bring together findings from recent developments in genome editing and computational approaches, which center around the possible convergence of clustered regularly interspaced short palindromic repeats (CRISPR) platforms and artificial intelligence (AI), towards precision neuroregeneration. We wished to outline possible ways in which CRISPR-based systems, including but not limited to Cas9 and Cas12 nucleases, RNA-targeting Cas13, base and prime editors, and transcriptional regulators such as CRISPRa/i, can be applied to potentially reactivate axon-growth programs, alter inhibitory extracellular signaling, reprogram or lineage transform glia to functional neurons, and block oncogenic pathways in glioblastoma. In addition, we wanted to highlight how AI approaches, such as single-cell multi-omics, radiogenomic prediction, development of digital twins, and design of adaptive clinical trials, will increasingly be positioned to act as system-level architects that allow translation of complex datasets into predictive and actionable therapeutic approaches. We examine convergence consumers in spinal cord injury and adaptive neuro-oncology and discuss expanse consumers in ischemic stroke, Alzheimer's disease, Parkinson's disease, and rare neurogenetic syndromes. Finally, we discuss the ethical and regulatory landscape around beyond off-target editing and genomic stability of CRISPR, algorithmic bias, explainability, and equitable access to advanced neurotherapies. Our intent was not to provide a comprehensive inventory of possibilities but rather to provide a conceptual tool where CRISPR acts as a molecular manipulator and AI as a computational integrator, converging to create pathways towards precision neuroregeneration, personalized medicine, and adaptive neurotherapeutics that are ethically sound.},
}
@article {pmid41096563,
year = {2025},
author = {Simoni, S and Fambrini, M and Pugliesi, C and Rogo, U},
title = {Genome Editing by Grafting.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
doi = {10.3390/ijms26199294},
pmid = {41096563},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Genome, Plant ; *Plants, Genetically Modified/genetics ; },
abstract = {Grafting is the process of joining parts of two plants, allowing the exchange of molecules such as small RNAs (including microRNAs and small interfering RNAs), messenger RNAs, and proteins between the rootstock and the scion. Genome editing by grafting exploits RNAs, such as tRNA-like sequences (TLS motifs), to deliver the components (RNA) of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system from transgenic rootstock to wild-type scion. The complex Cas9 protein and sgRNA-TLS produced in the scion perform the desired modification without the integration of foreign DNA in the plant genome, resulting in heritable transgene-free genome editing. In this review, we examine the current state of the art of this innovation and how it helps address regulatory problems, improves crop recovery and selection, exceeds the usage of viral vectors, and may reduce potential off-target effects. We also discuss the promise of genome editing by grafting for plants recalcitrant to in vitro culture and for agamic-propagated species that must maintain heterozygosity for plant productivity, fruit quality, and adaptation. Furthermore, we explore the limitations of this technique, including variable efficiency, graft incompatibility among genotypes, and challenges in large-scale application, while highlighting its considerable potential for further improvement and future broader applications for crop breeding.},
}
@article {pmid41093884,
year = {2025},
author = {Moon, J and Zhang, J and Guan, X and Yang, R and Guo, C and Schalper, KT and Avery, L and Banach, D and LaSala, R and Warrier, R and Liu, C},
title = {CRISPR anti-tag-mediated room-temperature RNA detection using CRISPR/Cas13a.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9142},
pmid = {41093884},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Hepacivirus/genetics/isolation &amp; purification ; *RNA, Viral/genetics/blood/analysis ; Temperature ; *CRISPR-Associated Proteins/metabolism/genetics ; HIV Infections/virology/diagnosis/blood ; Hepatitis C/diagnosis/virology ; HIV/genetics/isolation &amp; purification ; HIV-1/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The CRISPR/Cas13a enzyme serves as a powerful tool for RNA detection due to its RNA-targeting capabilities. However, simple and highly sensitive detection using Cas13a faces challenges, such as the need for pre-amplification and elevated reaction temperatures. In this study, we investigate the allosteric regulation mechanism of Cas13a activation by target RNAs with various structures containing the CRISPR anti-tag sequence. We discover that the target RNA secondary structure and anti-tag sequences inhibit the trans-cleavage reaction of Cas13a. By designing and introducing a specific CRISPR anti-tag hairpin, we develop CRISPR Anti-tag Mediated Room-temperature RNA Detection (CARRD) using a single CRISPR/Cas13a enzyme. This method enables one-step cascade signal amplification for RNA detection without the need for pre-amplification. We apply the CARRD method to detect human immunodeficiency virus (HIV) and hepatitis C virus (HCV), achieving a detection sensitivity of 10 aM. Furthermore, we validate its clinical feasibility by detecting HIV clinical plasma samples, demonstrating a simple, affordable, and efficient approach for viral RNA detection. Due to its simplicity, sensitivity, and flexible reaction temperature, the CARRD method is expected to have broad applicability, paving the way for the development of field-deployable diagnostic tools.},
}
@article {pmid41093851,
year = {2025},
author = {Zhou, SK and Luo, JT and Chen, YF and Lu, ZD and Jian, QH and Jiang, SQ and Li, J and Zhang, XQ and Tan, XY and Yang, XZ and Xu, CF and Wang, J},
title = {Muscle-specific gene editing therapy via mammalian fusogen-directed virus-like particles.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9145},
pmid = {41093851},
issn = {2041-1723},
support = {32430059//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32471434//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32271442//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Muscle, Skeletal/metabolism ; Mice ; *Muscular Dystrophy, Duchenne/therapy/genetics ; CRISPR-Cas Systems ; Dystrophin/genetics/metabolism ; *Genetic Therapy/methods ; Humans ; Disease Models, Animal ; CRISPR-Associated Protein 9/genetics/metabolism ; Male ; Ribonucleoproteins/genetics/metabolism ; Exons ; Mice, Inbred C57BL ; },
abstract = {Muscle genetic defects can lead to impaired movement, respiratory failure, and other severe symptoms. The development of curative therapies is challenging due to the need for the delivery of gene-editing tools into skeletal muscle cells throughout the body. Here, we use muscular fusogens (Myomaker and Myomerger) to engineer muscle-specific virus-like particles (MuVLPs) for the systemic delivery of gene-editing tools. We demonstrate that MuVLPs can be loaded with diverse payloads, including EGFP, Cre and Cas9/sgRNA ribonucleoproteins (Cas9 RNPs), and can be delivered into skeletal muscle cells via targeted membrane fusion. Systemic administration of MuVLPs carrying Cas9 RNPs enables skeletal muscle-specific gene editing, which excised the exon containing a premature terminator codon mutation in a mouse model for Duchenne muscular dystrophy (DMD). This treatment restores dystrophin expression in various skeletal muscle tissues, including the diaphragm, quadriceps, tibialis anterior, gastrocnemius, and triceps. As a result, the treated mice exhibit a significantly increased capacity for exercise and endurance. This study established a platform for precise gene editing in skeletal muscle tissues.},
}
@article {pmid41093849,
year = {2025},
author = {Shang, M and Li, Y and Cao, Q and Ren, J and Zeng, Y and Wang, J and Gonzalez, RVL and Zhang, X},
title = {A motif preferred adenine base editor with minimal bystander and off-targets editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9153},
pmid = {41093849},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; Animals ; Humans ; Mice ; *Adenine/metabolism/chemistry ; *Nucleotide Motifs/genetics ; HEK293 Cells ; CRISPR-Cas Systems ; },
abstract = {47% of hereditable diseases are caused by single C•G-to-T•A base conversions, which means efficient A-to-G base editing tools (ABEs) have great potential for the treatment of these diseases. However, the existing efficient ABEs, while catalyzing targeted A-to-G conversion, cause high A or C bystander editing and off-target events, which poses safety concerns for their clinical applications. To overcome this shortcoming, we have developed ABE8e-YA (ABE8e with TadA-8e A48E) for efficient and accurate editing of As in YA motifs with YAY > YAR (Y = T or C, R = A or G) hierarchy through structure-oriented rational design. Compared with ABE3.1, which is currently the only ABE version with a YAC motif preference, ABE8e-YA exhibits an average A-to-G editing efficiency improvement of an up to 3.1-fold increase in the indicated YA motif while maintaining reduced bystander C editing and minimized DNA or RNA off-targets. Additionally, we demonstrate that ABE8e-YA efficiently and precisely corrects pathogenic mutations in human cells, suggesting its high suitability for addressing 9.3% of pathogenic point mutations, higher than that of ABE8e and ABE9. Moreover, by using ABE8e-YA, we efficiently and precisely generate hypocholesterolemia and tail-loss mouse models mimicking human-associated disease, as well as performed in vivo mouse proprotein convertase subtilisin/kexin type 9 (Pcsk9) base editing for hypercholesterolemia gene therapy. Together these data indicate its great potential in broad applications for disease modeling and gene therapy.},
}
@article {pmid41093834,
year = {2025},
author = {Shibue, K and Kahraman, S and Castillo-Quan, JI and De Jesus, DF and Hu, J and Morita, H and Blackwell, TK and Yi, P and Kulkarni, RN},
title = {Genome-wide CRISPR Screen Identifies Sec31A as a Key Regulator of Alpha Cell Survival.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9159},
pmid = {41093834},
issn = {2041-1723},
support = {DK067536//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R35GM122610//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01AG054215//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; JP22K16404//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
mesh = {Animals ; Humans ; Mice ; Caenorhabditis elegans/metabolism/genetics ; *Vesicular Transport Proteins/genetics/metabolism ; *Glucagon-Secreting Cells/metabolism/cytology ; Cell Survival/genetics ; Endoplasmic Reticulum Stress/genetics ; CRISPR-Cas Systems ; Receptor, Insulin/metabolism ; Endoplasmic Reticulum/metabolism ; Signal Transduction ; Insulin/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Insulin-Secreting Cells/metabolism ; Glucagon/metabolism ; },
abstract = {Glucagon, secreted by pancreatic alpha cells, is essential for maintaining normal blood glucose levels. In type 1 and advanced type 2 diabetes, alpha cells often fail to respond to low glucose, yet the mechanisms underlying their stress resistance remain unclear. To investigate this, we performed a genome-wide CRISPR screen and identify Sec31A, a gene involved in transporting proteins from the endoplasmic reticulum (ER), as a key regulator of alpha cell survival under stress. We show that loss of Sec31A enhances survival in stressed mouse alpha cells and in C. elegans. In human islets, SEC31A expression increases in alpha cells under inflammatory stress, and this upregulation is reversed by reducing ER stress. Functional studies in lab-grown human islet clusters reveal distinct responses in alpha versus beta cells following Sec31A suppression. We also find that Sec31A interacts with the insulin receptor, suggesting a link between stress adaptation and insulin signaling in alpha cells.},
}
@article {pmid41093525,
year = {2025},
author = {Wei, Z and Luo, H and Huang, D and Wei, P and Zhang, J and Wei, J and Tang, Q and Huang, L and Zhang, K and Liao, X},
title = {Structure-specific electrochemiluminescent biosensor for FEN1 detection via dumbbell probe-mediated transcription and CRISPR/Cas13a-induced G-quadruplexes cleavage.},
journal = {Analytica chimica acta},
volume = {1377},
number = {},
pages = {344662},
doi = {10.1016/j.aca.2025.344662},
pmid = {41093525},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *G-Quadruplexes ; *Flap Endonucleases/analysis/blood/metabolism ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Luminescent Measurements/methods ; *DNA Probes/chemistry/genetics ; Limit of Detection ; Transcription, Genetic ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {Flap endonuclease 1 (FEN1) is crucial for DNA replication, repair, and telomere maintenance. Its dysregulation is linked to various cancers and diseases. Accurate detection of FEN1 is essential for early diagnosis and therapeutic monitoring. Thus, a novel electrochemiluminescent (ECL) biosensor has been developed for the structure-specific and detection of FEN1. The strategy integrates dumbbell DNA probe-mediated transcription and CRISPR/Cas13a-induced trans-cleavage of RNA G-quadruplexes. In the presence of FEN1, the 5'-flap structure of the probe was selectively cleaved and subsequently ligated by T4 DNA ligase to form a closed circular template. This enabled T7 RNA polymerase to transcribe crRNA-encoded RNA strands, which activated Cas13a to cleave surface-tethered G-quadruplexes/hemin complexes on a Ru(II)/Ti3C2/AuNPs-modified electrode, thereby restoring the quenched ECL signal. The biosensor exhibited an ultralow detection limit of 4.82 × 10[-9] U μL[-1] and a wide dynamic range (1 × 10[-8] to 1 × 10[-5] U μL[-1]), along with excellent specificity and stability. Successful application in human serum validated its reliability for complex biological samples. This work presents a powerful platform for sensitive FEN1 monitoring, holding potential for clinical diagnostics and enzymatic analysis.},
}
@article {pmid41093508,
year = {2025},
author = {Wei, Z and Huang, D and Luo, H and Zhang, J and Wei, J and Tang, Q and Huang, L and Zhang, K and Liao, X},
title = {A multi-level signal conversion architecture for enzyme sensing: Integrating MXene nanoplatforms with CRISPR-driven electrochemiluminescence.},
journal = {Analytica chimica acta},
volume = {1377},
number = {},
pages = {344636},
doi = {10.1016/j.aca.2025.344636},
pmid = {41093508},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; *CRISPR-Cas Systems ; Humans ; *Flap Endonucleases/analysis/metabolism ; Nanocomposites/chemistry ; Gold/chemistry ; Limit of Detection ; DNA/chemistry ; Nitrites ; Titanium ; Transition Elements ; },
abstract = {Precise and ultrasensitive detection of flap endonuclease 1 (FEN1), a key DNA repair enzyme implicated in cancer diagnostics, remains challenging due to its subtle structural cleavage activity. Herein, we present a cascade-amplified electrochemiluminescence (ECL) biosensor based on a Ti3C2 MXene-supported Ru (bpy)3[2+]/Au nanocomposite integrated with a CRISPR-Cas13a system and DNA walker circuitry. Upon specific recognition and cleavage of a 5'-flap substrate by FEN1, a nicked DNA product is circularized and transcribed via T7 RNA polymerase, yielding RNA activators that trigger Cas13a-mediated collateral cleavage. This event releases a blocked DNA walker, which reorganizes Fc-labeled DNA on the electrode surface and restores the ECL signal suppressed by resonance energy transfer. The system achieves a detection limit as low as 1.48 fU/mL and exhibits a dynamic range spanning five orders of magnitude. Compared to fluorescence-based CRISPR detection systems, the ECL-based platform offers low background, high signal-to-noise ratios, and operational simplicity using standard electrochemical instrumentation, supporting practical deployment in clinical diagnostics. Furthermore, the platform demonstrates high selectivity against other nucleases and proteins, along with excellent performance in spiked human serum samples. This work presents a robust and modular strategy for accurate enzyme activity profiling with promising applications in early-stage disease diagnostics.},
}
@article {pmid41092254,
year = {2025},
author = {Yin, K and Tsai, CJ},
title = {Turbo-charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70527},
pmid = {41092254},
issn = {1365-313X},
support = {//Georgia Research Alliance/ ; DE-SC0023166//U.S. Department of Energy/ ; DE-SC0023338//U.S. Department of Energy/ ; ERKP886//U.S. Department of Energy/ ; },
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; *Multifactorial Inheritance/genetics ; Plants, Genetically Modified/genetics ; *Genetic Engineering/methods ; },
abstract = {Multiplex CRISPR editing has emerged as a transformative platform for plant genome engineering, enabling the simultaneous targeting of multiple genes, regulatory elements, or chromosomal regions. This approach is effective for dissecting gene family functions, addressing genetic redundancy, engineering polygenic traits, and accelerating trait stacking and de novo domestication. Its applications now extend beyond standard gene knockouts to include epigenetic and transcriptional regulation, chromosomal engineering, and transgene-free editing. These capabilities are advancing crop improvement not only in annual species but also in more complex systems such as polyploids, undomesticated wild relatives, and species with long generation times. At the same time, multiplex editing presents technical challenges, including complex construct design and the need for robust, scalable mutation detection. We discuss current toolkits and recent innovations in vector architecture, such as promoter and scaffold engineering, that streamline workflows and enhance editing efficiency. High-throughput sequencing technologies, including long-read platforms, are improving the resolution of complex editing outcomes such as structural rearrangements-often missed by standard genotyping-when targeting repetitive or tandemly spaced loci. To fully realize the potential of multiplex genome engineering, there is growing demand for user-friendly, synthetic biology-compatible, and scalable computational workflows for gRNA design, construct assembly, and mutation analysis. Experimentally validated inducible or tissue-specific promoters are also highly desirable for achieving spatiotemporal control. As these tools continue to evolve, multiplex CRISPR editing is poised to become a foundational technology of next-generation crop improvement to address challenges in agriculture, sustainability, and climate resilience.},
}
@article {pmid41091748,
year = {2025},
author = {Yew, WN and Dean, CJ and Chan, DKH},
title = {STAG2 mutations in the normal colon induce upregulation of oncogenic pathways in neighbouring wildtype cells.},
journal = {PloS one},
volume = {20},
number = {10},
pages = {pone.0332499.exml},
pmid = {41091748},
issn = {1932-6203},
mesh = {Humans ; Organoids/metabolism ; *Colon/metabolism/pathology/cytology ; *Mutation ; Up-Regulation ; Signal Transduction/genetics ; CRISPR-Cas Systems ; Coculture Techniques ; Gene Editing ; Proto-Oncogene Proteins p21(ras)/metabolism/genetics ; *Carcinogenesis/genetics ; *Colorectal Neoplasms/genetics/pathology ; Tumor Necrosis Factor-alpha/metabolism ; },
abstract = {While driver mutations in the normal colon have been described, characterizing the role and function of these driver mutations in relation to colorectal oncogenesis remains incomplete. Here, we investigated the role of STAG2 mutants in the normal colon using patient-derived wildtype organoids. Using CRISPR-Cas9 gene editing, we generated STAG2 mutants, and co-cultured these mutants with wildtype organoids, mimicking the presence of such STAG2 mutants in the normal colon. We sought to determine the transcriptional impact of co-culture using scRNAseq. Surprisingly, we uncovered a possible cell-cell interaction between STAG2 mutants and wildtype organoids, in which wildtype organoids in co-culture with STAG2 mutants upregulated known oncogenic pathways. This included the upregulation of TNFα-signaling, as well as KRAS-signaling in wildtype organoids. These results suggested that STAG2 mutant cells exert a pro-oncogenic effect in a cell interactive manner, instead of via a cell autonomous approach. In conclusion, our findings demonstrate a novel mechanism of colorectal oncogenesis which can support further investigation.},
}
@article {pmid41091245,
year = {2025},
author = {Yang, S and Jiao, X and Liu, J and Liu, Y and Wang, M and Li, S and Qiao, J},
title = {CRISPR-Cas opens a new era of antimicrobial therapy as a powerful gene editing tool.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {10},
pages = {388},
pmid = {41091245},
issn = {1573-0972},
support = {X2024739//College Students' Innovation and Entrepreneurship Training Program of Shandong Second Medical University/ ; ZR2020MH305//Natural Science Foundation of Shandong Province, China/ ; },
}
@article {pmid41090767,
year = {2025},
author = {Slattery, JR and Naung, NY and Kalinna, BH and Pal, M},
title = {CRISPR-Powered Liquid Biopsies in Cancer Diagnostics.},
journal = {Cells},
volume = {14},
number = {19},
pages = {},
pmid = {41090767},
issn = {2073-4409},
mesh = {Humans ; Liquid Biopsy/methods ; *Neoplasms/diagnosis/genetics ; Biomarkers, Tumor/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; },
abstract = {Liquid biopsies promise major advantages for cancer screening and diagnosis. By detecting biomarkers in peripheral blood samples, liquid biopsies reduce the need for invasive techniques and provide important genetic information integral to the emerging molecular classification of cancers. Unfortunately, the concentrations of most biomarkers, particularly circulating tumour nucleic acids, are vanishingly small-beyond the sensitivity and specificity of most assays. Clustered Regularly Interspaced Short Palindromic Repeats diagnostics (herein labelled 'CRISPR-Dx') use gene editing tools to detect, rather than modify, nucleic acids with extremely high specificity. These tools are commonly combined with isothermal nucleic acid amplification to also achieve sensitivities comparable to high-performance laboratory-based techniques, such as digital PCR. CRISPR assays, however, are inherently well suited to adaptation for point-of-care (POC) use, and unlike antigen-based POC assays, are significantly easier and faster to develop. In this review, we summarise current CRISPR-Dx platforms and their analytical potential for cancer biomarker discovery, with an emphasis on enhancing early diagnosis, disease monitoring, point-of-care testing, and supporting cancer therapy.},
}
@article {pmid41090360,
year = {2025},
author = {Wei, W and Zhu, W and Silver, S and Armstrong, AM and Robbins, FS and Rameshbabu, AP and Walz, K and Quan, Y and Du, W and Kim, Y and Indzhykulian, AA and Shu, Y and Liu, XZ and Chen, ZY},
title = {Single-dose genome editing therapy rescues auditory and vestibular functions in adult mice with DFNA41 deafness.},
journal = {The Journal of clinical investigation},
volume = {135},
number = {20},
pages = {},
pmid = {41090360},
issn = {1558-8238},
mesh = {Animals ; *Gene Editing ; Mice ; *Genetic Therapy ; Humans ; Dependovirus/genetics ; *Deafness/therapy/genetics ; Disease Models, Animal ; *Vestibule, Labyrinth/physiopathology ; Mutation, Missense ; CRISPR-Cas Systems ; },
abstract = {Genome editing has the potential to treat genetic hearing loss. However, current editing therapies for genetic hearing loss have shown efficacy only in hearing rescue. In this study, we evaluated a rescue strategy using adeno-associated virus (AAV) type 2-mediated delivery of Staphylococcus aureus Cas9-sgRNA in the mature inner ear of the P2rx2V61L/+ mouse model of autosomal dominant deafness-41 (DFNA41), a dominant, delayed-onset, and progressive hearing loss in humans. We demonstrate that local injection in adult mice results in efficient and specific editing that abolishes the mutation without notable off-target effects or AAV genome integration. Editing effectively restores long-term auditory and vestibular function. Editing further protects P2rx2V61L/+ mice from hypersensitivity to noise-induced hearing loss, a phenotype also observed in patients with DFNA41. Intervention in mice at a juvenile stage broadens the frequency range rescued, highlighting the importance of early intervention. An effective and specific gRNA for the human P2RX2 V60L mutation has been identified. Our study establishes the feasibility of editing to treat DFNA41 caused by P2RX2 V60L mutation in humans and opens an avenue for using editing to rescue hearing and vestibular function while mitigating noise-induced hearing loss.},
}
@article {pmid40921807,
year = {2025},
author = {Zhang, Y and Zhao, Z and Liu, M and Yang, J and Yang, C and Su, N and Sun, J and Fang, Y and Wang, Y and Li, X and Chen, W and Wu, J and Bai, J},
title = {Asymmetric volume-mediated buffer control overcomes sensitivity limits in one-pot RAA-CRISPR/Cas12a visual detection.},
journal = {Analytical and bioanalytical chemistry},
volume = {417},
number = {26},
pages = {5971-5981},
pmid = {40921807},
issn = {1618-2650},
mesh = {*CRISPR-Cas Systems ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; Buffers ; *Recombinases/metabolism/chemistry ; Drug Resistance, Bacterial/genetics ; Colistin/pharmacology ; Animals ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Rapid, low-cost, and visual nucleic acid detection methods are highly attractive for curbing colistin resistance spread through the food chain. CRISPR/Cas12a combined with recombinase-aided amplification (RAA) offers a one-pot, aerosol-free approach for visual detection. However, traditional one-pot systems often run Cas12a trans-cleavage in a buffer suitable for RAA, thus limiting Cas12a cleavage efficiency. This study proposes an asymmetric volume-optimized RAA-CRISPR/Cas12a assay for ultrasensitive visual detection of mobile colistin resistance gene mcr-1. Unlike conventional one-pot systems constrained by buffer incompatibility, our design spatially segregates a minimal-volume RAA-MIX (lid) from a CRISPR-dominant buffer microenvironment (tube bottom). This architecture leverages RAA's exponential amplification power to ensure sufficient product yield from minimal reaction volumes, while enabling subsequent enhancement of Cas12a trans-cleavage through automatic buffer assimilation upon mixing. The results were able to be visually observed under UV light, achieving 63.1% cost reduction compared to standard one-pot methods. The sensitivity of the proposed method for the mcr-1 gene was 2.5 copies/reaction, with anti-interference against other plasmids or bacteria. This method was applied to the detection of mcr-1 in animal-derived foods, showing satisfactory practical performance. By fundamentally reengineering buffer microenvironments through volume asymmetry, this work provides a general strategy for one-pot molecular diagnostics, achieving dual optimization of amplification and cleavage without trade-offs.},
}
@article {pmid40914368,
year = {2025},
author = {Liu, X and Fu, Y and Li, M and Xiong, S and Huang, L and Zhang, S and Zhang, W and Liang, X and Wang, W and Tang, K and Shen, Q},
title = {Biofortification of tomatoes with beta-carotene through targeted gene editing.},
journal = {International journal of biological macromolecules},
volume = {327},
number = {Pt 2},
pages = {147396},
doi = {10.1016/j.ijbiomac.2025.147396},
pmid = {40914368},
issn = {1879-0003},
mesh = {*Solanum lycopersicum/genetics/metabolism/chemistry ; *beta Carotene/biosynthesis/genetics/metabolism ; *Biofortification/methods ; *Gene Editing/methods ; Fruit/genetics/chemistry ; Lycopene/metabolism ; Nutritive Value ; Carotenoids/metabolism ; CRISPR-Cas Systems ; },
abstract = {Vitamin A deficiency is one of the most severe micronutrient-related health issues worldwide. Tomatoes, a widely cultivated crop for their adaptability, nutritional value, and lycopene content (a beta-carotene precursor), are ideal candidates for biofortification. In this study, CRISPR-mediated knockout mutants (cr-SlLCYe and cr-SlBCH) were generated to enhance the precursor supply to the β-carotene biosynthetic pathway and reduce its degradation. Carotenoids profiling showed that β-carotene levels in the mutants were 1.7 to 2.5-fold higher than in the wild-type, whereas lycopene levels remained unaltered without altering lycopene content. To evaluate potential trade-offs, the characteristics of the mutant fruits were comprehensively assessed, including appearance quality (color, firmness), nutritional quality (sugars, organic acids, vitamin C), and postharvest traits (shelf life, resistance to Botrytis cinerea). These results provide a new strategy for elevating β-carotene without compromising fruit quality and offer new insights into combating vitamin A deficiency through targeted tomato breeding programs.},
}
@article {pmid40830415,
year = {2025},
author = {Bi, J and Mo, W and Liu, M and Song, Y and Xiao, Q and Fan, S and Wang, W and Shi, T and Zheng, Y and Lian, J and Liu, R and Chen, B and Huang, X and Li, P and Zhao, Z and Shi, J and Zhang, L and Su, G and Zhang, N and Lu, W},
title = {Systematic decoding of functional enhancer connectomes and risk variants in human glioma.},
journal = {Nature cell biology},
volume = {27},
number = {10},
pages = {1838-1847},
pmid = {40830415},
issn = {1476-4679},
support = {32130018//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Glioma/genetics/pathology/metabolism ; *Enhancer Elements, Genetic/genetics ; *Polymorphism, Single Nucleotide/genetics ; *Brain Neoplasms/genetics/pathology/metabolism ; Genetic Predisposition to Disease ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Myeloid Ecotropic Viral Integration Site 1 Protein/genetics/metabolism ; *Connectome/methods ; CRISPR-Cas Systems ; Disease Progression ; Risk Factors ; },
abstract = {Genetic and epigenetic variations contribute to the progression of glioma, but the mechanisms underlying these effects, particularly for enhancer-associated genetic variations in non-coding regions, still remain unclear. Here we performed high-throughput CRISPR interference screening to identify pro-tumour enhancers in glioma cells. By integrating genome-wide H3K27ac HiChIP data, we identified the target genes of these pro-tumour enhancers and revealed the essential role of enhancer connectomes in promoting glioma progression. Through systematic analysis of enhancers carrying glioma risk-associated single-nucleotide polymorphisms (SNPs), we found that these SNPs can promote glioma progression through the enhancer connectome. Using CRISPR-Cas9-mediated enhancer interference and SNP editing, we demonstrated that glioma-specific enhancer carrying the risk SNP rs2297440 regulates SOX18 expression by specifically recruiting transcription factor MEIS1 binding, thereby contributing to glioma progression. Our study sheds light on the molecular mechanisms underlying glioma susceptibility and provides potential therapeutic targets to treat glioma.},
}
@article {pmid40646310,
year = {2025},
author = {Rodschinka, G and Forcelloni, S and Kühner, FM and Wani, S and Riemenschneider, H and Edbauer, D and Behrens, A and Nedialkova, DD},
title = {Comparative CRISPRi screens reveal a human stem cell dependence on mRNA translation-coupled quality control.},
journal = {Nature structural &amp; molecular biology},
volume = {32},
number = {10},
pages = {1932-1946},
pmid = {40646310},
issn = {1545-9985},
mesh = {Humans ; *RNA, Messenger/genetics/metabolism ; *Protein Biosynthesis ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; Ribosomes/metabolism ; Cell Differentiation ; Myocytes, Cardiac/metabolism/cytology ; },
abstract = {The translation of mRNA into proteins in multicellular organisms needs to be carefully tuned to changing proteome demands in development and differentiation, while defects in translation often have a disproportionate impact in distinct cell types. Here we used inducible CRISPR interference screens to compare the essentiality of genes with functions in mRNA translation in human induced pluripotent stem cells (hiPS cells) and hiPS cell-derived neural and cardiac cells. We find that core components of the mRNA translation machinery are broadly essential but the consequences of perturbing translation-coupled quality control factors are cell type dependent. Human stem cells critically depend on pathways that detect and rescue slow or stalled ribosomes and on the E3 ligase ZNF598 to resolve a distinct type of ribosome collision at translation start sites on endogenous mRNAs with highly efficient initiation. Our findings underscore the importance of cell identity for deciphering the molecular mechanisms of translational control in metazoans.},
}
@article {pmid41090337,
year = {2025},
author = {Cherdantsev, AI and Kulagin, KA and Polyakova, AN and Karpov, VL and Sosnovtseva, AO and Karpov, DS},
title = {[DNA Double-Strand Break Repair System by a Mechanism of Non-Homologous End Joining Provides Resistance to DNA-Damaging and Oxidizing Stresses in the Yeast Debaryomyces hansenii].},
journal = {Molekuliarnaia biologiia},
volume = {59},
number = {4},
pages = {616-628},
doi = {10.31857/S0026898425040083},
pmid = {41090337},
issn = {0026-8984},
mesh = {*DNA End-Joining Repair ; *DNA Breaks, Double-Stranded ; *Oxidative Stress/genetics ; CRISPR-Cas Systems ; *Debaryomyces/genetics/metabolism ; *Fungal Proteins/genetics/metabolism ; Osmotic Pressure ; },
abstract = {The unconventional halotolerant yeast Debaryomyces hansenii is of great importance in biotechnology and the food industry, and in basic research it serves as a model for studying the molecular mechanisms of resistance to increased salinity and osmotic stress. We have previously established an efficient method for editing the D. hansenii genome using the CRISPR/Cas9 system. In turn, this has stimulated further investigation of the structure and physiological role of DNA double-strand break repair pathways in D. hansenii. The aim of the present work was to evaluate the involvement of key components of the DNA double-stranded break repair system by the non-homologous end joining (NHEJ) mechanism in the resistance of D. hansenii to DNA-damaging compounds and compounds that induce oxidative, high salinity, and osmotic stress. Using the CRISPR/Cas9 system, mutant strains with knockout of the DEHA2F10208g (DhKU70), DEHA2B01584g (DhKU80) , and DEHA2G04224g (DhLIG4) genes encoding key components of NHEJ were obtained. It was found that mutant strains, unlike the wild-type strain, are sensitive to chemical compounds that damage DNA, as well as to compounds that cause oxidative stress. Osmotic and high salinity stresses and vanillin do not cause significant changes in the rate of colony formation of mutant strains. Unexpectedly, mutant strains exhibit increased resistance to caffeine compared to the wild-type strain. The data indicate that the NHEJ systems of D. hansenii play a significant role in the response to DNA-damaging and oxidative types of stress. The importance of the NHEJ system in the processes of maintaining yeast cell homeostasis should be taken into account when creating strains producing valuable substances.},
}
@article {pmid41090155,
year = {2025},
author = {Li, JM and Huang, J and Liao, Y and Hu, T and Wang, CL and Zhang, WZ and Huang, CW},
title = {Gene and RNA Editing: Revolutionary Approaches to Treating Diseases.},
journal = {MedComm},
volume = {6},
number = {10},
pages = {e70389},
pmid = {41090155},
issn = {2688-2663},
abstract = {Gene editing and RNA editing technologies are advancing modern medicine by enabling precise manipulation of genetic information at the DNA and RNA levels, respectively. The third-generation gene editing tools, particularly Clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) system, have transformed genetic disease treatment with high efficiency, precision, and cost effectiveness, while RNA editing, via adenosine deaminase acting on RNA (ADAR) enzymes and CRISPR-Cas13, offers reversible regulation to avoid genomic integration risks. Despite advancements, challenges persist in delivery efficiency, tissue specificity, and long-term safety, limiting their clinical translation. This review systematically discusses the molecular mechanisms and technological evolution of these tools, focusing on their promising applications in treating nervous system disorders (e.g., Alzheimer's, Parkinson's), immune diseases (e.g., severe combined immunodeficiency, lupus), and cancers. It compares their technical attributes, analyzes ethical and regulatory issues, and highlights synergies between the two technologies. By bridging basic research and clinical translation, this review provides critical insights for advancing precision medicine, reshaping disease diagnosis, prevention, and treatment paradigms.},
}
@article {pmid41088816,
year = {2025},
author = {Mo, T and Ren, HY and Zhang, XX and Lu, YW and Teng, ZQ and Zhang, X and Dai, LP and Hou, L and Zhao, N and He, J and Qin, T},
title = {Phenotypic Function of Legionella pneumophila Type I-F CRISPR-Cas.},
journal = {Biomedical and environmental sciences : BES},
volume = {38},
number = {9},
pages = {1105-1119},
doi = {10.3967/bes2025.107},
pmid = {41088816},
issn = {2214-0190},
mesh = {*Legionella pneumophila/genetics/physiology/pathogenicity ; *CRISPR-Cas Systems ; Biofilms/growth &amp; development ; Phenotype ; Bacterial Proteins/genetics/metabolism ; Gene Deletion ; },
abstract = {OBJECTIVE: CRISPR-Cas protects bacteria from exogenous DNA invasion and is associated with bacterial biofilm formation and pathogenicity.<br><br>METHODS: We analyzed the type I-F CRISPR-Cas system of Legionella pneumophila WX48, including Cas1, Cas2-Cas3, Csy1, Csy2, Csy3, and Cas6f, along with downstream CRISPR arrays. We explored the effects of the CRISPR-Cas system on the in vitro growth, biofilm-forming ability, and pathogenicity of L. pneumophila through constructing gene deletion mutants.<br><br>RESULTS: The type I-F CRISPR-Cas system did not affect the in vitro growth of wild-type or mutant strains. The biofilm formation and intracellular proliferation of the mutant strains were weaker than those of the wild type owing to the regulation of type IV pili and Dot/Icm type IV secretion systems. In particular, Cas6f deletion strongly inhibited these processes.<br><br>CONCLUSION: The type I-F CRISPR-Cas system may reduce biofilm formation and intracellular proliferation in L. pneumophila.},
}
@article {pmid41086252,
year = {2025},
author = {Libri, AB and Wang, J and Marton, T and Yu, W and Dossin, F and Balmus, G and Reina-San-Martin, B and Frock, R and Lescale, C and Deriano, L},
title = {Senataxin promotes recombination fidelity during antigen receptor gene diversification.},
journal = {Science signaling},
volume = {18},
number = {908},
pages = {eadv8801},
doi = {10.1126/scisignal.adv8801},
pmid = {41086252},
issn = {1937-9145},
mesh = {Animals ; *V(D)J Recombination/genetics ; Mice ; DNA End-Joining Repair ; *RNA Helicases/genetics/metabolism ; Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; DNA Breaks, Double-Stranded ; Multifunctional Enzymes ; CRISPR-Cas Systems ; Mice, Knockout ; *DNA Helicases/genetics/metabolism ; Humans ; *Receptors, Antigen, T-Cell/genetics ; Precursor Cells, B-Lymphoid/metabolism/immunology ; },
abstract = {Antigen receptor diversity depends on the assembly of variable (V), diverse (D), and joining (J) exons in genes encoding immunoglobulins (Igs) and T cell receptors (TCRs). During V(D)J recombination, DNA double-strand breaks (DSBs) introduced by the RAG1/2 nuclease complex are repaired by the process of nonhomologous end-joining (NHEJ). We hypothesized that functional redundancies between NHEJ and the chromatin DSB response, which depends on the kinase ATM, potentially masked the activity of additional factors that regulate V(D)J recombination. We performed targeted CRISPR-Cas9 knockout screens for genes implicated in V(D)J recombination in pro-B cells that were either untreated or treated with an ATM inhibitor. We found that loss of the RNA/DNA helicase senataxin (SETX) impaired V(D)J recombination and led to the formation of aberrant hybrid joints between coding ends and signal ends, both in vitro and in mice. The loss of SETX in a background deficient in the NHEJ factor XLF or in which ATM was inhibited led to substantial impairment of V(D)J recombination and to the presence of unsealed coding ends. SETX limited aberrant activation-induced cytidine deaminase (AID)-induced DNA end-joining between Igh-containing alleles during the process of class-switch recombination. Together, our findings reveal a previously uncharacterized role for SETX in promoting recombination fidelity during antigen receptor gene diversification.},
}
@article {pmid41085803,
year = {2025},
author = {de Souza, HCA and Panzenhagen, P and Portes, AB and Dos Santos, AMP and Fidelis, J and Junior, CAC},
title = {CRISPR-Cas systems in combating antimicrobial resistance: which system to choose? A systematic review.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {10},
pages = {381},
pmid = {41085803},
issn = {1573-0972},
support = {FinanceCode001//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; 313119/2020-1//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; E26/202.227/2018//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Bacteria/genetics/drug effects ; *Drug Resistance, Bacterial/genetics ; Humans ; Anti-Bacterial Agents/pharmacology ; },
abstract = {Antimicrobial resistance (AMR) poses a growing threat to global public health, progressively compromising the efficacy of available antimicrobials. Technologies based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) have emerged as promising tools for controlling resistant pathogens, offering high specificity and versatility. However, a comprehensive and systematic synthesis of CRISPR strategies applied to AMR remains limited. From February 12, 2025, we conducted a systematic review of the PubMed, Embase, and Scopus databases, using the following search strategy: Population (resistant bacteria or plasmid-mediated resistance), Intervention (CRISPR, including variants such as CRISPR-Cas9, Cas3, Cas12, Cas13, and CRISPR interference [CRISPRi]), and Outcomes (bacterial resensitization or plasmid curing). The CRISPR-Cas9 system was the most frequently employed (75.7%), with conjugation identified as the primary delivery method. We identified the advantages and limitations of each system, highlighting CRISPRi and CRISPR-Cas13a as alternatives to overcome the constraints of direct genome editing. Delivery efficiency remains a central challenge, although nanocarrier- and bacteriophage-based methodologies show promising potential. We also propose a decision map that guides the selection of the most appropriate CRISPR-Cas system and delivery strategy, considering factors such as therapeutic objective, gene location, methodology efficiency, application environment, and clinical feasibility. This review provides an updated and structured synthesis of CRISPR strategies applied to AMR, emphasizing their potential translational and clinical applications. The findings can inform the development of CRISPR-based therapeutics, guide the design of preclinical studies, and support future strategies for combating multidrug-resistant infections in clinical settings.},
}
@article {pmid41085033,
year = {2025},
author = {Levassor, L and Whitford, CM and Petersen, SD and Blin, K and Weber, T and Frandsen, RJN},
title = {StreptoCAD: An Open-Source Software Toolbox Automating Genome Engineering Workflows in Streptomycetes.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00261},
pmid = {41085033},
issn = {2161-5063},
abstract = {Streptomycetes hold immense potential for discovering novel bioactive molecules for applications in medicine or sustainable agriculture. However, high-throughput exploration is hampered by the current Streptomyces genetic engineering methods that involve the manual design of complex experimental molecular biological engineering strategies for each targeted gene. Here, we introduce StreptoCAD, an open-source software toolbox that automates and streamlines the design of genome engineering strategies in Streptomyces, supporting various CRISPR-based and gene overexpression methods. Once initiated, StreptoCAD designs all necessary DNA primers and CRISPR guide sequences, simulates plasmid assemblies (cloning) and the resulting modification of the genomic target(s), and further summarizes the information needed for laboratory implementation and documentation. StreptoCAD currently offers six design workflows, including the construction of overexpression libraries, base-editing, including multiplexed CRISPR-BEST plasmid generation, and genome engineering using CRISPR-Cas9, CRISPR-Cas3, and CRISPRi systems. In addition to automating the design process, StreptoCAD further secures compliance with the FAIR principles, ensuring reproducibility and ease of data management via standardized output files. To experimentally demonstrate the design process and output of StreptoCAD, we designed and constructed a series of gene overexpression strains, and performed CRISPRi knockdowns in Streptomyces Gö40/10, underscoring the tool's efficiency and user-friendliness.. This tool simplifies complex genetic engineering tasks and promotes collaboration through standardized workflows and design parameters. StreptoCAD is set to transform genome engineering in Streptomyces, making sophisticated genetic manipulations accessible for all and accelerating natural product discovery.},
}
@article {pmid41084992,
year = {2025},
author = {Han, J and Ganguly, R and Yi, JY and Yun, H and Jung, SY and Sung, C and Lee, CS},
title = {Osmotically Tunable Microdroplets Enable Amplification-Free CRISPR Detection of Gene Doping.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e15861},
doi = {10.1002/advs.202515861},
pmid = {41084992},
issn = {2198-3844},
abstract = {Gene doping is an increasing challenge in sports, demanding highly sensitive and specific detection tools beyond the limitations of the current amplification-dependent methods. Here, an innovative amplification-free clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) 12a assay integrated with osmotically tunable double emulsion (DE) droplets is reported for rapid and ultrasensitive gene doping detection. Target DNA and CRISPR/Cas12a complexes are encapsulated within DE droplets, where osmotic shrinkage rapidly concentrates the reaction components, thereby enhancing the fluorescent signal intensity without nucleic acid amplification. This platform enables the detection of the human erythropoietin (hEPO) gene at unprecedented attomolar levels within 30 min, achieving a 25-fold improvement in sensitivity compared with that of nonshrinkable formats. Notably, the assay demonstrated a robust and specific performance in complex serum samples with minimal matrix interference. This novel approach offers a rapid, reliable, and inherently contamination-free solution for gene doping surveillance with broad potential for versatile amplification-free nucleic acid diagnostics.},
}
@article {pmid41083939,
year = {2025},
author = {Menelih, A and Girma, A and Aemiro, A},
title = {Advancing nutritional quality in oilseed crops through genome editing: a comprehensive review.},
journal = {GM crops &amp; food},
volume = {16},
number = {1},
pages = {709-732},
pmid = {41083939},
issn = {2164-5701},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics/metabolism ; *Nutritive Value ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Plant Oils/metabolism ; Seeds/genetics ; Fatty Acid Desaturases/genetics/metabolism ; Genome, Plant ; },
abstract = {Genome editing has emerged as a powerful approach to enhancing the nutritional quality of oilseed crops. Clustered regularly interspaced short palindromic repeats case9 (CRISPR/Cas9) is the predominant editing tool, while transcription activator-like effector nucleases (TALENs) and base editors are used less commonly. Key fatty acid desaturase genes such as FAD2 and FAD3 are prime targets because of their critical functions in fatty acid desaturation. This review summarizes recent progress in editing genes associated with oil composition and related traits across oilseed species. Visual data representations including, Sankey diagrams, heat maps, and crop-trait matrices illustrate shared editing priorities and emerging trait targets across crops. Despite its promise, genome editing still faces challenges in transformation efficiency, field-level validation, and regulatory acceptance. This review underscores the increasing impact of target gene editing on nutritional trait improvement and its potential to accelerate the development of healthier, more sustainable oilseed varieties.},
}
@article {pmid41002041,
year = {2025},
author = {Clark, MB and Funk, AT and Paporakis, A and Brown, GP and Beach, SJ and Tay, A and Deering, S and Cooper, C and Tizard, M and Jolly, CJ and Ward-Fear, G and Waddle, AW and Shine, R and Maselko, M},
title = {Efficient CRISPR-Cas9-Mediated Genome Editing of the Cane Toad (Rhinella marina).},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {321-332},
doi = {10.1177/25731599251382427},
pmid = {41002041},
issn = {2573-1602},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Monophenol Monooxygenase/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Bufo marinus/genetics ; Albinism/genetics ; Introduced Species ; },
abstract = {Invasive species inflict major ecological, economic, and cultural harm worldwide, highlighting the urgent need for innovative control strategies. Genome editing offers exciting possibilities for targeted control methods for invasive species. Here, we demonstrate CRISPR-Cas9 genome editing in the cane toad (Rhinella marina), one of Australia's most notorious invasive species, by targeting the tyrosinase gene to produce albino phenotypes as visual markers for assessing editing efficiency. Microinjection of Cas9 protein and guide RNAs into one-cell zygotes resulted in 87.6% of mosaic larvae displaying nearly complete albinism, with 2.3% exhibiting complete albinism. For completely albino individuals, genomic analysis confirmed predominantly frameshift mutations or large deletions at the target site, with no wild-type alleles detected. Germline transmission rates reflected the extent of albinism in the mosaic adult, with maternal transmission approaching 100%. This first application of CRISPR-Cas9 in the Bufonidae family opens possibilities for exploring basic research questions and population control strategies.},
}
@article {pmid40875132,
year = {2025},
author = {Skipper, TS and Dickson, KA and Denes, CE and Waller, MA and Du, TY and Neely, GG and Bowden, NA and Faiz, A and Marsh, DJ},
title = {Revealing genetic drivers of ovarian cancer and chemoresistance: insights from whole-genome CRISPR-knockout library screens.},
journal = {Cellular oncology (Dordrecht, Netherlands)},
volume = {48},
number = {5},
pages = {1245-1265},
pmid = {40875132},
issn = {2211-3436},
mesh = {Humans ; *Ovarian Neoplasms/genetics/drug therapy ; Female ; *Drug Resistance, Neoplasm/genetics ; *Gene Knockout Techniques ; *CRISPR-Cas Systems/genetics ; *Gene Library ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Understanding genetic dependencies in cancer is key to identifying novel actionable drug targets to advance precision medicine. Whole-genome CRISPR-knockout library screening methods have facilitated this goal. Pooled libraries of single guide RNAs (sgRNAs) targeting over 90% of the annotated protein coding genome are used to induce gene knockouts in pre-clinical cancer models. Novel genes of interest are identified by evaluating sgRNA dropout or enrichment following selection pressure application. This method is particularly beneficial for researching cancers where effective treatment strategies are limited. One example of a commonly chemoresistant cancer, particularly at relapse, is the low survival malignancy epithelial ovarian cancer (EOC), made up of multiple histotypes with distinct molecular profiles. CRISPR-knockout library screens in pre-clinical EOC models have demonstrated the ability to predict biomarkers of treatment response, identify targets synergistic with standard-of-care chemotherapy, and determine novel actionable targets which are synthetic lethal with cancer-associated mutations. Robust experimental design of CRISPR-knockout library screens, including the selection of strong pre-clinical cell line models, allows for meaningful conclusions to be made. We discuss essential design criteria for the use of CRISPR-knockout library screens to discover genetic dependencies in cancer and draw attention to discoveries with translational potential for EOC.},
}
@article {pmid40789680,
year = {2025},
author = {Sun, X and Li, M and Wang, H and Yang, Y and Kang, Y and Sun, P and Dong, J and Jin, M and Jin, W},
title = {Possible Reversion of CRISPR-Cas9-Edited Sequences in Octoploid Strawberry.},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {375-389},
doi = {10.1177/25731599251361374},
pmid = {40789680},
issn = {2573-1602},
mesh = {*Fragaria/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plant Leaves/genetics ; Polyploidy ; Gene Expression Regulation, Plant ; Plants, Genetically Modified/genetics ; Seedlings/genetics ; Plant Proteins/genetics ; },
abstract = {Gene editing is more challenging in octoploids due to the presence of multiple copies of each gene. However, the ability to edit genes in these plants would allow editing in commercial varieties. Here, we delivered sequences targeting FaMYB9 into octoploid strawberry "Honeoye" and identified several gene-edited lines. Among them, the heterozygous gene-edited line FaMYB9[CR]-15 had curved and wrinkled leaves at 3 months, whereas leaves of 3-month-old wild-type (WT) strawberry seedlings were elliptical with a smooth surface. At that stage, FaMYB9[CR]-15 leaves also had large patches of wax. We identified 11,402 differentially expressed genes, divided into four clusters, between WT and FaMYB9[CR]-15 seedlings at 3 months. Notably, cluster 4 genes-related to nonhomologous end joining, microhomology-mediated end joining repairs, homologous recombination, nucleotide excision repair, and mismatch repair-were more highly expressed in the gene-edited line than in the WT. Surprisingly, by 6 months of age, FaMYB9[CR]-15 leaves had become smooth with small patches of wax, and expression levels of cluster 4 genes were significantly lower than at 3 months. Over the same period, the percentage of FaMYB9 loci harboring the mutant allele decreased from 70.2% to 43.7%. These findings lead us to conclude that there could be reversion of mutated sequences in octoploid strawberry, emphasizing the challenges of gene editing high-ploidy materials.},
}
@article {pmid40675779,
year = {2025},
author = {Sherkow, JS},
title = {A "Bare Hope of A Result": The Second CRISPR Patent Appeal.},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {317-320},
doi = {10.1177/25731599251361362},
pmid = {40675779},
issn = {2573-1602},
mesh = {*Patents as Topic/legislation &amp; jurisprudence ; Humans ; *Gene Editing/legislation &amp; jurisprudence ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; United States ; Dissent and Disputes/legislation &amp; jurisprudence ; },
abstract = {On May 12, 2025, the US Court of Appeals for the Federal Circuit issued its second decision in the long-running CRISPR patent dispute between the Regents of the University of California and related institutions (CVC) and the Broad Institute. This Perspective recounts the principal dispute to date, reviews the Federal Circuit's recent opinion, and provides a critique of its analysis. In particular, this Perspective highlights how the decision is self-contradictory and in tension with patent law's conception doctrine-when an inventor has formed a "definite and permanent" idea of an invention in the mind or whether the invention was little more than a "bare hope" of a result. This Perspective briefly concludes with the implications of this recent decision and where the underlying dispute is likely headed.},
}
@article {pmid40659333,
year = {2025},
author = {Manuvera, VA and Bobrovsky, PA and Kharlampieva, DD and Grafskaia, EN and Brovina, KA and Serebrennikova, MY and Lazarev, VN},
title = {Bacterial Expression System with Deep Repression and Activation via CRISPR-Cas9.},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {353-365},
doi = {10.1177/25731599251358852},
pmid = {40659333},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; Plasmids/genetics ; *Gene Editing/methods ; Promoter Regions, Genetic ; *Gene Expression Regulation, Bacterial ; Green Fluorescent Proteins/genetics ; 5' Untranslated Regions ; Terminator Regions, Genetic ; },
abstract = {Incomplete repression of recombinant genes encoding toxic polypeptides can suppress cell growth even in the absence of a transcription inducer. To address this issue, we developed a CRISPR-Cas9-based genome editing approach that directly modifies the plasmid encoding the toxic peptide during Escherichia coli cultivation. The constructed plasmids contained a transcription terminator between the promoter and coding region, preventing full gene expression through abortive transcription. Upon CRISPR-Cas9 activation, this region is excised, thus restoring the functional gene. To implement this approach, we modified widely used pET-series expression plasmids by adding extra terminators in the 5'-untranslated region of the recombinant gene. Four antimicrobial peptides with strong bactericidal properties served as toxic gene products, while green fluorescent protein was used to assess the efficiency of expression repression. As a result, we developed an expression system with strong repression, which is activated by CRISPR-Cas9-mediated excision of a DNA fragment from the plasmids.},
}
@article {pmid40637801,
year = {2025},
author = {Han, J and Yu, B and Jing, J and He, X and Hua, Y and Xu, G},
title = {EGFR blockade confers sensitivity to pan-RAS inhibitors in KRAS-mutated cancers.},
journal = {Cellular oncology (Dordrecht, Netherlands)},
volume = {48},
number = {5},
pages = {1317-1335},
pmid = {40637801},
issn = {2211-3436},
mesh = {Humans ; *ErbB Receptors/antagonists &amp; inhibitors/metabolism ; Animals ; *Proto-Oncogene Proteins p21(ras)/genetics/antagonists &amp; inhibitors ; Cell Line, Tumor ; *Mutation/genetics ; *Protein Kinase Inhibitors/pharmacology/therapeutic use ; Mice ; *Neoplasms/genetics/drug therapy/pathology ; CRISPR-Cas Systems/genetics ; Cell Proliferation/drug effects ; Drug Resistance, Neoplasm/drug effects ; *ras Proteins/antagonists &amp; inhibitors ; },
abstract = {INTRODUCTION: KRAS is one of the most commonly occurring mutated oncogene in human cancers. Development of KRAS G12C or G12D inhibitors exhibit promising clinical activities, but patients harboring other hotspot KRAS mutations cannot benefit from those strategies. Recent development in pan-RAS inhibitors have broad therapeutic implications and merit clinical investigation. However, intrinsic and acquired drug resistance caused by tumor heterogeneity greatly limit the clinical application, posing a significant challenge in this field.<br><br>RESULTS: In this study, through CRISPR/Cas9 sgRNA screening using a human kinome sgRNA library, EGFR was discovered to correlate with the sensitivity of KRAS-mutated tumors to pan-RAS inhibitor RMC-7977. Through multiple in vitro cell proliferation or viability assays, EGFR loss or pharmacological EGFR inhibition significantly enhances the effectiveness of pan-RAS inhibitors in multiple KRAS[G12C] or KRAS[G12D] cancer cell lines, disregarding their cellular origins. Mechanistically, co-inhibition of EGFR and pan-RAS may further dampen the RTK-RAS-RAF-MEK-ERK pathway activation than either alone, thereby enhancing the anti-tumor activity of pan-RAS inhibitors. Strikingly, with the LL/2 syngeneic mice tumor model, the combination of pan-RAS inhibitors and EGFR inhibitors demonstrated more significant in vivo therapeutic efficacy compared to either single agent.<br><br>CONCLUSION: In conclusion, this study employed high-throughput CRISPR/Cas9 sgRNA screening to identify the enhanced anti-cancer effects when combining EGFR inhibitors with pan-RAS inhibitors in multiple human KRAS-mutated cancer cell lines as well as a mouse syngeneic tumor model. This synergy underscores the potential for a combinational therapy strategy, leveraging EGFR and pan-RAS inhibitors to improve treatment outcomes for patients with KRAS-driven cancers.},
}
@article {pmid40637619,
year = {2025},
author = {Guerra, I and Jensen, K and Perez-Pinera, P},
title = {Implementation of an Undergraduate Laboratory-Based Mammalian Genome Editing Course.},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {366-374},
doi = {10.1089/crispr.2025.0017},
pmid = {40637619},
issn = {2573-1602},
mesh = {*Gene Editing/methods ; Humans ; Curriculum ; Animals ; Genetic Engineering/methods ; Laboratories ; Students ; Mammals/genetics ; CRISPR-Cas Systems ; Universities ; },
abstract = {Genome engineering methods can be utilized to perform complex genetic manipulations in living cells with remarkable efficiency and precision. Given the transformative potential of these enabling technologies, their applications are steadily expanding into most biology and biomedical fields where they play a central role in many experimental frameworks. For these reasons, in order to effectively prepare future generations of biologists and bioengineers for successful careers, there is a high need to incorporate courses teaching genome editing fundamentals into existing curricula. To accomplish this objective, lecture-based courses are rapidly integrating genome editing concepts; however, there are few laboratory courses that teach the practical skills needed to successfully perform genome editing experiments. Here, we describe the development and implementation of a semester-long laboratory course that teaches students not only the techniques needed to perform gene knockout, gene activation, gene repression, and base editing in mammalian cells but also prepares them to design and troubleshoot experiments, write scientific manuscripts, as well as prepare and deliver scientific presentations. Course evaluations demonstrate that this class effectively equips students with the knowledge and hands-on experience needed to succeed in careers related to genome engineering, cell and tissue engineering, and, more broadly, biology.},
}
@article {pmid41083470,
year = {2025},
author = {McCallion, O and Du, W and Glaser, V and Milward, K and Short, S and Bilici, M and Cross, A and Stark, H and Franke, C and Kath, J and Valkov, M and Yang, M and Amini, L and Künkele, A and Polansky, JK and Schmueck-Henneresse, M and Volk, HD and Reinke, P and Wagner, DL and Hester, J and Issa, F},
title = {HLA matching or CRISPR editing of HLA class I/II enables engraftment and effective function of allogeneic human regulatory T cell therapy in a humanized mouse transplantation model.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9090},
pmid = {41083470},
issn = {2041-1723},
support = {825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 211122/Z/18//Wellcome Trust (Wellcome)/ ; FS/ 12/72/29754//British Heart Foundation (BHF)/ ; },
mesh = {Animals ; Humans ; *T-Lymphocytes, Regulatory/immunology/transplantation ; Mice ; Skin Transplantation ; Gene Editing/methods ; Graft Survival/immunology ; CRISPR-Cas Systems ; Transplantation, Homologous ; *Histocompatibility Antigens Class I/genetics/immunology ; Adoptive Transfer ; Forkhead Transcription Factors/metabolism/genetics ; CD8-Positive T-Lymphocytes/immunology ; },
abstract = {Regulatory T cells (Tregs) hold promise for treating autoimmune disease and transplant rejection, yet generation of autologous products for adoptive transfer can suffer donor variability and slow turnaround, limiting their use in urgent indications. We therefore examine whether allogeneic, pre-manufactured ('off-the-shelf') Tregs could overcome these barriers. In a human skin-xenograft model, HLA-mismatched Tregs are swiftly eliminated by recipient CD8[+] T cells and fail to protect grafts. Stringent matching of HLA class I and II restores efficacy but is clinically impractical. Using non-viral CRISPR editing we disrupt B2M and CIITA while inserting an HLA-E-B2M fusion, generating hypo-immunogenic Tregs that evade both T and NK cell attack. Engineered cells retain FOXP3 stability and potent in vitro suppression, and after a single low-dose infusion, prolong human skin graft survival in a humanized mouse model comparably to autologous Tregs. Histology and spatial transcriptomics reveal minimal cytotoxic infiltration and enrichment of immunoregulatory and tissue-repair programmes. Multiplex HLA engineering thus enables ready-to-use allogeneic Tregs that withstand host immune attack for adoptive transfer.},
}
@article {pmid41082405,
year = {2025},
author = {Yang, L and Fang, Y and Lian, Y and Kong, Z and Miao, J and Chen, Y and Li, W and Chen, F and Zhang, B and Chen, Y and Bian, Y},
title = {Aloe-Emodin Targeting FOXC2 Disrupts NETs Formation and EMT-Driven Postoperative Peritoneal Adhesion Through TGF-β1-Smad2/3 Pathway.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e11013},
doi = {10.1002/advs.202511013},
pmid = {41082405},
issn = {2198-3844},
support = {81704084//National Natural Science Foundation of China/ ; 0121/2022/A3//Science and Technology Development Fund, Macau SAR/ ; 0127/2023/RIA2//Science and Technology Development Fund, Macau SAR/ ; SJCX24_1123//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; SJCX25_0992//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; //333 High-Level Talents Cultivation Project of Jiangsu Province/ ; },
abstract = {Postoperative peritoneal adhesion (PPA) develops through TGF-β1-driven fibrotic remodeling, characterized by neutrophil extracellular trap (NETs)-induced aberrant epithelial-to-mesenchymal transition (EMT) deposition. Although aloe-emodin (AE) exhibits anti-fibrosis potential, its molecular mechanisms remain elusive. Forkhead box protein C2 (FOXC2) is a critical regulator of fibrotic tissue formation, yet its role in PPA is unknown. Here, it is demonstrated that FOXC2 expression is elevated in human ileostomy tissue, PPA rodent model, and TGF-β1-exposed peritoneal mesothelial cells (PMCs), where it orchestrates NETs formation and extracellular matrix (ECM) remodeling. Mechanically, CRISPR/Cas-based knockdown and overexpression of FOXC2 alter EMT changes in PMCs, which is achieved via TGF-β1-Smad2/3 signaling. FOXC2 functions as a dual mediator and amplifier through the TGF-β1-Smad2/3 pathway feedback loop to drive EMT alterations. Its overexpression further induces neutrophil recruitment and NETs formation, exacerbating EMT in PMCs. Notably, AE ameliorates FOXC2-driven peritoneal fibrosis by impeding NETs formation and EMT changes through the TGF-β1-Smad2/3 pathway. Moreover, AE binds directly to FOXC2, and the Ser125 residue is critical for the binding of FOXC2 to AE. These findings identify FOXC2 as a pivotal effector in fibrotic responses during PPA formation and reveal that AE targeting the Ser125 residue of FOXC2 may be a promising therapeutic approach to attenuate PPA.},
}
@article {pmid41082121,
year = {2026},
author = {Kim, WD and Huber, RJ},
title = {Modeling Lysosomal Disease in Dictyostelium discoideum: Examining the Trafficking and Secretion of Lysosomal Enzymes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2976},
number = {},
pages = {189-207},
pmid = {41082121},
issn = {1940-6029},
mesh = {*Dictyostelium/genetics/metabolism/enzymology ; *Lysosomes/enzymology/metabolism ; Protein Transport ; *Lysosomal Storage Diseases/genetics/metabolism/enzymology ; Gene Editing/methods ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Protozoan Proteins/metabolism/genetics ; },
abstract = {Non-mammalian models are powerful systems for enhancing our understanding of lysosomal function and lysosomal storage diseases. The social amoeba Dictyostelium discoideum is an excellent model organism for studying lysosomal function, as its genome encodes many proteins associated with lysosomal disease. Methods for gene knockout are straightforward in D. discoideum and include restriction enzyme-mediated integration (REMI) mutagenesis, homologous recombination via the Cre-loxP system, and CRISPR/Cas9-mediated gene editing, which collectively allow researchers to study protein function (e.g., lysosomal enzymes) in a genetically tractable biomedical model system. Additionally, activity assays for conserved lysosomal enzymes are well-established in D. discoideum. In this chapter, we outline methods for studying the intracellular localization and secretion of conserved lysosomal proteins in D. discoideum.},
}
@article {pmid41082119,
year = {2026},
author = {Chear, S and Chiam, A and Talbot, J and Thorne, BN and Wilkinson, EJ and Hewitt, AW and Cook, AL},
title = {Generation of Donor-Specific iPSC for Modelling Lysosomal Storage Disorders.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2976},
number = {},
pages = {151-173},
pmid = {41082119},
issn = {1940-6029},
mesh = {Humans ; *Lysosomal Storage Diseases/pathology/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; Fibroblasts/metabolism/cytology ; *Cell Culture Techniques/methods ; CRISPR-Cas Systems ; Cells, Cultured ; Tissue Donors ; },
abstract = {iPSC technology has enabled the generation of human cell-based models of lysosomal storage disorders and has provided disease-relevant systems to undertake drug discovery or pre-clinical testing of gene- or cell-based therapies. Here, we provide a protocol to generate iPSCs derived from people with lysosomal storage disorders and illustrate expected results using a CLN2 disease donor-specific skin fibroblast culture. Protocol steps include lipofection of episomal plasmids, picking of putative iPSC colonies following live cell TRA-1-60 immunofluorescence, and quality control steps such as immunofluorescence for expression of undifferentiated cell markers, germ layer differentiation, and confirmation of pathological variant genotype. The iPSC generated by this protocol can be differentiated to several cell lineages and can be used with CRISPR/Cas technology to generate isogenic disease models.},
}
@article {pmid41082037,
year = {2025},
author = {Kim, HJ and Kwon, MY and Song, S and Cheon, SW and Kim, HJ},
title = {Engineered Lactiplantibacillus plantarum and Levilactobacillus brevis utilizing ribonucleoprotein-mediated editing for inactivation of hemolysin gene.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {10},
pages = {373},
pmid = {41082037},
issn = {1573-0972},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/metabolism ; *Hemolysin Proteins/genetics ; Probiotics ; Bacterial Proteins/genetics ; Plasmids/genetics ; *Lactobacillus plantarum/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Lactobacillus/genetics ; },
abstract = {Lactiplantibacillus plantarum and Levilactobacillus brevis are widely used probiotics with significant potential as chassis organisms for probiotic engineering. However, their bioengineering remains underdeveloped compared to that of other probiotic bacteria due to the limited availability of genetic tools. Although CRISPR-Cas systems have shown promise for genome editing in Lactobacillus species, strain- or site-specific targeting challenges must be overcome to enhance their broader applicability. This study aimed to develop a novel editing system with reduced dependency on plasmids and antibiotics in L. plantarum WCFS1, L. plantarum SPC 72 - 1 and L. brevis SPC-SNU 70 - 2 using a Cas9-gRNA ribonucleoprotein (RNP) complex. Although the hlyIII gene has been annotated as a hemolysin-related gene in several Lactobacillus genomes, no functional hemolytic activity has been definitively demonstrated to date. In this study, hlyIII was selected as a target to evaluate genome editing efficiency and to assess its potential relevance to strain safety. To construct ΔhlyIII strains, the RNP complex targeting hlyIII was separately transformed with recombinase RecE/T and double-stranded donor DNA. As a result, ΔhlyIII mutants were obtained under optimized electroporation conditions. Sequencing analysis revealed a 50 bp deletion and the introduction of a stop codon in hlyIII across all mutant strains. The hemolytic activity test showed a reduction in free hemoglobin levels in the ΔhlyIII strains compared to the wild type: 27.0%, 74.3%, and 5.0% in L. plantarum WCFS1, L. plantarum SPC 72 - 1, and L. brevis SPC-SNU 70 - 2, respectively. These results suggest strain-dependent differences in hemolytic activity and indicate that inactivation of hlyIII may contribute to reduced hemolysis, although further validation is needed to clarify its functional role. In conclusion, the hlyIII gene was successfully edited in L. plantarum and L. brevis using Cas9-gRNA ribonucleoprotein-mediated editing, demonstrating the feasibility of this genome editing platform for application in probiotic strains.},
}
@article {pmid41039923,
year = {2025},
author = {Xiao, J and Hu, X and Chen, H and Diao, B and Huang, X and Liu, L},
title = {CRISPR-Programmed CuO Nanocatalyst Release for Ultrasensitive Detection of Pathogens in Sterile Body Fluids.},
journal = {Analytical chemistry},
volume = {97},
number = {40},
pages = {22427-22435},
doi = {10.1021/acs.analchem.5c05043},
pmid = {41039923},
issn = {1520-6882},
mesh = {*Copper/chemistry ; Humans ; Catalysis ; *CRISPR-Associated Proteins/metabolism/chemistry ; Benzidines/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; Limit of Detection ; RNA, Ribosomal, 16S/genetics/analysis ; *Body Fluids/microbiology ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; Bacterial Proteins/metabolism/chemistry ; Colorimetry ; },
abstract = {Treatment of sterile body fluids (SBFs) infections is delayed by conventional methods that require up to 72 h to detect pathogens. Here, we present a CRISPR-associated protein 12a (Cas12a)-programmed nanocatalyst release (CNR) method for culture-free diagnostics. To enhance both sensitivity and coverage, three starter DNA (sDNA)-complementary DNA (cDNA) probe pairs were designed for conserved regions and additional three pairs for variable regions of bacterial 16S or fungal 18S rRNA. Upon target recognition, cDNA undergoes strand displacement, releasing sDNA to activate Cas12a. The activated Cas12a cleaves copper oxide nanoparticle (CuONPs)-loaded magnetic probes, releasing tandem CuONPs. Upon acid dissolution, each CuONP generates Cu[2+] ions that catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), producing a visible colorimetric signal. This quadruple signal amplification strategy integrates high-copy rRNA targets, multi-cDNA recognition, Cas12a-mediated continuous release of tandem CuONPs, and Cu[2+]-driven chromogenic amplification. This nucleic acid amplification-free assay detects pathogens at 0.69 CFU mL[-1] in original SBFs samples (after 10-fold centrifugation) within 70 min. In 64 clinical samples, it achieved 100% sensitivity and 100% specificity versus culture. Notably, one culture-negative but clinically confirmed case was correctly identified. Overall, the CNR method offers a rapid, ultrasensitive, and accessible diagnostic solution for resource-limited settings.},
}
@article {pmid40980924,
year = {2025},
author = {Deng, L and He, X and Zhou, S and Gu, T and Dong, J and Zhu, S and Luo, X and Huo, D and Hou, C},
title = {A sticky end-driven PAM-free RPA-CRISPR/Cas12a dual amplification system for ultrasensitive detection of KRAS G12C.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {83},
pages = {16282-16285},
doi = {10.1039/d5cc04401d},
pmid = {40980924},
issn = {1364-548X},
mesh = {*Proto-Oncogene Proteins p21(ras)/genetics ; *CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; Limit of Detection ; DNA/chemistry/genetics ; *Nucleic Acid Amplification Techniques ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Herein, a fluorescent biosensing platform was constructed for KRAS G12C single base mutation detection by CRISPR/Cas12a-coupled RPA without the PAM site. The KRAS G12C gene sequence was cleaved into double-stranded DNA containing a sticky end using HindIII enzyme cleavage site specificity. Sticky end dsDNA activated the trans-cleavage activity of Cas12a and generates an intense fluorescent signal. This strategy detected KRAS G12C targets in a linear range of 10 aM-10 pM with a detection limit of 1.5 aM. What's more, the method was able to distinguish 0.1% KRAS G12C mutation in a total of 10 pM gene concentration and demonstrated excellent detection performance in real samples.},
}
@article {pmid40721863,
year = {2025},
author = {Rybarikova, M and Rey, M and Hasanovic, E and Sipion, M and Rambousek, L and Déglon, N},
title = {Gene editing for Spinocerebellar ataxia type 3 taking advantage of the human ATXN3L paralog as replacement gene.},
journal = {Gene therapy},
volume = {32},
number = {5},
pages = {462-474},
pmid = {40721863},
issn = {1476-5462},
support = {FN 310030_184761/1//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; No. 31ER30_179594//EC | EU Framework Programme for Research and Innovation H2020 | H2020 European Institute of Innovation and Technology (H2020 The European Institute of Innovation and Technology)/ ; },
mesh = {*Machado-Joseph Disease/therapy/genetics ; Animals ; *Ataxin-3/genetics ; Humans ; *Gene Editing/methods ; Mice, Transgenic ; Mice ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Exons ; Cerebellum/metabolism ; Disease Models, Animal ; Repressor Proteins ; },
abstract = {Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by a CAG expansion of the ataxin-3 gene (ATXN3). SCA3 patients suffer from ataxia, spasticity and dystonia in mid-adulthood, with spinocerebellar dysfunction and degeneration. As a monogenic disease for which only symptomatic treatment is available, ATXN3 is an attractive target for gene editing. We used the KamiCas9, a self-inactivating gene editing system, to explore gene editing strategies suitable for all SCA3 patients. We first tested the deletion of exon 10 or the introduction of a premature stop codon into exon 9. High editing events were observed in vitro, but efficiency was very low in SCA3 transgenic mice. We then evaluated an ablate-and-replace strategy. The ablate experiments resulted in 55 ± 18% cerebellar editing of the ATXN3 gene. A human ATXN3L paralog, expressed in the brains of SCA3 patients, may act as a natural, CRISPR-resistant replacement gene. In a proof-of-principle study, ablate and ablate-and-replace strategies were evaluated in SCA3 transgenic mice. Two months after injection, similar editing efficiencies were obtained in the ablate and ablate-and-replace groups. Immunofluorescence and RT-qPCR analyses of cerebellar markers support the development of this strategy for SCA3 treatment.},
}
@article {pmid40581821,
year = {2025},
author = {Luo, Y and Zhan, X and Zhang, Y and Wang, B and Wang, G and Zhang, Y and Li, G and Liu, Q and Shen, X and Chen, D and Hong, Y and Wu, W and Ye, G and Cheng, S and Pan, G and Cao, L},
title = {CRISPR-Cas9-mediated knockup of OsDREB1C enhances rice yield without compromising grain quality.},
journal = {Plant communications},
volume = {6},
number = {10},
pages = {101433},
doi = {10.1016/j.xplc.2025.101433},
pmid = {40581821},
issn = {2590-3462},
mesh = {*Oryza/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Edible Grain/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified/genetics ; Gene Editing ; Gene Knock-In Techniques ; },
abstract = {This study presents a CRISPR-Cas9-based strategy for engineering structural variations in the OsDREB1C gene in rice, leading to a yield increase of over 20% without compromising grain quality. The resulting homozygous plants are transgene-free, highlighting the potential of this approach for precise and effective crop improvement.},
}
@article {pmid39572737,
year = {2025},
author = {Yan, RE and Corman, A and Katgara, L and Wang, X and Xue, X and Gajic, ZZ and Sam, R and Farid, M and Friedman, SM and Choo, J and Raimondi, I and Ganesan, S and Katsevich, E and Greenfield, JP and Dahmane, N and Sanjana, NE},
title = {Pooled CRISPR screens with joint single-nucleus chromatin accessibility and transcriptome profiling.},
journal = {Nature biotechnology},
volume = {43},
number = {10},
pages = {1628-1634},
pmid = {39572737},
issn = {1546-1696},
support = {DP2HG010099//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; GM136573//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R03OD034499//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Humans ; *Chromatin/genetics/metabolism ; *Gene Expression Profiling/methods ; Single-Cell Analysis/methods ; *CRISPR-Cas Systems/genetics ; Transcriptome/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Cell Nucleus/genetics ; },
abstract = {Pooled single-cell CRISPR screens have profiled either gene expression or chromatin accessibility but not both modalities. Here we develop MultiPerturb-seq, a high-throughput CRISPR screening platform with joint single-nucleus chromatin accessibility, transcriptome and guide RNA capture using combinatorial indexing combined with droplet microfluidics to scale throughput and integrate all three modalities. We identify key differentiation genes in a rare pediatric cancer and establish ZNHIT1 as a potential target for cancer reprogramming therapy.},
}
@article {pmid39482449,
year = {2025},
author = {Jabalera, Y and Tascón, I and Samperio, S and López-Alonso, JP and Gonzalez-Lopez, M and Aransay, AM and Abascal-Palacios, G and Beisel, CL and Ubarretxena-Belandia, I and Perez-Jimenez, R},
title = {A resurrected ancestor of Cas12a expands target access and substrate recognition for nucleic acid editing and detection.},
journal = {Nature biotechnology},
volume = {43},
number = {10},
pages = {1663-1672},
pmid = {39482449},
issn = {1546-1696},
mesh = {*Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Humans ; *CRISPR-Cas Systems/genetics ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; DNA/metabolism/genetics ; *Bacterial Proteins/genetics/metabolism ; Substrate Specificity ; },
abstract = {The properties of Cas12a nucleases constrict the range of accessible targets and their applications. In this study, we applied ancestral sequence reconstruction (ASR) to a set of Cas12a orthologs from hydrobacteria to reconstruct a common ancestor, ReChb, characterized by near-PAMless targeting and the recognition of diverse nucleic acid activators and collateral substrates. ReChb shares 53% sequence identity with the closest Cas12a ortholog but no longer requires a T-rich PAM and can achieve genome editing in human cells at sites inaccessible to the natural FnCas12a or the engineered and PAM-flexible enAsCas12a. Furthermore, ReChb can be triggered not only by double-stranded DNA but also by single-stranded RNA and DNA targets, leading to non-specific collateral cleavage of all three nucleic acid substrates with similar efficiencies. Finally, tertiary and quaternary structures of ReChb obtained by cryogenic electron microscopy reveal the molecular details underlying its expanded biophysical activities. Overall, ReChb expands the application space of Cas12a nucleases and underscores the potential of ASR for enhancing CRISPR technologies.},
}
@article {pmid38472508,
year = {2025},
author = {Gould, SI and Wuest, AN and Dong, K and Johnson, GA and Hsu, A and Narendra, VK and Atwa, O and Levine, SS and Liu, DR and Sánchez Rivera, FJ},
title = {High-throughput evaluation of genetic variants with prime editing sensor libraries.},
journal = {Nature biotechnology},
volume = {43},
number = {10},
pages = {1648-1662},
pmid = {38472508},
issn = {1546-1696},
support = {V2022-028//V Foundation for Cancer Research (V Foundation)/ ; P30-CA14051//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; U01AI142756//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; RM1HG009490//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; *Genetic Variation/genetics ; *Gene Editing/methods ; *Tumor Suppressor Protein p53/genetics ; *High-Throughput Screening Assays/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; Neoplasms/genetics ; },
abstract = {Tumor genomes often harbor a complex spectrum of single nucleotide alterations and chromosomal rearrangements that can perturb protein function. Prime editing has been applied to install and evaluate genetic variants, but previous approaches have been limited by the variable efficiency of prime editing guide RNAs. Here we present a high-throughput prime editing sensor strategy that couples prime editing guide RNAs with synthetic versions of their cognate target sites to quantitatively assess the functional impact of endogenous genetic variants. We screen over 1,000 endogenous cancer-associated variants of TP53-the most frequently mutated gene in cancer-to identify alleles that impact p53 function in mechanistically diverse ways. We find that certain endogenous TP53 variants, particularly those in the p53 oligomerization domain, display opposite phenotypes in exogenous overexpression systems. Our results emphasize the physiological importance of gene dosage in shaping native protein stoichiometry and protein-protein interactions, and establish a framework for studying genetic variants in their endogenous sequence context at scale.},
}
@article {pmid41081710,
year = {2025},
author = {Serreze, DV and Tousey-Pfarrer, M and Racine, JJ},
title = {Humanized Mouse Models for Type 1 Diabetes.},
journal = {Current protocols},
volume = {5},
number = {10},
pages = {e70224},
doi = {10.1002/cpz1.70224},
pmid = {41081710},
issn = {2691-1299},
mesh = {*Diabetes Mellitus, Type 1/immunology/genetics ; Animals ; *Disease Models, Animal ; Mice, Inbred NOD ; Humans ; Mice ; CRISPR-Cas Systems ; },
abstract = {T cell-mediated autoimmune type 1 diabetes (T1D) is under complex polygenic control in both humans and the NOD mouse model. However, in both species, particular major histocompatibility complex (MHC; designated HLA in humans) haplotypes provide the primary T1D risk factor. Both MHC/HLA class I and II variants interactively contribute to T1D by respectively driving autoreactive CD8 and CD4 T cell responses that cooperatively destroy insulin-producing pancreatic β cells. While NOD mice have provided important insights to the pathogenic basis of T1D, the model has so far provided only a limited means to identify possible clinically translatable disease intervention approaches. This highlights a need to humanize NOD mice in ways that their pathogenic basis of T1D development becomes more similar to that characterizing the disease course in patients. In this review, we discuss the use of CRISPR/Cas9-generated murine-MHC-deficient NOD mice as a platform for introduction of patient-relevant HLA and T cell receptor molecules. These mice provide ever-improving models for development of clinically applicable interventions for T1D and other autoimmune diseases. © 2025 The Author(s) Current Protocols published by Wiley Periodicals LLC.},
}
@article {pmid41081480,
year = {2025},
author = {Ma, F and Zheng, Q and Sun, Y and Zhang, N and Xu, W},
title = {Toward Tissue-Free Plant Engineering: Emerging Platforms for Sustainable Horticultural Transformation.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c06803},
pmid = {41081480},
issn = {1520-5118},
abstract = {Genetic transformation in horticultural crops is being reshaped by the emergence of nontissue culture technologies that bypass entrenched barriers of genotype dependence, regeneration inefficiency, and sterile culture requirements. This review surveys recent in planta methods, including regenerative activity-dependent in Planta injection delivery (RAPID), cut-dip-budding (CDB), virus-based delivery, nanoparticle-mediated transformation, and Agrobacterium rhizogenes-induced regeneration, and evaluates their operational versatility across species. We further examine their integration with developmental regulators (BABY BOOM [BBM], WUSCHEL [WUS]), visual markers (RUBY), and CRISPR/Cas systems to enhance transformation efficiency and precision. Case studies across fruit, vegetable, and ornamental crops illustrate broad applicability and growing technical maturity. Despite these advances, unresolved challenges in biosafety, reproducibility, and regulatory alignment remain. We advocate a new transformation paradigm that is rapid, genotype-independent, and environmentally compatible, enabling scalable and more accessible broadly applicable crop improvement in horticultural biotechnology.},
}
@article {pmid41080519,
year = {2025},
author = {Chaturvedi, A and Ranjan, R},
title = {Strategies for plant-virus disease management from gene editing to nanotechnology.},
journal = {Physiology and molecular biology of plants : an international journal of functional plant biology},
volume = {31},
number = {8},
pages = {1293-1308},
pmid = {41080519},
issn = {0971-5894},
abstract = {Plant viruses are a global agricultural threat and can result in large financial losses. The globalization of agriculture and its international trading are the major causes of viruses and their vectors expanding to new environmental niches. Conventional methods are not effective in managing virus infection. To mitigate the virus spread, one of the cutting-edge biotechnological approaches, CRISPR/Cas is a robust tool. CRISPR/Cas is a powerful genome editing technology, and provides a highly specific viral genome targeting. Additionally, nanotechnology is a cutting-edge method for mitigating plant viruses. Nanoparticles in biosensors aid in the early identification of plant viruses, hence preventing the spread of disease in the future. Moreover, nanoparticles can also be used as a flexible delivery system. Nanoparticle-mediated delivery of dsRNA ensures minimal off-target while maintaining biosafety. This review explores the genome editing approach and nanotechnological strategies for ensuring sustainable agriculture practices for virus disease management, focusing on biosafety, efficacy, and practical applicability. It also aims to provide a clear insight into the limitations and strengths of each approach.},
}
@article {pmid41076478,
year = {2025},
author = {Wu, M and Wang, F and Wang, Y and Wu, YX and Tian, BY and Ou, XY and Xu, QC and Wu, XY and Han, C and Liu, WL and Xing, S},
title = {Mismatch-introduced crRNA guided PCR-CRISPR/Cas12a platform improves EGFR point mutation detection in single tumor cell.},
journal = {Mikrochimica acta},
volume = {192},
number = {11},
pages = {727},
pmid = {41076478},
issn = {1436-5073},
support = {2021B1515230006//the Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {Humans ; ErbB Receptors/genetics ; *Point Mutation ; *CRISPR-Cas Systems ; *Polymerase Chain Reaction/methods ; *Single-Cell Analysis/methods ; Cell Line, Tumor ; Neoplastic Cells, Circulating ; DNA Mutational Analysis/methods ; Base Pair Mismatch ; },
abstract = {Dynamic monitoring of epidermal growth factor receptor (EGFR) mutations is essential for the early identification of resistance and treatment adaptation. Single-cell heterogeneity analysis is crucial for precision cancer medicine, yet sensitive and specific detection methods for individual tumor cells remain challenging. Here, we develop a PCR-CRISPR/Cas12a platform enhanced by the incorporation of mismatched base in crRNA at specific site for single-cell point mutation detection. This platform demonstrated high specificity and sensitivity, detecting point mutation at a frequency of 0.1% and in as low as 1.02 ng of genomic DNA, which represents an improvement over the amplification-refractory mutation system PCR (ARMS-PCR). Notably, the accuracy of the platform is highly consistent with next-generation sequencing (NGS), as evidenced by Kappa test values surpassing 0.9. By utilizing a conical-pore membrane with optimized porosity for single circulating tumor cell (CTC) enrichment, our platform enables point mutations detection in individual tumor cells, offering potential enhancements in precision and reliability for EGFR mutation analysis. This novel methodology holds potential for more accurate and personalized cancer treatment strategies.},
}
@article {pmid41075940,
year = {2025},
author = {Zhang, J and Dai, P and Weng, Z and Xu, R and Li, Y and Liu, X and Lei, J},
title = {Efficient CRISPR/Cas-based gene editing in cotton induced by cotton leaf crumple virus.},
journal = {Journal of biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jbiotec.2025.10.001},
pmid = {41075940},
issn = {1873-4863},
abstract = {Plant viral vectors can replicate autonomously and spread within host cells, making them an ideal tool for the delivery of CRISPR/Cas gene-editing elements. Here, we constructed a cotton CRISPR/Cas system mediated by cotton leaf crumple virus (CLCrV) as a delivery vector. We first inoculated Pro35s::Cas9 and ProUbi::Cas9 cotton with sgRNAs designed to knock out GhAGL16, GhPDS, and GhCLA1 target genes via the CLCrV vector and then compared the effects of these two transformation receptors on the editing efficiency of the same target genes. We next explored the feasibility of simultaneous multi-target editing in cotton via pooled virus inoculation. Finally, we used a cotton line overexpressing nCas9-TadA7.10 as the transformation receptor to explore the feasibility of CLCrV-mediated adenine base editing and verify the specificity of gene editing in this system. Mutation detection and deep sequencing revealed that the Pro35s::Cas9 and ProUbi::Cas9 cotton lines did not differ significantly in editing efficiency, and both could be used as successful receptors for the CLCrV-mediated Cas9 system. Pooled inoculation with CLCrV-sgRNAs enabled the simultaneous editing of multiple target genes in Pro35s::Cas9 and ProUbi::Cas9 cotton, although this approach had somewhat lower editing efficiency than inoculation with single sgRNAs. The CLCrV-mediated adenine base-editing system enabled A-to-G conversion at target sites in cotton GhPEBP and showed high gene-editing specificity. In summary, this study establishes an efficient CLCrV-mediated CRISPR system in cotton, providing a powerful technical tool for editing of multiple target genes and base editing.},
}
@article {pmid41074985,
year = {2025},
author = {Gupta, I and Sharma, JG and Kaul, T},
title = {Nanoparticle-driven CRISPR-Cas9 genome editing: a new frontier in crop improvement.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {1015},
pmid = {41074985},
issn = {1573-4978},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; *Nanoparticles/chemistry ; Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; Nanotechnology/methods ; Genetic Engineering/methods ; Agriculture/methods ; },
abstract = {The agricultural sector has experienced unpredictable and extreme climatic aberrations, which have severely hampered food production. However, applying advanced nanotechnological approaches in agriculture will be crucial for ensuring more secure and sustainable food production. The revolutionizing phyto-nanotechnology enables the precise delivery of biomolecules i.e., nucleotides and proteins, and the modulated release of agrochemicals, including fungicides and pesticides. In addition, CRISPR-Cas-based genetic engineering holds great promise for food security, agriculture, and environmental sustainability. However, its application in plants faces challenges, including cargo delivery, germline transformation, species independence, HDR efficiency, and overall editing effectiveness. Nanomaterials offer innovative and effective solutions to overcome these challenges by enhancing genome-editing tools precision, efficiency, and delivery mechanisms. This review examines the key limitations of CRISPR-mediated plant genome editing and how nanoparticle technologies can overcome them. We highlight essential nanotech innovations that enhance genome modification, paving the way for a faster, more versatile genomic toolbox in plant biotechnology.},
}
@article {pmid41074026,
year = {2025},
author = {Frey, T and Kandolf-Zumpf, C and Kaempf, A and Schaffer, K and Hollenstein, M and Lampl, A and Kovarik, JJ and Strobl, J and Stary, G and Eckl-Dorna, J and Schmidt, R and Schmetterer, KG},
title = {T cell receptor associated transmembrane adaptor 1 (TRAT1) modulates human Th17 and Treg responses via PI3-kinase and STAT dependent mechanisms.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {431},
pmid = {41074026},
issn = {1478-811X},
support = {P34728-B//Austrian Science Funds/ ; P34728-B//Austrian Science Funds/ ; P34728-B//Austrian Science Funds/ ; },
mesh = {Humans ; *T-Lymphocytes, Regulatory/metabolism/immunology/cytology ; *Th17 Cells/metabolism/immunology/cytology ; Signal Transduction ; *Phosphatidylinositol 3-Kinases/metabolism ; *STAT Transcription Factors/metabolism ; *Adaptor Proteins, Signal Transducing/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Adaptor proteins associated with the T cell receptor (TCR) play critical roles in regulating immune responses by Translating receptor engagement into intracellular signals. T cell Receptor Associated Transmembrane Adaptor 1 (TRAT1) has been implicated in modulating TCR complex stability, but its functional role in human effector and regulatory CD4[+] T cell subsets remains poorly understood. This study aimed to elucidate the role of TRAT1 in regulating T cell activation and differentiation, particularly in helper T cells function and regulatory T cells.<br><br>METHODS: Primary human CD4&#x207a; T cells, including thymus-derived and induced regulatory T cells (Treg), were genetically modified by CRISPR/Cas9-mediated gene deletion or retro-/lentiviral overexpression of TRAT1. Functional assays, flow cytometry, cytokine quantification, and RNA sequencing were performed to evaluate modulation of T cell functions. Mechanistic studies included pathway inhibition using small molecules and phospho-protein analysis. The influence of TRAT1 on Treg function was further assessed in a CAR Treg context in an immune organoid model of allo-rejection.<br><br>RESULTS: Thymus-derived, TGFb-induced and FOXP3-transgenic Treg displayed reduced expression of TRAT1 compared to effector T cells, which showed pronounced up-regulation of TRAT1 following activation. In effector T cells, deletion of TRAT1 led to increased signaling through the phosphoinositide 3-kinase pathway resulting in enhanced proliferation and increased expression of activation markers. However, this was accompanied by reduced production of interleukin-17, which was linked to elevated activity of STAT6 as shown by inhibition experiments using small molecule inhibitors. Overexpression and CRISPR/Cas9-mediated knockout of TRAT1 in Treg enhanced suppression of CD4&#x207a; target cells via up-regulation of LAP/GARP but reduced suppression of CD8&#x207a; target cells, an effect confirmed in HLA-A2-specific CAR Treg in a human organoid model of allo-rejection.<br><br>CONCLUSIONS: TRAT1 acts as a dual regulator of human CD4&#x207a; T cell function, limiting effector activation through modulation of intracellular signaling and supporting regulatory T cell-mediated suppression. These findings reveal a novel mechanism of immune regulation with potential implications for the development of cell-based immunotherapies.},
}
@article {pmid41073589,
year = {2025},
author = {Das, A and Debnath, S and Pramanik, S and Monshi, FI and Rahimi, M},
title = {Bio-digital feedback loop systems: a synergistic integration of predictive genomics, genome editing, and AI-driven phenomic synthesis for next-generation edible and medicinal mushroom breeding.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {11},
pages = {168},
pmid = {41073589},
issn = {1572-9699},
mesh = {*Gene Editing/methods ; *Genomics/methods ; *Agaricales/genetics ; *Artificial Intelligence ; CRISPR-Cas Systems ; *Phenomics/methods ; *Breeding/methods ; },
abstract = {Edible mushrooms face persistent challenges in yield optimization, bioactive compound production, and climate resilience that conventional breeding methods struggle to address. Traditional approaches such as cross-breeding, protoplast fusion, and mutagenesis are limited by genetic noise, laborious screening, and unstable trait inheritance. This review proposes a transformative paradigm built upon converging advances in molecular biology and data science: the bio-digital feedback loop (BDFL) framework, integrating multi-omics, CRISPR-engineered chassis strains, and predictive phenomics for precision mushroom breeding. Our framework employs multi-omics to decipher gene networks governing critical traits, such as substrate degradation enzymes, developmental synchrony regulators, and secondary metabolite pathways. CRISPR-Cas9 and synthetic biology tools then deploy these insights to verify and design modular gene circuits in pre-engineered "plug-and-play" chassis strains, enabling conflict-free stacking of desirable traits. Artificial intelligence serves as the linchpin, not only automating high-throughput phenotyping through advanced imaging but also accelerating the entire breeding cycle by predicting trait heritability from omics data and optimizing the design of CRISPR guide RNAs and genetic constructs for efficient editing. The BDFL we describe iteratively refines strains by feeding phenomics data back into AI algorithms, enabling rapid trait optimization cycles. This transcends the trial-and-error limitations of classical methods, accelerating development of climate-smart mushrooms for circular bioeconomies including strains engineered to thrive on agricultural waste, overproduce immunomodulatory compounds, or resist emerging pathogens. The integration of predictive genomics, AI-driven phenomics, and CRISPR-edited chassis strains heralds a new era of precision mycology, where mushrooms are computationally designed as sustainable solutions for global food security, pharmaceutical innovation, and ecological resilience, ultimately transforming fungi into programmable biological factories tailored to address pressing agricultural and ecological challenges.},
}
@article {pmid40959957,
year = {2025},
author = {Chen, T and Hu, G and Fu, J and Tu, J},
title = {Structural Basis of PAM-Induced Conformational Changes in SpCas9: A Molecular Dynamics Study.},
journal = {Journal of chemical information and modeling},
volume = {65},
number = {19},
pages = {10624-10633},
doi = {10.1021/acs.jcim.5c01626},
pmid = {40959957},
issn = {1549-960X},
mesh = {*Molecular Dynamics Simulation ; *CRISPR-Associated Protein 9/chemistry/metabolism ; Protein Conformation ; DNA/metabolism/chemistry ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry ; CRISPR-Cas Systems ; },
abstract = {As the most widely utilized CRISPR gene-editing enzyme, SpCas9 has been extensively studied and applied. However, its strict dependence on the canonical NGG PAM sequence significantly restricts its targeting scope. Although recent research has successfully engineered SpCas9 variants capable of recognizing noncanonical (non-NGG) PAMs, these variants still exhibit limitations when binding noncanonical PAMs, including substantially reduced cleavage efficiency. To elucidate the molecular mechanisms underlying noncanonical PAM recognition by SpCas9, we employed molecular dynamics simulations to compare the structural differences within the Cas9-gRNA-DNA ternary complex when bound to various PAM sequences. Our analysis revealed significant conformational changes within SpCas9 upon engagement with noncanonical PAMs and uncovered the regulatory mechanisms underpinning these changes. We further identified key dynamic determinants governing the extensive conformational transitions occurring during the noncanonical PAM binding process. These findings provide insights into the dynamic landscape of noncanonical PAM recognition, offering crucial mechanistic guidance for designing efficient, PAM-compatible Cas9 variants.},
}
@article {pmid40925496,
year = {2025},
author = {Yamazaki, M and Ueta, A and Nakanishi, T and Tachikawa, K and Kawai, M and Ozono, K and Michigami, T},
title = {Involvement of impaired phosphate production and aberrant extracellular ATP signaling in the pathogenesis of hypophosphatasia: Analysis of ALPL-Knockout human iPS cell models.},
journal = {Bone},
volume = {201},
number = {},
pages = {117629},
doi = {10.1016/j.bone.2025.117629},
pmid = {40925496},
issn = {1873-2763},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *Hypophosphatasia/metabolism/pathology/genetics ; *Alkaline Phosphatase/metabolism/genetics/deficiency ; *Phosphates/metabolism ; *Adenosine Triphosphate/metabolism ; *Signal Transduction ; Gene Knockout Techniques ; Osteogenesis ; Cell Differentiation ; Osteoblasts/metabolism/pathology ; CRISPR-Cas Systems/genetics ; *Models, Biological ; *Extracellular Space/metabolism ; },
abstract = {Hypophosphatasia (HPP) is caused by inactivating variants of ALPL, the gene encoding tissue non-specific alkaline phosphatase (TNSALP). In order to deepen our understanding of the pathogenic mechanisms of HPP, we herein generated ALPL-knockout (KO) human induced pluripotent stem (iPS) cells by applying CRISPR/Cas9-mediated gene deletion to an iPS clone derived from a healthy subject. We analyzed two ALPL-KO clones, one ALPL-hetero KO clone, and a control clone isogenic except for ALPL. In an osteogenic culture using β-glycerophosphate, which generates inorganic phosphate (Pi) by TNSALP-mediated degradation, ALPL-KO clones showed impaired mineralization, elevated levels of extracellular pyrophosphate (PPi), and reduced levels of extracellular Pi. Osteogenic induction using 3 mM Pi instead of β-glycerophosphate rescued the decreased content of hydroxyapatite in ALPL-KO cells despite the still high levels of extracellular PPi; however, abnormal distribution of hydroxyapatite was noted. Osteoblast lineage cells differentiated from ALPL-KO iPS clones showed the up-regulation of SPP1 and the down-regulation of ANKH and the genes for type III sodium/phosphate co-transporters in the culture using β-glycerophosphate, but not when 3 mM Pi was used. Extracellular ATP levels were elevated in osteoblast lineage cells derived from ALPL-KO iPS clones in both culture conditions, which was associated with the down-regulation of P2X7 encoding a purinergic receptor. Moreover, osteoblast lineage cells differentiated from ALPL-KO iPS clones in the culture using β-glycerophosphate showed a change in cellular response to extracellular Pi. These results suggest that the reduced local production of extracellular Pi and aberrant ATP signaling play substantial roles in the pathogenesis of HPP.},
}
@article {pmid40884048,
year = {2025},
author = {Mikdar, M and Shabani, E and Grüring, C and Chaand, M and Kanjee, U and Goldberg, JM and Azouzi, S and Tennessen, JA and Elsworth, B and Keutcha, C and Barteneva, NS and Doench, JG and Duraisingh, MT},
title = {Sialyl-T Antigen: A Novel Red Blood Cell Determinant for Plasmodium falciparum Invasion.},
journal = {American journal of hematology},
volume = {100},
number = {11},
pages = {1952-1962},
doi = {10.1002/ajh.70037},
pmid = {40884048},
issn = {1096-8652},
support = {P300P3_151146//Schweizerischer Nationalfonds zur Frderung der Wissenschaftlichen Forschung/ ; PBSKP3_140144//Schweizerischer Nationalfonds zur Frderung der Wissenschaftlichen Forschung/ ; 5R01AI140751//National Institute of Allergy and Infectious Diseases/ ; 5R01HL139337//National Heart Lung and Blood Institute/ ; 23POST1017743//American Heart Association/ ; },
mesh = {*Plasmodium falciparum/pathogenicity/physiology ; Humans ; *Erythrocytes/parasitology/metabolism ; *Malaria, Falciparum/parasitology/blood/genetics ; *Galactosyltransferases/genetics/metabolism ; Animals ; N-Acetylneuraminic Acid/metabolism ; CRISPR-Cas Systems ; },
abstract = {Malaria continues to pose significant health challenges globally despite advances in control measures. Plasmodium falciparum, the parasite responsible for most severe malaria cases, uses multiple redundant invasion pathways to enter the red blood cell (RBC) during the blood stage of infection. Through a combination of RNA interference screening in erythroid cells and validation by CRISPR/Cas9-mediated knockout in primary human hematopoietic stem cells, we identified the glycosyltransferase Core 1 Synthase Glycoprotein-N-Acetylgalactosamine 3-Beta-Galactosyltransferase 1 (C1GALT1) as a novel host determinant for P. falciparum invasion. Analyses of C1GALT1-deficient cultured reticulocytes and RBCs with the glycophorin A/B-null MkMk blood group phenotype demonstrated that the C1GALT1-dependent α(2-3) sialic acid structures within mucin-type O-glycans are crucial for efficient invasion of both sialic acid-dependent and sialic acid-independent P. falciparum strains, but not the primate malaria parasite Plasmodium knowlesi. However, different P. falciparum parasite strains exhibit variable dependencies on distinct sialic acid configurations on the RBC surface. Overall, our findings highlight a key role for RBC glycans in malaria infection.},
}
@article {pmid40992249,
year = {2025},
author = {Greisle, T and Kunze, I and Wang, X and Malinowski, AR and Böttcher, A and Lickert, H and Burtscher, I},
title = {Generation of a Flattop-T2A-H2B-Venus x C-peptide-mCherry double reporter human iPSC line to monitor WNT/Planar cell polarity pathway activity.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103838},
doi = {10.1016/j.scr.2025.103838},
pmid = {40992249},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cell Polarity ; *Wnt Signaling Pathway ; Cell Line ; CRISPR-Cas Systems ; Insulin-Secreting Cells/metabolism/cytology ; Genes, Reporter ; C-Peptide/metabolism/genetics ; Cell Differentiation ; },
abstract = {Deriving functional β-cells from human induced pluripotent stem cells (hiPSCs) holds potential for cell replacement therapy, disease modeling, and drug testing in diabetes research. Wnt/Planar cell polarity (PCP) signaling is crucial for endocrine cell development and β-cell maturation in murine models and can be tracked by the expression of the tissue-specific effector gene Flattop. Here, we report the generation of a human fluorescent FLTP/CFAP126 (Flattop-T2A-H2B-Venus) and FLTP-Insulin (Flattop-T2A-H2B-Venus x C-peptide-mCherry) double reporter by CRISPR/Cas9 gene editing. These hiPSC reporter lines allow monitoring of WNT/PCP signaling during endocrine cell formation and studying its role in β-cells in a human model system.},
}
@article {pmid40983220,
year = {2025},
author = {Madhusudhan, K and Padmanaban, A and Parvathi, VD},
title = {Early detection of Parkinson's disease via aptamer-CRISPR platform.},
journal = {Neuroscience},
volume = {586},
number = {},
pages = {163-195},
doi = {10.1016/j.neuroscience.2025.09.027},
pmid = {40983220},
issn = {1873-7544},
mesh = {Humans ; *Parkinson Disease/diagnosis/genetics ; *Aptamers, Nucleotide ; Early Diagnosis ; *CRISPR-Cas Systems ; Biomarkers ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Parkinson's disease (PD) is a neurodegenerative disorder with a worldwide prevalence of around 9.4 million that is expected to double by 2040. It's extended prodromal phase allows irreversible neuronal loss to occur before manifestation of symptoms. Current diagnostic approaches, primarily based on clinical assessment and neuroimaging, are often delayed and lack sensitivity in the early stages, highlighting the need for an early, conclusive, and minimally invasive test. This review focuses on the integration of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) diagnostics with aptamers to detect PD-associated biomarkers. CRISPR systems utilising Cas12 and Cas13 enzymes offer high specificity and collateral cleavage activity that can be harnessed for signal amplification. Aptamers are short, single-stranded oligonucleotides that can be designed to identify nucleic and non-nucleic acid targets. Their fusion with CRISPR may enable the sensitive detection of key PD biomarkers such as α-Syn, dopa decarboxylase, glial fibrillary acidic protein, and neurofilament light chain in biological fluids like blood, CSF, urine, saliva, and sweat. We explore various strategies for aptamer-CRISPR integration, detection, and multiplexing techniques for parallel biomarker detection. We also examine existing diagnostic platforms and discuss barriers to clinical translation. Ultimately, aptamer-CRISPR diagnostics could represent a powerful, next-generation approach for early PD detection.},
}
@article {pmid40953792,
year = {2025},
author = {Watts, JL and Willeke, L and Stottmann, RW},
title = {Mouse variants in Taf1c result in reduced survival to birth.},
journal = {Developmental biology},
volume = {528},
number = {},
pages = {143-151},
doi = {10.1016/j.ydbio.2025.09.011},
pmid = {40953792},
issn = {1095-564X},
mesh = {Animals ; Mice ; *TATA-Binding Protein Associated Factors/genetics/metabolism ; Humans ; *Transcription Factor TFIID/genetics ; Mutation, Missense ; Female ; Craniofacial Abnormalities/genetics ; Phenotype ; Ribosomes/metabolism/genetics ; Male ; CRISPR-Cas Systems ; },
abstract = {Ribosome biogenesis is a key cellular function and disruptions in this process can lead to congenital anomalies or "ribosomopathies" with varying phenotypes including craniofacial malformations and neurodevelopment symptoms. Classically, the mouse is a robust model to understand the molecular mechanisms underlying ribosomopathies to further elucidate human pathogenesis. We identified novel compound heterozygous missense variants in the TATA-box binding protein associated factor, RNA polymerase I subunit C (TAF1C) locus in a patient with some phenotypes consistent with ribosomopathies. TAF1C encodes a subunit of the SL1 complex which is critical for the RNA PolI complex to initiate ribosomal RNA transcription. We hypothesized that functional TAF1C is required at developmental stages critical for craniofacial and neurodevelopment. To test this hypothesis, we created mouse Taf1c variants orthologous to the human variants using CRISPR-CAS9 technology (Taf1c[R202Q] and Taf1c[S428A]). We also created an 11bp deletion to complement the missense variants (Taf1c[11bpdel]). We created multiple allelic combinations to determine the roles for Taf1c in survival and craniofacial development. Homozygous mice for any of these novel variants were underrepresented at organogenesis stages. We did not observe craniofacial anomalies in any surviving mice. Our results suggest that these specific TAF1C variants are not the cause of any human phenotype present in the patient motivating the study. However, we showed that Taf1c is required for embryonic survival and our studies contribute to knowledge about the role of ribosome biogenesis machinery throughout organogenesis.},
}
@article {pmid40929753,
year = {2025},
author = {Herbrich, S and Ramachandran, H and Seibt, A and Tolle, I and Zink, A and Prigione, A and Rossi, A and Distelmaier, F},
title = {CRISPR/Cas9-mediated editing of COQ4 in induced pluripotent stem cells: A model for investigating COQ4-associated human coenzyme Q10 deficiency.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103825},
doi = {10.1016/j.scr.2025.103825},
pmid = {40929753},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *CRISPR-Cas Systems/genetics ; *Ubiquinone/deficiency/analogs &amp; derivatives/genetics/metabolism ; *Gene Editing/methods ; *Mitochondrial Diseases/genetics/pathology/metabolism ; *Ataxia/genetics/pathology/metabolism ; *Mitochondrial Proteins/genetics/metabolism ; *Muscle Weakness/genetics/pathology/metabolism ; Cell Line ; },
abstract = {Pathogenic variants in the gene COQ4 cause primary coenzyme Q10 deficiency, which is associated with symptoms ranging from early epileptic encephalopathy up to adult-onset ataxia-spasticity spectrum disease. We genetically modified commercially available wild-type iPS cells by using a CRISPR/Cas9 approach to create heterozygous and homozygous isogenic cell lines carrying the disease-causing COQ4 variants c.458C > T, p.Ala153Val and c.437T > G, p.Phe146Cys, respectively. All iPSCs lines exhibited a normal cell morphology, expression of pluripotency markers, and the ability to differentiate into the three primary germ layers. The COQ4-deficient cell lines will provide a helpful tool to investigate the disease mechanism and to develop therapeutic strategies.},
}
@article {pmid40925290,
year = {2025},
author = {Raabe, J and Lewandowski, V and Fuchs, S and Hammerschmidt, A and Piasecki, A and Orthey, E and Krämer, E and Ehler, E and Cuello, F},
title = {Generation of a biallelic NRAP-knockout mutant from a human iPSC line.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103829},
doi = {10.1016/j.scr.2025.103829},
pmid = {40925290},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; Cell Line ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Alleles ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; Myocytes, Cardiac/metabolism/cytology ; Mutation ; },
abstract = {Cardiomyopathies, a leading cause of mortality, are associated with dysfunctional intercalated discs, which connect neighbouring cardiomyocytes and ensure proper contractility. In human cardiac diseases, loss-of-function mutations of the intercalated disc-associated protein Nebulin-Related Anchoring Protein (NRAP) have been reported. NRAP plays a crucial role in myofibril assembly and mechanotransduction, however, its regulatory functions remain unclear. To investigate the effects of NRAP loss-of-function in cardiac disease, a human induced pluripotent stem cell (hiPSC) line was generated carrying a biallelic NRAP-knockout (KO) using the CRISPR-Cas9 technology. Control and mutant cell lines were assessed for karyotype integrity, pluripotency, off-target effects, mycoplasma contamination, and differentiation into ectoderm, mesoderm, and endoderm. This hiPSC line provides a valuable tool to study how NRAP modulates cardiac function and contributes to disease progression.},
}
@article {pmid40902326,
year = {2025},
author = {Ran, Y and Ruan, J and Wang, Y and Feng, X and Tan, P and Guan, Y and Guo, X},
title = {Generation of a PHF19 knockout human embryonic stem cell line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103824},
doi = {10.1016/j.scr.2025.103824},
pmid = {40902326},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Transcription Factors/genetics/metabolism/deficiency ; Cell Line ; *Gene Knockout Techniques ; *DNA-Binding Proteins/genetics/metabolism/deficiency ; },
abstract = {PHD finger protein 19 (PHF19) is a polycomb protein that promoted cardiac hypertrophy via epigenetic targeting SIRT2. To determine the role of PHF19 in myocardial hypertrophy, we established a large fragment knockout model of PHF19 gene in human embryonic stem cells (hESCs-H7) using the CRISPR/Cas9 system based on a vector. This PHF19-KO cell line has a normal karyotype, classical human pluripotent stem cell morphology, strong pluripotency, and significantly reduced PHF19 gene expression, which will become a useful tool for further in-depth research on the pathogenesis of PHF19 gene deficiency induced myocardial hypertrophy.},
}
@article {pmid40850232,
year = {2025},
author = {Kim, JW and Jo, S and Kang, EH and Ryu, JH and Noh, H and Park, HJ and Kim, H},
title = {Generation of human embryonic stem cell line expressing dCas9-TET1 fusion protein for epigenetic editing.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103811},
doi = {10.1016/j.scr.2025.103811},
pmid = {40850232},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *Proto-Oncogene Proteins/genetics/metabolism ; *Mixed Function Oxygenases/genetics/metabolism ; *Epigenesis, Genetic ; CRISPR-Cas Systems ; Cell Line ; *CRISPR-Associated Protein 9/metabolism/genetics ; Epigenome Editing ; },
abstract = {CRISPR-based epigenome editing systems can induce site-specific transcriptional activation or repression of target genes. Ten-eleven translocation methylcytosine dioxygenase 1 (TET1) is a transcriptional activation effector involved in the cytosine demethylation of CpG dinucleotides in gene regulatory regions. In this study, we generated a human embryonic stem cell line that stably expresses catalytically dead Cas9 (dCas9) fused to the catalytic domain of TET1 via lentiviral transduction. This cell line can be used for locus-specific transcriptional activation in combination with single guide RNAs and serves as a valuable tool for epigenetic regulation in stem cell and organoid models.},
}
@article {pmid40848400,
year = {2025},
author = {Cota-Coronado, A and Manning, M and Kim, DH and Lee, J and Gibbons, A and Rosenbluh, J and Hill, RA and Sundram, S},
title = {Generation of two Betacellulin CRISPR-Cas9 knockout hiPSC lines to study the affected EGF system paradigm in schizophrenia.},
journal = {Stem cell research},
volume = {88},
number = {},
pages = {103808},
doi = {10.1016/j.scr.2025.103808},
pmid = {40848400},
issn = {1876-7753},
mesh = {*Schizophrenia/genetics/metabolism/pathology ; Humans ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Betacellulin/genetics/metabolism ; *Epidermal Growth Factor/metabolism/genetics ; Gene Knockout Techniques ; Cell Differentiation ; Cell Line ; },
abstract = {Several members of the epidermal growth factor (EGF) family have been implicated in the biology of schizophrenia (Ketharanathan et al., 2024). The EGF-related ligand, Betacellulin (BTC), plays an important role in the proliferation and differentiation of neural stem cells and our group found markedly reduced BTC levels in patients with schizophrenia. Nevertheless, the interplay of affected BTC and its participation in neural specification and neurodevelopment remains elusive. We generated Knockout (KO) - BTC clones from an existing hiPSC line through CRISPR/Cas9-mediated modification. Furthermore, we validated BTC-KO through genotyping/sequencing, FACS and Western Blot. Finally, we demonstrated trilineage differentiation potential in vitro.},
}
@article {pmid41073543,
year = {2025},
author = {Shi, Z and Cheng, TL},
title = {UGI relocation inside Cas9 reduces Cas9 dependent off target effects in cytosine base editors.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {35518},
pmid = {41073543},
issn = {2045-2322},
support = {20ZR1403100//Shanghai Natural Science Foundation/ ; },
mesh = {*Gene Editing/methods ; *Cytosine/metabolism ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; *Uracil-DNA Glycosidase/antagonists &amp; inhibitors/metabolism ; Humans ; HEK293 Cells ; },
abstract = {Cytosine base editors (CBEs) achieve precise C-to-T conversions by addition of uracil DNA glycosylase inhibitor (UGI) with Cas9 nickase (nCas9) and cytidine deaminase, and the conventional fusion at the nCas9 carboxyl terminus effectively inhibits uracil excision repair to enhance editing efficiency. However, despite potent on-target activity, classical CBEs exhibit significant Cas9-dependent DNA off-target effects that necessitate optimization for future applications. Here we present a strategic UGI relocation through internal fusion within the nCas9 architecture. This spatial reorganization maintains comparable on-target editing efficiency while substantially reducing Cas9-dependent DNA off-target activity. Our findings establish an alternative engineering paradigm to develop high-fidelity CBEs, offering an improved platform for widespread genome editing applications.},
}
@article {pmid41073413,
year = {2025},
author = {Zhang, H and Li, M and Wang, G and Zhu, K and Guo, G and Fu, H and Hu, C and Chu, Z and Hu, J and Wu, Q and Chen, Y and Qiu, D and Xie, J and Li, D and Li, B and Li, W and Dong, L and Hou, Y and Cui, X and Huang, B and Liu, Y and Li, Y and Li, H and Yuan, C and Dong, L and Liu, Z and Lu, P},
title = {Paired NLRs originated from Triticum dicoccoides coordinately confer resistance to powdery mildew in wheat.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9040},
pmid = {41073413},
issn = {2041-1723},
mesh = {*Triticum/genetics/microbiology/immunology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; *Ascomycota/physiology/pathogenicity ; *Plant Proteins/genetics/metabolism ; *NLR Proteins/genetics/metabolism ; Haplotypes ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Wheat has evolved diverse resistance genes against powdery mildew, typically controlled by single-gene-encoded proteins. Here, we report the map-based cloning of PmWR183, a resistance locus encoding two adjacent NLR proteins (PmWR183-NLR1 and PmWR183-NLR2) from wild emmer wheat. Stable transformation and CRISPR/Cas9 knockout experiments demonstrate that the two NLRs function cooperatively: neither gene alone confers resistance, but their co-expression restores immunity, while disruption of either gene abolishes resistance. PmWR183 mediates a developmental stage-dependent response, with susceptibility at the seedling stage and strong resistance at the adult stage. Protein interaction assays reveal constitutive association of PmWR183-NLR1 and PmWR183-NLR2, supporting their cooperative role. Geographical and haplotype analyses show the locus originates from wild emmer and is rare in cultivated wheat, exhibiting at least nine haplotypes. Together, our findings uncover a rare NLR gene pair conferring effective resistance to powdery mildew, providing valuable resources for wheat breeding.},
}
@article {pmid41073016,
year = {2025},
author = {Nie, Y and Wang, W and Wang, N and Yuan, M and Huang, L and Sun, Y and Li, K and Liu, Z and Mu, Y},
title = {PCR-CRISPR/Cas12a-based fluorescence and lateral flow dipstick platforms for efficient screening of CD71 biallelic mutants.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344622},
doi = {10.1016/j.aca.2025.344622},
pmid = {41073016},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Receptors, Transferrin/genetics ; *Polymerase Chain Reaction/methods ; *Mutation ; *Antigens, CD/genetics ; *Alleles ; Fluorescence ; *CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR/Cas9 technology plays a pivotal role in gene editing and has been extensively utilized in gene function studies, disease modeling, and gene therapy. However, efficient and accurate detection of CRISPR/Cas9-induced mutants remains a challenge due to the complexity, time-consuming nature, and high cost of existing detection methods. Meanwhile, CRISPR/Cas12a systems have been widely applied in molecular diagnostics due to the non-specific trans-cleavage activity of Cas12a, yet their application in detecting CRISPR/Cas9-induced mutations remains limited. In this study, we developed a PCR-CRISPR/Cas12a-based method to enable the rapid and accurate screening of CD71 biallelic mutants. The detection system was mainly composed of CRISPR RNA specific to the CD71 gene-editing site, Cas12a protein, target DNA, and ssDNA probes for fluorescence or lateral flow dipstick assays. The system demonstrated high specificity in distinguishing CD71 biallelic mutants, with validation through TA cloning confirming its accuracy. Additionally, the method exhibited high sensitivity, establishing it as an efficient tool for biallelic mutated cell clone screening. These findings underscore the potential of PCR-CRISPR/Cas12a as a rapid, sensitive, and cost-effective approach for the precise identification of biallelic mutants, contributing to advancements in gene-editing research and molecular diagnostics.},
}
@article {pmid41073014,
year = {2025},
author = {Cao, R and Wang, S and Guo, Q and Xie, H and Wang, Y and Li, T and Chang, F and Shi, H and Ding, S and Min, X and Duan, X},
title = {DNAzyme-driven SDA reaction regulates CRISPR/Cas12a for highly sensitive and selective analysis of underexpressed miRNA.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344620},
doi = {10.1016/j.aca.2025.344620},
pmid = {41073014},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics/metabolism ; *DNA, Catalytic/metabolism/chemistry ; *CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Male ; Prostatic Neoplasms/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Underexpressed microRNA (miRNA) exerts a pivotal influence across a spectrum of physiological and pathological processes, with their role being particularly pronounced in the incipient stages of tumorigenesis. However, there are several challenges to analyzing these underexpressed miRNAs for their lower abundance and relative decreases in some cancers. Here, we developed a novel fluorescence biosensor based on the DNAzyme-driven strand displacement amplification (SDA) regulating CRISPR/Cas12a for the sensitive and selective detection of underexpressed miRNA, using prostate cancer-associated miR-222 as a proof-of-concept. This study innovatively expanded the application of DNAzyme substrates, designed as templates to trigger SDA and CRISPR/Cas12a reaction, which could effectively generate a positive signal output for detecting underexpressed miRNA. In the absence of miR-222, DNAzyme formation was blocked, allowing the complete substrate to activate SDA, which generated ssDNA that triggered CRISPR/Cas12a trans-cleavage activity to produce a strong fluorescent signal. In contrast, intact DNAzymes (in the presence of miR-222) cleaved the substrates into short DNA fragments, preventing SDA and CRISPR/Cas12a activation, thereby maintaining the sensor in a low fluorescent state. The biosensor demonstrated a linear detection range spanning from 0.1 pmol/L to 1 nmol/L, accompanied by a detection limit of 33.5 fmol/L. Moreover, it exhibited excellent specificity and anti-interference capacity, enabling the successful detection of miR-222 in blood samples. This "DNAzyme-SDA-CRISPR" fluorescence strategy offers a effective, programmability and scalable solution for detecting underexpressed miRNAs in early cancer screening, which is expected to become a powerful tool in early tumor diagnostics and precision therapy.},
}
@article {pmid41043311,
year = {2026},
author = {Hu, M and Zhou, C and Li, M and Zhao, J},
title = {From 3D culture to clinical decision-making: Systematic innovations in breast cancer organoids.},
journal = {Biomaterials advances},
volume = {179},
number = {},
pages = {214528},
doi = {10.1016/j.bioadv.2025.214528},
pmid = {41043311},
issn = {2772-9508},
mesh = {Humans ; *Organoids/pathology ; *Breast Neoplasms/pathology/therapy/genetics ; Female ; *Clinical Decision-Making ; Animals ; CRISPR-Cas Systems ; *Cell Culture Techniques, Three Dimensional/methods ; Tumor Microenvironment ; Gene Editing ; Precision Medicine ; },
abstract = {Breast cancer is a malignant tumour with high heterogeneity. Traditional research models rely mainly on 2D cell culture and patient-derived tumour xenografts (PDXs). However, these models have limited use in clinical trials because of their shortcomings in mimicking the tumour microenvironment and preserving the genetic background. In recent years, organoids, emerging models capable of self-organizing to form 3D structures in vitro, have become key tools for overcoming the traditional dilemma and are promising alternatives for breast cancer research. This review integrates cutting-edge technologies such as organ-on-a-chip and CRISPR/Cas9 gene editing to summarize the multidimensional generation strategy of breast cancer organoids and discusses the clinical value of translation from diagnosis to therapy. Compared with existing studies, this review provides a systematic solution from "model generation" to "precision medicine" for breast cancer research, and the hope is that this review will pave the way for the further development of organoids.},
}
@article {pmid41015100,
year = {2025},
author = {Deng, D and Yi, X and Wen, W and He, L and Peng, W},
title = {The role of the transformer gene in sex determination and its employment in CRISPR/Cas9-based homing gene drive in the global fruit pest Drosophila suzukii.},
journal = {Insect biochemistry and molecular biology},
volume = {184},
number = {},
pages = {104406},
doi = {10.1016/j.ibmb.2025.104406},
pmid = {41015100},
issn = {1879-0240},
mesh = {Animals ; *Sex Determination Processes/genetics ; CRISPR-Cas Systems ; Female ; Male ; *Drosophila Proteins/genetics/metabolism ; *Drosophila/genetics/growth &amp; development ; *Gene Drive Technology ; Nuclear Proteins ; },
abstract = {Sex determination of Diptera is established by the cascade genes such as transformer (tra), though the primary signals for sex determination differ among different insects. Here, we report the isolation, expression and function of tra gene in an invasive pest, Drosophila suzukii, and study the potential use of the D. suzukii tra (Dstra) gene in CRISPR/Cas9-based homing gene drive for genetic-based pest management. The Dstra gene is highly conserved in structure and has a sex-specific transcript. To test the function of this gene in sex determination, Dstra dsRNA was injected into embryos. Almost all XX embryos developed into masculinized phenotypic male adults with intersex morphology. Abnormal ovaries were revealed in XX pseudomales upon dissection. Based on the necessary role of Dstra for female development, we developed and evaluated a homing gene drive that targets Dstra in D. suzukii. The drive component consisting of multiplex Dstra single guide RNAs and Cas9 with Dsvasa promoter was introduced into the Dstra locus. Abnormal development of both the external genitalia and gonads was observed in G0 and G1 chromosomal female adults that expressed the male-specific doublesex (dsx) transcript. Interestingly, knocking out Dstra led to significantly reduced fertility in adults of corresponding sex and moderate transmission rates of the DsRed gene (63.54 %) were observed. Our results not only confirm the conserved function of the Dstra gene in sex determination, but also highlight the potential of sex conversion-based suppression gene-drive strategy targeting the Dstra gene in controlling of D. suzukii populations.},
}
@article {pmid41072992,
year = {2025},
author = {Shi, S and Qin, F and Wu, J and Yang, J and Zhang, X and Wang, S and Wen, W and Wu, Z},
title = {Ultrasensitive single-particle collision electrochemical platform employing CRISPR/Cas12a for ctDNA biosensing.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344590},
doi = {10.1016/j.aca.2025.344590},
pmid = {41072992},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *Circulating Tumor DNA/blood/analysis/genetics ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; Silver/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; },
abstract = {Circulating tumor DNA (ctDNA) is a characteristic tumor biomarker used for cancer diagnosis, treatment, and prognosis. However, the low concentration of ctDNA in peripheral blood and the interference of complex matrices with signals make the detection of ctDNA extremely challenging. Single-particle collision electrochemistry (SPCE) has been widely used in bioanalysis due to its advantages such as high throughput, simple operation, high sensitivity, and low detection limit. In this work, a novel SPCE biosensor for the rapid detection of ctDNA was developed by combining CRISPR/Cas12a with excellent cleavage activity and magnetic beads (MBs) with good separation and enrichment capabilities. The trans-cleavage ability of CRISPR/Cas12a can only be triggered in the presence of target ctDNA to effectively cleave the ssDNA2 on the surface of Ag NPs-ssDNA2 within 1 h, thereby activating the collision activity of silver nanoparticles (Ag NPs). ctDNA was quantified by the collision frequency of Ag NPs. The detection limit of the developed SPCE biosensor for ctDNA was as low as 4.2 fM, and the linear range was 10 fM-1 nM. In addition, MBs allow the biosensor to detect ctDNA in complex samples by directly sampling from complex matrices, with excellent sensitivity and specificity, demonstrating the great potential of the developed SPCE biosensor in the detection of patient samples.},
}
@article {pmid41072860,
year = {2025},
author = {Patel, RR and Arun, PP and Singh, SK and Singh, M},
title = {Overcoming Antimicrobial Resistance: Phage Therapy as a Promising Solution to Combat ESKAPE Pathogens.},
journal = {International journal of antimicrobial agents},
volume = {},
number = {},
pages = {107640},
doi = {10.1016/j.ijantimicag.2025.107640},
pmid = {41072860},
issn = {1872-7913},
abstract = {The global escalation of antimicrobial resistance (AMR) has intensified the search for alternative therapies, with bacteriophage (phage) therapy re-emerging as a promising solution. This review critically examines the therapeutic potential of phage therapy against multidrug-resistant (MDR) ESKAPE pathogens which are among the leading causes of hospital-acquired infections. The review discusses the distinct antibacterial strategies of phage namely, targeted lysis, enzymatic biofilm disruption, and synergy with antibiotics. It also explores the molecular regulation of phage life cycles, highlighting the therapeutic importance of the lytic-lysogenic switch. A central focus is the interplay between advanced delivery systems such as liposomes, hydrogels, nanofibers, and nanoemulsions, and specific administration routes including oral, topical, intravenous, intranasal, and intravesical approaches. These delivery strategies are essential for overcoming key physiological barriers such as gastric acidity, enzymatic degradation, and immune clearance, thereby enhancing phage stability, retention, and therapeutic efficacy. Recent innovations in phage engineering are also explored, particularly the use of CRISPR-Cas systems, synthetic biology, and continuous evolution platforms to broaden host range and optimize lytic function. The review further evaluates emerging clinical evidence, including outcomes from compassionate use cases and early-phase trials, which emphasize both the safety and therapeutic potential of phage therapy in real-world settings. Despite these advances, significant challenges persist, including bacterial resistance to phages, the need for regulatory clarity, and scalability of personalized treatments. With the integration of microbiology, nanotechnology, and clinical practice, phage therapy bridges the gap between ecological solutions and modern medicine, positioning itself as a versatile, sustainable pillar in the post-antibiotic era.},
}
@article {pmid41072406,
year = {2025},
author = {Henriques, WS and Bowman, J and Hall, LN and Gauvin, CC and Wei, H and Kuang, H and Zimanyi, CM and Eng, ET and Santiago-Frangos, A and Wiedenheft, B},
title = {Structures reveal how the Cas1-2/3 integrase captures, delivers, and integrates foreign DNA into CRISPR loci.},
journal = {Structure (London, England : 1993)},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.str.2025.09.007},
pmid = {41072406},
issn = {1878-4186},
abstract = {Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify and determine structures of Cas1 and the Cas2/3 fusion proteins from Pseudomonas aeruginosa at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome.},
}
@article {pmid41071884,
year = {2025},
author = {Guo, Y and Xu, M and Xue, H and Ding, X and Wong, AM and Lin, N and Pu, D and Wong, AM and Wang, X and Zhao, H and Wong, N},
title = {Genome-wide CRISPR screen identifies splicing factor SF3B4 in driving hepatocellular carcinoma.},
journal = {Science advances},
volume = {11},
number = {41},
pages = {eadw7181},
doi = {10.1126/sciadv.adw7181},
pmid = {41071884},
issn = {2375-2548},
mesh = {*Carcinoma, Hepatocellular/genetics/pathology/metabolism/drug therapy ; Humans ; *Liver Neoplasms/genetics/pathology/metabolism/drug therapy ; Animals ; Mice ; *RNA Splicing Factors/genetics/metabolism ; Ferroptosis/genetics ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Phenylurea Compounds/pharmacology ; Drug Resistance, Neoplasm/genetics ; T-Box Domain Proteins/genetics/metabolism ; Quinolines ; },
abstract = {Although genome sequencings have recognized many cancer-associated genes in hepatocellular carcinoma (HCC), distinguishing their functional effect remains challenging. Leveraging on a genome-wide CRISPR knockout (KO) screening, we uncovered spliceosome factors as major survival essential genes in HCC and up-regulations of ferroptosis suppressors [particularly glutamate-cysteine ligase catalytic subunit (GCLC)] in lenvatinib resistance. Our KO screen in patient-derived HCC organoid showed splicing factor 3b subunit 4 (SF3B4) to be top-ranked, conferring prosurvival signal in HCC organoid and driving tumorigenic potentials in both hepatic progenitor organoids and hydrodynamic tail vein injection HCC murine model. The combined RNA immunoprecipitation sequencing, long-read isoform sequencing, and transcriptome revealed characteristic splicing landscape regulated by SF3B4 and identified T-box transcription factor 3 (TBX3) variant TBX3+2a as a potent downstream effector. Our findings highlighted vital roles of SF3B4 in HCC cell survival and tumor progression, and the phenomenon of ferroptosis resistance in patients unresponsive to first-line agent lenvatinib.},
}
@article {pmid41070887,
year = {2025},
author = {Zhang, Y and Wu, Y and Guo, A and Liu, Y and Sun, Q and Zou, X and Sun, Z},
title = {Fluorescent biosensors for the detection of foodborne pathogenic bacteria in food: a comprehensive review.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay01025j},
pmid = {41070887},
issn = {1759-9679},
abstract = {Foodborne pathogenic bacterial contamination poses a major challenge to global food safety and public health, making the development of rapid, sensitive, and specific detection technologies critically important. Conventional methods are limited by their long turnaround time, complex operations, and reliance on large-scale instruments, making them unsuitable for on-site rapid detection. Fluorescent biosensors, which combine highly specific biological recognition elements with highly sensitive fluorescent signal output, demonstrate significant advantages in detecting foodborne pathogens. This review systematically summarizes recent advances in fluorescent biosensors for the detection of common foodborne pathogenic bacteria, with a focus on the application of signal amplification strategies such as functional nanomaterials, amplification techniques, CRISPR/Cas systems, and Argonaute proteins. Furthermore, it analyzes performance metrics including multiplex pathogen detection, real-time quantification, anti-interference capability, and on-site applicability. Finally, future development trends and challenges are discussed, aiming to provide insights for the innovation of food safety monitoring technologies.},
}
@article {pmid41069986,
year = {2025},
author = {Pandey, V and Sharma, S and Pokharel, YR},
title = {Exploring CRISPR-Cas: The transformative impact of gene editing in molecular biology.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102717},
pmid = {41069986},
issn = {2162-2531},
abstract = {This review traces the evolution of clustered regularly interspaced short palindromic repeats (CRISPR) technology from a prokaryotic immune mechanism to a versatile tool for precise genome engineering. We compare CRISPR with traditional gene-editing methods like RNA interference (RNAi), zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), emphasizing its advantages in target specificity, multiplexing, and ease of design. We examine various Cas enzyme classes, engineered variants, and their applications in dissecting genetic alterations at the cellular level. The review further explores CRISPR's expanding role in developing disease models using tissues, organoids, and animal systems, enhancing our understanding of disease mechanisms. Finally, we discuss CRISPR's emerging applications in diagnostics and its transformative impact on immunotherapy and cell-based cancer treatments.},
}
@article {pmid41069170,
year = {2025},
author = {Zhu, Z and Xue, J and Cao, J and Zhang, Z and Gu, T and Sun, Y and Wang, H},
title = {One-pot assay for rapid detection of heterozygous herbicide resistance in Digitaria ciliaris var. chrysoblephara by combining CRISPR/Cas and LAMP.},
journal = {Pest management science},
volume = {},
number = {},
pages = {},
doi = {10.1002/ps.70279},
pmid = {41069170},
issn = {1526-4998},
support = {CX(22)5003//Jiangsu Agricultural Science and Technology Innovation Fund/ ; 2023YFD1401100//National Key R&amp;D Program of China/ ; },
abstract = {BACKGROUND: Resistance to the acetyl-CoA carboxylase (ACCase) inhibitor herbicide cyhalofop-butyl in Digitaria ciliaris var. chrysoblephara is mainly caused by a mutation at the W2027C or W2027S site; however, the existing methods for this mutation site have insufficient detection performance and are difficult to achieve integrated detection in the field.<br><br>RESULTS: In this work, we have developed and optimized a One-Pot single-nucleotide polymorphism (SNP) detection for herbicide resistance based on CRISPR/Cas recognition coupled with the loop-mediated isothermal amplification (LAMP), named OpCas-LAMP. By designing specific CRISPR/Cas guide RNAs and LAMP primers, the OpCas-LAMP can accurately identify with 1% heterozygous mutants of the W2027S or W2027C mutations ACCase gene in D.ciliaris var. chrysoblephara. The optimized reaction system exhibits optimal amplification efficiency at 65&#xb0;C, effectively distinguishing within 60 min (30-min LAMP detection after 30 min CRISPR/Cas pre-cleavage) between homozygous mutant (HM), heterozygous mutant (HT) and wild-type (WT).<br><br>CONCLUSION: This method enables real-time one-pot field detection by integrated with miniaturized detection devices, significantly enhancing its practicality and potential for widespread application. This work provides a novel technical approach for detecting herbicide resistance for global weed resistance monitoring and management. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid41068986,
year = {2025},
author = {Wu, J and Jang, H and Kwak, H and Son, M and Jiang, W and Hwang, HY and Jo, DH and Kim, D and Kim, HH and Kim, JH},
title = {Engineered virus-like particle-assembled Vegfa-targeting Cas9 ribonucleoprotein treatment alleviates neovascularization in wet age-related macular degeneration.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {346},
pmid = {41068986},
issn = {1474-760X},
support = {2022M3A9F3017506//National Research Foundation of Korea/ ; 2022M3A9E4017127//National Research Foundation of Korea/ ; 202200004004//Kun-hee Lee Child Cancer &amp; Rare Disease Project/ ; 18-2023-0010//Seoul National University Hospital/ ; GTL24021-000//National Research Council of Science and Technology/ ; },
mesh = {Animals ; *Vascular Endothelial Growth Factor A/genetics/metabolism ; Mice ; Gene Editing/methods ; *Ribonucleoproteins/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Choroidal Neovascularization/therapy/genetics ; CRISPR-Cas Systems ; NIH 3T3 Cells ; Disease Models, Animal ; Humans ; *Wet Macular Degeneration/therapy/genetics ; Genetic Therapy ; *Macular Degeneration/therapy/genetics ; },
abstract = {BACKGROUND: Age-related macular degeneration, particularly the wet form, is a leading cause of vision loss, characterized by vascular endothelial growth factor A (VEGFA) overproduction. Engineered virus-like particles (eVLPs) combine the efficiency of viral systems with the transient nature of non-viral platforms to offer a potential solution for delivering VEGFA-targeting genome editing enzymes in a safe and efficient manner. Here, we investigate the therapeutic efficacy of eVLPs for transient delivery of Vegfa-targeting Cas9 ribonucleoprotein in a laser-induced choroidal neovascularization mouse model of wet age-related macular degeneration.<br><br>RESULTS: We find that Cas9-eVLPs enables efficient intracellular delivery in vitro, achieving up to 99% insertion and deletion frequency at Vegfa target locus and significant VEGFA protein downregulation in NIH/3T3 cells. A single subretinal injection of Cas9-eVLPs into the mouse retinal pigment epithelium effectively disrupts Vegfa expression, achieving an average indel efficiency of 16.7%. Compared to control groups, the laser-induced choroidal neovascularization mouse model exhibits significantly reduced choroidal neovascularization formation following Cas9-eVLPs intervention, and decreased VEGFA protein levels are detected in the retinal pigment epithelium. Furthermore, the retinal anatomical and functional toxicity are not affected after treatment.<br><br>CONCLUSIONS: eVLPs exhibit the potential as a safe and efficient delivery platform for Cas9 ribonucleoproteins, achieving precise Vegfa downregulation and significant reduction in choroidal neovascularization in a mouse model of wet age-related macular degeneration. With transient delivery of gene editing enzymes, high editing efficiency, and minimal risk of genomic integration, eVLPs present a promising alternative to conventional delivery systems for advancing genome editing therapies in retinal diseases.},
}
@article {pmid41066575,
year = {2025},
author = {Groessl, S and Kalis, R and Snaebjornsson, MT and Wambach, L and Haider, J and Andersch, F and Schulze, A and Palm, W and Zuber, J},
title = {Acidosis orchestrates adaptations of energy metabolism in tumors.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6769},
pages = {eadp7603},
doi = {10.1126/science.adp7603},
pmid = {41066575},
issn = {1095-9203},
mesh = {*Energy Metabolism/genetics ; *Acidosis/metabolism/genetics ; Humans ; *Pancreatic Neoplasms/metabolism/genetics ; Mitochondria/metabolism ; Cell Line, Tumor ; *Adaptation, Physiological/genetics ; Animals ; Mice ; Stress, Physiological ; CRISPR-Cas Systems ; MAP Kinase Signaling System ; Mitochondrial Dynamics ; },
abstract = {Malignant tumors are characterized by diverse metabolic stresses, including nutrient shortages, hypoxia, and buildup of metabolic by-products. To understand how cancer cells adapt to such challenges, we conducted sequential CRISPR screens to identify genes that affect cellular fitness under specific metabolic stress conditions in cell culture and to then probe their relevance in pancreatic tumors. Comparative analyses of hundreds of fitness genes revealed that cancer metabolism in vivo was shaped by bioenergetic adaptations to tumor acidosis. Mechanistically, acidosis suppressed cytoplasmic activity of extracellular signal-regulated kinase (ERK), thereby preventing oncogene-induced mitochondrial fragmentation and promoting fused mitochondria. The resulting boost in mitochondrial respiration supported cancer cell adaptations to various metabolic stresses. Thus, acidosis is an environmental factor that alters energy metabolism to promote stress resilience in cancer.},
}
@article {pmid41065057,
year = {2025},
author = {Anjali, and Punetha, M and Kumar, A and Tripathi, MK and Kishor Kumar, DG and Khanna, S and Nanda, R and Yadav, P and Sharma, S and Maurya, VP and Singh, G and Chouhan, VS},
title = {Application of CRISPR/Cas9 for GDF9 Gene Editing in Caprine Granulosa Cells: Effects on Receptor Signalling and FGF2 Response.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {60},
number = {10},
pages = {e70128},
doi = {10.1111/rda.70128},
pmid = {41065057},
issn = {1439-0531},
support = {NASF/GTR-8004/2019-20//ICAR - National Agricultural Science Fund/ ; },
mesh = {Animals ; Female ; *Granulosa Cells/metabolism ; *Growth Differentiation Factor 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/veterinary ; *Goats/genetics ; *Fibroblast Growth Factor 2/pharmacology/metabolism ; Signal Transduction ; Receptors, FSH/metabolism/genetics ; },
abstract = {Fecundity-related genes, such as GDF9, play a critical role in regulating ovulation, fertilisation and early embryonic development. This study aimed to elucidate the functional role of GDF9 in caprine granulosa cells by employing CRISPR/Cas9-mediated gene editing. The CRISPR/Cas9 system, incorporating single guide RNA (sgRNA) and Cas9 endonuclease, was used to specifically disrupt the GDF9 gene. Successful GDF9 knockout was confirmed via the T7 Endonuclease I (T7E1) cleavage assay. Subsequent analyses assessed the impact of GDF9 disruption on the expression of GDF9 and its associated receptors-BMPR-1A, BMPR-1B and BMPR-II. Additionally, the study examined the modulatory effects of fibroblast growth factor 2 (FGF2) on receptor expression. FGF2 treatment led to increased mRNA expression of BMPR-1A, BMPR-1B and BMPR-II in wild-type granulosa cells. Furthermore, follicle-stimulating hormone receptor (FSHR) levels were significantly upregulated, whereas luteinising hormone receptor (LHR) expression decreased following FGF2 stimulation in wild-type cells. In contrast, GDF9-knockout cells showed elevated expression of both FSHR and LHR. The study also investigated the impact of GDF9 deletion on the expression of key steroidogenic genes, particularly StAR. The combined presence of GDF9 and FGF2 synergistically enhanced StAR expression. Cellular responses to FGF2 included a downregulation of CASPASE 3, indicating reduced apoptosis and an upregulation of PCNA, suggesting increased cell proliferation. In conclusion, this study provides novel insights into the regulatory role of GDF9 in ovarian granulosa cell function and highlights the utility of CRISPR/Cas9 technology for functional genomics in caprine species. The findings have significant implications for enhancing reproductive performance through targeted gene modulation.},
}
@article {pmid41064997,
year = {2025},
author = {Chen, X and Ye, Q and Liang, Q and Li, J and Huang, Y and Xia, Q and Xiao, J and Liao, C and Lau, CH and Zhu, H},
title = {CRISPR-based platforms for detecting tumor-associated genetic materials in clinical samples.},
journal = {Bioanalysis},
volume = {},
number = {},
pages = {1-15},
doi = {10.1080/17576180.2025.2571023},
pmid = {41064997},
issn = {1757-6199},
abstract = {Tumor-associated genetic markers are useful for early cancer screening, diagnosis, and treatment monitoring. However, traditional detection methods are complex in operation procedures, time-consuming, and the equipment costs are expensive. CRISPR/Cas systems are becoming emerging detection tools for tumor detection due to their programmability, rapid reaction, high targeting specificity, and the ability to amplify the signals. CRISPR/Cas has made breakthroughs in the detection of tumor-associated genetic materials including gene mutations, DNA methylation, miRNA, lncRNA, and circRNA detection. Herein, we critically discuss these advancements and describe the key concepts of each CRISPR/Cas system for detecting tumor-associated genetic materials. The significance of these tumor-associated genetic materials in cancer diagnosis and prognosis is highlighted.},
}
@article {pmid41064763,
year = {2025},
author = {Ragulakollu, S and Loganathan, A and Swaminatham, M and Chellappan, G and Veeraswamy, R and Jegadeesan, R},
title = {Molecular breeding approaches for sustainable rice blast management: recent advances and challenges.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1551018},
pmid = {41064763},
issn = {1664-462X},
abstract = {Rice (Oryza sativa. L) is a staple crop globally, but blast disease caused by fungal pathogens Magnaporthe oryzae is one of the most devastating and results in severe economic losses in rice production worldwide. Recent technological advancements have opened new possibilities for developing blast resistance. The dynamic and highly adaptable nature of M. oryzae allows it to overcome plant defense mechanisms rapidly, posing a major threat to global food security and agricultural sustainability. While foundational to early resistance development, traditional breeding approaches have been limited by their time-consuming nature and reliance on phenotypic selection. These methods often require several generations to establish stable resistance traits. However, with the emergence of molecular breeding technologies, resistance breeding has experienced significant acceleration and precision. Tools such as marker-assisted selection (MAS), marker-assisted backcross breeding (MABB), and quantitative trait locus (QTL) mapping allow for the identification and introgression of resistance genes (R genes) more efficiently and accurately. Recent advances in genome engineering techniques, particularly CRISPR-Cas 9, have transformed the capability to manipulate resistance genes directly, enabling targeted editing and stacking of multiple genes (gene pyramiding) for durable resistance. Moreover, omics technologies-including genomics, transcriptomics, proteomics, and metabolomics-offer a comprehensive understanding of the molecular interactions between host and pathogen, facilitating the discovery of novel resistance mechanisms and regulatory pathways. The integration of allele mining with advanced biotechnological tools has further promoted the development of cisgenic and intragenic plants, where resistance genes from related cultivars or wild species are introduced without foreign DNA, thus addressing public concerns over transgenic crops. These strategies enhance resistance and help retain the desirable agronomic traits of elite rice varieties. Despite these advancements, the high mutation rate and genetic plasticity of M. oryzae enable it to evolve and overcome resistance provided by single R genes. Therefore, understanding host-pathogen interactions at the molecular and cellular levels remains essential. Emerging technologies such as nanotechnology show promise in developing targeted fungicide delivery systems and innovative diagnostic tools. Synthetic biology opens avenues for constructing synthetic resistance pathways or deploying plant biosensors. Additionally, machine learning and artificial intelligence (AI) algorithms are increasingly used to predict disease outbreaks, model gene interactions, and optimize breeding strategies based on large datasets. Thus, managing rice blast disease necessitates a holistic approach combining conventional breeding wisdom with modern molecular tools and emerging technologies. The synergy among these approaches holds promise to enhance resistance durability and protect global rice production against evolving fungal threats. This review emphasizes recent advancements in managing rice blast disease, offering valuable insights to sustain resilient breeding programs against this pathogen.},
}
@article {pmid41064460,
year = {2025},
author = {Rentz, L and Hellwig, L and Schneider, S and Schmitz, RA},
title = {Functional insights into Solo-Cas4 in Methanosarcina mazei Gö1.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf024},
pmid = {41064460},
issn = {2633-6693},
abstract = {Solo-Cas4 homologs are Cas4-family proteins found outside of canonical CRISPR-Cas operons. Here, we present the biochemical characterization of Solo-Cas4 from Methanosarcina mazei Gö1. We found significantly upregulated solo-cas4 transcript levels during stationary phase, while remaining constant under oxygen exposure, temperature shifts, high salt conditions or virus challenge. Heterologously expressed as a SUMO-fusion, the purified tag-free protein displays an absorption peak at 420 nm, indicative of a [4Fe-4S]-cluster . Size-exclusion-chromatography revealed that Solo-Cas4 forms a higher oligomeric complex, with an apparent molecular mass of 318 kDa. In vitro nuclease activity assays demonstrated that Solo-Cas4 cleaves metal-dependent linear dsDNA, with highest cleavage activity in the presence of Mn[2+], followed by Mg[2+], while Ca[2+] and Cu[2+] result in negligible cleavage. Isoleucine169 was identified to be crucial for catalysis, mutating it to alanine completely abolished nuclease activity . Mutating any of the four conserved cysteines-proposed to coordinate the [4Fe-4S]-cluster did not affect nuclease activity; however, it abolishes metal cluster binding. Supercoiled circular dsDNA was preferentially nicked by Solo-Cas4 in the presence of Mg[2+], whereas Mn[2+] also led to linearization followed by complete degradation. Besides, ssDNA was cleaved by Solo-Cas4 but with lower activity. In agreement, Microscale thermophoresis analysis revealed strong dsDNA binding with highest affinity to supercoiled circular DNA, and weak ssDNA binding. Overall, these findings indicate that M. mazei Solo-Cas4 is a high oligomeric Cas4-family nuclease that preferentially targets supercoiled dsDNA and is upregulated during stationary growth.},
}
@article {pmid40988554,
year = {2025},
author = {Li, H and Gui, P and Li, X and Lin, Y and Ma, Z and Yu, H and Ma, F},
title = {CRISPR/Cas9-Mediated Construction of a YPS Gene-Deficient Komagataella phaffii Strain for Enhanced Expression of BIAP Ⅱ.},
journal = {Yeast (Chichester, England)},
volume = {42},
number = {8-10},
pages = {195-205},
doi = {10.1002/yea.70002},
pmid = {40988554},
issn = {1097-0061},
support = {//This work was supported by Youth Innovation Promotion Association Fellowship Program, CAS (2022327), Shandong Provincial Laboratory Project (SYS202209), Key Technologies R&amp;D Program of Guangdong Province (2022B1111050001), Natural Science Foundation of Shandong Province, China (ZR2021QB155)./ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Alkaline Phosphatase/genetics/metabolism ; *Saccharomycetales/genetics/metabolism/enzymology ; Cattle ; *Aspartic Acid Endopeptidases/genetics/metabolism ; Recombinant Proteins/genetics/metabolism ; Gene Editing ; Proteolysis ; Gene Expression ; },
abstract = {Multiple isoforms of bovine intestinal alkaline phosphatase (BIAP) have been identified, among which type Ⅱ (BIAP Ⅱ) exhibits the highest specific activity. While Komagataella phaffii has been successfully employed for the secretory expression of recombinant BIAP Ⅱ, substantial proteolytic degradation during the secretion and expression processes has been observed, leading to reduced protein yield and challenging purification procedures. Our investigation demonstrates that the proteolytic cleavage of BIAP Ⅱ is predominantly mediated by secretory pathway proteases, particularly the aspartic protease yapsin (Yps), with Yps1 playing a crucial role. Genetic disruption of the YPS1 gene resulted in a remarkable 2.5-fold increase in BIAP Ⅱ production yield compared to the parental strain, accompanied by significantly reduced proteolytic degradation. Through detailed analysis, we have identified the Yps1 cleavage site within the BIAP Ⅱ peptide chain, located between Lys137 and Lys138. To further minimize BIAP Ⅱ proteolysis, we developed a YPS multigene-deficient engineered strain using CRISPR/Cas9-mediated triple gene editing technology. Additionally, we have established a novel dual-color quantitative PCR (DC-qPCR) method that enables rapid and precise determination of target gene dosage, thereby enhancing screening efficiency while reducing experimental errors associated with repeated sample processing. The strategies and methodologies developed in this study may serve as a valuable reference for optimizing the expression of various secretory heterologous proteins in Komagataella phaffii.},
}
@article {pmid40962170,
year = {2025},
author = {He, H and Huang, Z and Wen, F and Ge, N and Lin, X and Pan, J},
title = {PSPC1 knockout promotes radiosensitivity, inhibits EMT, and metastasis of nasopharyngeal carcinoma cells.},
journal = {Experimental cell research},
volume = {452},
number = {2},
pages = {114755},
doi = {10.1016/j.yexcr.2025.114755},
pmid = {40962170},
issn = {1090-2422},
mesh = {Humans ; *Radiation Tolerance/genetics ; *Nasopharyngeal Carcinoma/genetics/pathology/radiotherapy/metabolism ; Animals ; *Nasopharyngeal Neoplasms/pathology/genetics/radiotherapy ; Cell Line, Tumor ; *Epithelial-Mesenchymal Transition/genetics/radiation effects ; Cell Movement/genetics/radiation effects ; Apoptosis/genetics/radiation effects ; Mice ; Mice, Nude ; *RNA-Binding Proteins/genetics/metabolism ; Cell Proliferation/genetics ; *Nuclear Proteins/genetics/metabolism ; Gene Expression Regulation, Neoplastic/genetics ; Neoplasm Metastasis ; Mice, Inbred BALB C ; Gene Knockout Techniques ; CRISPR-Cas Systems ; },
abstract = {PURPOSE: Paraspeckle component 1 (PSPC1) is upregulated in numerous cancers and is associated with reduced patient survival rates. Our previous research indicated that elevated PSPC1 levels in nasopharyngeal carcinoma (NPC) are positively linked to radiation resistance and tumor metastasis, two primary clinical challenges in NPC treatment. However, the precise role of PSPC1 in radiation resistance and metastasis of NPC cells remains unclear. This study aimed to explore the molecular mechanisms by which PSPC1 influences radiation resistance and metastasis in NPC.<br><br>METHODS: Using the radiation-resistant R743 and radiosensitive CNE2 cell lines of NPC, we examined the impact of PSPC1 expression on post-radiation survival, cell cycle progression, apoptosis, migration, invasion, and tumor growth. CRISPR/Cas9 genome editing was employed to generate PSPC1 knockout (KO) lines in R743&#xa0;cells, while PSPC1 overexpression (pcD-PSPC1) was achieved in CNE2 cells via pcDNA3.1(+)-PSPC1 plasmid transfection.<br><br>RESULTS: PSPC1 knockout converted R743&#xa0;cells from radioresistant to radiosensitive, whereas PSPC1 overexpression decreased radiosensitivity in CNE2 cells. Cell cycle analysis revealed that PSPC1 KO arrested R743&#xa0;cells in the G2/M phase post-irradiation, while PSPC1 overexpression prevented G2/M phase arrest in CNE2 cells. PSPC1 KO increased irradiation-induced apoptosis in R743&#xa0;cells, whereas PSPC1 overexpression decreased it in CNE2 cells. Post-radiation, PSPC1 KO cells showed significantly reduced migration and invasion abilities. Bioinformatics analysis identified SFPQ as a PSPC1-interacting protein, with PSPC1 KO resulting in SFPQ downregulation. Additionally, PSPC1 KO enhanced the radiosensitivity of xenografted tumors in nude mice.<br><br>CONCLUSION: Our findings suggest that PSPC1 is a pivotal factor in enhancing the survival and spread of NPC cells post-radiation. Targeting PSPC1 or its downstream pathways could offer novel strategies to overcome radiation resistance and metastasis in NPC cells.},
}
@article {pmid40952014,
year = {2025},
author = {Llanga, T and Bush, K and Sun, Y and Yan, A and Zhou, J and Gorodkin, J and Sullenger, BA},
title = {Binding and Ligand Activation Driven Enrichment-Directed Evolution of SaCas9 gRNAs Improves Gene Editing Efficiency.},
journal = {Nucleic acid therapeutics},
volume = {35},
number = {5},
pages = {209-219},
doi = {10.1177/21593337251370553},
pmid = {40952014},
issn = {2159-3345},
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Staphylococcus aureus/genetics/enzymology ; *Directed Molecular Evolution/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Ligands ; Humans ; },
abstract = {Clustered regularly interspaced short palindromic repeats-based editing is inefficient at over two-thirds of genetic targets. A primary cause is ribonucleic acid (RNA) misfolding that can occur between the spacer and scaffold regions of the gRNA, which hinders the formation of functional Cas9 ribonucleoprotein (RNP) complexes. Here, we uncover hundreds of highly efficient gRNA variant scaffolds for Staphylococcus aureus (Sa)Cas9 utilizing an innovative binding and ligand activation driven enrichment (BLADE) methodology, which leverages asymmetrical product dissociation over rounds of evolution. SaBLADE-derived gRNA scaffolds contain 7%-42% of nucleotide variation relative to wild type. gRNA variants are able to improve gene editing efficiency at all targets tested, and they achieve their highest levels of editing improvement (>400%) at the most challenging DNA target sites for the wild-type SaCas9 gRNA. This arsenal of SaBLADE-derived gRNA variants showcases the power and flexibility of combinatorial chemistry and directed evolution to enable efficient gene editing at challenging, or previously intractable, genomic sites.},
}
@article {pmid40935921,
year = {2025},
author = {Roh, H and Shen, SP and Hu, Y and Kwok, HS and Siegenfeld, AP and Lee, C and Zepeda, MU and Guo, CJ and Roseman, SA and Comenho, C and Sankaran, VG and Buenrostro, JD and Liau, BB},
title = {Coupling CRISPR scanning with targeted chromatin accessibility profiling using a double-stranded DNA deaminase.},
journal = {Nature methods},
volume = {22},
number = {10},
pages = {2083-2093},
pmid = {40935921},
issn = {1548-7105},
support = {R35GM153476//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; F31HL174076//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01DK103794//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; DGE1745303//National Science Foundation (NSF)/ ; },
mesh = {Humans ; *Chromatin/genetics/metabolism ; Hematopoietic Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Cytidine Deaminase/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Genome editing enables sequence-function profiling of endogenous cis-regulatory elements, driving understanding of their mechanisms. However, these approaches lack direct, scalable readouts of chromatin accessibility across long single-molecule chromatin fibers. Here we leverage double-stranded DNA cytidine deaminases to profile chromatin accessibility at endogenous loci of interest through targeted PCR and long-read sequencing, a method we term targeted deaminase-accessible chromatin sequencing (TDAC-seq). With high sequence coverage at targeted loci, TDAC-seq can be integrated with CRISPR perturbations to link genetic edits and their effects on chromatin accessibility on the same single chromatin fiber at single-nucleotide resolution. We employed TDAC-seq to parse CRISPR edits that activate fetal hemoglobin in human CD34[+] hematopoietic stem and progenitor cells (HSPCs) during erythroid differentiation as well as in pooled CRISPR and base-editing screens tiling an enhancer controlling the globin locus. We further scaled the method to interrogate 947 variants in a GFI1B-linked enhancer associated with myeloproliferative neoplasm risk in a single pooled CRISPR experiment in CD34[+] HSPCs. Together, TDAC-seq enables high-resolution sequence-function mapping of single-molecule chromatin fibers by genome editing.},
}
@article {pmid40921108,
year = {2026},
author = {Zhang, Z and Wang, J and Li, C and Sun, H and Bu, S and Jia, Q and Wan, Y and Zhao, Y and Zhou, H and Hao, Z and Li, N and Yu, S and Wang, L and Wan, J},
title = {An ultrasensitive biosensor for H1N1 virus coupled with 3D spherical DNA nanostructure and CRISPR-Cas12a.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {346},
number = {},
pages = {126905},
doi = {10.1016/j.saa.2025.126905},
pmid = {40921108},
issn = {1873-3557},
mesh = {*Influenza A Virus, H1N1 Subtype/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *Nanostructures/chemistry ; *CRISPR-Cas Systems ; Limit of Detection ; *DNA/chemistry ; Animals ; *CRISPR-Associated Proteins/metabolism/chemistry ; Humans ; Aptamers, Nucleotide/chemistry ; Cattle ; Milk/virology ; Chickens ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {To achieve ultrasensitive and real-time detection of the H1N1 influenza virus, this study designed a nucleic acid-free fluorescent biosensor based on 3D spherical DNA nanostructure and CRISPR/Cas12a (3D-SDNC). The biosensor constructs a rigid 3D nano-framework via self-assembly of six oligonucleotide chains, with H1N1-specific nucleic acid aptamers and Cas12a activator strands strategically positioned at multi-spined vertices for precise spatial coupling between viral recognition and signal transduction. Upon aptamer-virus binding, the induced conformational change liberates the activator strand, thereby activating the trans-cleavage activity of the Cas12a/crRNA complex to efficiently cleave the HEX/BHQ1 double-labeled fluorescent probe and initiate cascade signal amplification. Experimental results demonstrate a detection limit of 0.17 copies/μL (S/N = 3), achieving qPCR-comparable sensitivity, with spike recovery rates of 91.89 % to 104.03 % (RSD < 5.12 %) in chicken serum, bovine serum, and milk matrices. The innovative nucleic acid-free extraction design reduces the total detection time to 40 min; its efficiency is three times higher than qPCR. Notably, we not only discovered the ultra-high sensitivity of the sensor to H1N1 but also unexpectedly found that the rigid structure of the 3D spherical DNA nanostructure conferred enhanced stability under storage conditions. This work establishes a groundbreaking molecular engineering paradigm for rapid pathogen diagnosis, combining ultrahigh sensitivity, fast response, and clinical utility.},
}
@article {pmid40886669,
year = {2026},
author = {Liu, W and Xu, L and Lyu, Y and Yang, C},
title = {MXene-integrated CRISPR/Cas12a biosensor with Split activators for direct and rapid fluorescent detection of MicroRNA.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {346},
number = {},
pages = {126850},
doi = {10.1016/j.saa.2025.126850},
pmid = {40886669},
issn = {1873-3557},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; Limit of Detection ; Spectrometry, Fluorescence/methods ; *Endodeoxyribonucleases/metabolism ; DNA, Single-Stranded/chemistry ; Bacterial Proteins ; Nitrites ; Transition Elements ; CRISPR-Associated Proteins ; },
abstract = {Early and accurate cancer diagnosis is essential for reducing cancer-related mortality, and miRNA-21 has emerged as a critical biomarker for the early detection of various malignancies In this study, we developed a novel fluorescence biosensor, termed the MXene-SNA-Cas12a, that enables direct and amplification-free detection of miRNA-21 by integrating the CRISPR/Cas12a system with a chimeric split nucleic acid (SNA) activator and MXene-assisted fluorescence modulation. Specifically, a split activator comprising S12 ssDNA hybridized with miRNA-21 was employed to activate the trans-cleavage activity of Cas12a, effectively overcoming the system's inherent limitation in RNA recognition. Simultaneously, MXene nanosheets served as efficient quenchers by adsorbing FAM-labeled ssDNA reporters through non-covalent interactions and facilitating target-induced strand release, enabling a robust fluorescence "on/off" mechanism. This biosensor demonstrated excellent linearity over a miRNA-21 concentration range of 50 pM-25 nM, with a detection limit as low as 16 pM. It exhibited high specificity and strong resistance to interference, making it well-suited for complex biological environments. Moreover, the programmable nature of the split activator allows for easy adaptation to detect other RNA targets through rational sequence redesign, offering a versatile platform for CRISPR/Cas12a-based RNA diagnostics.},
}
@article {pmid40851311,
year = {2025},
author = {Kittock, CM and Karia, K and Kc, P and Evans, C and Wollman, J and Meyerink, BL and Pilaz, LJ},
title = {Modeling MPPH syndrome in vivo using Breasi-CRISPR.},
journal = {HGG advances},
volume = {6},
number = {4},
pages = {100497},
pmid = {40851311},
issn = {2666-2477},
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Gene Editing/methods ; Humans ; *Hydrocephalus/genetics/pathology ; *Megalencephaly/genetics/pathology ; *Polydactyly/genetics/pathology ; Neural Stem Cells/metabolism ; },
abstract = {The increasing availability and affordability of genetic testing has resulted in the identification of numerous novel variants associated with neurodevelopmental disorders. There remains a need for methods to analyze the functional impact of these variants. Some methods, like expressing these variants in cell culture, may be rapid, but they lack physiologic context. Other methods, like making a whole-mouse model, may provide physiologic accuracy, but these are costly and time-consuming. We recently developed a technique, Breasi-CRISPR (Brain Easi-CRISPR), which results in efficient genome editing of neural precursor cells via electroporation of CRISPR-Cas9 reagents into developing mouse brains. Since Breasi-CRISPR is extremely rapid and enables the analysis of targeted genes in vivo, we wondered whether this technique would accelerate the study of monogenic neurodevelopmental disorders. Here, we use Breasi-CRISPR to model megalencephaly postaxial polydactyly polymicrogyria hydrocephalus (MPPH) syndrome. We found that 2 days after Breasi-CRISPR, we were able to see neurodevelopmental phenotypes known to be associated with MPPH syndrome, including increased cyclin D2 protein abundance and an increase in neural progenitor proliferation. Thus, Breasi-CRISPR can efficiently model MPPH syndrome and may be a powerful method to add to the toolbox of those investigating the functional impact of patient variants in neurodevelopmental disorders.},
}
@article {pmid40706707,
year = {2025},
author = {Davydova, S and Meccariello, A},
title = {Engineering new clustered regularly interspaced short palindromic repeats-mediated population control for tephritid pests.},
journal = {Current opinion in insect science},
volume = {72},
number = {},
pages = {101415},
doi = {10.1016/j.cois.2025.101415},
pmid = {40706707},
issn = {2214-5753},
mesh = {Animals ; *Tephritidae/genetics ; *Pest Control, Biological/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; Male ; Population Control ; },
abstract = {Tephritid fruit flies threaten the agricultural industry with a rising intensity on a worldwide scale. The application of clustered regularly interspaced short palindromic repeats (CRISPR) in insects has resulted in a current boost of CRISPR studies in tephritid pests. One of the primary pathways toward more efficient population management lies in genetic improvements to the Sterile Insect Technique. Herein, we review the pivotal advances in CRISPR application in non-model tephritid fruit flies in recent years. This consists of proof-of-principle studies to optimise CRISPR tools, applications for female elimination and male sterility, and the existing CRISPR-based systems for population control.},
}
@article {pmid40658346,
year = {2025},
author = {Uchigashima, M and Mikuni, T},
title = {Single-cell endogenous protein labeling via CRISPR-Cas9-mediated genome editing in the mouse brain.},
journal = {Anatomical science international},
volume = {100},
number = {4},
pages = {579-590},
pmid = {40658346},
issn = {1447-073X},
support = {20H05914//Japan Society for the Promotion of Science/ ; 20H05918//Japan Society for the Promotion of Science/ ; 23K18160//Japan Society for the Promotion of Science/ ; 24K02130//Japan Society for the Promotion of Science/ ; 22K21353//Japan Society for the Promotion of Science/ ; 23H04672//Japan Society for the Promotion of Science/ ; 23H02574//Japan Society for the Promotion of Science/ ; 23K27265//Japan Society for the Promotion of Science/ ; 24H01229//Japan Society for the Promotion of Science/ ; 24K22000//Japan Society for the Promotion of Science/ ; 25H02490//Japan Society for the Promotion of Science/ ; JP19dm0207080//Japan Agency for Medical Research and Development/ ; JP24wm0625117//Japan Agency for Medical Research and Development/ ; JPMJFR231M//Fusion Oriented REsearch for disruptive Science and Technology/ ; JPMJPR16F9//Precursory Research for Embryonic Science and Technology/ ; CDA00043/2019-C//Human Frontier Science Program/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; *Brain/metabolism/cytology ; *Single-Cell Analysis/methods ; Electroporation ; Somatosensory Cortex/cytology/metabolism ; Pyramidal Cells/metabolism ; },
abstract = {High-precision mapping of endogenous proteins is essential for understanding the molecular mechanism underlying neuronal functions in the brain. The SLENDR (single-cell labeling of endogenous proteins by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-mediated homology-directed repair) technique provides single-cell endogenous protein labeling with genetically encoded tags within the mammalian brain through precise genome editing via homology-directed repair (HDR). This technique is based on the introduction of HDR-mediated genome editing into neuronal progenitors in embryonic brains by in utero electroporation. Subsequent histological analyses enable high-resolution interrogation of the subcellular distribution of endogenous proteins within a single neuron using conventional fluorescent microscopy. Here, we describe a step-by-step protocol for the SLENDR technique to label endogenous proteins with genetically encoded tags in single pyramidal cells of the mouse primary somatosensory cortex. This protocol would be helpful to visualize the molecular organization underlying biological processes at single-neuron levels in the brain, such as signal processing from synaptic inputs to neuronal outputs across different scales.},
}
@article {pmid41063348,
year = {2025},
author = {Newman, A and Saha, A and Starrs, L and Arantes, PR and Palermo, G and Burgio, G},
title = {CRISPR-Cas12a REC2-Nuc interactions drive target-strand cleavage and constrain trans cleavage.},
journal = {Nucleic acids research},
volume = {53},
number = {18},
pages = {},
doi = {10.1093/nar/gkaf988},
pmid = {41063348},
issn = {1362-4962},
support = {//National Health and Medical Research Council/ ; //The Gordon and Gretel Bootes/ ; //National Computing Infrastructure/ ; //NCRIS/ ; //Australian Government Research Training Program/ ; R01GM141329/NH/NIH HHS/United States ; CHE-2144823//National Science Foundation/ ; 2138259//National Science Foundation/ ; 2138286//National Science Foundation/ ; 2138307//National Science Foundation/ ; 2137603//National Science Foundation/ ; 2138296//National Science Foundation/ ; FG-2023-20431//Alfred P. Sloan Foundation/ ; TC-24-063//Camille and Henry Dreyfus Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA Cleavage ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Escherichia coli/genetics ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Gene Editing ; Molecular Dynamics Simulation ; DNA/metabolism/chemistry/genetics ; *Bacterial Proteins/genetics/metabolism/chemistry ; Kinetics ; DNA, Single-Stranded/metabolism/genetics ; Mutation ; },
abstract = {CRISPR-Cas12a mediates RNA-guided cleavage of double-stranded DNA in cis, after which it remains catalytically active and non-specifically cleaves single-stranded DNA in trans. Native host defence by Cas12a employs cis cleavage, which can be repurposed for the genome editing of other organisms, and trans cleavage can be used for in vitro DNA detection. Cas12a orthologues have high structural similarity and a conserved mechanism of DNA cleavage, yet highly different efficacies when applied for genome editing or DNA detection. By comparing three well-characterized Cas12a orthologues (FnCas12a, LbCas12a, and AsCas12a), we sought to determine what drives their different cis and trans cleavage and how this relates to their applied function. We integrated in vitro DNA cleavage kinetics with molecular dynamics simulations, plasmid interference in Escherichia coli, and genome editing in human cell lines. We report large differences in cis cleavage kinetics between orthologues, which may be driven by dynamic REC2-Nuc interactions. We generated and tested REC2 and Nuc mutants, including a hitherto unstudied 'Nuc-loop', integrity of which is critical for the function of Cas12. In total, our in vitro, in vivo, and in silico survey of Cas12a orthologues highlights key properties that drive their function in biotechnology applications.},
}
@article {pmid41062879,
year = {2025},
author = {Paul, K and Raman K, V and Baaniya, M and Jadhav, I and Bhattacharjee, S and Tilgam, J and Saakre, M and Kumari, P and Das, S and Vijayan, J and Sreevathsa, R and Pattanayak, D},
title = {A novel recombinant CRISPR/Cas9 vector system for genome editing in plants.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {45},
pmid = {41062879},
issn = {1573-9368},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genetic Vectors/genetics ; Solanum tuberosum/genetics ; *Plants, Genetically Modified/genetics/growth &amp; development ; Genome, Plant ; Arabidopsis/genetics ; Nicotiana/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic ; },
abstract = {Genome editing employing CRISPR/Cas9 systems has found widespread applications for knocking out targeted genes. In spite of exponential applications in plants for trait improvement, low editing efficiency in plants is a major concern. We report construction of a pCAMBIA2300 based binary vector cassette (pCR) harbouring novel recombinant CRISPR/Cas9 system for efficient genome editing in plants. The Cas9 cDNA with sequence encoding nuclear localization signals at the N-terminal and C-terminal ends had been codon optimized for better expression in plants. Undesirable internal restriction sites were removed. Small stretch of 5' UTR sequence of Rubisco small subunit (rcbS) of potato, harbouring in between potato granule bound starch synthase (GBSS) intron, was added at the 5' end of the Cas9 cDNA to function as 5' UTR. The recombinant Cas9 gene (rdCas9) was placed under the transcriptional control of CaMV 35S promoter and NOS terminator. The single guide RNA cassette (sgRNA) was comprised of Arabidopsis U6 promoter, 20-21 nucleotide (nt) spacer sequence, sgRNA scaffold sequence and potato U6 RNA Pol-III termination sequence. The 20-21 nt sgRNA spacer sequence could be added to the sgRNA construct by AarI or PaqCI digestion. The sgRNA construct had been designed in such a way so that single or multiplexed sgRNA could be cloned into the pCR vector cassette in a single step. Moreover, modular nature of this vector system can help to derive different combination of promoter, terminator with Cas9 and sgRNA constructs. The efficacy of the pCR vector system had been validated in Nicotiana tabacum and Solanum tuberosum by knocking out phytoene desaturase gene (PDS), through Agrobacterium-mediated transformation. The pCR binary vector system can be utilized as a versatile tool box for efficient genome editing of plant to improve agriculturally important traits.},
}
@article {pmid41062549,
year = {2025},
author = {Sofras, D and Carolus, H and Subotić, A and Lobo Romero, C and Ennis, CL and Cuypers, L and Lagrou, K and Hernday, AD and Nobile, CJ and Rybak, JM and Van Dijck, P},
title = {A systematic comparison of CRISPR-Cas9 allele editing in Candida auris demonstrates unreliable cassette integration and effective episomal plasmid-based editing.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {35105},
pmid = {41062549},
issn = {2045-2322},
support = {11J8122N//Fonds Wetenschappelijk Onderzoek/ ; 11D7620N//Fonds Wetenschappelijk Onderzoek/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; G0L1622N//Joint Programming Initiative on Antimicrobial Resistance/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; PDMT2/23/032//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; C3/22/007//Onderzoeksraad, KU Leuven/ ; R35GM124594/GM/NIGMS NIH HHS/United States ; R35GM124594/GM/NIGMS NIH HHS/United States ; F31DE028488/DE/NIDCR NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Plasmids/genetics ; *Candida auris/genetics ; Alleles ; Genome, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Candidozyma (Candida) auris is an emergent fungal pathogen of significant interest for molecular research. A handful of CRISPR-Cas9 based allele editing tools have been optimized for C. auris. Nonetheless, allele editing in this species remains a significant challenge, and different systems have different advantages and disadvantages. In this work, we compare four systems to introduce the genetic elements necessary for the production of Cas9 and the guide RNA molecule in the genome of C. auris, replacing the ENO1, LEU2 and HIS1 loci respectively, while the fourth system makes use of an episomal plasmid. We observed that the editing efficiency of all four systems was significantly different and strain-dependent. However, we did not detect correct integration of linear CRISPR cassette constructs in integration-based systems, in over 4,900 screened transformants. Still, all transformants, whether correctly edited or not, grew on selective nourseothricin media, suggesting ectopic integration of the CRISPR cassette, which was confirmed by long-read whole genome sequencing. The plasmid-based system showed the highest editing efficiency with an average of 41.9% correct transformants, despite yielding fewer transformants compared to the other systems. Transformation of protoplasts or silencing the non-homologous end joining (NHEJ) DNA repair pathway, by deleting two main NHEJ factors, KU70 and LIG4, did not improve the editing efficiency. While our research highlights important challenges in precise genome editing of C. auris by quantitatively evaluating the editing and targeting efficiencies of different methods, it also clearly shows the safety and usefulness of plasmid-based systems like EPIC, which we recommend for molecular work in this enigmatic fungal pathogen.},
}
@article {pmid41062528,
year = {2025},
author = {Smidler, AL and Marrogi, EA and Scott, S and Mameli, E and Abernathy, D and Akbari, OS and Church, GM and Catteruccia, F and Esvelt, K},
title = {Engineering gene drive docking sites in a haplolethal locus in Anopheles gambiae.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {35074},
pmid = {41062528},
issn = {2045-2322},
support = {AI120480-02//Foundation for the National Institutes of Health/ ; R01 AI104956/AI/NIAID NIH HHS/United States ; R01 AI104956/AI/NIAID NIH HHS/United States ; R01AI151004//Foundation for the National Institutes of Health/ ; R01 AI104956/AI/NIAID NIH HHS/United States ; R00-DK102669-04//Foundation for the National Institutes of Health/ ; IRSA1016432//Burroughs Wellcome Fund/ ; IRSA1016432//Burroughs Wellcome Fund/ ; IRSA1016432//Burroughs Wellcome Fund/ ; OPP1158190/HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {*Anopheles/genetics ; Animals ; *Gene Drive Technology/methods ; Proteasome Endopeptidase Complex/genetics ; *Genetic Engineering/methods ; Mosquito Vectors/genetics ; *Genetic Loci ; CRISPR-Cas Systems ; },
abstract = {Gene drives are selfish genetic elements which promise to be powerful tools in the fight against vector-borne diseases such as malaria. We previously proposed population replacement gene drives designed to better withstand the evolution of resistance by homing through haplolethal loci. Because most mutations in the wild-type allele that would otherwise confer resistance are lethal, only successful drive homing and functional r1 alleles permits the cell to survive. Here we outline the development and characterization of two ΦC31-Recombination mediated cassette exchange gene drive docking lines with these features in Anopheles gambiae, a first step towards construction of robust gene drives in this important malaria vector. We outline adaption of the technique HACK (Homology Assisted CRISPR knockin) to knock-in two docking site sequences into a paired putative haplolethal-haplosufficient (Ribosome-Proteasome) locus, and confirm that these docking lines permit insertion of drive-relevant transgenes. We report the first anopheline proteasome knockouts, and identify ribosome mutants in the process reveal a major lethality and infertility hurdle that such designs must overcome to develop robust drives in the future. Although we do not achieve drive, this work provides a new tool for constructing future evolution-robust drive systems and reveals critical challenges that must be overcome for development of future gene drives designed to target haplolethal loci in anophelines and, potentially, other metazoans.},
}
@article {pmid41062469,
year = {2025},
author = {Han, J and Min, Y and Hu, L and Chen, JJ and Zhang, SX and Li, XF and Cheng, ZH and Liu, DF and Yu, HQ},
title = {Tailoring Cas12a functionality with a user-friendly and versatile crRNA variant toolbox.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8939},
pmid = {41062469},
issn = {2041-1723},
support = {52322002//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Mutation ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Escherichia coli/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Cas12a, with its unique targeting and cleavage activity towards DNA, has been widely applied in gene editing and molecular diagnostics. However, there currently lacks an activity regulation strategy that combines flexibility and simplicity to adapt Cas12a to different demands across various application scenarios. In this study, we present a simple yet effective strategy, wherein we systematically mutate the crRNA direct repeat (DR) sequence to uncover a range of distinct crRNA mutants, which are then compiled into a crRNA toolbox to enable flexible regulation of Cas12a activity. By harnessing the complementarity and synergy between these mutants, we successfully enhance Cas12a performance across various application scenarios. Our crRNA toolbox enables fine-tuned control over expression levels, improves base editing accuracy, enhances transformation and editing efficiency in prokaryote homologous recombination-mediated gene editing, and facilitates rapid, accurate, one-pot, semi-quantitative nucleic acid diagnostics. In summary, the DR sequence mutation strategy provides simple, flexible, and diverse options for Cas12a activity regulation and functional optimization.},
}
@article {pmid41061057,
year = {2025},
author = {Oonuma, K and Kuroda, R and Uchida, T and Zhang, SM},
title = {CRISPR/Cas9-germline editing of Biomphalaria glabrata: A breakthrough in genetic modification of snails that transmit schistosomiasis.},
journal = {Science advances},
volume = {11},
number = {41},
pages = {eadx5889},
pmid = {41061057},
issn = {2375-2548},
mesh = {Animals ; *Biomphalaria/genetics/parasitology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Schistosoma mansoni ; *Schistosomiasis/transmission/parasitology ; Humans ; },
abstract = {Human schistosomiasis remains one of the most devastating parasitic diseases worldwide, and the development of genetically modified vector snails has long been a goal in the field. Here, we report the successful creation of genetically modified Biomphalaria glabrata, an important intermediate host, using CRISPR/Cas9 gene editing. We targeted the fibrinogen-related protein 3.1 (FREP3.1) gene, confirmed stable germline transmission of the mutated gene, and established two different homozygous FREP3.1-edited lines. Disruption of the FREP 3.1 gene did not alter snail susceptibility to Schistosoma mansoni infection, possibly due to a limited role of FREP3.1 in resistance or to functional redundancy and/or compensatory expression within the highly diverse FREP gene family. Our study demonstrates successful germline editing, effective ex ovo culture of decapsulated embryos, and the generation of viable, genetically modified B. glabrata snails, thereby establishing a foundation for future genetic strategies to control schistosomiasis.},
}
@article {pmid41060691,
year = {2025},
author = {Gao, F and Colles, FM and Ko, S and Luo, J and Sheppard, SK and Chen, M},
title = {Genomic epidemiology and the evolution of erm(B)-mediated macrolide resistance in Campylobacter.},
journal = {Microbial genomics},
volume = {11},
number = {10},
pages = {},
doi = {10.1099/mgen.0.001528},
pmid = {41060691},
issn = {2057-5858},
mesh = {*Campylobacter/genetics/drug effects/isolation &amp; purification/classification ; *Macrolides/pharmacology ; Humans ; *Anti-Bacterial Agents/pharmacology ; Phylogeny ; Animals ; *Campylobacter Infections/epidemiology/microbiology ; *Drug Resistance, Bacterial/genetics ; China/epidemiology ; *Methyltransferases/genetics ; Genome, Bacterial ; Microbial Sensitivity Tests ; *Bacterial Proteins/genetics ; Gene Transfer, Horizontal ; Poultry/microbiology ; Evolution, Molecular ; },
abstract = {Campylobacter is a major foodborne bacterial pathogen that has become increasingly resistant to clinically important antimicrobials. Of particular concern is the emergence of erm(B)-mediated macrolide resistance, which has been increasingly documented across Campylobacter isolates from diverse ecological reservoirs. In this study, we investigated the genomic characteristics and epidemiology of erm(B)-carrying clinical Campylobacter isolates from Shanghai, alongside a globally representative dataset of all publicly available strains. Among clinical isolates obtained from a diarrhoeal outpatient surveillance programme between 2020 and 2023 in Shanghai, China, 16% (80/500) were erythromycin-resistant, with 23.8% (19/80) testing positive for erm(B). The genomes of these isolates were sequenced to identify erm(B) gene alleles. Phylogenetic analyses, pairwise comparisons of core and accessory genomes and examination of shared alleles revealed horizontal gene transfer as the predominant mechanism driving the transmission of erm(B) between isolates from various sources. Poultry was identified as a key reservoir for human infections caused by erm(B)-positive Campylobacter isolates. Comparative pangenome analyses of erm(B)-positive and negative isolates identified multiple accessory elements associated with erm(B) acquisition, among which the IS607 family transposon-associated tnpB gene exhibited sequence and structural homology to functional progenitors of CRISPR-Cas nucleases. These findings expand our understanding of the epidemiology of erm(B)-mediated macrolide resistance in Campylobacter and underscore the urgent need for enhanced antimicrobial stewardship in poultry production and targeted surveillance programmes to curb the spread of resistance.},
}
@article {pmid41042795,
year = {2025},
author = {Shen, Y and Fan, K and Gökbağ, B and Sun, N and Yang, C and Cheng, L and Li, L},
title = {A multi-layer encoder prediction model for individual sample specific gene combination effect (MLEC-iGeneCombo).},
journal = {PLoS computational biology},
volume = {21},
number = {10},
pages = {e1013547},
doi = {10.1371/journal.pcbi.1013547},
pmid = {41042795},
issn = {1553-7358},
mesh = {Humans ; Gene Knockout Techniques ; CRISPR-Cas Systems/genetics ; Computational Biology/methods ; Deep Learning ; *Models, Genetic ; Systems Biology/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Using data from gene combination double knockout (CDKO) experiments, top ranked synthetic lethal (SL) gene pairs were highly inconsistent among different SL scores. This leads to a significant concern that SL prediction models highly depend on SL scores. In this paper, we introduce a new gene combination effect (GCE) measurement, log-fold change of dual-gRNA expression before and after CRISPR-cas9 lentivirus transfection. We show it is a direct and highly consistent measurement of GCE in all CDKO experiments. We therefore develop a multi-layer encoder model for individual sample specific GCE prediction, MLEC-iGeneCombo. Under a deep learning framework, MLEC-iGeneCombo is a systems biology model that contains sample specific multi-omics encoder, network encoder and cell-line encoder. For the first time, MLEC-iGeneCombo predicts GCE for a new cell. Using data from 18 CDKO experiments, MLEC-iGeneCombo achieves an average GCE prediction performance, 71.9%. All three encoders significantly improve the model's prediction performance (p[Formula: see text]), and their combined use yields the best GCE prediction performance. Our source code is available at https://github.com/karenyun/MLEC-iGeneCombo.},
}
@article {pmid40930101,
year = {2025},
author = {Vučićević, D and Hsu, CW and Lopez Zepeda, LS and Burkert, M and Hirsekorn, A and Bilić, I and Kastelić, N and Landthaler, M and Lacadie, SA and Ohler, U},
title = {Sensitive dissection of a genomic regulatory landscape using bulk and targeted single-cell activation.},
journal = {Cell genomics},
volume = {5},
number = {10},
pages = {100984},
doi = {10.1016/j.xgen.2025.100984},
pmid = {40930101},
issn = {2666-979X},
mesh = {Humans ; *Single-Cell Analysis/methods ; Transcription Factors/genetics/metabolism ; Neuroblastoma/genetics ; Homeodomain Proteins/genetics ; Gene Regulatory Networks/genetics ; Enhancer Elements, Genetic/genetics ; CRISPR-Cas Systems/genetics ; *Genomics/methods ; Cell Line, Tumor ; },
abstract = {Enhancers are known to spatiotemporally regulate gene transcription, yet the identification of enhancers and their target genes is often indirect, low resolution, and/or assumptive. To identify and functionally perturb enhancers at their endogenous sites, we performed a pooled tiling CRISPR activation (CRISPRa) screen surrounding PHOX2B, a master regulator of neuronal cell fate and a key player in neuroblastoma, and found many CRISPRa-responsive elements (CaREs) that alter cellular growth. To determine CaRE target genes, we developed TESLA-seq (targeted single-cell activation), which combines CRISPRa screening with targeted single-cell RNA sequencing and enables the parallel readout of the effect of hundreds of enhancers on all genes in the locus. While most TESLA-revealed CaRE-gene relationships involved neuroblastoma-related regulatory elements, we found many CaREs and target connections normally active only in other tissues. This highlights the power of TESLA-seq to reveal gene regulatory networks, including edges active outside of a given experimental system.},
}
@article {pmid40888415,
year = {2025},
author = {Zhao, T and Yu, L and Yin, M and Huang, S and Tian, R and Zhong, C and Nan, F and Zhang, H and Tian, X and Hu, Z},
title = {Enhanced One-Pot Cas12a-Based Nucleic Acid Detection via Epitope Insertion and Recruitment of Rad51.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {40},
pages = {e02417},
doi = {10.1002/smll.202502417},
pmid = {40888415},
issn = {1613-6829},
support = {32171465//National Natural Science Foundation of China/ ; 32371541//National Natural Science Foundation of China/ ; 82102392//National Natural Science Foundation of China/ ; 82172584//National Natural Science Foundation of China/ ; 2023M734091//China Postdoctoral Science Foundation/ ; 2023M734090//China Postdoctoral Science Foundation/ ; 2023M744121//China Postdoctoral Science Foundation/ ; 2024BCB057//Key Technology R&amp;D Program of Hubei/ ; 0820250//Guangdong Special Support Plan Young Top-notch Talent/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Epitopes/metabolism ; *Rad51 Recombinase/metabolism ; Humans ; *Nucleic Acids/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The CRISPR-Cas12a system has emerged as a promising tool for nucleic acid-based diagnostics. However, its multi-step workflow and limited sensitivity hinder its integration into point-of-care testing (POCT). Here, the ECOT system (Engineered Cas12a for One-pot Test), a novel approach that combines protein engineering with one-pot detection, offering high sensitivity, specificity, and rapid response is introduced. By introducing GCN4 epitope insertions into LtCas12a and LbCas12a variants, their cis-cleavage activity, promoting efficient accumulation of amplification products is reduced. Additionally, the inclusion of scFv-Rad51 (single-chain variable fragment-Rad51) enhances Cas12a's trans-cleavage activity, amplifying signal intensity. The ECOT-Lb system demonstrated superior sensitivity in detecting low-copy HPV DNA samples, outperforming traditional qPCR in clinical tests. Achieving detection limits as low as 3 copies in under 30 min, the ECOT-Lb system is well-suited for home-based self-testing and widespread clinical diagnostics. This work provides a versatile and scalable protein engineering strategy that enhances the performance of CRISPR-based diagnostic tools, offering a promising platform for rapid molecular detection in diverse applications.},
}
@article {pmid40811280,
year = {2025},
author = {Chen, H and Jiang, Z},
title = {CrisprDA: A Data Augmentation Method Enhancing the Efficiency of sgRNA on-Target Activity Prediction.},
journal = {IEEE transactions on computational biology and bioinformatics},
volume = {22},
number = {5},
pages = {2313-2319},
doi = {10.1109/TCBBIO.2025.3591871},
pmid = {40811280},
issn = {2998-4165},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Computational Biology/methods ; *Deep Learning ; Neural Networks, Computer ; },
abstract = {The CRISPR/Cas9 system has emerged as a revolutionary technology in genome editing, yet the efficiency of this system is often limited by the activity level of single-guide RNAs (sgRNAs). In recent years, deep learning models have been increasingly utilized to predict sgRNA targeting activity. Notably, data scarcity rather than model architecture has become the predominant bottleneck in accurately predicting sgRNA activity. To overcome this challenge and enhance the performance of deep learning models, we propose Automix, a straightforward yet effective data augmentation method grounded in autoencoder technology. This method is complemented by CNLC (Confidence-based Nearest Label Correction), a pseudo-label correction technique designed to improve both the quality and quantity of training data. Additionally, we develop CrisprDA, a novel parallel architecture that integrates convolutional neural networks (CNNs) with attention mechanisms, for the precise prediction of sgRNA activity. Comprehensive experiments conducted on nine high-throughput datasets and eight functional datasets demonstrate that CrisprDA outperforms five compared methods, showing its superior predictive ability. Moreover, the application of Automix and CNLC to other comparative methods in our experiments further validates the effectiveness and generalizability of the proposed data augmentation strategy.},
}
@article {pmid41058711,
year = {2025},
author = {Barraclough, A and Bär, I and van Duijl, T and Fijnvandraat, K and Eikenboom, JCJ and Leebeek, FWG and Bierings, R and Voorberg, J and Trasanidou, D},
title = {Rewriting the script: gene therapy and genome editing for von Willebrand Disease.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1620438},
pmid = {41058711},
issn = {2673-3439},
abstract = {In recent years gene therapy has emerged as a powerful technology for treatment of a large variety of inherited disorders. With the FDA approval of in vivo gene therapy of hemophilia A and B using AAV-mediated transgene delivery to hepatocytes, the path towards a new treatment era seemed paved. Also, CRISPR-Cas based approaches have reached the clinic, as in the ex vivo treatment of hematopoietic stem cells for sickle cell disease and thalassemia patients. The question arises whether these innovative strategies will also be suitable for patients with von Willebrand Disease (VWD). Whilst in and ex vivo delivery to endothelial cells (ECs) has been demonstrated, and CRISPR-Cas9 gene editing has been successful in ECs, there are currently no gene therapy options available for VWD. The wide variety of pathogenic VWF mutations makes development of broadly applicable, cost-effective gene therapies challenging. While delivery of von Willebrand factor (VWF) as a therapeutic transgene would be optimal, the size of VWF challenges efficient delivery. Therefore, treatment of VWD requires targeted, personalized gene therapy; for instance by using the newest CRISPR-Cas technologies which can be tailored to facilitate alteration and restoration of various pathogenic VWD variants. This review describes the inherited bleeding disorder VWD and potential gene therapy approaches for management of the disease. Thereby we are exploring different CRISPR-Cas technologies and recent developments in the field. Moreover, we will discuss the ongoing advances of in vivo delivery systems, all with the scope on ECs.},
}
@article {pmid41057395,
year = {2025},
author = {Osgood, JA and Brown, AC and Burnham, KL and Mielczarek, O and Migliorini, G and Tay, C and Zhang, P and Palmer, MH and Davies, B and Cowley, SA and Knight, JC},
title = {Evidence for enhancer activity in intron 1 of TNFRSF1A using CRISPR/Cas9 in human induced pluripotent stem cell-derived macrophages.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {34885},
pmid = {41057395},
issn = {2045-2322},
support = {20773/VAC_/Versus Arthritis/United Kingdom ; 204969/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; 2018-I2M-2-002//Chinese Academy of Medical Sciences Innovation 537 Fund for Medical Science/ ; LC0910-004//Oxford Martin School, University of Oxford/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Macrophages/metabolism/cytology ; *Introns/genetics ; *Enhancer Elements, Genetic ; *Receptors, Tumor Necrosis Factor, Type I/genetics ; Gene Editing ; Cell Differentiation ; Genome-Wide Association Study ; Gene Expression Regulation ; },
abstract = {TNFα is a common drug target in the treatment of autoimmune diseases, with pro-inflammatory functions that are primarily mediated through its receptor, TNFRSF1A. TNFRSF1A has been genetically associated with many immune-mediated diseases including ankylosing spondylitis, multiple sclerosis, and inflammatory bowel disease. Many of the genetic variants within or near TNFRSF1A that have been associated with disease through genome-wide association studies (GWAS) lie in non-coding regions of the genome. Understanding the functional consequences of these genetic variants is limited by incomplete understanding of TNFRSF1A gene regulation, including for specific cellular contexts relevant to inflammation and immunity such as macrophages. This work used CRISPR/Cas9 in human induced pluripotent stem cells followed by differentiation into macrophages to investigate putative regulatory elements in the TNFRSF1A gene locus. Through gene editing, with functional genomic readouts including the assay for transposase-accessible chromatin using sequencing (ATAC-Seq), chromatin immunoprecipitation with sequencing (ChIP-Seq), and RNA-Seq to assess the consequences of these edits, we present evidence for an enhancer of TNFRSF1A contained within an intron of the gene. Understanding gene regulation and the genomic context in which GWAS variants lie could bring us closer to deconvoluting the genetic basis of common disease aetiology and uncover effective drug targets.},
}
@article {pmid41057263,
year = {2025},
author = {Yang, B and Wang, K},
title = {CRISPR-Cas-Directed Genome Editing in Maize.},
journal = {Cold Spring Harbor protocols},
volume = {},
number = {},
pages = {},
doi = {10.1101/pdb.top108448},
pmid = {41057263},
issn = {1559-6095},
abstract = {Genetic engineering techniques are essential for both plant science and agricultural biotechnology, enabling functional genomics studies, dissection of complex traits, and targeted crop improvement. Among the various genetic tools currently in use, clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based genome editing has emerged as a transformative technology due to its precision, versatility, and ease of use. In particular, CRISPR-Cas9 has become the most widely adopted platform for genome manipulation in plant systems, including maize, owing to its high editing efficiency, multiplexing capabilities, and scalability for diverse applications. This review highlights the biological significance and technical considerations necessary to implement CRISPR-Cas9 in maize. We discuss critical components for successful editing, including the selection of strong and tissue-appropriate promoters for Cas gene and guide RNA expression, codon optimization of Cas nuclease genes, effective guide RNA design, and multiplexing strategies using RNA polymerase III (Pol III)- or Pol II-dependent promoter-driven polycistronic expression systems. Additionally, we provide insights into vector construction methodologies and reliable genotyping techniques to detect and validate genome edits. Together, these elements constitute a practical framework for deploying genome editing in maize research and breeding. By optimizing these parameters, researchers can enhance the efficiency and accuracy of CRISPR-mediated genome modifications, accelerating functional genomic discovery and the development of improved maize varieties tailored to meet future agricultural demands.},
}
@article {pmid41056201,
year = {2025},
author = {Weber, VJ and Reschigna, A and Gerhardt, MJ and Heigl, T and Hinrichsmeyer, KS and van den Engel, S and Otify, DY and Gavrilov, Z and Blaser, F and Meneau, I and Betz, C and Bolz, HJ and Biel, M and Michalakis, S and Becirovic, E},
title = {CRISPR/Cas-mediated activation of genes associated with inherited retinal dystrophies in human cells for diagnostic purposes.},
journal = {JCI insight},
volume = {},
number = {},
pages = {},
doi = {10.1172/jci.insight.189615},
pmid = {41056201},
issn = {2379-3708},
abstract = {Many patients suffering from inherited diseases do not receive a genetic diagnosis and are therefore excluded as candidates for treatments, such as gene therapies. Analyzing disease-related gene transcripts from patient cells would improve detection of mutations that have been missed or misinterpreted in terms of pathogenicity during routine genome sequencing. However, the analysis of transcripts is complicated by the fact that a biopsy of the affected tissue is often not appropriate, and many disease-associated genes are not expressed in tissues or cells that can be easily obtained from patients. Here, using CRISPR/Cas-mediated transcriptional activation (CRISPRa) we developed a robust and efficient approach to activate genes in skin-derived fibroblasts and in freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals. This approach was successfully applied to blood samples from patients with inherited retinal dystrophies (IRD). We were able to efficiently activate several IRD-linked genes and detect the corresponding transcripts using different diagnostically relevant methods such as RT-qPCR, RT-PCR and long- and short-read RNA sequencing. The detection and analysis of known and unknown mRNA isoforms demonstrates the potential of CRISPRa-mediated transcriptional activation in PBMCs. These results will contribute to ceasing the critical gap in the genetic diagnosis of IRD patients and other inherited diseases.},
}
@article {pmid41055650,
year = {2025},
author = {Che, X and Wei, Y and Wang, X and Wang, X and Wu, Z and Deng, J and Ge, S and Liu, X and Cai, Z and Zhang, H and He, L and Xu, J},
title = {Lipoxygenase ZmLOX3 Enhances Salt Tolerance of Maize Under the Regulation of ZmNAC032.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c02349},
pmid = {41055650},
issn = {1520-5118},
abstract = {Lipoxygenase (LOX) plays a critical role in plant biotic and abiotic stress responses by mediating lipid peroxidation and the production of jasmonic acid (JA). In this study, maize ZmLOX3 was identified as a positive regulator in salt stress tolerance. Overexpression of ZmLOX3 enhanced the salt tolerance of Arabidopsis. When maize seedlings were subjected to salt stress, the ZmLOX3[OE] lines exhibited a better growth phenotype than the control (B104) and the zmlox3[CR] (CRISPR/Cas) knockout mutants. Overexpression of ZmLOX3 improved ROS scavenging, Na[+]/K[+] homeostasis, and cell membrane stability. Transcriptome analyses revealed that ZmLOX3[OE] triggered the expression of genes involved in both the JA synthesis and signaling pathways. A transcription factor ZmNAC032 was identified via Y1H screening and was able to bind to the C[A/G]CG[T/G] sequence in the ZmLOX3 promoter and activate its expression. These findings are helpful for deciphering the function and regulatory status of ZmLOX in improving salt tolerance.},
}
@article {pmid41054568,
year = {2025},
author = {Xia, B and Wang, Z and Fei, T and Ma, Y and Guo, Y and Fei, D and Shu, X and Zhao, G and Ma, M and Yuan, H},
title = {Development and application of a CRISPR/Cas12a-based reverse transcription-recombinase polymerase amplification assay with lateral flow dipstick and fluorescence detection for Getah virus.},
journal = {PeerJ},
volume = {13},
number = {},
pages = {e20119},
pmid = {41054568},
issn = {2167-8359},
mesh = {*CRISPR-Cas Systems ; *Alphavirus/isolation &amp; purification/genetics ; Animals ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Fluorescence ; *Alphavirus Infections/diagnosis/virology/veterinary ; Swine ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Getah virus (GETV), a mosquito-borne alphavirus classified as a zoonotic disease, primarily infects livestock, particularly pigs and horses. In recent years, it has re-emerged in multiple Asian countries, posing a potential threat to animal husbandry and public health. In this study, we developed a rapid and sensitive GETV detection method based on reverse transcription-recombinase polymerase amplification (RT-RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system combined with a lateral flow dipstick (LFD) for visual readout. By leveraging sequence conservation in the GETV E2 envelope protein-coding regions, we engineered matched crRNA guides and amplification primers to develop a rapid CRISPR-Cas12a diagnostic workflow. The optimized platform combines RT-RPA (42 °C/20 min) with Cas12a's trans-nuclease activity, permitting multiplex detection via real-time fluorescence quantification or immunochromatographic strip visualization. Analytical evaluation demonstrated a detection capability of 10 copies/µL and exclusive specificity against four pathogen controls, including Japanese encephalitis virus and pseudorabies virus. Validation performed using simulated clinical samples revealed 100% concordance between the results of RT-RPA-CRISPR/Cas12a-LFD and quantitative polymerase chain reaction (PCR), while reducing the total detection time to 50 minutes. This approach eliminated the need for advanced instrumentation owing to its simplified operational design, enabling field-deployable rapid detection capabilities that establish essential technical infrastructure for initiating timely GETV containment measures. This approach has broad application potential in the fields of food safety, clinical diagnostics, and environmental science.},
}
@article {pmid41026092,
year = {2025},
author = {Toyonishi, G and Nakazawa, T and Koshi, D and Horii, M and An, GH and Kawauchi, M and Honda, Y},
title = {CRISPR/Cas9-directed disruption of wc-2 leads to the absence of fruiting body development in Pleurotus ostreatus.},
journal = {FEMS microbiology letters},
volume = {372},
number = {},
pages = {},
doi = {10.1093/femsle/fnaf104},
pmid = {41026092},
issn = {1574-6968},
support = {18KK0178//Japan Society for the Promotion of Science/ ; 22H00380//Japan Society for the Promotion of Science/ ; 22KK0090//Japan Society for the Promotion of Science/ ; PJ0175072025//Rural Development Administration/ ; },
mesh = {*Pleurotus/genetics/growth &amp; development/radiation effects ; *Fruiting Bodies, Fungal/growth &amp; development/genetics/radiation effects ; *CRISPR-Cas Systems ; *Fungal Proteins/genetics/metabolism ; Light ; },
abstract = {Light, particularly blue light, is a key environmental factor that induces fruiting in certain agaricomycetes. In this study, we characterized mutant strains of Pleurotus ostreatus with disrupted wc-2, which encodes one of the white-collar proteins, Wc-2, to investigate the role of light in fruiting in P. ostreatus. We introduced two different plasmids containing expression cassettes for Cas9 and two different gRNAs targeting wc-2 separately into the dikaryotic P. ostreatus strain PC9×#64. Among the 11 dikaryotic hygromycin-resistant transformants, six strains did not form fruiting bodies. Genomic PCR followed by sequencing analysis suggested that all six fruitless strains were dikaryotic wc-2 disruptants. Small aggregate structures were not observed in the dikaryotic wc-2 disruptants grown under light conditions, as in PC9×#64 grown in a red box. These results suggest that Wc-2 is essential for the initiation of blue light-induced fruiting in P. ostreatus.},
}
@article {pmid41002250,
year = {2025},
author = {Gillmore, JD and Gane, E and Täubel, J and Pilebro, B and Echaniz-Laguna, A and Kao, J and Litchy, W and Shahda, S and Haagensen, A and Walsh, L and Smith, D and Kachadourian, J and Ward, JH and Lebwohl, D and Zhu, P and Xu, Y and Leung, A and Sonderfan, A and Gutstein, DE and Manvelian, G and Adams, D},
title = {Nexiguran Ziclumeran Gene Editing in Hereditary ATTR with Polyneuropathy.},
journal = {The New England journal of medicine},
volume = {393},
number = {14},
pages = {1375-1386},
doi = {10.1056/NEJMoa2510209},
pmid = {41002250},
issn = {1533-4406},
mesh = {Humans ; *Amyloid Neuropathies, Familial/therapy/genetics ; Male ; Female ; Middle Aged ; *Prealbumin/genetics/metabolism/antagonists &amp; inhibitors ; Adult ; Aged ; *Gene Editing ; *Genetic Therapy/adverse effects/methods ; CRISPR-Cas Systems ; Neurofilament Proteins/blood ; Body Mass Index ; },
abstract = {BACKGROUND: Hereditary transthyretin amyloidosis with polyneuropathy (ATTRv-PN) is a rare, multisystem, progressive, debilitating, and fatal disease characterized by tissue deposition of misfolded transthyretin (TTR) in peripheral nerves. Nexiguran ziclumeran (nex-z) is an investigational in vivo therapy based on CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats and associated Cas9 endonuclease) that is designed to reduce serum TTR levels through selective inactivation of TTR in the liver.<br><br>METHODS: In this phase 1, open-label study, we administered one infusion of nex-z to patients with ATTRv-PN. Primary objectives included assessment of the safety and pharmacodynamics of nex-z. Secondary end points included changes in the familial amyloid polyneuropathy stage, polyneuropathy disability score, serum neurofilament light chain (NfL) level, modified body-mass index (modified BMI, defined as the conventional BMI [weight in kilograms divided by square of height in meters] multiplied by the albumin level in grams per liter), and modified Neuropathy Impairment Score+7 (mNIS+7; range, 0 to 304, with higher scores indicating more impairment).<br><br>RESULTS: A total of 36 patients received nex-z; the mean follow-up was 27 months. The mean percent change from baseline in the serum TTR level was -90% at day 28, which was sustained through month 24 (-92%). Treatment-related adverse events included transient infusion-related reactions (in 21 patients), decreased thyroxine level without hypothyroidism or elevated thyrotropin level (in 8), and headache (in 4). One participant died from cardiac amyloidosis, and one withdrew owing to progressive decline in motor function. Serious adverse events were reported in 11 patients. At month 24, the familial amyloid polyneuropathy stage and polyneuropathy disability score remained stable in 29 and 27 patients, respectively; improved in 2 and 5, respectively; and worsened in 2 and 2, respectively. The mean change in the serum NfL level was -9.0 pg per milliliter, and the change in the modified BMI was 24.7. The mean change from baseline in the mNIS+7 was -8.5.<br><br>CONCLUSIONS: A single administration of nex-z in patients with ATTRv-PN was associated with rapid, deep, and durable reductions in serum TTR levels. The results support further investigation of nex-z to treat ATTRv-PN. (Funded by Intellia Therapeutics and Regeneron Pharmaceuticals; ClinicalTrials.gov number, NCT04601051.).},
}
@article {pmid40990297,
year = {2025},
author = {Sheng, J and Dong, Y and Sun, S and Zhang, Y and Li, C and Xu, X and Wang, H},
title = {Construction of a Sensing Platform Integrated with a CRISPR/Cas12a-Triggered Colorimetric Strategy for the Quantitative Detection of Meat Freshness.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {40},
pages = {25604-25614},
doi = {10.1021/acs.jafc.5c04851},
pmid = {40990297},
issn = {1520-5118},
mesh = {*Colorimetry/methods/instrumentation ; *Meat/analysis/microbiology ; CRISPR-Cas Systems ; Animals ; *Pseudomonas/genetics/isolation &amp; purification/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Biosensing Techniques/instrumentation/methods ; *Endodeoxyribonucleases/genetics/metabolism ; CRISPR-Associated Proteins ; },
abstract = {Monitoring microbial determinants, such as Pseudomonas spp., is thus essential for assessing meat freshness. Here, a novel colorimetric sensing platform based on magnetic enzyme-labeled nanoparticles combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a without nucleic acid molecule preamplification was developed for detecting meat freshness. Under optimal conditions, a high-specificity crRNA was systematically verified, and the colorimetric sensor could accurately quantify Pseudomonas spp. loads with levels ranging from 1 × 10[3.7] to 1 × 10[8.7] CFU/mL, with a color change from colorless to yellow. A smart colorimetric platform, including a self-designed image acquisition device and self-programmed image analysis software, was developed and applied to the integrated determination of meat freshness by using the B-value in the RGB channel. The platform has been applied to both consumers and producers and has been validated by 48 actual samples of chilled meat. These findings provide new insights into the exploration of reliable tools for monitoring meat freshness.},
}
@article {pmid40985907,
year = {2025},
author = {Fei, S and Zhang, C and Zhang, X and Xie, Y and Fu, S and Wu, J},
title = {An Ultrasensitive Immunocapture (IC)-RPA-CRISPR/Cas12a Assay with Three Readout Modes for Detecting Xanthomonas oryzae pv. oryzicola of Rice Bacterial Leaf Streak.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {40},
pages = {25664-25675},
doi = {10.1021/acs.jafc.5c04360},
pmid = {40985907},
issn = {1520-5118},
mesh = {*Oryza/microbiology ; *Xanthomonas/genetics/isolation &amp; purification ; *Plant Diseases/microbiology ; CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; Plant Leaves/microbiology ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Xanthomonas oryzae pv oryzicola (Xoc) is the causal agent of rice bacterial leaf streak (BLS) and causes enormous losses of rice yields in many countries every year. Development of sensitive diagnostic techniques is crucial for its prevention and control. Here, we developed an ultrasensitive IC-RPA-CRISPR/Cas12a assay with three readout modes [qPCR machine, UV lamp, and lateral flow strip (LFS)] for Xoc detection in rice, which combined advantages of immunocapture, recombinase polymerase amplification (RPA), and CRISPR/Cas12a-based cleavage. Especially, the immunocapture step allows to capture and enrich Xoc from samples, which minimizes the interference from rice debris to benefit nucleic acid release and amplification and enhances the specificity and sensitivity of this assay. The detection limits of its three readout modes for Xoc bacterial suspension is 2, 6, and 60 CFU/mL, respectively. Collectively, this study provides a specific, ultrasensitive, practical approach for quarantine and detection of Xoc that will benefit the prevention and control of BLS.},
}
@article {pmid40970715,
year = {2025},
author = {Zhao, C and Li, G and Shen, C and Xie, Y and Chen, Y and Ying, X and Chen, Y and Zhang, C},
title = {An extraction-free and one-pot two-temperature CRISPR/Cas12b system for visual detection of Group B Streptococcus by integrating with RPA.},
journal = {Journal of clinical microbiology},
volume = {63},
number = {10},
pages = {e0081925},
doi = {10.1128/jcm.00819-25},
pmid = {40970715},
issn = {1098-660X},
support = {2024KY1444//Medical and Health Research Project of Zhejiang Province/ ; LTGC23H200004//Zhejiang Provincial Natural Science Foundation of China/ ; },
mesh = {*Streptococcus agalactiae/isolation &amp; purification/genetics ; Humans ; *Streptococcal Infections/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Female ; Recombinases/metabolism ; Temperature ; Vagina/microbiology ; },
abstract = {UNLABELLED: Group B Streptococcus (GBS) is a major cause of neonatal infections, and rapid detection is essential for timely clinical intervention. In this study, we developed an extraction-free, one-pot CRISPR/Cas12b assay for visual detection of GBS by combining with isothermal amplification, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). The results showed that LAMP-CRISPR/Cas12b outperformed RPA-CRISPR/Cas12b system across all template concentrations, especially in low-copy template (30 and 10 copies/test) detection. To enhance the detection performance of RPA-CRISPR/Cas12b, we introduced a two-temperature protocol, with RPA reaction at 39°C followed by Cas12b activation at 62°C. Through the two-temperature approach, the detection rate of RPA-CRISPR/Cas12b system was significantly improved even in low-copy samples, achieving a sensitivity of 10 copies/test (1 copy/μL). Clinical validation using 60 vaginal-rectal swab samples showed 96.7% and 98.3% of concordance when compared to culture and qPCR methods, respectively. This assay offers a rapid (<1 h), highly sensitive, and user-friendly solution without requiring nucleic acid extraction or sophisticated instruments. Its compatibility with visual signal detection makes it ideal for point-of-care testing, especially in low-resource or time-sensitive settings. The platform can be adapted for broader pathogen detection in future field diagnostics.<br><br>IMPORTANCE: This study presents a rapid, convenient, and highly accurate method for Group B Streptococcus (GBS) detection by integrating the CRISPR/Cas12b system with recombinase polymerase amplification, an isothermal nucleic acid amplification technique. To streamline the workflow, we established a one-pot, extraction-free assay that significantly reduces the detection time. Through the systematic optimization of the dual-temperature conditions, we enhanced the amplification efficiency of target DNA, thereby improving the sensitivity of the CRISPR/Cas12b system. Additionally, the incorporation of a UV-visible detection system enables visual readout, facilitating instrument-free testing suitable for point-of-care (POC) applications.},
}
@article {pmid40899880,
year = {2025},
author = {Liu, P and Zhang, J and Gong, Y and Liu, W and Xiao, G and Liang, J and Wang, X and Bi, J and Zhang, G},
title = {Application of engineered CRISPR/Cas12a variants with altered protospacer adjacent motif specificities for the detection of isoniazid resistance mutations in Mycobacterium tuberculosis.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0016525},
pmid = {40899880},
issn = {2165-0497},
mesh = {*Isoniazid/pharmacology ; *Mycobacterium tuberculosis/genetics/drug effects/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Antitubercular Agents/pharmacology ; Bacterial Proteins/genetics ; Humans ; *Drug Resistance, Bacterial/genetics ; Mutation ; Tuberculosis, Multidrug-Resistant/microbiology/diagnosis ; *Endodeoxyribonucleases/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Catalase/genetics ; Sensitivity and Specificity ; CRISPR-Associated Proteins/genetics ; Microbial Sensitivity Tests ; },
abstract = {UNLABELLED: Drug-resistant tuberculosis (TB) is a major global public health concern. Although isoniazid is currently considered one of the most effective first-line drugs for TB treatment, its efficacy is limited by the emergence of resistance. Therefore, it is imperative to develop new methods for detecting drug-resistant TB. In this study, we developed a nucleic acid detection system based on the clustered regularly interspaced short palindromic repeat (CRISPR) Cas12a_RR protein. The system combines recombinase polymerase amplification with an engineered CRISPR/Cas12a_RR protein to enable rapid and specific detection of the katG G944C mutation in isoniazid-resistant Mycobacterium tuberculosis (Mtb). It could detect the target DNA at concentrations as low as 1% in a mixed sample. Compared with TaqMan quantitative polymerase chain reaction and DNA sequencing, the CRISPR/Cas12a_RR system demonstrated superior detection performance in terms of sensitivity, specificity, and cost-effectiveness. Furthermore, it effectively differentiated between drug-resistant Mtb strains from wild-type Mtb strains in clinically isolated samples, with the entire detection process completed in 60 min. In conclusion, the CRISPR/Cas12a_RR detection system offers a novel, rapid, simple, sensitive, and specific approach for identifying isoniazid-resistant Mtb, with significant potential for clinical application, particularly in resource-limited settings.<br><br>IMPORTANCE: This study presents a novel method for detecting isoniazid-resistant Mycobacterium tuberculosis (Mtb) using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a mutants, offering rapid detection, cost-effectiveness, and high specificity, and thereby providing a promising new avenue for detecting isoniazid-resistant Mtb.},
}
@article {pmid40866699,
year = {2025},
author = {Pacesa, M and Nickel, L and Schellhaas, C and Schmidt, J and Pyatova, E and Kissling, L and Barendse, P and Choudhury, J and Kapoor, S and Alcaraz-Serna, A and Cho, Y and Ghamary, KH and Vinué, L and Yachnin, BJ and Wollacott, AM and Buckley, S and Westphal, AH and Lindhoud, S and Georgeon, S and Goverde, CA and Hatzopoulos, GN and Gönczy, P and Muller, YD and Schwank, G and Swarts, DC and Vecchio, AJ and Schneider, BL and Ovchinnikov, S and Correia, BE},
title = {One-shot design of functional protein binders with BindCraft.},
journal = {Nature},
volume = {646},
number = {8084},
pages = {483-492},
pmid = {40866699},
issn = {1476-4687},
support = {R35 GM138368/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; Gene Editing ; Protein Binding ; CRISPR-Cas Systems/genetics ; Allergens/immunology/metabolism/chemistry ; Immunoglobulin E/immunology/metabolism ; *Protein Engineering/methods ; *Proteins/metabolism/chemistry ; Receptors, Cell Surface/metabolism/chemistry ; Binding Sites ; Models, Molecular ; CRISPR-Associated Protein 9/metabolism ; Animals ; },
abstract = {Protein-protein interactions are at the core of all key biological processes. However, the complexity of the structural features that determine protein-protein interactions makes their design challenging. Here we present BindCraft, an open-source and automated pipeline for de novo protein binder design with experimental success rates of 10-100%. BindCraft leverages the weights of AlphaFold2 (ref. [1]) to generate binders with nanomolar affinity without the need for high-throughput screening or experimental optimization, even in the absence of known binding sites. We successfully designed binders against a diverse set of challenging targets, including cell-surface receptors, common allergens, de novo designed proteins and multi-domain nucleases, such as CRISPR-Cas9. We showcase the functional and therapeutic potential of designed binders by reducing IgE binding to birch allergen in patient-derived samples, modulating Cas9 gene editing activity and reducing the cytotoxicity of a foodborne bacterial enterotoxin. Last, we use cell-surface-receptor-specific binders to redirect adeno-associated virus capsids for targeted gene delivery. This work represents a significant advancement towards a 'one design-one binder' approach in computational design, with immense potential in therapeutics, diagnostics and biotechnology.},
}
@article {pmid40862592,
year = {2025},
author = {Shen, Y and Yi, C and Wang, H and Tang, Y and Li, J},
title = {Development of a rapid and sensitive RPA-CRISPR/Cas12a-based assay for the detection of Brucella melitensis.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0099825},
pmid = {40862592},
issn = {2165-0497},
mesh = {*Brucellosis/diagnosis/microbiology ; *Brucella melitensis/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases/genetics/metabolism ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; Reproducibility of Results ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Brucellosis, a zoonotic disease caused by Brucella species, presents significant public health challenges due to its complex diagnosis and the limited availability of rapid detection methods. To address these challenges, we developed a novel detection method that integrates recombinase polymerase amplification (RPA) with the CRISPR/Cas12a system, enabling dual readout through fluorescence (FL) and lateral flow strip (LFS) detection. The RPA-CRISPR/Cas12a-FL assay demonstrated an impressive detection limit of 1 copy/μL, which is 10 times more sensitive than quantitative polymerase chain reaction, while the RPA-CRISPR/Cas12a-LFS method achieved a detection limit of 10 copies/μL, comparable to nested PCR. Specificity testing confirmed the robustness of the assay, as it produced strong signals exclusively for Brucella without cross-reactivity with other bacterial species. Clinical validation using serum samples from 24 confirmed brucellosis patients and six healthy controls demonstrated a 100% concordance with serological results, underscoring the reliability of this method for clinical applications. This assay provides a rapid, sensitive, and specific tool for Brucella detection, suitable for both laboratory and field settings, and holds significant potential for enhancing the diagnosis and control of brucellosis.IMPORTANCEBrucellosis is a significant zoonotic disease, and rapid and accurate diagnosis is crucial for its treatment and control. To address this need, we developed a novel detection method that combines recombinant enzyme polymerase amplification with a CRISPR/Cas12a system, achieving dual readout through fluorescence and lateral flow strips. The test demonstrates excellent sensitivity and specificity, with clinical validation confirming complete concordance with serological results. This approach offers a fast, reliable, and field-deployable solution for brucellosis diagnosis, significantly enhancing disease management and public health outcomes.},
}
@article {pmid40856920,
year = {2025},
author = {Thiel, J and Sürün, D and Brändle, DC and Teichert, M and Künzel, SR and Friedrich, U and Dahl, A and Schubert, K and Rzagalinski, I and Shevchenko, A and Traikov, S and Mirtschink, P and Wagenführ, L and Buchholz, F and Hölig, K and Tonn, T and Kronstein-Wiedemann, R},
title = {Knock Out of miRNA-30a-5p and Reconstitution of the Actin Network Dynamics Partly Restores the Impaired Terminal Erythroid Differentiation during Blood Pharming.},
journal = {Stem cell reviews and reports},
volume = {21},
number = {8},
pages = {2637-2653},
pmid = {40856920},
issn = {2629-3277},
support = {530364326//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Cell Differentiation/genetics ; *Actins/metabolism ; *Erythroid Cells/metabolism/cytology ; *Erythropoiesis/genetics ; Proto-Oncogene Mas ; Hematopoietic Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Erythrocytes/metabolism/cytology ; bcl-X Protein/metabolism/genetics ; Gene Knockout Techniques ; Cell Line ; },
abstract = {In vitro red blood cell (RBC) production offers a promising complement to conventional blood donation, particularly for patients with rare blood types. Previously, we developed imBMEP-A, the first erythroid cell line derived from reticulocyte progenitors, which maintains robust hemoglobin expression and erythroid differentiation in the presence of erythropoietin (EPO) despite its immortalized state. However, clinical translation remains hindered by the inability to scale up production due to impaired in vitro enucleation of RBC progenitor cell lines. Enhancing enucleation efficiency in imBMEP-A cells involved CRISPR/Cas9-mediated knockout (K.O.) of miR-30a-5p, a key enucleation inhibitor, moderately increasing rates to 3.3 ± 0.4%- 8.9 ± 1.7%. Further investigation of enucleation inefficiencies led to transcriptome and proteome comparisons between imBMEP-miR30a-K.O. cells and hematopoietic stem cells (HSCs). These analyses revealed altered gene expression and protein abundances linked to metabolic transitions, apoptosis promotion, and cytoskeletal regulation. Notably, forced expression of the proto-oncogene c-Myc, required for cell immortalization, emerged as a key driver of these physiological changes. Counteracting these effects required optimization of imBMEP-A cells by activating BCL-XL transcription and knocking out SCIN, which encodes the actin-severing protein scinderin. While BCL-XL is upregulated in normal erythropoiesis, it is downregulated in imBMEP-A. Conversely, SCIN, typically absent in erythroid cells, is highly expressed in imBMEP-A, disrupting actin organization. These interventions improved viability, restored actin network formation, and increased terminal erythropoiesis, yielding 22.1 ± 1.7% more orthochromatic erythroblasts. These findings establish a foundation for optimizing imBMEP-A cells for therapeutic use and advancing the understanding the pathophysiology of erythroleukemia.},
}
@article {pmid40833103,
year = {2025},
author = {Omachi, R and Imai, K and Sato, A and Tanaka, M and Mizushina, H and Takeuchi, K and Maeda, T},
title = {Development and clinical evaluation of a novel SHERLOCK test for Mycoplasma genitalium.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0044525},
pmid = {40833103},
issn = {2165-0497},
support = {24K10540//KAKEN/ ; 2024 Research grant//Charitable Trust Laboratory Medicine Research Foundation of Japan/ ; Grant-in-Aid for Young Researchers//Saitama Medical University Hospital/ ; },
mesh = {*Mycoplasma genitalium/genetics/isolation &amp; purification ; Humans ; *Mycoplasma Infections/diagnosis/microbiology/urine ; Male ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Urethritis/microbiology/diagnosis ; *Molecular Diagnostic Techniques/methods ; Japan ; Point-of-Care Testing ; DNA, Bacterial/genetics ; CRISPR-Cas Systems ; },
abstract = {Mycoplasma genitalium (MG) is a sexually transmitted pathogen associated with urethritis. Nucleic acid amplification tests are the gold standard for its diagnosis but often require specialized equipment, which limits their use in point-of-care testing. This study aimed to develop a rapid, sensitive detection method for MG using a specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) test, which combines isothermal recombinase polymerase amplification and a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a reaction. We developed a novel SHERLOCK test targeting the Mg219 gene in MG. The SHERLOCK method was evaluated using 128 first-void urine samples collected from male patients who were suspected of MG urethritis in Japan. The results of SHERLOCK were compared to those of the cobas TV/MG test and in-house quantitative PCR. SHERLOCK was optimized for use with crude DNA extracted from clinical urine samples. The results were detected via a lateral flow assay, allowing for visual interpretation within 60 min. The method demonstrated a limit of detection of 10 copies/reaction and showed no cross-reactivity with other pathogens. In clinical evaluations of 128 urine samples, SHERLOCK showed an overall agreement rate of 91.4% with the cobas TV/MG PCR test; the positive and negative agreement rates were 79.6 and 100%, respectively. SHERLOCK showed superior performance to quantitative PCR. This study demonstrates that the novel SHERLOCK assay for MG has potential as a point-of-care test in the clinical setting. Further evaluation in prospective studies is needed to confirm its clinical value.IMPORTANCEMycoplasma genitalium (MG) is a causative agent of sexually transmitted infections and is associated with urethritis and prostatitis in men. To prevent the transmission of MG, it is essential to identify infected individuals through diagnostic testing and provide appropriate treatment. Nucleic acid amplification tests are commonly used for MG diagnosis in the clinical setting, but the point-of-care testing (POCT) for MG remains limited. In this study, we developed a novel nucleic acid amplification test-specific high-sensitivity enzymatic reporter unlocking (SHERLOCK)-for MG, combining crude DNA extraction with a lateral flow assay. Our SHERLOCK assay successfully detected MG in approximately 1 h, with a detection limit of 10 copies/reaction. Clinical evaluations using urine samples showed a high agreement rate with the cobas TV/MG test. SHERLOCK is expected to be a useful tool for POCT for MG.},
}
@article {pmid40819286,
year = {2025},
author = {Beck, CW and Reily-Bell, M and Bicknell, LS},
title = {Unilateral loss of recql4 function in Xenopus laevis tadpoles leads to ipsilateral ablation of the forelimb, hypoplastic Meckel's cartilage, and vascular defects.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {10},
pages = {},
doi = {10.1093/g3journal/jkaf179},
pmid = {40819286},
issn = {2160-1836},
support = {PRG1732//Neurological Foundation of New Zealand/ ; },
mesh = {Animals ; *Xenopus laevis/genetics ; Larva/genetics ; *RecQ Helicases/genetics/metabolism ; *Forelimb/abnormalities ; *Cartilage/abnormalities/metabolism ; *Xenopus Proteins/genetics/metabolism ; Gene Knockdown Techniques ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {RECQL4 encodes a RecQ helicase, one of a family of DNA unwinding enzymes with roles in DNA replication, double-strand break repair, and genomic stability. Pathogenic variants in RECQL4 are clinically associated with 3 rare autosomal recessive conditions: Rothmund-Thomson syndrome type II, Baller-Gerold syndrome, and RAPADILINO syndrome. These 3 syndromes show overlapping growth retardation, low bone density, and skeletal defects affecting the arms and hands. Here, we take advantage of the ability to generate one-sided CRISPR knockdowns of recql4 in Xenopus laevis tadpoles. Tadpoles develop normally until feeding starts, after which growth slows on the edited side, leading to a curved posture, smaller eyes (microphthalmia), and reduced head size (microcephaly). Forelimb buds fail to develop, leading to complete absence of the forelimb on the edited side. Additionally, Meckel's cartilage (lower jaw) ossification is absent or reduced and the hyoid cartilage is smaller, but this is not due to deficiencies in cranial neural crest migration on the edited side. Knockdown of recql4 also results in hypoplastic vasculature, with reduced branching from the aorta on the edited side. Taken together, our results clearly show the utility of unilateral CRISPR editing in Xenopus for understanding the specific phenotypic developmental effects of mutations affecting cell proliferation.},
}
@article {pmid40794772,
year = {2025},
author = {Després, PC and Gervais, NC and Fogal, M and Rogers, RKJ and Cuomo, CA and Shapiro, RS},
title = {Targeted loss of heterozygosity in Candida albicans using CRISPR-Cas9 reveals the functional impact of allelic variation.},
journal = {Genetics},
volume = {231},
number = {2},
pages = {},
doi = {10.1093/genetics/iyaf154},
pmid = {40794772},
issn = {1943-2631},
support = {//FRQS/ ; //NSERC/ ; //N.C.G./ ; //M.F./ ; PJT 162195//CIHR/ ; //R.S.S./ ; //CIFAR/ ; //Fungal Kingdom/ ; //C.A.C./ ; U19 AI110818/AI/NIAID NIH HHS/United States ; //Canada Research Chair/ ; },
mesh = {*Candida albicans/genetics/drug effects ; *Loss of Heterozygosity ; *CRISPR-Cas Systems ; Alleles ; Genome, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; Drug Resistance, Fungal/genetics ; Gene Editing ; Genetic Variation ; Antifungal Agents/pharmacology ; },
abstract = {The diploid genome of the fungal pathogen Candida albicans is highly heterozygous, with most allele pairs diverging at either the coding or regulatory level. When faced with selection pressure like antifungal exposure, this hidden genetic diversity can provide a reservoir of adaptive mutations through loss of heterozygosity (LOH) events. Validating the potential phenotypic impact of LOH events observed in clinical or experimentally evolved strains can be difficult due to the challenge of precisely targeting one allele over the other. Here, we show that a CRISPR-Cas9 system can be used to overcome this challenge. By designing allele-specific guide RNA sequences, we can induce targeted, directed LOH events, which we validate by whole-genome long-read sequencing. Using this approach, we efficiently recapitulate a recently described LOH event that increases resistance to the antifungal fluconazole. Additionally, we find that the recombination tracts of these induced LOH events have similar lengths to those observed naturally. To facilitate future use of this method, we provide a database of allele-specific sgRNA sequences for Cas9 that provide near genome-wide coverage of heterozygous sites through either direct or indirect targeting. This approach will be useful in probing the adaptive role of LOH events in this important human pathogen.},
}
@article {pmid40758833,
year = {2025},
author = {Stuecker, TN and Hood, SE and Molina Pineda, J and Lenaduwe, S and Winter, J and Sadhu, MJ and Lewis, JA},
title = {Improved vectors for retron-mediated CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.},
journal = {G3 (Bethesda, Md.)},
volume = {15},
number = {10},
pages = {},
doi = {10.1093/g3journal/jkaf175},
pmid = {40758833},
issn = {2160-1836},
support = {P20 GM103429/GM/NIGMS NIH HHS/United States ; MCB-1941824//National Science Foundation/ ; /HG/NHGRI NIH HHS/United States ; 1ZIAHG200401/GF/NIH HHS/United States ; //Arkansas IDeA Network of Biomedical Research Excellence/ ; //Summer Research Fellowship/ ; P20 GM103429/GM/NIGMS NIH HHS/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Vectors/genetics ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Fungal ; Escherichia coli/genetics ; },
abstract = {In vivo site-directed mutagenesis is a powerful genetic tool for testing the effects of specific alleles in their normal genomic context. While the budding yeast Saccharomyces cerevisiae possesses classical tools for site-directed mutagenesis, more efficient recent CRISPR-based approaches use Cas "cutting" combined with homologous recombination of a "repair" template that introduces the desired edit. However, current approaches are limited for fully prototrophic yeast strains and rely on relatively low-efficiency cloning of short gRNAs. We were thus motivated to simplify the process by combining the gRNA and its cognate repair template in cis on a single oligonucleotide. Moreover, we wished to take advantage of a new approach that uses an Escherichia coli retron (EcRT) to amplify repair templates as multi-copy single-stranded (ms)DNA in vivo, which are more efficient templates for homologous recombination. To this end, we have created a set of plasmids that express Cas9-EcRT, allowing for co-transformation with the gRNA-repair template plasmid in a single step. Our suite of plasmids contains different antibiotic (Nat, Hyg, Kan) or auxotrophic (HIS3, URA3) selectable markers, allowing for editing of fully prototrophic wild yeast strains. In addition to classic galactose induction, we generated a β-estradiol-inducible version of each plasmid to facilitate editing in yeast strains that grow poorly on galactose. The plasmid-based system results in >95% editing efficiencies for point mutations and >50% efficiencies for markerless deletions, in a minimum number of steps and time. We provide a detailed step-by-step guide on how to use this system.},
}
@article {pmid40495695,
year = {2025},
author = {Wang, H and Zhan, H and Pan, B and Zeng, L and Chen, Z and Liu, S and Zhang, Q and Hong, X and Lu, J and Lin, X and Zhao, X and Lai, J and Jie, K and Li, Y and Zhong, J and Peng, S and Chen, S and Chen, C and Zhong, W and Wu, S and Pan, Y and Lin, T and Chen, X},
title = {Engineering CRISPR System-Based Bacterial Outer Membrane Vesicle Potentiates T Cell Immunity for Enhanced Cancer Immunotherapy.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {37},
number = {39},
pages = {e2501565},
doi = {10.1002/adma.202501565},
pmid = {40495695},
issn = {1521-4095},
support = {2018YFA0902800//National Key Research and Development Program of China/ ; 82322056//National Natural Science Foundation of China/ ; 82341018//National Natural Science Foundation of China/ ; 82072827//National Natural Science Foundation of China/ ; 82273421//National Natural Science Foundation of China/ ; 82303405//National Natural Science Foundation of China/ ; 82472137//National Natural Science Foundation of China/ ; 2023A03J0718//Science and Technology Program of Guangzhou/ ; 2024B03J1234//Science and Technology Program of Guangzhou/ ; 2024A04J6558//Science and Technology Program of Guangzhou/ ; //Fundamental Research Funds for the Central Universities/ ; 23ykbj002//Sun Yat-sen University/ ; 2020B1111170006//Guangdong Provincial Clinical Research Centre for Urological Diseases/ ; 2020B1212060018//Guangdong Science and Technology Department/ ; 2018B030317001//Guangdong Science and Technology Department/ ; 2017B030314026//Guangdong Science and Technology Department/ ; },
mesh = {Animals ; Mice ; *Immunotherapy/methods ; *T-Lymphocytes/immunology ; Humans ; Cell Line, Tumor ; *Bacterial Outer Membrane/metabolism ; *CRISPR-Cas Systems ; *Neoplasms/therapy/immunology ; Escherichia coli/genetics ; Interleukin-12/genetics ; Chemokine CXCL9/genetics ; Genetic Engineering ; },
abstract = {Immune checkpoint blockade (ICB) therapy has revolutionized cancer treatment but only benefits a subset of patients because of insufficient infiltration and inactivation of effector T cells. Bacterial outer membrane vesicles (OMVs) can activate immunity and deliver therapeutic agents for immunotherapy. However, efficiently targeting and packaging therapeutic molecules into OMVs remains challenging. Here, the engineered E. coli BL21-derived OMVs enable the packaging of multiple genes, resulting in a 7-fold increase in DNA enrichment efficiency and gene silencing in vitro. Moreover, the engineered OMVs carrying genes encoding CXCL9 and IL12 (OMV-C9I12) reprogram tumor cells to secrete these factors, significantly enhancing T-cell chemotaxis and activation. More importantly, this system markedly inhibits tumors, extends survival, and synergizes with anti-PD-1/PD-L1 therapy in murine MB49 and B16F10 tumor models. Single-cell RNA sequencing (scRNA-seq) further reveals significant upregulation of T-cell chemotaxis and activation-related pathways following OMV-C9I12 treatment. Finally, OMV-C9I12 potentiates T cell-mediated immunotherapy and suppresses the growth of bladder and breast cancer tumors in humanized mouse models. These findings highlight the potential of this engineered OMV platform for cancer gene therapy and provide novel strategies to overcome resistance to immunotherapy.},
}
@article {pmid40436751,
year = {2025},
author = {Bold-Erdene, A and Miura, K and Yamasaki, N and Miura, S and Ogata, S and Sasatani, M and Yamamoto, T and Kaminuma, O},
title = {Effect of gamma-ray exposure on the genome-editing efficiency of improved genome-editing via oviductal nucleic acids delivery (i-GONAD).},
journal = {Experimental animals},
volume = {74},
number = {4},
pages = {457-462},
doi = {10.1538/expanim.25-0036},
pmid = {40436751},
issn = {1881-7122},
mesh = {Animals ; Female ; *Gamma Rays/adverse effects ; *Gene Editing/methods ; Pregnancy ; Mice ; *Oviducts ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded/radiation effects ; *Nucleic Acids/administration &amp; dosage ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {DNA double-strand breaks (DSBs) are among the most hazardous cellular damages, potentially leading to cell death or oncogenesis if unrepaired. Genome editing methods, such as the CRISPR/Cas9 system, induce DSBs and utilize these repair pathways for gene knockout and knock-in. Although ionizing radiation also induces DSBs, it is not clear whether the efficiency of genome editing is affected by ionizing radiation. This study investigated the impact of gamma-ray exposure on the genome editing efficiency of the improved genome editing via oviductal nucleic acid delivery (i-GONAD) method. Gamma-rays were exposed to pregnant mice receiving i-GONAD targeting the Hr gene, whose mutation causes hair loss in mice. The exposure on the fertilization day (Day 0) decreased natural delivery rates and litter sizes, with notable effects at 0.3 Gy or higher. Although the proportions of hairless offspring obtained by i-GONAD differed greatly between single-guide RNAs (sgRNAs) used, total mutation rates, including hairless, mosaic, and indel, were equivalent. Gamma-ray exposure on Day 0 and the day after fertilization (Day 1) similarly and almost dose-dependently enhanced the genome editing efficiency evaluated by the total mutation rate. This study suggests the improvement of genome editing efficiency by gamma-ray exposure, at least in i-GONAD method, potentially facilitating the creation of diverse experimental animal models.},
}
@article {pmid40029006,
year = {2025},
author = {Mattoscio, D and Baeza, LA and Bai, H and Colangelo, T and Castagnozzi, S and Marzotto, M and Cufaro, MC and Lotti, V and Yuan, YC and Mucci, M and Si, L and Zuccarini, M and Tredicine, M and D'Orazio, S and Pieragostino, D and Del Boccio, P and Sorio, C and Trerotola, M and Romano, M and Plebani, R},
title = {Inflammation and epithelial-mesenchymal transition in a CFTR-depleted human bronchial epithelial cell line revealed by proteomics and human organ-on-a-chip.},
journal = {The FEBS journal},
volume = {292},
number = {19},
pages = {5086-5104},
doi = {10.1111/febs.70050},
pmid = {40029006},
issn = {1742-4658},
support = {AT2021 to Roberto Plebani//Italian Ministry of Health (fund ex60%)/ ; AT2022 to Roberto Plebani//Italian Ministry of Health (fund ex60%)/ ; AIRC; #19548 to Tommaso Colangelo//Associazione Italiana per la Ricerca sul Cancro/ ; MFAG 2022 - ID. 27060 to Domenico Mattoscio//Associazione Italiana per la Ricerca sul Cancro/ ; //Fondazione Umberto Veronesi/ ; GMSG#01/2023 to Roberto Plebani//Fondazione per la Ricerca sulla Fibrosi Cistica/ ; FFC#11/2022 to Domenico Mattoscio//Fondazione per la Ricerca sulla Fibrosi Cistica/ ; },
mesh = {Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; Proteomics/methods ; *Epithelial-Mesenchymal Transition/genetics ; *Epithelial Cells/metabolism/pathology ; *Inflammation/pathology/genetics/metabolism ; *Cystic Fibrosis/pathology/genetics/metabolism ; *Bronchi/pathology/metabolism/cytology ; Cell Line ; Cell Movement/genetics ; Lab-On-A-Chip Devices ; CRISPR-Cas Systems ; Neutrophils/metabolism/pathology ; Signal Transduction ; Microphysiological Systems ; },
abstract = {Cystic fibrosis (CF) is a genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, leading to chronic, unresolved inflammation of the airways due to uncontrolled recruitment of polymorphonuclear leukocytes (PMNs). Evidence indicates that CFTR loss-of-function, in addition to promoting a pro-inflammatory phenotype, is associated with an increased risk of developing cancer, suggesting that CFTR can exert tumor-suppressor functions. Three-dimensional (3D) in vitro culture models, such as the CF lung airway-on-a-chip, can be suitable for studying PMN recruitment, as well as events of cancerogenesis, that is epithelial cell invasion and migration, in CF. To gather insight into the pathobiology of CFTR loss-of-function, we generated CFTR-knockout (KO) clones of the 16HBE14o- human bronchial cell line by CRISPR/Cas9 gene editing, and performed a comparative proteomic analysis of these clones with their wild-type (WT) counterparts. Systematic signaling pathway analysis of CFTR-KO clones revealed modulation of inflammation, PMN recruitment, epithelial cell migration, and epithelial-mesenchymal transition. Using a latest-generation organ-on-a-chip microfluidic platform, we confirmed that CFTR-KO enhanced PMN recruitment and epithelial cell invasion of the endothelial layer. Thus, a dysfunctional CFTR affects multiple pathways in the airway epithelium that ultimately contribute to sustained inflammation and cancerogenesis in CF.},
}
@article {pmid41053552,
year = {2025},
author = {Qin, Z and Surnido, W and Mizuta, H and Uji, T},
title = {Stable transgene expression and CRISPR-mediated knock-in system of a bacteria-derived antibiotic selection gene in the green alga Ulva prolifera.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1323},
pmid = {41053552},
issn = {1471-2229},
support = {JPMJOP1851//Japan Science and Technology Agency/ ; },
mesh = {*Ulva/genetics/drug effects ; *Gene Knock-In Techniques/methods ; *Transgenes/genetics ; Kanamycin Kinase/genetics ; CRISPR-Cas Systems ; Hygromycin B/pharmacology ; Anti-Bacterial Agents/pharmacology ; Edible Seaweeds ; },
abstract = {Ulva prolifera is a fast-growing green seaweed that has garnered considerable interest in both fundamental and applied research. Here, we established a molecular tool by employing a selectable marker gene that allowed the isolation of U. prolifera cells integrating exogenous DNA. We developed a modular plasmid for expressing exogenous genes in U. prolifera based on the bacterial antibiotic-resistance marker, aminoglycoside phosphotransferase gene (aph7"). Integration of aph7" in macroalgae can generate transformants resistant to hygromycin B. In addition, we characterized the promoter region of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase gene (pUpRbcS) to drive the expression of aph7". The transcripts were consistently confirmed from antibiotic-selected transformants, stably retaining the exogenous gene in the succeeding generations. Subsequently, a CRISPR-based knock-in system was established, facilitating the integration of aph7" cassette in the endogenous selection gene encoding for adenine phosphoribosyltransferase (UpAPT). APT gene can serve as an endogenous marker in algae that exhibits a lethal phenotype under cultivation with 2-fluoroadenine. The resulting knock-in mutants could resist the co-selection of the antibiotic hygromycin B and 2-fluoroadenine. Our results advance U. prolifera as a genetic platform, enabling functional research to elucidate Ulva biology, and to bring forth biotechnological utilization of algal resources.},
}
@article {pmid41053297,
year = {2025},
author = {Vanderperre, B and Muraleedharan, A and Dorion, MF and Larroquette, F and Del Cid Pellitero, E and Rajakulendran, N and Chen, CX and Larivière, R and Michaud-Tardif, C and Goiran, T and Chidiac, R and Lipuma, D and MacLeod, G and Thomas, R and Wang, Z and Reintsch, WE and Luo, W and Shlaifer, I and Zhang, F and Xia, K and Steinhart, Z and Linhardt, RJ and Trempe, JF and Liu, J and Durcan, TM and Angers, S and Fon, EA},
title = {Novel regulators of heparan sulfate proteoglycans modulate cellular uptake of α-synuclein fibrils.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1426},
pmid = {41053297},
issn = {2399-3642},
support = {021129//Michael J. Fox Foundation for Parkinson's Research (Michael J. Fox Foundation)/ ; MFE-152571//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/ ; Canada Research Chair (Tier 1) in Parkinson's disease//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/ ; },
mesh = {*alpha-Synuclein/metabolism/genetics ; Humans ; *Heparan Sulfate Proteoglycans/metabolism ; Golgi Apparatus/metabolism ; Animals ; CRISPR-Cas Systems ; Cation Transport Proteins/genetics/metabolism ; Mice ; },
abstract = {Synucleinopathies are characterized by the accumulation and propagation of α-synuclein (α-syn) aggregates throughout the brain, leading to neuronal dysfunction and death. In this study, we used an unbiased FACS-based genome-wide CRISPR/Cas9 knockout screening to identify genes that regulate the entry and accumulation of α-syn preformed fibrils (PFFs) in cells. We identified key genes and pathways specifically implicated in α-syn PFFs intracellular accumulation, including heparan sulfate proteoglycans (HSPG) biosynthesis and Golgi trafficking. All confirmed hits affected heparan sulfate (HS), a post-translational modification known to act as a receptor for proteinaceous aggregates including α-syn and tau. Intriguingly, deletion of SLC39A9 and C3orf58 genes, encoding respectively a Golgi-localized exporter of Zn[2+], and the Golgi-localized putative kinase DIPK2A, specifically impaired the uptake of α-syn PFFs, by preventing the binding of PFFs to the cell surface. Mass spectrometry-based analysis of HS chains in SLC39A9[-/-] and C3orf58[-/-] cells indicated major defects in HS homeostasis. Additionally, Golgi accumulation of NDST1, a prime HSPG biosynthetic enzyme, was detected in C3orf58[-/-] cells. Interestingly, C3orf58[-/-] human iPSC-derived microglia and dopaminergic neurons exhibited a strong reduction in their ability to internalize α-syn PFFs. Altogether, our data identifies new modulators of HSPGs that regulate α-syn PFFs cell surface binding and uptake.},
}
@article {pmid41051826,
year = {2025},
author = {McCallum, GE and Ho, SFS and Cummins, EA and Wildsmith, AJ and McInnes, RS and Weigel, C and Tong, LYS and Quick, J and van Schaik, W and Moran, RA},
title = {The Kocurious case of Noodlococcus: genomic insights into Kocuria rhizophila from characterisation of a laboratory contaminant.},
journal = {Microbial genomics},
volume = {11},
number = {10},
pages = {},
doi = {10.1099/mgen.0.001526},
pmid = {41051826},
issn = {2057-5858},
mesh = {*Genome, Bacterial ; Phylogeny ; Genomics ; *Micrococcaceae/genetics/classification/isolation &amp; purification ; Whole Genome Sequencing ; Sequence Analysis, DNA ; },
abstract = {The laboratory contaminant strain Noodlococcus was named for its coccoid cells and unusual colony morphology, which resembled a pile of noodles. Along with laboratory characterisation and electron microscopy, we generated a complete Noodlococcus genome sequence using Illumina and Oxford Nanopore data. The genome consisted of a single, circular, 2,732,108 bp chromosome that shared 97.5% average nucleotide identity (ANI) with the Kocuria rhizophila type strain TA68. We identified genomic features involved in replication (oriC), carotenoid synthesis (crt) and genome defence (CRISPR-Cas) and discovered four novel mobile elements (ISKrh4-7). Despite its environmental ubiquity and relevance to food production, bioremediation and human medicine, there have been few genomic studies of the Kocuria genus. We conducted a comparative, phylogenetic and pangenomic examination of all 257 publicly available Kocuria genomes, with a particular focus on the 56 that were identified as K. rhizophila. We found that there are two phylogenetically distinct clades of K. rhizophila, with within-clade ANI values of 96.7-100.0% and between-clade values of 89.5-90.4%. The second clade, which we refer to as Kocuria pseudorhizophila, exhibited ANI values of <95% relative to TA68 and should constitute a separate species. Delineation of the two clades would be consistent with the rest of the genus, where all other species satisfy the 95% ANI threshold criteria. Differences in the K. rhizophila and K. pseudorhizophila pangenomes likely reflect phenotypic as well as evolutionary divergence. This distinction is relevant to clinical and industrial settings, as strains and genomes from both clades are currently used interchangeably, which may lead to reproducibility issues and phenotype-genotype discordance. Investigating an innocuous laboratory contaminant has therefore provided useful insights into the understudied species K. rhizophila, prompting an unexpected reassessment of its taxonomy.},
}
@article {pmid41051673,
year = {2026},
author = {Kumar, P and Verma, V and Irfan, M},
title = {Target-Specific Single Guide RNA (sgRNA) Design and In Vitro Validation of Target-Specific sgRNAs for CRISPR/Cas9-Mediated Editing of a Plant Stress Memory-Associated Gene.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2988},
number = {},
pages = {145-156},
pmid = {41051673},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Arabidopsis/genetics ; *Stress, Physiological/genetics ; Arabidopsis Proteins/genetics ; Transcription Factors/genetics ; },
abstract = {CRISPR/Cas9 genome editing has emerged as a transformative tool in plant biology, enabling precise manipulation of genes involved in stress responses. In the context of plant stress memory, where prior exposure to environmental stress enhances subsequent stress tolerance. CRISPR-based approaches offer a powerful means to dissect and engineer underlying regulatory genes. A critical factor determining the success of CRISPR/Cas9 editing is the careful design and validation of single guide RNAs (sgRNAs), which guide the Cas9 nuclease to specific genomic targets. This chapter provides a detailed, step-by-step protocol for the design, in vitro transcription, and in vitro cleavage assay to check efficiency of target-specific sgRNAs for plant genome editing applications. As a case study, we describe the design and validation of sgRNAs targeting the Arabidopsis thaliana DREB2A gene, a key transcription factor associated with drought stress memory. Emphasis is placed on strategies to maximize on-target efficiency, minimize off-target effects, and assess sgRNA functionality in vitro prior to in planta applications. This chapter serves as a practical guide for researchers aiming to functionally characterize stress memory-associated genes using CRISPR/Cas9 technology.},
}
@article {pmid41051363,
year = {2025},
author = {Zhytnik, L and Ventura, L and Sclocco, A and Verhage, M and Bakker, AD and Shim, JH and Beaino, W and Pereira, PM and Hoogeland, ME and Heine, VM and Maas, H and Jaspers, RT and Niehoff, A and Zaucke, F and de Waard, V and Eekhoff, EMW and Micha, D},
title = {New Lens On Congenital Mild Bone Fragility: a Novel Col1a1 Knockout Mouse Model for Osteogenesis Imperfecta Type 1.},
journal = {Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research},
volume = {},
number = {},
pages = {},
doi = {10.1093/jbmr/zjaf138},
pmid = {41051363},
issn = {1523-4681},
abstract = {Osteogenesis imperfecta (OI) is a genetic disorder characterized by bone fragility. It is one of the most prevalent rare skeletal dysplasias. The mildest form, OI type 1, predominantly results from collagen type I haploinsufficiency due to pathogenic variants in the COL1A1 gene, leading to reduced collagen type I. Despite OI type 1 representing approximately half of the OI population, the lack of an effective mouse model has hindered research and therapy development(1). To address this gap, we developed a genetically engineered mouse model harbouring a heterozygous deletion of the Col1a1 allele using the CRISPR/Cas system. The bone phenotype was characterised in 8- and 24-week-old mice, assessing transcriptomics and serum markers for bone formation (procollagen type I N-terminal propeptide) and resorption (tartrate-resistant acid phosphatase 5b). Bone volume, microarchitecture, and strength were evaluated by micro-computed tomography, histomorphometry and three-point bending test. We showed that the decreased Col1a1 to Col1a2 mRNA ratio determines reduced collagen type I production in OI mice bones as the underlying mechanism of haploinsufficient OI. This was supported by COL1A1 to COL1A2 mRNA ratio findings in human OI cell models, including fibroblasts and induced mesenchymal stem cells, as well as in induced pluripotent and mesenchymal stem cell models that were edited to carry a heterozygous COL1A1 allele. Our findings indicate for the first time that reduced bone volume and altered bone microarchitecture in haploinsufficient OI depends on the Col1a1 to Col1a2 mRNA ratio regulation. This novel mouse model faithfully recapitulates OI type 1 and provides a vital tool for investigating the disease mechanism and developing targeted therapeutic strategies for this large neglected OI patient population.},
}
@article {pmid41048399,
year = {2025},
author = {Huang, S and Qin, H and Dai, B and Liu, M and Shen, J},
title = {Establishment and evaluation of a circAdpgk-0001 knockdown method using CRISPR-Cas13d RNA-targeting technology.},
journal = {PeerJ},
volume = {13},
number = {},
pages = {e20123},
pmid = {41048399},
issn = {2167-8359},
mesh = {*RNA, Circular/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Knockdown Techniques/methods ; RNA, Small Interfering/genetics ; Humans ; Cell Line ; Actins/genetics/metabolism ; },
abstract = {BACKGROUND: The small interfering RNA (siRNA) method has been used to knock down circular RNAs (circRNAs). However, issues such as low efficiency and off-target effects have become increasingly recognized. Recent studies have demonstrated that CRISPR-Cas13 can specifically target and cleave RNA. In this study, we established a CRISPR-Cas13d-based RNA-targeting method to specifically knock down circRNAs, such as circAdpgk-0001, and compared its performance with the siRNA method.<br><br>METHODS: Four clustered regularly interspaced short palindromic repeats (CRISPR) RNAs (crRNAs) of different nucleotide lengths spanning the back-splicing junction (BSJ) of circAdpgk-0001 were designed. A CRISPR-RfxCas13d plasmid capable of specifically cleaving circAdpgk-0001 was constructed and transfected into the JS-1 cell line. Knockdown efficiency was assessed using quantitative real-time PCR (qRT-PCR) and compared with that of the siRNA method. The expression of activation-related factors alpha-smooth muscle actin (&#x3b1;-SMA) and collagen I in JS-1 cells was further evaluated using qRT-PCR and Western blot.<br><br>RESULTS: CRISPR-Cas13d with a 24-nucleotide crRNA showed the highest knockdown efficiency (&#x223c;50%). After further optimization, the knockdown efficiency of CRISPR-Cas13d reached 70%, significantly higher than that of the siRNA method (40%). Knockdown of circAdpgk-0001 using Cas13d reduced the expression of collagen I and &#x3b1;-SMA by approximately 40%, which was greater than the reduction achieved by siRNA-mediated knockdown.<br><br>CONCLUSION: CRISPR-Cas13d demonstrated higher efficiency than the siRNA method in knocking down circRNAs, providing a promising tool for investigating circRNA functions.},
}
@article {pmid41020566,
year = {2025},
author = {Xue, J and Mao, K and Tang, Z and Hu, J and Zhang, H},
title = {Machine-Learning-Assisted CRISPR/Cas12a Biosensors for Monitoring Organophosphorus Pesticide Degradation.},
journal = {Analytical chemistry},
volume = {97},
number = {39},
pages = {21491-21501},
doi = {10.1021/acs.analchem.5c03596},
pmid = {41020566},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Pesticides/analysis/metabolism ; *CRISPR-Cas Systems ; *Organophosphorus Compounds/analysis/metabolism ; *Machine Learning ; *Water Pollutants, Chemical/analysis/metabolism ; Smartphone ; Acetylcholinesterase/metabolism ; Manganese Compounds/chemistry ; Oxides/chemistry ; Bacterial Proteins/metabolism/genetics ; Limit of Detection ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Owing to the severe environmental and health issues posed by organophosphorus pesticides (OPs), a dual-enzyme cascade biosensing platform based on manganese dioxide (MnO2) and CRISPR/Cas12a was developed in this study. Smartphones were innovatively integrated with a stacked ensemble learning (SEL) model for ultrasensitive detection and dynamic monitoring of OPs in environmental water samples. A dual-enzyme cascade signal amplification strategy was used to construct this sensing platform. Acetylcholinesterase (AChE) catalyzes the generation of thiocholine (TCh), which, in turn, regulates the degradation of MnO2 nanosheets, releasing Cas12a activators and generating a fluorescence signal. Owing to the irreversible inhibition of AChE activity by OPs, dichlorvos (DDVP) was successfully detected, with a detection limit as low as 4.62 pg/mL. Additionally, the SEL model, integrated into the smartphone biosensing platform and incorporating random forest (RF), XGBoost, and ridge regression algorithms, exhibited strong performance in detecting OPs after optimization (R[2] = 0.9985). In real water samples, the SEL model achieved a recovery rate of 93.1-103.1%, and the degradation kinetics of DDVP were successfully monitored over 24 h, revealing significant differences in DDVP degradation rates across various water matrices. This study is the first to report the integration of CRISPR/Cas12a biosensing technology with an SEL model-driven smartphone detection platform, providing a novel approach for sensitive, portable, and intelligent monitoring of OPs and offering new insights for water quality monitoring and early detection of environmental risks.},
}
@article {pmid40923283,
year = {2025},
author = {Man, Y and Posey, RR and Bai, H and Jiang, A and Dosta, P and Ocampo-Alvarado, D and Plebani, R and Ji, J and Belgur, C and Artzi, N and Ingber, DE},
title = {Preclinical assessment of pan-influenza A virus CRISPR RNA therapeutics in a human lung alveolus chip.},
journal = {Lab on a chip},
volume = {25},
number = {20},
pages = {5240-5254},
doi = {10.1039/d5lc00156k},
pmid = {40923283},
issn = {1473-0189},
mesh = {Humans ; *RNA, Viral/genetics ; *Pulmonary Alveoli/virology/cytology ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Influenza A virus/genetics ; *Influenza A Virus, H3N2 Subtype/genetics ; *Lab-On-A-Chip Devices ; Influenza, Human/therapy ; },
abstract = {CRISPR technology offers an entirely new approach to therapeutic development because it can target specific nucleotide sequences with high specificity, however, preclinical animal models are not useful for evaluation of their efficacy and potential off-target effects because of high gene sequence variations between animals and humans. Here, we explored the potential of using the CRISPR effector Cas13 to develop a new therapeutic approach for influenza A virus (IAV) infections based on its ability to specifically and robustly cleave single-strand viral RNA using a complementary CRISPR RNA (crRNA). We engineered crRNAs to target highly conserved regions in the IAV genome to create a potential pan-viral treatment strategy. A human lung alveolus chip (Lung Chip) lined by human primary alveolar epithelial cells interfaced with human primary pulmonary microvascular endothelial cells and infected with a pandemic IAV H3N2 strain was used to evaluate the on-target and off-target effects of these antiviral crRNA therapeutics. Our data show that the crRNAs targeting highly conserved regions in the IAV genome potently reduced viral replication in the alveolar airspace in the Lung Chip, and this was accompanied by suppression of the human host inflammatory response as indicated by a significant reduction in cytokine production and recruitment of immune cells. Importantly, only minimal off-target effects were observed based on transcriptomic analyses. As these crRNAs inhibit replication of influenza H1N1 and H3N2 in A549 cells as well as H3N2 in Lung Chips, these findings support use of CRISPR-Cas13 as a potentially viable approach to develop pan-IAV therapeutics for combating future influenza pandemics. The results also demonstrate that human Organ Chips be useful as more clinically relevant preclinical models for testing the efficacy and safety of crRNA therapeutics.},
}
@article {pmid40910953,
year = {2025},
author = {Feng, D and Guo, J and Yan, J and Chen, J and Ding, L and Zhu, X and Chen, Z and Hu, Y and Zhang, M and Liu, J and Zhu, C and Liu, M and Zhao, C and Zhang, X and Xu, J},
title = {COG6 is an essential host factor for influenza A virus infection.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0136225},
doi = {10.1128/spectrum.01362-25},
pmid = {40910953},
issn = {2165-0497},
support = {2022YFC2604100//National Key Research and Development Program of China/ ; 2023YFC2605602//National Key Research and Development Program of China/ ; 82072273//National Natural Science Foundation of China/ ; },
mesh = {*Influenza A virus/physiology/genetics ; Humans ; Virus Replication ; Golgi Apparatus/metabolism ; Lysosomes/metabolism ; Animals ; *Influenza, Human/virology/metabolism ; *Host-Pathogen Interactions ; *Adaptor Proteins, Vesicular Transport/metabolism/genetics ; Viral Proteins/metabolism ; CRISPR-Cas Systems ; Madin Darby Canine Kidney Cells ; Dogs ; HEK293 Cells ; A549 Cells ; Virus Internalization ; },
abstract = {Influenza A virus (IAV) relies on the host cellular machinery to support its replication. Understanding these host dependencies can inform the development of novel antiviral strategies. In this study, we identified conserved oligomeric Golgi complex subunit 6 (COG6) as a novel host factor critical for IAV replication through a genome-wide clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) knockout screen. Disruption of COG6 significantly impaired viral replication. Mechanistically, COG6 supports IAV replication via two distinct means. First, consistent with the role of the COG complex in Golgi homeostasis, COG6 is required for the proper presentation of surface sialic acids, the primary receptor for IAV entry. Second, COG6 deficiency unexpectedly led to lysosome-dependent degradation of viral proteins. Notably, lysosomal activity was also upregulated in IAV-infected wild-type cells, albeit to a lesser extent than in COG6-deficient cells. Treatment with lysosomal inhibitors rescued viral protein stability in COG6 knockout cells. Protein interaction analysis further demonstrated that COG6-mediated stabilization of viral proteins did not rely on viral protein-COG6 interaction, refuting the hypothesis that COG6 acts as a shield factor to protect viral protein from lysosomal degradation. Moreover, knockout of other COG subunits produced similar antiviral effects, suggesting that an intact COG complex is required for IAV replication. Together, these findings uncover a critical role of the COG complex in regulating IAV replication and highlight a previously unappreciated functional link between the Golgi and lysosomes that could be exploited for treating IAV infections.IMPORTANCEDespite advances in virology, numerous host determinants facilitating influenza A virus (IAV) pathogenesis remain uncharacterized. Our study establishes conserved oligomeric Golgi complex subunit 6 (COG6) as a critical host factor promoting IAV infection through complementary mechanisms: receptor modulation and viral protein stabilization. This represents the first demonstration that the COG complex regulates viral pathogenesis through proteostasis mechanisms, fundamentally expanding our understanding of host-virus interactions at the organelle interface. These findings not only provide new perspectives on viral exploitation of Golgi trafficking networks but also identify potential therapeutic targets against evolving influenza strains.},
}
@article {pmid40865226,
year = {2025},
author = {Zhang, X and Zhao, J and Dong, Y and Zhang, C and Hu, X and Wang, S and Chen, Y},
title = {Magnetic relaxation switching biosensor based on CRISPR-mediated cascade reaction for the amplification-free detection of Salmonella.},
journal = {Journal of hazardous materials},
volume = {497},
number = {},
pages = {139664},
doi = {10.1016/j.jhazmat.2025.139664},
pmid = {40865226},
issn = {1873-3336},
mesh = {*Biosensing Techniques/methods ; *Salmonella typhimurium/isolation &amp; purification/genetics ; Alkaline Phosphatase/chemistry ; *CRISPR-Cas Systems ; Food Microbiology ; Limit of Detection ; Magnetite Nanoparticles/chemistry ; DNA, Single-Stranded ; DNA, Bacterial/analysis ; Food Contamination/analysis ; },
abstract = {Sensitive and accurate detection of foodborne pathogens is crucial for preventing foodborne outbreaks and ensuring public health safety, but challenged by extremely low infectious doses of many foodborne pathogens and matrix interference effects. In this study, we developed a magnetic relaxation switching biosensor boosted by CRISPR/Cas12a-mediated enzymatic cascade reaction (CMCR-MRS) for amplification-free detection of Salmonella typhimurium (S. typhimurium). CRISPR/Cas12a was designed to precisely target the pathogen-specific DNA and efficiently cleaved single-stranded DNA (ssDNA) immobilized on the magnetic nanoparticle-alkaline phosphatase (MNP-ALP) probes through trans-cleavage activity. Subsequently, the released ALP facilitated the conversion of paramagnetic Mn(VII) to Mn(II), which resulted in the change of transverse relaxation time (T2), achieving a high signal-to-background ratio with superior biocompatibility and minimal background interference, making them highly advantageous for sensitive detection in complex biological samples. Our assay showed a broad dynamic range from 40 to 10[7] CFU/mL and a limit of detection (LOD) of 10 CFU/mL for S. typhimurium without amplification. Furthermore, it has been successfully validated in real food samples, demonstrating strong consistency (R[2] = 0.989) with the quantitative real-time polymerase chain reaction (qPCR) test. CMCR-MRS can serve as a highly effective and reliable strategy for achieving sensitive and accurate pathogen detection.},
}
@article {pmid40843899,
year = {2025},
author = {Stuible, M and Alpuche-Lazcano, SP and Gervais, C and Ouimet, M and Lippens, J and Pagé, M and Morasse, A and Moraitis, AN and Durocher, Y},
title = {Endogenous Retrovirus-Like Particle-Deficient CHO Cells Can be Generated by CRISPR or shRNA and Enriched Based on Cell-Surface Expression of Retroviral Envelope Protein.},
journal = {Biotechnology and bioengineering},
volume = {122},
number = {11},
pages = {3192-3204},
doi = {10.1002/bit.70043},
pmid = {40843899},
issn = {1097-0290},
support = {//The authors received no specific funding for this work./ ; },
mesh = {CHO Cells ; Cricetulus ; Animals ; *Viral Envelope Proteins/genetics/metabolism ; *RNA, Small Interfering/genetics/metabolism ; *Endogenous Retroviruses/genetics ; *CRISPR-Cas Systems ; Cricetinae ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Despite evidence that they are not functional or infective, retrovirus-like particles (RVLPs), originating from endogenous proviral sequences in Chinese hamster ovary (CHO) cells, present a safety risk for biotherapeutics manufactured using this cell line due to their resemblance to other mammalian leukemia viruses. Here, we demonstrate that CRISPR- and shRNA-based cell engineering strategies can be used to disrupt RVLP production by targeting the RVLP nucleotide sequences. Additionally, specific antibodies were generated to monitor RVLP protein expression, including RVLP envelope (Env) protein localized on the surface of CHO cells, greatly facilitating selection of RVLP-deficient clones. These modified CHO cells showed reduced RVLP production while maintaining or enhancing the ability to produce recombinant virus-like particles (VLPs), highlighting their potential application in biomanufacturing, especially for complex biologics that are incompatible with standard RVLP mitigation procedures, namely viral inactivation and nanofiltration.},
}
@article {pmid40829398,
year = {2025},
author = {Song, D and Xu, C and Sang, P and Liu, Y and Huang, X},
title = {Rapid and contamination-free detection of cucumber green mottle mosaic virus as a viral indicator in wastewater via UDG-RT-LAMP combined with CRISPR/Cas12a.},
journal = {Journal of hazardous materials},
volume = {497},
number = {},
pages = {139571},
doi = {10.1016/j.jhazmat.2025.139571},
pmid = {40829398},
issn = {1873-3336},
mesh = {*Wastewater/virology ; CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Tobamovirus/isolation &amp; purification/genetics ; Uracil-DNA Glycosidase/genetics ; Biosensing Techniques ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; Molecular Diagnostic Techniques ; },
abstract = {The removal of viruses by wastewater treatment plants plays a pivotal role in ensuring water environment safety, where precise evaluation of elimination efficiency is essential for controlling viral dissemination. Current bacterial indicators exhibit limited correlations with virological safety parameters, whereas conventional viral detection methods face practical constraints such as high instrumentation requirements and long detection cycles. To overcome these limitations, this study presents an on-site detection method for cucumber green mottle mosaic virus (CGMMV) in wastewater as a viral indicator, integrating uracil-DNA glycosylase (UDG)-reverse transcription loop-mediated isothermal amplification (RT-LAMP) with CRISPR/Cas12a (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 12a)-mediated biosensing system. The developed method achieved effective prevention of aerosol contamination from residual amplicons via the dUTP-UDG system, rapid amplification via RT-LAMP, and improved sensitivity and visualization by CRISPR/Cas12a-mediated biosensing system. The potential of CGMMV as a viral indicator in wastewater treatment process was demonstrated, and the rapid detection was realized using the proposed method. This integrated approach achieves sensitive CGMMV detection (limit of detection of 1.13 copies/μL) within 35 min, demonstrating field applicability through equipment independence, contamination resistance, and rapid operation. The proposed assay offers a promising tool for rapid viral monitoring in wastewater treatment system for resource-limited settings.},
}
@article {pmid40810567,
year = {2025},
author = {Ji, C and Ru, L and Han, T and Mai, G and Zheng, L and Jiang, Y},
title = {VGRCOT: a one-tube visual detection method for group B Streptococcus combining RPA and CRISPR/Cas12a for point-of-care testing in reproductive health.},
journal = {Microbiology spectrum},
volume = {13},
number = {10},
pages = {e0139525},
doi = {10.1128/spectrum.01395-25},
pmid = {40810567},
issn = {2165-0497},
support = {2025ZNSFSC1561//Natural Science Foundation of Sichuan Province/ ; 2024SZY001//Key Research and Developmet Guidance Projects of Deyang City/ ; },
mesh = {Humans ; *Streptococcal Infections/diagnosis/microbiology ; *Streptococcus agalactiae/genetics/isolation &amp; purification ; *Point-of-Care Testing ; *CRISPR-Cas Systems ; Female ; Pregnancy ; Sensitivity and Specificity ; Pregnancy Complications, Infectious/diagnosis/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Group B Streptococcus (GBS) is a significant pathogen that causes perinatal infections, seriously threatening the health of pregnant women and newborns. Prophylactic antibiotic treatment for pregnant women who screen positive for GBS can notably reduce the incidence and fatality of neonatal infections. Herein, we developed a visual nucleic acid method for GBS that integrates RPA and CRISPR/Cas12a in a one-tube setup, termed VGRCOT. The VGRCOT method achieved one-tube detection by adding the appropriate reagents to the bottom and lid of the EP tube, respectively. By rigorous optimization of ssDNA-FQ reporter concentration, crRNA concentration, RPA reaction time, and CRISPR/Cas12a cleavage time, VGRCOT can exhibit fluorescence under ultraviolet light, enabling visual detection. Under optimal conditions, VGRCOT has a satisfactory selectivity, and the detection limit was determined as 10[1] copies/reaction. Finally, VGRCOT also showed good performance comparable to qPCR in the actual detection of clinical specimens. Due to its ease of operation and convenient signal acquisition, VGRCOT shows promise for point-of-care testing in reproductive health.IMPORTANCEThis study presents a convenient, sensitive, and accurate visual detection method (VGRCOT) for GBS, combining RPA and CRISPR/Cas12a in a single reaction vessel. Through optimization of experimental conditions, VGRCOT enables detection within 60 min, with a minimum detection limit of 10[1] copies per reaction. VGRCOT offers several advantages by adding the appropriate reagents to the bottom and lid of the EP tube. The one-tube visualization method effectively prevents aerosol contamination, simplifies procedures, and enables visual detection without complex instruments, making it ideal for resource-limited environments. Additionally, its editable crRNA and the use of commonly available laboratory reagents allow for easy reprogramming to detect various pathogens, supporting scalable and low-cost batch production.},
}
@article {pmid40645879,
year = {2025},
author = {Liang, Y and Tong, S and Zhang, J and Tan, GY and Zhang, L and Lee, SY and Tong, Y},
title = {Expanding horizons of CRISPR applications beyond genome editing.},
journal = {Trends in genetics : TIG},
volume = {41},
number = {10},
pages = {934-953},
doi = {10.1016/j.tig.2025.06.003},
pmid = {40645879},
issn = {0168-9525},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Biosensing Techniques ; Single-Cell Analysis ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technologies have rapidly evolved beyond genome editing, transforming fields such as molecular diagnostics, biosensing, transcriptional regulation, molecular imaging, protein interaction mapping, and single-cell analysis. Emerging CRISPR-based diagnostics harness the collateral cleavage activity of CRISPR-associated (Cas) enzymes for rapid nucleic acid detection. Advanced biosensors extend CRISPR's capabilities to detect ions, metabolites, and proteins by integrating synthetic biology components. Catalytically inactive Cas proteins enable precise gene regulation and live-cell imaging of nucleic acids, whereas CRISPR-guided proximity labeling has revolutionized the mapping of biomolecular interactions. Recent single-cell CRISPR screens provide unprecedented resolution of cellular heterogeneity. Future research will focus on overcoming current limitations. The integration of CRISPR technologies with artificial intelligence (AI), spatial omics, and microfluidics is expected to further amplify their impact.},
}
@article {pmid40032999,
year = {2025},
author = {Sahu, SU and Castro, M and Muldoon, JJ and Asija, K and Wyman, SK and Krishnappa, N and de Oñate, L and Eyquem, J and Nguyen, DN and Wilson, RC},
title = {Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) in primary human immune cells and hematopoietic stem cells.},
journal = {Nature protocols},
volume = {20},
number = {10},
pages = {2735-2770},
pmid = {40032999},
issn = {1750-2799},
support = {K08 AI153767/AI/NIAID NIH HHS/United States ; L40 AI140341/AI/NIAID NIH HHS/United States ; K08AI153767//U.S. Department of Health &amp; Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; UG3AI150552//U.S. Department of Health &amp; Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; L40AI140341//U.S. Department of Health &amp; Human Services | NIH | Office of Extramural Research, National Institutes of Health (OER)/ ; },
mesh = {Humans ; *Hematopoietic Stem Cells/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Peptides/chemistry ; T-Lymphocytes/metabolism ; Cells, Cultured ; },
abstract = {Peptide-enabled ribonucleoprotein delivery for CRISPR engineering (PERC) is a new approach for ex vivo genome editing of primary human cells. PERC uses a single amphiphilic peptide reagent to mediate intracellular delivery of the same pre-formed CRISPR ribonucleoprotein enzymes that are broadly used in research and therapeutics, resulting in high-efficiency editing of stimulated immune cells and cultured hematopoietic stem and progenitor cells (HSPCs). PERC facilitates nuclease-mediated gene knockout, precise transgene knock-in and base editing. The protocol involves mixing the CRISPR ribonucleoprotein enzyme with peptide and then incubating with cultured cells. For efficient transgene knock-in, adeno-associated virus (AAV) homology-directed repair template (HDRT) DNA may be included. In contrast to electroporation, PERC is appealing because it needs no dedicated hardware and has less impact on cell phenotype and viability. Because of the gentle nature of PERC, delivery can be performed multiple times without substantial impact to cell health or phenotype. Editing efficiencies can surpass 90% when using either Cas9 or Cas12a in primary T cells or HSPCs. After 3 h dedicated to reagent preparation, the PERC delivery step can be completed in 1 h, with the associated cell culture steps taking 3-7 d total. Because the protocol calls for only three readily available reagents (protein, RNA and peptide) and does not require dedicated hardware for any step, PERC demands no special expertise and is exceptionally straightforward to adopt. The inherent compatibility of PERC with established cell engineering pipelines makes the protocol appealing for rapid deployment in research and clinical settings.},
}
@article {pmid41046988,
year = {2025},
author = {Chen, Y and Qi, ZD and Ji, R and Shi, N and Chen, H and Wei, DX},
title = {Synthetic biology for scalable production of medical polyhydroxyalkanoates: Advances and applications.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108722},
doi = {10.1016/j.biotechadv.2025.108722},
pmid = {41046988},
issn = {1873-1899},
abstract = {Polyhydroxyalkanoates (PHAs), characterized by their biodegradability and biocompatibility, present a promising, sustainable alternative to conventional synthetic polymers for biomedical applications. This study highlights the diversity of PHA monomers and structures, controllable biodegradability, and excellent biocompatibility, emphasizing their suitability for tissue engineering (bone, skin, cardiovascular, oral), anti-hair loss treatments, and drug delivery systems. Significant advancements in synthetic biology, encompassing CRISPR/Cas genome editing, promoter engineering, ribosome binding site optimization, metabolic pathway fine-tuning, and morphology engineering, have led to substantial improvements in PHA production efficiency and a reduction in associated costs. The adoption of next-generation industrial biotechnology (NGIB) using halophiles further enhances economic viability and simplifies the production process. The current commercial landscape and the future prospects of medical-grade PHAs, poised to become mainstream biodegradable materials, are also critically discussed.},
}
@article {pmid41046905,
year = {2025},
author = {Jaballah, SA and Ali, LM and Jehad, MA and Akhlaq, S and Rizvi, TA and Mustafa, F},
title = {Retroviral Vector Technology for Gene Therapy: History, Current Landscape, and Future Prospects.},
journal = {Journal of molecular biology},
volume = {},
number = {},
pages = {169473},
doi = {10.1016/j.jmb.2025.169473},
pmid = {41046905},
issn = {1089-8638},
abstract = {The concept of gene therapy and its practice has been prevalent for over five decades. The first successful retroviral vector-based gene therapy trial took place ∼35 years ago, followed by several setbacks. However, recent years have seen a surge in successes, offering new hope to patients with genetic and other disorders once deemed untreatable. Over the past decade, rapid advancements in molecular biology have led to the development of safer and more effective gene therapy strategies with various gene delivery systems now in use. Among these, viral vectors such as retroviruses, adenoviruses, and adeno-associated viruses are the most widely employed in both research and clinical settings. This is due to their natural efficiency in delivering genetic material into target cells. Among these viral vectors, retroviruses stand out for their unique ability to reverse-transcribe and integrate their genetic material into the host genome, ensuring stable and long-term gene expression. This review highlights advances in retroviral vector development, examining both their therapeutic potential and associated challenges. It also explores strategies for vector production, including transient and stable systems tailored to meet clinical and regulatory demands. Significant progress is discussed in mitigating insertional mutagenesis and vector silencing. As a result, next-generation retroviral vectors with improved safety and efficacy have made it past regulatory-approval and are commercially available. Current innovations include replication-competent, non-integrating, integration-re-targeted, and hybrid CRISPR/Cas-expressing retroviral vectors undergoing pre-clinical and clinical investigations. This reflects a new era in gene therapy, with retroviral vectors reimagined for greater precision, control, and therapeutic impact.},
}
@article {pmid41045024,
year = {2025},
author = {Thomson, G and Mermaz, B and Sagawa, CHD and Lin, CI and Tachev, M and Joly, V and Irish, VF and Jacob, Y},
title = {Enzymatic depletion of transposable elements in sequencing libraries and its application for genotyping multiplexed CRISPR-edited plants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70501},
doi = {10.1111/tpj.70501},
pmid = {41045024},
issn = {1365-313X},
support = {2023-70029-41277//U.S. Department of Agriculture/ ; },
mesh = {*DNA Transposable Elements/genetics ; Zea mays/genetics ; *Gene Editing/methods ; Genome, Plant/genetics ; DNA Methylation ; *Citrus/genetics ; Gene Library ; CRISPR-Cas Systems/genetics ; *Genotyping Techniques/methods ; Genotype ; High-Throughput Nucleotide Sequencing/methods ; Sequence Analysis, DNA/methods ; },
abstract = {Whole-genome sequencing has become a common strategy to genotype individual plants of interest. Although a limited number of genomic regions usually need to be surveyed with this strategy, excess sequencing information is almost always generated at an appreciable financial cost. Repetitive sequences (e.g., transposons), which can account for more than 80% of the genome of some plants, are often not required in these genotyping projects. Therefore, strategies that enrich DNA coding for the protein-coding genes prior to sequencing can lower the cost to obtain sufficient sequence information. Here, we present the development and application of methylation-sensitive reduced representation sequencing (MsRR-Seq), which relies on the cytosine methylation-sensitive restriction enzyme MspJI to deplete constitutive heterochromatic DNA before library construction. By applying MsRR-Seq to citrus and maize, we show that protein-coding genes can be enriched in sequencing datasets. We then describe the application of MsRR-Seq to facilitate the identification of complex mutants from populations of citrus plants resulting from multiplex CRISPR/Cas9 editing of four genes. Overall, this work demonstrates an easy and low-cost method to enrich non-repetitive DNA in high-throughput sequencing libraries, an approach that is especially useful for large plant genomes with an excessively high proportion of methylated repetitive sequences.},
}
@article {pmid41044600,
year = {2025},
author = {Conery, M and Pippin, JA and Wagley, Y and Trang, K and Pahl, MC and Villani, DA and Favazzo, LJ and Ackert-Bicknell, CL and Zuscik, MJ and Katsevich, E and Wells, AD and Zemel, BS and Voight, BF and Hankenson, KD and Chesi, A and Grant, SFA},
title = {GWAS-informed data integration and non-coding CRISPRi screen illuminate genetic etiology of bone mineral density.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {331},
pmid = {41044600},
issn = {1474-760X},
support = {Gates Grubstake Award//University of Colorado/ ; DMS 2113072//National Science Foundation, United States/ ; R01DK122586//National Institute of Diabetes and Digestive and Kidney Diseases,United States/ ; UM1 DK126194/DK/NIDDK NIH HHS/United States ; R01AI154773//National Institute of Allergy and Infectious Diseases/ ; R01 HD100406/HD/NICHD NIH HHS/United States ; UL1 TR001878/TR/NCATS NIH HHS/United States ; Henry Ruppenthal Family Professorship for Bioengineering and Orthopaedic Surgery//University of Michigan/ ; R01 AG072705/AG/NIA NIH HHS/United States ; Daniel B. Burke Endowed Chair for Diabetes Research//Children's Hospital of Philadelphia/ ; },
mesh = {Humans ; *Bone Density/genetics ; *Genome-Wide Association Study ; Osteoblasts/metabolism ; *CRISPR-Cas Systems ; Single-Cell Analysis ; Quantitative Trait Loci ; },
abstract = {BACKGROUND: Over 1100 independent signals have been identified with genome-wide association studies (GWAS) for bone mineral density (BMD), a key risk factor for mortality-increasing fragility fractures; however, the effector gene(s) for most remain unknown.<br><br>RESULTS: We execute a CRISPRi screen in human fetal osteoblasts (hFOBs) with single-cell RNA-seq read-out for 89 non-coding elements predicted to regulate osteoblast gene expression at BMD GWAS loci. The BMD relevance of hFOBs is supported by heritability enrichment from stratified LD-score regression involving 98 cell types grouped into 15 tissues. Twenty-three genes show perturbation in the screen, with four (ARID5B, CC2D1B, EIF4G2, and NCOA3) exhibiting consistent effects upon siRNA knockdown on three measures of osteoblast maturation and mineralization. Lastly, additional heritability enrichments, genetic correlations, and multi-trait fine-mapping unexpectedly reveal that many BMD GWAS signals are pleiotropic and likely mediate their effects via non-bone tissues.<br><br>CONCLUSIONS: Our results provide a roadmap for how single-cell CRISPRi screens may be applied to the challenging task of resolving effector gene identities at all BMD GWAS loci. Extending our CRISPRi screening approach to other tissues could play a key role in fully elucidating the etiology of BMD.},
}
@article {pmid41041610,
year = {2025},
author = {de Mello Fiallos, N and Irfan, M and Solbiati, J and R Walker, A and Frias-Lopez, J and Gibson, FC},
title = {CRISPR cas7 influences the host-pathogen interaction of Porphyromonas gingivalis.},
journal = {Journal of oral microbiology},
volume = {17},
number = {1},
pages = {2561790},
pmid = {41041610},
issn = {2000-2297},
abstract = {INTRODUCTION: Porphyromonas gingivalis, a Gram-negative anaerobe, is a key contributor to periodontal disease. Emerging evidence suggests a role for the P. gingivalis CRISPR-Cas system in disease progression, although the specific roles of its components remain unclear.<br><br>OBJECTIVES: Here we investigate the role of cas7, a Class 1 type I-B CRISPR-Cas system component, in P. gingivalis physiology and host interaction.<br><br>METHODS: We compared P. gingivalis wild-type and &#x2206;cas7 strains for growth, biofilm formation, oxidative stress resistance, and hemagglutination. Host interactions were assessed using THP-1 macrophage-like cells to evaluate intracellular survival and cytokine response. Dual RNA-seq enabled host and microbe transcriptomic profiling during cellular infection, and Galleria mellonella was used to assess virulence.<br><br>RESULTS: The &#x2206;cas7 mutant showed similar planktonic growth and biofilm formation compared to wild-type but was more sensitive to oxidative stress and had reduced hemagglutination. Although intracellular survival was unaffected, &#x2206;cas7 altered the host cytokine production profile. Transcriptomic analysis revealed differential gene expression linked to oxidative stress and disease progression. In vivo, &#x2206;cas7 infection led to a trend of increased larval mortality.<br><br>CONCLUSION: These findings reveal a previously unrecognized role for cas7 in modulating P. gingivalis virulence, offering new insights into CRISPR-Cas system functions in bacterial pathogenesis.},
}
@article {pmid40974079,
year = {2025},
author = {Mariki, A and Kohlmeier, KA and Mousavi, SM and Shabani, M},
title = {CRISPR and Myelin regeneration: a systematic review of applications in demyelinating CNS Disorders, with a focus on MS.},
journal = {Regenerative medicine},
volume = {20},
number = {9},
pages = {431-443},
doi = {10.1080/17460751.2025.2561451},
pmid = {40974079},
issn = {1746-076X},
mesh = {Humans ; Animals ; *Myelin Sheath/metabolism ; *Multiple Sclerosis/therapy/genetics ; *CRISPR-Cas Systems ; Gene Editing ; *Demyelinating Diseases/therapy/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Regeneration/genetics ; },
abstract = {AIMS: Current treatments for demyelinating disorders focus on slowing progression but fail to repair damaged myelin. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -based technology has the potential to address key challenges in myelin repair by targeting genetic dysfunctions, modulating immune responses, and promoting oligodendrocyte differentiation. This systematic review aimed to evaluate CRISPR applications for myelin regeneration.<br><br>METHODS: A comprehensive search of PubMed, Scopus, and other databases identified 48 studies. The included studies employed CRISPR in diverse experimental models, targeting genes associated with immune regulation and astrocyte activity, as well as correcting RNA splicing dysfunctions linked to neurodegeneration.<br><br>RESULTS: CRISPR-edited stem cells showed significant potential in promoting myelin regeneration, with enhanced functional recovery in animal models of multiple sclerosis (MS). While most research focused on MS, promising applications were also observed in neuromyelitis optica spectrum disorder (NMOSD), such as reducing astrocytic damage via AQP4 targeting, and in progressive multifocal leukoencephalopathy (PML), where CRISPR disrupted JC polyomavirus replication.<br><br>CONCLUSIONS: Despite its promise, challenges remain. Future research should prioritize optimizing CRISPR delivery systems, expanding applications to underexplored disorders, and conducting long-term safety assessments. Early results are encouraging, but further studies are essential to translate preclinical success into clinical therapies.},
}
@article {pmid40972450,
year = {2026},
author = {Eom, KH and Yum, SY and Gim, GM and Kim, YC and Moon, B and Jang, G},
title = {SpCas9-mediated gene editing in bovine embryo via single adeno-associated virus infection using a novel micro-sized promoter.},
journal = {Theriogenology},
volume = {249},
number = {},
pages = {117676},
doi = {10.1016/j.theriogenology.2025.117676},
pmid = {40972450},
issn = {1879-3231},
mesh = {Animals ; Cattle/embryology/genetics ; *Gene Editing/veterinary/methods ; *Promoter Regions, Genetic ; *Dependovirus/genetics ; Genetic Vectors ; Embryo, Mammalian ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Fertilization in Vitro/veterinary ; },
abstract = {Genome editing in livestock offers practical solutions to address challenges related to land use, climate change, and food production. However, conventional delivery methods such as electroporation and microinjection impose physical stress on embryos, limiting scalability. This study aimed to develop a simplified, non-invasive, and scalable genome editing system for bovine embryos by designing an all-in-one adeno-associated virus (AAV) vector. A novel micro-sized promoter (50 bp), derived from the core regulatory region upstream of the bovine MSTN gene, was constructed to enable expression of Streptococcus pyogenes Cas9 (spCas9) within the AAV packaging limit (∼4.7 kb). This promoter was incorporated into an AAV cassette containing spCas9, a polyadenylation signal, a U6 promoter, and a single-guide RNA (sgRNA) targeting the bovine ALB gene. After confirming editing activity in bovine fibroblasts, the AAV6 vector was added directly to in vitro fertilization (IVF) cultures without physical manipulation. Genome editing was successfully induced, with insertion/deletion (indel) mutations detected in 33.8 ± 23.2 % of the blastocysts. Although blastocyst development was moderately reduced, gene editing was achieved without invasive techniques. These results demonstrate that a micro-promoter-based AAV system can support spCas9-mediated genome editing in bovine embryos through a single-vector infection strategy. The system presents a promising platform for producing gene-edited livestock and may contribute to more efficient and less labor-intensive applications in animal biotechnology.},
}
@article {pmid40650655,
year = {2025},
author = {Li, Y and Zhang, Y and Li, C and Chen, G and Muhammad, P and Yao, Y and Gao, L and Liu, Z and Wang, Y},
title = {Advanced Cancer Immunotherapy via SMARCAL1 Blockade Using a Glucose-Responsive CRISPR Nanovaccine.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {37},
pages = {e02929},
doi = {10.1002/advs.202502929},
pmid = {40650655},
issn = {2198-3844},
support = {81901684//National Natural Science Foundation of China/ ; },
mesh = {*Immunotherapy/methods ; Animals ; Mice ; *Glucose/metabolism ; Humans ; *Cancer Vaccines/immunology/genetics ; *Neoplasms/therapy/immunology ; CRISPR-Cas Systems/genetics ; Glucose Oxidase ; Gene Editing/methods ; Membrane Proteins/metabolism ; Nanovaccines ; },
abstract = {Cancer immunotherapy that activates the stimulator of interferon genes (STING) signaling pathway to resist tumors has recently attracted considerable attention. However, STING activation can induce opposing interferon functions that contribute to T-cell exhaustion via programmed death-ligand 1 (PD-L1). In particular, effectively using the immune system to combat tumors remains a substantial challenge due to tumor immunosuppressive factors such as SMARCAL1. Here, a glucose-responsive CRISPR nanovaccine is developed for enhancing STING signaling while inhibiting interferon-mediated immunosuppressive feedback. The formulation encapsulates a bimetallic zeolitic imidazolate framework with glucose oxidase (GOx) and CRISPR-mediated SMARCAL1 gene-editing plasmids. The dual enzyme-driven cascade reactions of peroxidase and GOx generate reactive oxygen species (ROS) and gluconic acid, which release and activate the genome-editing system. The silencing of SMARCAL1 enhances STING activity and inhibits PD-L1 expression, resulting in the termination of PD-L1-mediated opposing functions of interferon. Zinc ions and double-stranded DNA formed via ROS further activate the STING pathway, effectively inducing dendritic cell maturation and immune system activation. This is a critical report of in situ CRISPR nanovaccination driven by dual enzymes. The work highlights the potential of glucose-responsive CRISPR nanovaccination in bolstering antitumor immunity and extends the implementation of gene editing in cancer immunotherapy.},
}
@article {pmid40642954,
year = {2025},
author = {Fu, X and Wang, N and Li, L and Qiao, D and Qi, X and Liu, C and Gao, Z and Xie, C and Zhu, J},
title = {Development of cytosine and adenine base editors for maize precision breeding.},
journal = {Journal of integrative plant biology},
volume = {67},
number = {10},
pages = {2731-2743},
doi = {10.1111/jipb.13964},
pmid = {40642954},
issn = {1744-7909},
support = {No. 2023YFD1202901//National Key Research and Development Program of China Stem Cell and Translational Research/ ; 241111112300//Henan Province key research and development project/ ; //Xinjiang "Leading the Charge with Open Competition" project/ ; No. 2022ZD04006//Science and Technology Innovation 2030 Major projects/ ; },
mesh = {*Zea mays/genetics/drug effects ; *Gene Editing/methods ; *Adenine/metabolism ; *Cytosine/metabolism ; *Plant Breeding/methods ; Plants, Genetically Modified ; Mutation/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Base editing technologies can improve crops, but their efficiency in maize remains suboptimal. This study attempts to overcome these limitations by examining optimized cytosine and adenine base editors (CBEs and ABEs), namely evoAPOBEC1, evoFERNY, evoCDA1, TadA8.20, and TadA8e, for precise genome editing in transient and stable expression maize cells. Employing a seed fluorescence reporter (SFR) system for rapid screening of BE transformants and transgene-free progenies, we enhanced editing efficiencies and heritability. Notably, TadA8.20 and evoCDA1 attained multiplexed editing efficiencies of up to 100.0% and 79.0% at the tested loci, respectively, with some homozygous and bi-allelic mutants exceeding 72.4% and 73.7%. Precise editing of ZmACC1/2 (acetyl-CoA carboxylase) improved herbicide resistance, with ZmACC2 mutants displaying improved performance. This study advances crop genetic engineering by facilitating robust, multi-locus modifications without altered agronomic performance, enhancing herbicide tolerance in maize. The successful utilization of these BE is a significant step forward in agricultural biotechnology and precision breeding.},
}
@article {pmid39994413,
year = {2025},
author = {Oliynyk, RT and Church, GM},
title = {Circular Vectors as an efficient, fully synthetic, cell-free approach for preparing small circular DNA as a plasmid substitute for guide RNA expression in CRISPR-Cas9 genome editing.},
journal = {Nature protocols},
volume = {20},
number = {10},
pages = {2942-2959},
pmid = {39994413},
issn = {1750-2799},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *Plasmids/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Vectors/genetics ; *DNA, Circular/genetics ; Cell-Free System ; Humans ; },
abstract = {Robust expression of guide RNA (gRNA) is essential for successful implementation of CRISPR-Cas9 genome-editing methods. The gRNA components, such as an RNA polymerase promoter followed by the gRNA coding sequence and an RNA polymerase terminator sequence, and the Cas9 protein are expressed either via an all-in-one plasmid or separate dedicated plasmids. The preparation of such plasmids involves a laborious multi-day process of DNA assembly, bacterial cloning, validation, purification and sequencing. Our Circular Vector (CV) protocol introduces an efficient, fully synthetic, cell-free approach for preparing gRNA expression templates suitable for transfection, marking a significant advancement over traditional plasmid-based approaches. This protocol consists of the circularization and purification of linear double-stranded DNA (dsDNA) containing gRNA expression elements into compact, bacterial-backbone-free circular DNA expression vectors in as little as 3 h. We provide a guide to the design of the dsDNA template coding for gRNA elements for CRISPR-Cas9 base and prime editing, along with step-by-step instructions for the efficient preparation of gRNA-expressing CVs. In addition to rapid preparation, CVs created via this protocol offer several key advantages: a compact size, absence of a bacterial backbone, absence of bacterial endotoxins and no contamination by bacterial RNA or DNA fragments. These features make gRNA-expressing CVs a superior choice over plasmid-based gRNA expression templates.},
}
@article {pmid41039221,
year = {2025},
author = {Ilmi, AFN and Kaewsapsak, P and Rotcheewaphan, S},
title = {Repression of mab_1999 impairs growth and alters cellular morphology of Mycobacterium abscessus.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {599},
pmid = {41039221},
issn = {1471-2180},
mesh = {*Mycobacterium abscessus/growth &amp; development/genetics/drug effects/cytology/metabolism ; *Bacterial Proteins/genetics/metabolism ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Cell Division/genetics ; Gene Expression Regulation, Bacterial ; Mycobacterium smegmatis/genetics ; Gene Knockdown Techniques ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Cell division is essential for bacterial survival and represents a promising target for the development of novel antibiotics, particularly in mycobacteria. The role of the division protein FtsL in Mycobacterium abscessus remains poorly understood. This study investigated the effects of MAB_1999, a predicted homolog of FtsL, on the growth and cell division of M. abscessus.<br><br>METHOD: To investigate the function of mab_1999, a knockdown mutant was generated via CRISPR interference (CRISPRi). The phenotypic impact of mab_1999 suppression was evaluated, with a focus on its effects on M. abscessus growth, cellular morphology, and antibiotic susceptibility.<br><br>RESULTS: The putative homolog of FtsL in M. abscessus (MAB_1999) shares 54% amino acid sequence identity with FtsL from M. smegmatis (MSMEG_4234). CRISPRi-mediated repression of mab_1999 expression resulted in cell elongation and growth defects, although complete growth arrest was not observed. Furthermore, reduced mab_1999 expression increased the susceptibility of M. abscessus to &#x3b2;-lactam antibiotics, including ceftriaxone and imipenem.<br><br>CONCLUSIONS: Our findings suggest that mab_1999 is involved in cell division and cell wall integrity in M. abscessus. However, further investigation is necessary to confirm its identity as FtsL and to fully elucidate its role in the cell division process and cell wall synthesis.},
}
@article {pmid41039127,
year = {2025},
author = {Safenkova, IV and Kamionskaya, MV and Ivanov, AV and Zherdev, AV and Dzantiev, BB},
title = {A novel tripod probe and lateral flow test to improve CRISPR/Cas12a assay: benefits of branched probe based on trebler phosphoramidite modification.},
journal = {Mikrochimica acta},
volume = {192},
number = {11},
pages = {711},
pmid = {41039127},
issn = {1436-5073},
support = {23-46-10011//Russian Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA Probes/chemistry ; *Biosensing Techniques/methods ; *Organophosphorus Compounds/chemistry ; Fluorescent Dyes/chemistry ; Limit of Detection ; Gold/chemistry ; *CRISPR-Associated Proteins/metabolism ; Metal Nanoparticles/chemistry ; *Bacterial Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; },
abstract = {CRISPR/Cas12a-based assays, when integrated with lateral flow tests (LFTs), provide highly specific nucleic acid detection in a simple, rapid, and equipment-free format. Nevertheless, traditional DNA probes utilized for cleavage by Cas12a have limitations as the cleaved probe only has one label. To overcome this challenge, we engineered a novel type of DNA probe with multiple fluorescein (FAM) labels and a biotin-labeled single-stranded DNA fragment (polyFAM probe). The cleaved polyFAM parts of the probes were detected using a specially designed sandwich LFT, where FAM-specific antibodies were immobilized in the test zone and conjugated with gold nanoparticles. The LFT ensured accurate recognition of the cleaved polyFAM fragments within 10 min. A comparison of five distinct polyFAM probes revealed that the highest signal-to-noise ratio was achieved with a tripod-branched probe synthesized via trebler phosphoramidite modification. Each arm of the tripod probe consists of a hexaethylene glycol spacer ending in a FAM label. Upon Cas12a cleavage, the tripod structure carrying three FAMs is released and detected by LFT. A rapid magnetic separation strategy was subsequently implemented, facilitating the efficient removal of uncleaved probes via biotin-streptavidin capture within 5 min. The CRISPR/Cas12a-tripod-LFT strategy demonstrated excellent sensitivity without preamplification, with a detection Limit of 1.4 pM for DNA target of Salmonella Typhimurium. The CRISPR/Cas12a-tripod-LFT with preliminary loop-mediated isothermal amplification enabled the detection of as few as 0.3 cells per reaction. This innovative tripod probe with corresponding LFT creates a universal, sensitive, rapid, and equipment-free biosensing platform for CRISPR/Cas12a-based diagnostics in point-of-care applications.},
}
@article {pmid40847019,
year = {2025},
author = {Ramani, B and Rose, IVL and Teyssier, N and Pan, A and Danner-Bocks, S and Sanghal, T and Yadanar, L and Tian, R and Ma, K and Palop, JJ and Kampmann, M},
title = {CRISPR screening by AAV episome-sequencing (CrAAVe-seq): a scalable cell-type-specific in vivo platform uncovers neuronal essential genes.},
journal = {Nature neuroscience},
volume = {28},
number = {10},
pages = {2129-2140},
pmid = {40847019},
issn = {1546-1726},
support = {R01 AG082141/AG/NIA NIH HHS/United States ; K99 AG062776/AG/NIA NIH HHS/United States ; RF1 AG062234/AG/NIA NIH HHS/United States ; R25 NS070680/NS/NINDS NIH HHS/United States ; K08 NS133300/NS/NINDS NIH HHS/United States ; T32 NS115706/NS/NINDS NIH HHS/United States ; EDUC4-12812//California Institute for Regenerative Medicine (CIRM)/ ; UCSF Hillblom/BARI Graduate Fellowship Award//Larry L. Hillblom Foundation (Larry L. Hillblom Foundation, Inc.)/ ; R01 AG082141/AG/NIA NIH HHS/United States ; K99 AG062776/AG/NIA NIH HHS/United States ; RF1 AG062234/AG/NIA NIH HHS/United States ; R25 NS070680/NS/NINDS NIH HHS/United States ; K08 NS133300/NS/NINDS NIH HHS/United States ; T32 NS115706/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Dependovirus/genetics ; *Neurons/metabolism ; Mice ; *CRISPR-Cas Systems/genetics ; *Genes, Essential/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mice, Inbred C57BL ; *Genetic Testing/methods ; Humans ; },
abstract = {There is a substantial need for scalable CRISPR-based genetic screening methods that can be applied in mammalian tissues in vivo while enabling cell-type-specific analysis. Here we developed an adeno-associated virus (AAV)-based CRISPR screening platform, CrAAVe-seq, that incorporates a Cre-sensitive sgRNA construct for pooled screening within targeted cell populations in mouse tissues. We used this approach to screen two large sgRNA libraries, which collectively target over 5,000 genes, in mouse brains and uncovered genes essential for neuronal survival, of which we validated Rabggta and Hspa5. We highlight the reproducibility and scalability of the platform and show that it is sufficiently sensitive for screening in a restricted subset of neurons. We systematically characterize the impact of sgRNA library size, mouse cohort size, the size of the targeted cell population, viral titer, and coinfection rate on screen performance to establish general guidelines for large-scale in vivo screens.},
}
@article {pmid41038958,
year = {2025},
author = {Chen, J and Huang, H and Chen, C and Xia, G and Huang, H and Xiong, Y and Luo, P and Chen, Y and Li, J and Wen, L and Li, L and Lin, J and Xu, G and Ji, C and Tian, W and Zhou, J and Wei, P and Shen, C and Wang, X},
title = {ABCC4 impairs the clearance of plasma LDL cholesterol through suppressing LDLR expression in the liver.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1414},
pmid = {41038958},
issn = {2399-3642},
support = {8217051361//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Receptors, LDL/metabolism/genetics ; Animals ; *Liver/metabolism ; Mice ; *Cholesterol, LDL/blood/metabolism ; *Multidrug Resistance-Associated Proteins/metabolism/genetics ; Humans ; Male ; Proprotein Convertase 9/metabolism/genetics ; Hepatocytes/metabolism ; Mice, Inbred C57BL ; Hypercholesterolemia/metabolism/genetics ; CRISPR-Cas Systems ; Signal Transduction ; Hep G2 Cells ; },
abstract = {Low expression level of low-density lipoprotein receptor (LDLR) in hepatocytes leads to hypercholesterolemia and eventually contributes to atherosclerotic cardiovascular disease (ASCVD). Here, we report that inhibition of hepatocyte ABCC4, identified as a top hit from large-scale CRISPR/Cas9 screens, significantly increases hepatic LDLR abundance and enhances LDL cholesterol clearance. As a hepatic transporter for cAMP efflux, ABCC4 silencing alters its intracellular distribution and activates the downstream Epac2/Rap1a signaling pathway, which ultimately blocks PCSK9 protein expression, thereby preventing lysosomal degradation of LDLR. Furthermore, in both male mice and cell models, we demonstrate that liver-specific disruption and pharmacological inhibition of ABCC4 elevate hepatic plasma membrane LDLR levels and reduce plasma LDL cholesterol through ABCC4-cAMP-PCSK9 pathway. Collectively, our genome-wide CRISPR screening offers a valuable resource for identifying LDLR modifiers, providing potential insights for therapeutic strategies in hypercholesterolemia and atherosclerosis.},
}
@article {pmid41038616,
year = {2025},
author = {Aliciaslan, M and Erbasan, E and Erendor, F and Sanlioglu, S},
title = {Prime Editing: The Next Frontier in Precision Gene Therapy.},
journal = {The journal of gene medicine},
volume = {27},
number = {10},
pages = {e70040},
doi = {10.1002/jgm.70040},
pmid = {41038616},
issn = {1521-2254},
support = {//Akdeniz &#xdc;niversitesi/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; *Precision Medicine/methods ; CRISPR-Cas Systems ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Prime editing (PE) represents a significant advancement in genome editing, offering high precision for diverse genetic modifications without inducing double-strand breaks or requiring exogenous donor DNA templates. This "search-and-replace" technology employs a Cas9 nickase-reverse transcriptase fusion protein, guided by a PE guide RNA (pegRNA), to directly install specified edits including all 12 base-to-base conversions and targeted insertions/deletions with high fidelity. Since its introduction, PE systems have undergone rapid evolution (e.g., PE2-PE6, PEmax), markedly improving editing efficiency, product purity, and targeting scope. Although PE efficacy is context dependent, influenced by pegRNA design, cellular milieu, and DNA repair pathway engagement, ongoing research focuses on comprehensive system optimization. These efforts include engineering the Cas9 nickase and reverse transcriptase components for enhanced performance and processivity, alongside developing improved pegRNA architectures and chemical modifications to increase their stability and editing efficiency. Furthermore, strategies to modulate the cellular environment, such as transiently altering DNA repair pathway activities, particularly mismatch repair, are being explored to boost the accuracy and yield of precise edits. PE holds substantial promise for basic research, including precise disease modeling, and has demonstrated successful correction of pathogenic mutations in preclinical models of various genetic disorders like sickle cell disease, cystic fibrosis, and inherited retinal diseases. A significant milestone was the US Food and Drug Administration's granting of Investigational New Drug (IND) clearance for the first clinical trial of PM359, a therapeutic based on PE. This agent employs an ex vivo strategy, correcting the NCF1 gene in patient-derived hematopoietic stem cells for the treatment of chronic granulomatous disease. Despite considerable progress, unlocking the complete therapeutic promise of PE requires overcoming significant hurdles, particularly in developing effective in vivo delivery systems for its sizable components, with ongoing research actively investigating diverse viral and nonviral approaches. The translation of this versatile platform into transformative precision gene therapies is critically dependent upon its continued responsible advancement under robust ethical and regulatory oversight.},
}
@article {pmid41037600,
year = {2025},
author = {de Alba, EL and Salguero, I and Giménez-Llorente, D and Montes-Torres, J and Fernández-Sanromán, &#xc1; and Casajús-Pelegay, E and Terrón-Bautista, J and Barroso-González, J and Bernal, JA and Macintyre, G and Fernández-Leiro, R and Losada, A and Cortés-Ledesma, F},
title = {A comprehensive genetic catalog of human double-strand break repair.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6768},
pages = {eadr5048},
doi = {10.1126/science.adr5048},
pmid = {41037600},
issn = {1095-9203},
mesh = {Humans ; *DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; *DNA Repair/genetics ; Von Hippel-Lindau Tumor Suppressor Protein/genetics ; INDEL Mutation ; Gene Editing ; DNA End-Joining Repair/genetics ; Kidney Neoplasms/genetics ; DNA-Binding Proteins/genetics ; Transcription Factors/genetics ; CRISPR-Associated Protein 9 ; Genome, Human ; Gene Knockout Techniques ; Carcinoma, Renal Cell/genetics ; },
abstract = {The analysis of DNA sequence outcomes provides molecular insights into double-strand break (DSB) repair mechanisms. Using parallel in-pool profiling of Cas9-induced insertions and deletions (indels) within a genome-wide knockout library, we present a comprehensive catalog that assesses the influence of nearly every human gene on DSB repair outcomes. This REPAIRome resource uncovers uncharacterized mechanisms, pathways, and factors involved in DSB repair, including opposing roles for XLF and PAXX, a molecular explanation for Cas9-induced multinucleotide insertions, HLTF functions in Cas9-induced DSB repair, the involvement of the SAGA complex in microhomology-mediated end joining, and an indel mutational signature linked to VHL loss, renal carcinoma, and hypoxia. These results exemplify the potential of REPAIRome to drive future discoveries in DSB repair, CRISPR-Cas gene editing and the etiology of cancer mutational signatures.},
}
@article {pmid41035653,
year = {2025},
author = {Naumovas, D and Rojas-Araya, B and Polanco, CM and Andrade, V and Čekauskienė, R and Valatkaitė-Rakštienė, B and Laurinaitytė, I and Jakubauskas, A and Stoškus, M and Griškevičius, L and Nalvarte, I and Inzunza, J and Baltriukienė, D and Arias, J},
title = {Identification of HLA-A, HLA-B, and HLA-C triple homozygous and double homozygous donors: a path toward synthetic superdonor advanced therapeutic medicinal products.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1626787},
pmid = {41035653},
issn = {1664-3224},
mesh = {Humans ; *Homozygote ; *HLA-B Antigens/genetics ; *HLA-C Antigens/genetics ; *HLA-A Antigens/genetics ; Alleles ; Gene Frequency ; Induced Pluripotent Stem Cells/immunology ; *Tissue Donors ; Genotype ; CRISPR-Cas Systems ; Female ; Male ; },
abstract = {Human-induced pluripotent stem cells with broad immune compatibility are highly desirable for regenerative medicine applications. Human leukocyte antigen (HLA) class I homozygous cell sources are ideal for immune compatibility modeling. Here, we profile HLA-A, HLA-B, and HLA-C alleles in 3,496 Lithuanian donors genotyped at three-field resolution. The five most frequent alleles constitute 74.6% of HLA-A, 43.2% of HLA-B, and 59.2% of HLA-C, with HLA-A*02:01:01, HLA-B*07:02:01, and HLA-C*07:02:01 being the most common. Lithuanian allele frequencies closely resemble those of European-American and British populations. We identified 153 double homozygotes and 51 triple homozygotes for HLA-A, HLA-B, and HLA-C. Compatibility modeling showed that triple homozygous profiles match 60.5% of Lithuanians, 13.4% of the British population, and 7.4% of European-Americans. CRISPR-Cas9 guide RNA design yielded 54 candidates predicted to disrupt HLA-A or HLA-B while preserving HLA-C, producing edited profiles matching over 97.9% of Lithuanians, 95.7% of European-Americans, and 95.5% of the British population. Finally, we established 15 fibroblast lines from triple homozygotes as a bioresource for the derivation of human-induced pluripotent stem cells and immune compatibility studies.},
}
@article {pmid41034738,
year = {2025},
author = {Devkar, V and Ghose, K and D'Agostino, L and Patil, GB},
title = {Exonuclease-fused CRISPR-cas system enhances targeted genome editing for functional genomics in soybean.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1283},
pmid = {41034738},
issn = {1471-2229},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Glycine max/genetics ; *Genome, Plant ; *Exonucleases/genetics/metabolism ; Genomics/methods ; },
abstract = {CRISPR/Cas technologies have revolutionized plant genome editing, yet their inherent bias toward small insertions or deletions (indels) limits their utility for dissecting regulatory elements and generating impactful allelic variants. Here, we report the development and systematic evaluation of exonuclease-fused CRISPR/Cas systems in soybean to overcome this limitation. We engineered fusions of Cas9 and Cas12a with bacteriophage T5 exonuclease and human TREX2 and assessed their editing performance at the GmWOX5 locus using Agrobacterium rhizogenes-mediated transformation and deep amplicon sequencing. While native Cas9 and Cas12a predominantly generated micro-size deletions (1-10 bp), T5-Exo fusions shifted the mutation spectrum, producing a high frequency of moderate (26-50 bp) and large (> 50 bp) deletions. TREX2 fusions preferentially enhanced the generation of small (11-25 bp) to moderate deletions (26-50 bp). Fusion of exonucleases to Cas9 substantially reduced insertion frequencies and promoted more precise deletion patterns, as observed in T5-Exo-Cas9 and TREX2-Cas9. Deletions from both exonuclease fusions were biased toward the PAM-proximal region, reflecting altered repair outcomes likely driven by directional exonuclease activity and enhanced end resection. These results demonstrate that exonuclease fusions effectively expand the CRISPR toolkit by enabling efficient, targeted generation of larger deletions, which are often required for targeting cis-regulatory elements and microRNAs.},
}
@article {pmid40974621,
year = {2025},
author = {Zheng, X and Yao, S and Yin, C and Zhao, H and Wang, J and Su, T and Li, H and Wang, J and Zhao, C},
title = {CRISPR-integrated nanoconfined interparticle catalytic hairpin assembly for enhanced dual-mode SARS-CoV-2 detection in wastewater.},
journal = {Biosensors &amp; bioelectronics},
volume = {290},
number = {},
pages = {118008},
doi = {10.1016/j.bios.2025.118008},
pmid = {40974621},
issn = {1873-4235},
mesh = {*SARS-CoV-2/isolation &amp; purification/genetics ; *Wastewater/virology ; *Biosensing Techniques/methods/instrumentation ; *COVID-19/diagnosis/virology ; *CRISPR-Cas Systems ; Limit of Detection ; Humans ; *RNA, Viral/analysis/genetics/isolation &amp; purification ; Colorimetry/methods ; Metal-Organic Frameworks/chemistry ; Nanoparticles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Accurate monitoring of pathogenic viruses in wastewater is critical for early outbreak and risk assessment. This study presented a novel biosensing platform that combined an interparticle magnetic covalent organic framework (MCOF)-assisted mismatched catalytic hairpin assembly (iMMCHA) with CRISPR/Cas12a-activated colorimetric-photothermal dual-mode detection of SARS-CoV-2 RNA. The system strategically immobilized CHA reactants (H1 and mismatched H2) on separate MCOF nanoparticles, creating a spatially confined and collision-enhanced interparticle MCHA that achieved 270-fold higher local reactant concentration and 20-min faster kinetics than solution-phase CHA. Upon target recognition, the iMMCHA system generated dsDNA activators that triggered Cas12a-mediated cleavage of ssDNA linkers on magnetic bead-glucose oxidase conjugates. This cleavage event reduced the TMB-oxidizing activity of the magnetically isolated integrated enzyme system, producing inversely correlated colorimetric and photothermal signals. This iMMCHA-CRISPR dual-mode assay allowed for the rapid and sensitive detection of SARS-CoV-2 pseudovirus in sanitary wastewater samples, with detection limits of 100 and 120 copies/μL (colorimetric mode) and 100 and 140 copies/μL (photothermal mode) for S and N genes, respectively. This work established a powerful platform for aqueous environmental virus monitoring that combined the specificity of CRISPR with the signal enhancement and kinetics acceleration of nanoconfined interparticle CHA and the reliability of dual-mode detection.},
}
@article {pmid40961645,
year = {2025},
author = {He, Y and Zhang, Y and Xiang, H and Ren, K and Yin, Y and Gao, Y and Yang, Y and Zhang, W and Liu, L and Han, H and Wang, W},
title = {Magnetic bead-assisted one-pot RCA-activated CRISPR/Cas12a electrochemiluminescence biosensor for the detection of citrus Huanglongbing pathogen.},
journal = {Biosensors &amp; bioelectronics},
volume = {290},
number = {},
pages = {117986},
doi = {10.1016/j.bios.2025.117986},
pmid = {40961645},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Citrus/microbiology ; *Plant Diseases/microbiology ; Luminescent Measurements/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; *Liberibacter/isolation &amp; purification/genetics/pathogenicity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Huanglongbing (HLB) poses a catastrophic threat to the global citrus industry, necessitating early detection of pathogen for disease control and minimize economic losses. Herein, we reported a one-pot electrochemiluminescence (ECL) biosensor for integrating rolling circle amplification (RCA)-activated CRISPR/Cas12a dual cleavage activity, and engineered magnetic beads-based quenched ECL emitter. Target-initiated RCA generated amplicons that activated Cas12a, simultaneously leveraging cis-cleavage for template recycling and trans-cleavage to degrade single stranded DNA attached on Ru(bpy)3[2+]-loaded magnetic beads. This dual-amplification strategy restored ECL signals, enabling ultrasensitive detection of Candidatus Liberibacter asiaticus (CLas) ribonucleotide-diphosphate reductase subunit beta gene fragments with high specificity. A linear range 10 fM-1 nM with the detection limit of 2 fM was obtained. The integrated platform eliminated multi-step incubations, and exhibited satisfactory performance in citrus leaf samples, offering a powerful tool for HLB diagnostics.},
}
@article {pmid40945113,
year = {2025},
author = {Che, R and Tang, D and Fu, B and Wen, T and Wang, Z and Feng, D and Huang, KJ and Xu, J},
title = {Smartphone-integrated tri-mode RCA-CRISPR/Cas12a biosensor with Fe3O4@Au nanozyme for on-site detection of sugarcane smut at attomolar level.},
journal = {Biosensors &amp; bioelectronics},
volume = {290},
number = {},
pages = {117985},
doi = {10.1016/j.bios.2025.117985},
pmid = {40945113},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *Saccharum/microbiology ; Smartphone ; Gold/chemistry ; CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; Limit of Detection ; *Plant Diseases/microbiology ; Colorimetry ; Electrochemical Techniques ; Metal Nanoparticles/chemistry ; },
abstract = {The devastating sugarcane smut causes up to 70 % sugar yield loss and secondary infections, but field-deployable diagnostics remain challenging due to the limitations of lab-dependent methods. Herein, we report a portable CRISPR/Cas12a-powered biosensor integrated with tri-functional Fe3O4@Au nanozymes and triple-modal signal readout for precise and on-site pathogen detection. By synergizing rolling circle amplification (RCA) with CRISPR/Cas12a trans-cleavage activity, the system achieves ultrasensitive target recognition (detection limit: 32.11 aM for electrochemical mode). The Fe3O4@Au@GOD bioconjugates simultaneously enables magnetic separation, optimizes GOD-mediated colorimetric signals (visual LOD: 49.28 fM), and enhances photothermal responses (LOD: 42.17 fM) via precise biocatalyst-catalyzed TMB oxidation. A smartphone-coupled 3D-printed device integrates electrochemical, colorimetric, and photothermal detection modes, providing cross-validated results that eliminate false positives in complex matrices (recovery: 98-104 %). This field-portable platform completes detection within 2.5-4.5 h (80 % cost reduction vs. qPCR) and demonstrates high specificity against non-target pathogens. The fusion of nanozyme engineering, CRISPR amplification, and multi-modal sensing offers a transformative tool for precision agriculture.},
}
@article {pmid40939269,
year = {2025},
author = {Zhou, C and Jiang, F and Chen, W and Nugen, SR and Huang, C},
title = {Synthetic biology meets diagnostics: Engineering biosensing platforms for rapid and accurate pathogen and viral detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {290},
number = {},
pages = {117946},
doi = {10.1016/j.bios.2025.117946},
pmid = {40939269},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods/instrumentation ; *Synthetic Biology/methods/instrumentation ; Humans ; *Viruses/isolation &amp; purification/genetics/pathogenicity ; *Virus Diseases/diagnosis/virology ; Nanotechnology/methods ; CRISPR-Cas Systems ; },
abstract = {The integration of synthetic biology with biosensor technologies has catalyzed a paradigm shift in the development of programmable, field-deployable diagnostic systems for precision detection of pathogens and viral threats. This review provides a comprehensive overview of current synthetic biology toolkits, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-based) systems, argonaute proteins, and modular genetic circuits. These tools were integrated into biosensors and in vitro diagnostic devices. The applications of cell-free systems, modular genetic circuits, and nanomaterial-enhanced platforms have further expanded the versatility of these tools, which include infectious disease diagnostics, public health monitoring, and food safety. Recent studies integrate synthetic biology with artificial intelligence (AI) and nanotechnology, enabling the development of automated, low-cost, and high-throughput diagnostic systems. This review provides a comprehensive overview of current technologies, emerging trends, future directions, and challenges, which offers valuable insights for advancing pathogen detection and in vitro diagnostics through synthetic biology.},
}
@article {pmid40914019,
year = {2025},
author = {Ding, Y and Zhang, J and Li, K and Wang, X and Shi, X and Zhao, C and Dai, J and Wang, Q and Yao, S and Wang, J},
title = {A cascade amplification platform integrating entropy-driven DNA nanomachine with CRISPR/Cas12a for microRNA-21 and Listeria monocytogenes detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {290},
number = {},
pages = {117947},
doi = {10.1016/j.bios.2025.117947},
pmid = {40914019},
issn = {1873-4235},
mesh = {*Listeria monocytogenes/isolation &amp; purification/genetics ; CRISPR-Cas Systems/genetics ; *MicroRNAs/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; Entropy ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Animals ; DNA/chemistry ; Swine ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {As one of enzyme-free amplification strategies, entropy-driven catalytic (EDC) based on toehold-mediated strand displacement reaction could achieve efficient amplification without cumbersome temperature changing and expensive enzymes, which shows great potential in biological sensing. However, the limitations in reaction velocity and sensitivity need to be further improved. Herein, a cascade platform integrating entropy-driven DNA nanomachine with CRISPR/Cas12a was proposed. Benefiting from the increased local concentration of DNA on AuNPs, the reaction velocity was enhanced 2-fold compared to solution-based EDC efficiently and the signal was cascade amplified through specific recognition by the designed CRISPR/Cas12a with high sensitivity and selectivity. Impressively, utilizing the flexible design capabilities of DNA molecules, the proposed method achieved both nucleic acid and non-nucleic acid targets detection. The platform achieved a low limit of 6.1pM for microRNA-21 detection and 6 CFU/mL for Listeria monocytogenes detection. Moreover, it showed good performance in 10-fold diluted serum with 98.6-102.3 % recovery. And it has achieved good consistency with traditional plate culture methods in pork. Here, a rapid and sensitive platform based on entropy-driven DNA nanomachine coupled with CRISPR/Cas12a is proposed with great potential of application for early disease detection and food safety screening.},
}
@article {pmid40914018,
year = {2025},
author = {Deng, Z and Mao, X and Yang, Y and Wu, G and Meng, H and Hou, J and Yu, XF and Zhou, W and Mao, G and Ma, Y},
title = {Amplification-free CRISPR/Cas12a biosensor integrating AuNPs-mediated surface plasmon resonance for human papillomavirus detection and genotyping.},
journal = {Biosensors &amp; bioelectronics},
volume = {290},
number = {},
pages = {117960},
doi = {10.1016/j.bios.2025.117960},
pmid = {40914018},
issn = {1873-4235},
mesh = {Humans ; Gold/chemistry ; *Surface Plasmon Resonance/methods ; CRISPR-Cas Systems/genetics ; *Papillomavirus Infections/virology/diagnosis ; Metal Nanoparticles/chemistry ; *Papillomaviridae/genetics/isolation &amp; purification ; *DNA, Viral/genetics/isolation &amp; purification ; *Biosensing Techniques ; Limit of Detection ; Genotyping Techniques ; Female ; Alkaline Phosphatase/chemistry ; Genotype ; Human Papillomavirus Viruses ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Screening for high-risk human papillomavirus (hrHPV) infection is essential for cervical cancer prevention. However, developing a simple, portable, and low-cost hrHPV genotyping method remains challenging, particularly in resource-limited settings. Herein, we present an innovative amplification-free, point-of-care hrHPV genotyping platform integrating CRISPR/Cas12a with alkaline phosphatase (ALP)-mediated surface plasmon effect. The platform detects HPV DNA through the Cas12a-crRNA complex recognition, activating the cleavage of ALP-labeled oligonucleotides within microwells and releasing ALP. The output signal is generated by changes in surface plasmon resonance of gold nanoparticles (AuNPs) induced by the ALP-mediated reaction of AuNPs with p-aminophenyl phosphate. This ALP-integrated CRISPR/Cas12a biosensing strategy enhances sensitivity by 10,000-fold compared to Cas12a-based detection integrating ALP-mediated p-nitrophenyl phosphate (p-NPP) hydrolysis. This approach allows the sensitive detection of HPV DNA with a detection limit of 300 aM. Moreover, integration with microplate separation allows specifically screen for the nine HPV subtypes targeted by the nine-valent HPV vaccine within 2.5 h. The platform's performance is validated using cervical swab samples, confirming its accuracy for HPV genotyping. Overall, this strategy provides a simple, portable, and cost-effective solution for multiplex nucleic acid targets detection without preamplification or instrumentation.},
}
@article {pmid40907452,
year = {2025},
author = {Hu, D and Lin, K and Xu, X and Chen, P and Wang, G and Parvin, R and Chen, X and Wang, D and Zhang, L and Ye, F},
title = {SEE-phAST: Spatially encapsulated emulsions for phenotypic antibiotic susceptibility testing via sequential digital RAA-CRISPR.},
journal = {Biosensors &amp; bioelectronics},
volume = {290},
number = {},
pages = {117937},
doi = {10.1016/j.bios.2025.117937},
pmid = {40907452},
issn = {1873-4235},
mesh = {*Anti-Bacterial Agents/pharmacology ; *Biosensing Techniques ; Emulsions/chemistry ; Microbial Sensitivity Tests ; Humans ; DNA Copy Number Variations/genetics ; CRISPR-Cas Systems/genetics ; Escherichia coli/genetics/drug effects ; Pseudomonas aeruginosa/genetics/drug effects ; Nucleic Acid Amplification Techniques ; Recombinases/chemistry ; Klebsiella pneumoniae/genetics/drug effects ; DNA, Bacterial/genetics ; },
abstract = {The escalating threat of antimicrobial resistance is exacerbated by delayed diagnostics and improper antibiotics use, underscoring an urgent demand for rapid, versatile AST tools to support evidence-based prescribing. In this study, we present an innovative, generalizable phenotypic AST approach by quantifying bacterial gDNA copy number variations (CNVs) following 0.5-h-brief culturing with antibiotic exposure, termed spatially encapsulated emulsions (SEE)-phAST. It employed gelatin-PEG biomimetic phase separation and microfluidic technology to one-step fabricate spatial-confined microcarriers that enabled on-demand separation and mixing of recombinase-aided amplification (RAA) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) reactions in a sequentially orchestrated manner. Importantly, it retained the superiority of droplet-based digitalization framework to identify 3-4 folds CNVs while synergistically enhancing signal-to-noise ratio and detection speed through RAA pre-amplification. By utilizing a phase diagram for precise separation, coupled with channel design and rate regulation, we controllably synthesized 180-210 μm microcarriers with 90-100 μm cores, achieving a 30-min RAA amplification that boosted sensitivity from 10[11] to 10[1] aM. The artificial urinary infection samples were prepared by introducing common bacteria, specifically Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa, into urinary samples obtained from healthy individuals. We demonstrated the system's capability to classify susceptibility and resistance to three specific drugs following a 30-min pre-culture, with epigenetic changes quantified as reduced to 0.5 for inhibition and maintained at 2 for unaffected growth. This strategy presents valuable potential for AST diagnosis and versatility in other sequence recognition scenarios.},
}
@article {pmid40902593,
year = {2025},
author = {Chan, BKC and Zhang, C and Poon, CH and Lee, MHY and Chu, HY and Wang, B and Chen, SG and Yan, HHN and Leung, SY and Wong, ASL},
title = {A combined enteric neuron-gastric tumor organoid reveals metabolic vulnerabilities in gastric cancer.},
journal = {Cell stem cell},
volume = {32},
number = {10},
pages = {1595-1613.e10},
doi = {10.1016/j.stem.2025.08.006},
pmid = {40902593},
issn = {1875-9777},
mesh = {*Organoids/metabolism/pathology/drug effects ; *Stomach Neoplasms/metabolism/pathology ; Humans ; Animals ; *Neurons/metabolism/pathology/drug effects ; Mice ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Lipid Metabolism/drug effects ; Acetyl-CoA Carboxylase/antagonists &amp; inhibitors/metabolism ; },
abstract = {The discrepancy between organoid and immortalized cell line cultures for cancer target discovery remains unclear. Here, our multi-tiered clustered regularly interspaced short palindromic repeats (CRISPR) screens reveal in vivo-relevant metabolic dependencies and synthetic lethal pairs that can be uncovered with tumor organoids but not cell lines or even three-dimensional (3D) spheroids. These screens identify lanosterol synthase and acetyl-coenzyme A (CoA) carboxylase inhibitors as effective treatments that impede xenografted tumor growth in mice. These lipid metabolic inhibitors exhibit nanomolar half-maximal inhibitory concentration (IC50) values across diverse human gastric cancer organoids resistant to first-line treatments. Mechanistically, gastric cancer organoids and in vivo tumors exhibit lipid metabolic adaptations not seen in two-dimensional (2D) in vitro cultures. Additionally, enteric neurons modulate lipid metabolism in tumor organoids, altering drug sensitivity by up to two orders of magnitude. A neuron-cocultured CRISPR screen further reveals that acetyl-CoA carboxylase expression determines lanosterol synthase inhibitor efficacy. These findings highlight the critical roles of organoid environment and neuronal interaction in cancer lipid reliance.},
}
@article {pmid40674704,
year = {2025},
author = {Yamaguchi, K and Koya, J and Mizuno, K and Mizukami, Y and Yoshifuji, K and Saito, Y and Tabata, M and Shingaki, S and Yuasa, M and Ito, Y and Nakashima, K and Dreval, K and Morin, RD and Chiba, K and Okada, A and Shiraishi, Y and Murakami, K and Kogure, Y and Ohshima, K and Kataoka, K},
title = {In vivo CRISPR screening reveals cooperation of KMT2D and TP53 deficiencies in B-cell lymphomagenesis.},
journal = {Blood advances},
volume = {9},
number = {19},
pages = {5040-5055},
doi = {10.1182/bloodadvances.2024015519},
pmid = {40674704},
issn = {2473-9537},
mesh = {*Tumor Suppressor Protein p53/genetics/deficiency ; Humans ; Animals ; Mice ; *Lymphoma, Large B-Cell, Diffuse/genetics/pathology ; *DNA-Binding Proteins/genetics/deficiency ; *CRISPR-Cas Systems ; *Lymphoma, B-Cell/genetics ; *Neoplasm Proteins/genetics/deficiency ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line, Tumor ; },
abstract = {Although recent genetic studies have identified numerous genetic alterations in diffuse large B-cell lymphoma (DLBCL), their biological relevance remains elusive. Here, we performed in vivo CRISPR loss-of-function screening targeting 86 genes recurrently altered in DLBCL to examine oncogenicity of single-guide RNA (sgRNA)-targeted genes, association between genotype and lineage, occurrence of second-hit alterations, and cooperability among sgRNA-targeted genes and second-hit alterations. Transplantation of the CRISPR library-transduced hematopoietic stem/progenitor cells induces various hematologic malignancies, including B-cell lymphomas in mice. Enrichment analysis of sgRNA-targeted genes demonstrates significant overrepresentation of Kmt2d, Pax5, and Trp53 in B-cell lymphomas. Whole-exome sequencing identifies recurrent second-hit driver alterations, showing significant enrichment of Trp53 alterations in sgKmt2d-targeted B-cell lymphomas. Importantly, KMT2D and TP53 mutations are found to be the most prevalent co-occurring combination in human DLBCL, which is more prominent in relapsed/refractory DLBCL. Moreover, this combination confers significantly worse prognosis independent of clinical factors. Transcriptomic sequencing identifies overexpression of Yap1, the Hippo pathway component, in double sgKmt2d-targeted/Trp53-altered B-cell lymphomas. Furthermore, chromatin accessibility analysis demonstrates enrichment of transcriptional enhanced associate domain 1 binding motifs in regions that gained accessibility and increased expression of their nearest genes in these B-cell lymphomas. Most importantly, genetic and pharmacological inhibition of YAP1 suppresses in vitro cell proliferation and in vivo tumor growth of a human KMT2D/TP53-altered DLBCL cell line and prolongs survival of mice transplanted with double sgKmt2d-targeted/Trp53-altered B-cell lymphoma cells. Our findings demonstrate the utility of in vivo CRISPR screening to integrate human cancer genomics with mouse modeling and highlight the functional interplay between KMT2D and TP53 aberrations, providing insights into therapeutic strategies in DLBCL.},
}
@article {pmid41033726,
year = {2025},
author = {Gupta, Y and Chosdol, K},
title = {Practical approaches to advanced molecular biology techniques.},
journal = {Methods in cell biology},
volume = {198},
number = {},
pages = {73-101},
doi = {10.1016/bs.mcb.2025.02.022},
pmid = {41033726},
issn = {0091-679X},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Molecular Biology/methods ; Single-Cell Analysis/methods ; Animals ; Chromatin Immunoprecipitation/methods ; Proteomics/methods ; },
abstract = {The field of molecular biology has undergone tremendous advancements in recent years, with the development of powerful techniques that allow for in-depth exploration of cellular processes at the molecular level. This chapter, "Advanced Molecular Biology Techniques," provides a detailed protocol of the molecular techniques. We begin with CRISPR-Cas9 genome editing, a transformative tool for precise and efficient gene manipulation, enabling targeted mutations and gene knockouts in various organisms. Gene amplification via Real-Time PCR is then discussed, highlighting its ability to quantify gene expression and detect rare genetic variants with high sensitivity. Flowcytometry follows, offering a robust platform for analyzing cellular populations based on specific markers, enabling the study of immune cells, cancer diagnostics, and cell cycle analysis. Chromatin Immunoprecipitation Sequencing (ChIP-Seq) is explored as a method for mapping protein-DNA interactions, providing insights into gene regulation and epigenetic modifications. The chapter also covers Single-cell RNA sequencing (scRNA-Seq), a groundbreaking technique for profiling gene expression at the single-cell level, allowing for the discovery of cell heterogeneity and complex biological processes. Next, we explore into proteomics through Mass Spectrometry-Based Analysis, which offers detailed proteome characterization and biomarker discovery by identifying and quantifying proteins in complex samples. Finally, Fluorescence In Situ Hybridization (FISH) is discussed as a method for visualizing the spatial localization of specific nucleic acid sequences within intact cells or tissues. Together, these advanced molecular biology techniques offer unparalleled precision and insight into the molecular mechanisms underlying health, disease, and cellular function.},
}
@article {pmid41033723,
year = {2025},
author = {Hasan, N and Palungan, J and Ullah, M},
title = {Gene editing techniques in cancer research.},
journal = {Methods in cell biology},
volume = {198},
number = {},
pages = {287-312},
doi = {10.1016/bs.mcb.2025.03.002},
pmid = {41033723},
issn = {0091-679X},
mesh = {*Gene Editing/methods ; Humans ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems/genetics ; Zinc Finger Nucleases/genetics ; Transcription Activator-Like Effector Nucleases/genetics ; Animals ; },
abstract = {The process of editing genes has emerged as a game-changing instrument in the field of cancer research. It has the potential to provide a whole new understanding of the biology of tumors and to facilitate the creation of tailored medicines. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system are the three basic methods of gene editing techniques that are discussed in this chapter. We investigate the protocol modifications that are specific to each approach, focusing on high-prevalence tumors, and we investigate the utility, efficiency, and application issues that are associated with each technique in oncology. In addition, we describe current developments in improving these methods to successfully target oncogenes and tumor suppressor genes, with the goal of driving forward advances in precision cancer therapy.},
}
@article {pmid41033719,
year = {2025},
author = {Tiwari, PC and Chaudhary, MJ and Pal, R and Nath, R},
title = {In vivo cancer modeling using mouse models.},
journal = {Methods in cell biology},
volume = {198},
number = {},
pages = {221-250},
doi = {10.1016/bs.mcb.2025.02.013},
pmid = {41033719},
issn = {0091-679X},
mesh = {Animals ; Mice ; *Disease Models, Animal ; *Neoplasms/pathology/genetics ; Humans ; Tumor Microenvironment ; Xenograft Model Antitumor Assays/methods ; CRISPR-Cas Systems ; },
abstract = {Mouse models have contributed to a better understanding of cancer biology and the development of new treatments. This chapter elaborates on the various types of mouse models applied in cancer research, such as xenograft, syngeneic, and humanized models, together with the state-of-the-art techniques of genetic engineering involved in their generation. We described the methodologies of tumor induction and engraftment procedures and these model applications in drug development, efficacy testing, and studies on immuno-oncology. Further, the chapter covers ethical considerations and regulatory requirements on the use of animals in research, essentially aligned with international guidelines and those in India. The chapter illustrates that mouse models will not become outdated in preclinical testing any time soon but continue to be relevant for the study of tumor biology and the tumor microenvironment besides their use for investigating genetic and molecular pathways in cancer. Emerging technologies, such as CRISPR/Cas9 and organoid integration, are also highlighted for their work in improving the accuracy and translational potential of models. These developments combined with initiatives on collaborative and open science that enable the sharing of data and resources, hold great promise for the future of in vivo cancer modeling. The mouse models will continue to be one of the prime movers in advancing cancer research and formulating individual medication strategies that lead to improved patient outcomes through their integration of classical approaches with modern technologies.},
}
@article {pmid41032194,
year = {2025},
author = {Naderian, R and Alibabaei, F and Paraandavaji, E and Dehghan, P and Eslami, M},
title = {Phage-Microbiota Interactions in the Gut: Implications for Health and Therapeutic Strategies.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41032194},
issn = {1867-1314},
abstract = {The diversified ecology of microorganisms, including bacteria, archaea, fungi, protozoa, and viruses known collectively as the gut microbiota, which includes bacteriophages, is crucial to human health because it affects functions like immune system regulation, vitamin production, and pathogen protection. Bacteriophages are viruses that infect bacteria and are increasingly recognized as a viable treatment option for antibiotic-resistant strains, owing to their high host specificity, which enables precise targeting of drug-resistant bacteria while sparing commensal microbiota. The complex relationships between bacteriophages and gut microbiota are examined, with emphasis on their roles in maintaining health and contributing to disease. Gut microbiota homeostasis is influenced by a number of factors, including age, nutrition, and drugs. Bacteriophages, via lytic cycles and lysogenic conversion, influence the gut microbiota composition and microbial community structure. Gaining an understanding of these processes is crucial to appreciating their contribution to the stability and variety of microbes. Recent research highlights the gut phageome's potential for therapeutic interventions by demonstrating its substantial influence on immunological responses and metabolic problems. The study of phage-microbiota interactions has been transformed by cutting-edge technologies, including high-throughput sequencing, CRISPR-Cas systems, and viral metagenomics, which allow for thorough research and the creation of new therapeutics. Even though tailored medicine and pathogen management hold great potential, obstacles such as regulatory difficulties and bacterial resistance call for additional investigation. Phage-based therapeutic strategies are rapidly advancing, ranging from genetically engineered phages and phages with modified capsid proteins designed to enhance efficacy to phage cocktails that target multiple bacterial strains.},
}
@article {pmid40803956,
year = {2025},
author = {Murata, S and Kushiyama, N and Yabu, Y and Watanabe, K and Fujii, T and Yasui, R and Nojima, D and Maeda, Y and Yoshino, T and Matsuda, Y and Tanaka, T},
title = {Establishment of genome editing techniques in the marine oleaginous diatom Fistulifera solaris for improved oil accumulation.},
journal = {Journal of bioscience and bioengineering},
volume = {140},
number = {5},
pages = {271-276},
doi = {10.1016/j.jbiosc.2025.07.008},
pmid = {40803956},
issn = {1347-4421},
mesh = {*Diatoms/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems ; Biofuels ; Lipase/genetics/metabolism ; Gene Knockout Techniques ; *Oils/metabolism ; Microalgae/genetics/metabolism ; Triglycerides/metabolism ; *Lipid Metabolism/genetics ; },
abstract = {Biofuel production using microalgae has attracted considerable attention owing to high growth rate and lipid accumulation properties. However, further enhancement in lipid productivity is required to render this economically feasible. CRISPR/Cas9, which is one of the powerful genome editing tools, is an essential technique that may solve this problem. The marine diatom Fistulifera solaris JPCC DA0580 is a promising candidate of the biofuel production, since it accumulates significant amount of lipids. However, genome editing techniques have not yet been established for F. solaris, which prevent the construction of valuable strains. In this study, CRISPR/Cas9-mediated specific gene knockout technique was established in F. solaris, through targeting adenine phosphoribosyl transferase gene (apt) and triacylglycerol (TAG) lipase gene (tgl1). Mutations in the target sequence were detected in apt- and tgl1-edited mutants. Moreover, the mutants showed distinct phenotypes, such as suppression of TAG degradation and resistance to 2-fluoroadenine. These results indicate the successful demonstration of CRISPR/Cas9-mediated genome editing in the oleaginous marine diatom F. solaris. Furthermore, oil degradation was successfully suppressed by knocking-out tgl1. The CRISPR/Cas9-mediated genome editing established in this study provides key molecular tools for both the basic biology and the future biotechnological applications of F. solaris, such as biofuel production.},
}
@article {pmid40774862,
year = {2025},
author = {Karashima, T and Oda, K and Futagami, T and Hokazono, H and Takashita, H},
title = {Ribonucleoprotein-based CRISPR/Cas9 genome co-editing in Aspergillus luchuensis mut. kawachii.},
journal = {Journal of bioscience and bioengineering},
volume = {140},
number = {5},
pages = {298-305},
doi = {10.1016/j.jbiosc.2025.07.006},
pmid = {40774862},
issn = {1347-4421},
mesh = {*CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Gene Editing/methods ; *Aspergillus/genetics/metabolism ; Gene Knockout Techniques ; *Genome, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sulfate Adenylyltransferase/genetics ; },
abstract = {In this study, we established a ribonucleoprotein-based clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome co-editing method for the white koji fungus, Aspergillus luchuensis mut. kawachii. To introduce the single guide RNA-Cas9 ribonucleoprotein complex into protoplast cells of A. luchuensis mut. kawachii, we investigated the conditions for protoplast preparation using Yatalase -Plus-. Subsequently, we employed the ribonucleoprotein-based method to knockout the ATP sulfurylase-encoding sC gene, which imparts selenate resistance in the model strain NBRC 4308 and the industrial strain No. 8046. Furthermore, we explored genome co-editing by simultaneously targeting sC along with either the orotidine 5'-phosphate decarboxylase-encoding pyrG gene or the transcriptional activator of protease genes-encoding prtR gene in NBRC 4308. The transformants were selected in medium containing selenate, resulting in the successful generation of pyrG- and prtR-knockout strains. Similarly, transformants were selected on medium containing selenate, resulting in the successful generation of prtR-knockout strain in No. 8046. These results demonstrate that the ribonucleoprotein-based genome co-editing method is applicable not only to the model strain but also to industrial strains, making it a promising approach for manipulating A. luchuensis mut. kawachii.},
}
@article {pmid40634141,
year = {2025},
author = {Shan, L and Verstrepen, KJ and Wang, Q and Dai, Z},
title = {A homologous recombination-proficient Yarrowia lipolytica chassis for multiplex genome manipulation.},
journal = {Trends in biotechnology},
volume = {43},
number = {10},
pages = {2627-2645},
doi = {10.1016/j.tibtech.2025.06.009},
pmid = {40634141},
issn = {1879-3096},
mesh = {*Yarrowia/genetics ; *Homologous Recombination/genetics ; *Gene Editing/methods ; DNA End-Joining Repair/genetics ; *Genome, Fungal/genetics ; CRISPR-Cas Systems ; },
abstract = {Homologous recombination (HR) greatly facilitates precise genome editing. However, most organisms prefer error-prone non-homologous end joining (NHEJ) for DNA double-strand break (DSB) repair. Here, the NHEJ-proficient Yarrowia lipolytica was transformed into a HR-proficient strain by systematic engineering of recombination machinery, regulating the multiinvasion-induced rearrangement (MIR) process, and expressing cognate single-stranded DNA-annealing protein (SSAP)-single-stranded DNA-binding protein (SSB) pairs. These strategies improved HR efficiency by 38.9, 1.6, and 1.2-fold compared with the NHEJ-deficient strain for multifragment multisite integration, and multi- and single-fragment single-site integration, respectively. Moreover, HR efficiency remained high at 58% even with 50-base pair (bp) homology arms (HAs) and reached 11% for simultaneously integrating two mega-DNA fragments (18.0 kb and 13.5 kb) at two genome sites. This strain also enabled simultaneous editing, repression, and activation of multiple genes, while cellular robustness parameters showed marked increases over the NHEJ-deficient strain. Our work provides a HR-proficient Y. lipolytica chassis allowing efficient and precise genome editing of this increasingly important microbe.},
}
@article {pmid40605271,
year = {2025},
author = {Stigzelius, V and Cavallo, AL and Chandode, RK and Nitsch, R},
title = {Peeling back the layers of immunogenicity in Cas9-based genomic medicine.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {10},
pages = {4714-4730},
doi = {10.1016/j.ymthe.2025.06.045},
pmid = {40605271},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems/immunology ; Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; *CRISPR-Associated Protein 9/immunology/genetics ; *Genomics/methods ; },
abstract = {The CRISPR-Cas9 genome editing system is rewriting the treatment of genetic disorders, offering unprecedented potential for detrimental and previously untreatable diseases. As this technology advances toward wider utilization in clinical applications, the immunogenicity of Cas9 nuclease has emerged as a potential challenge for in vivo therapies. Immune recognition of CRISPR-Cas9 components can trigger both innate and adaptive responses. The complex interactions between Cas9, delivery vectors, and host immune reactivity play a crucial role in determining the safety and efficacy of CRISPR-based treatments. Recent advances in mitigating Cas9 immunogenicity include epitope engineering, optimized delivery systems, and nucleic acid modifications. These strategies, explored across various tissue contexts and delivery methods, show promise in enhancing the tolerability of CRISPR-based therapies. However, pre-existing immunity to Cas9 and the potential for long-term adaptive immune responses remain important considerations. Addressing these immunological challenges requires an integrated approach, combining insights from immunology with innovative engineering solutions. As the field progresses, overcoming Cas9 immunogenicity will be crucial for realizing the full therapeutic potential of the CRISPR-Cas9 system in diverse clinical applications.},
}
@article {pmid40368676,
year = {2025},
author = {Zhang, S and Xu, D and Li, F and Wang, J},
title = {CRISPR-based non-nucleic acid detection.},
journal = {Trends in biotechnology},
volume = {43},
number = {10},
pages = {2494-2508},
doi = {10.1016/j.tibtech.2025.04.012},
pmid = {40368676},
issn = {1879-3096},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Nucleic Acids/genetics/analysis ; Gene Editing/methods ; Humans ; },
abstract = {Characterization of clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) trans-cleavage activities has initiated the era of next-generation CRISPR diagnostics. By using the trans-cleavage reaction for signal output, CRISPR systems have been engineered to detect non-nucleic acids (NNAs), including ions, inorganic small molecules, organic compounds, proteins, and bacteria. Diverse strategies are being used to specifically recognize NNAs and regulate Cas trans-cleavage activities, via generation or depletion of output signals. In this review, we introduce the principles and advantages of CRISPR-based NNA detection. We then classify CRISPR-based NNA detection strategies into three classes: the generation or depletion of free activators, synthesis of crRNAs, and reconstruction of active Cas effectors. Finally, we discuss the challenges and potential strategies to advance both clinical and nonclinical applications of CRISPR-based NNA detection.},
}
@article {pmid41032193,
year = {2025},
author = {Yetiman, AE and Horzum, M and Kanbur, E and Çadir, M and Bahar, D and Gürbüz, &#x15e; and Karaman, MZ and Fidan, &#xd6; and Kaya, M and Yetiman, S and Doğan, M and Akbulut, M},
title = {Pangenome Analysis and Genome-Guided Probiotic Evaluation of Cyclic Dipeptides Producing Levilactobacillus brevis DY55bre Strain from a Lactic Acid Fermented Shalgam to Assess Its Metabolic, Probiotic Potentials, and Cytotoxic Effects on Colorectal Cancer Cells.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41032193},
issn = {1867-1314},
support = {FBA-2025-14779//Bilimsel Ara&#x15f;t&#x131;rma Projeleri, Erciyes &#xdc;niversitesi/ ; },
abstract = {This study investigates the genetic, metabolic, and probiotic characteristics of Levilactobacillus brevis DY55bre, a strain isolated from the traditional Turkish fermented beverage, shalgam. Whole-genome sequencing revealed a circular genome of 2.485 Mb with a GC content of 45.72%, predicted 2791 genes, and multiple CRISPR-Cas systems. Pangenome analysis demonstrated an open structure, with 18.9% core genes and 103 strain-specific genes, highlighting its genetic diversity. The DY55bre exhibits heterofermentative carbohydrate metabolism due to the presence of the araBAD operon and the lack of 1-phosphofructokinase (pfK) and fructose-1,6-bisphosphate aldolase enzymes. Probiotic evaluation revealed firm survival under simulated gastrointestinal conditions, including resistance to acidic pH (as low as 3.0) and bile salts (up to 1%), along with significant adhesion to intestinal epithelial cell lines (HT29;59.3%, Caco-2;87%, and DLD-1;60.8%). The strain exhibited high auto-aggregation (84.55%) and cell surface hydrophobicity (56.69%), essential for gut colonization. Safety assessments confirmed its non-hemolytic nature and absence of horizontally acquired antibiotic resistance genes. Notably, GC-MS analysis identified bioactive cyclic dipeptides, Cyclo(D-Phe-L-Pro) and Cyclo(L-Leu-L-Pro), which demonstrated cytotoxic effects against colorectal cancer cell lines, with IC50 values of 7.71 mg/mL for HT29 and 3.19 mg/mL for DLD-1. The cell-free supernatant exhibited antimicrobial activity against pathogens, likely due to the synergistic effects of cyclic dipeptides, organic acids, and other metabolites. Antioxidant assays revealed significant ABTS[+] (76.63%) and DPPH (34.25%) radical scavenging activities, while cholesterol assimilation tests showed a 27.29% reduction. These findings position the DY55bre as a promising candidate for functional foods, nutraceuticals, and therapeutic applications, warranting further in vivo validation.},
}
@article {pmid41032156,
year = {2025},
author = {Meshram, V and Jadhav, SK and Chandrawanshi, NK},
title = {Strain improvement of Cordyceps militaris for optimized bioactive metabolite biosynthesis: current progress and prospective approaches.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {11},
pages = {162},
pmid = {41032156},
issn = {1572-9699},
support = {201610136180//University Grants Commission/ ; },
mesh = {*Cordyceps/metabolism/genetics ; *Metabolic Engineering/methods ; Gene Editing ; CRISPR-Cas Systems ; Biosynthetic Pathways ; },
abstract = {Cordyceps militaris is a rare and highly valued medicinal fungus that has attracted considerable attention due to its production of diverse bioactive compounds, including nucleosides such as cordycepin, polysaccharides, lovastatin, carotenoids, etc., all of which exhibit significant nutritional and therapeutic potential. However, the large-scale utilization of C. militaris is constrained by several critical challenges. A major limitation is the progressive degeneration of strains over successive subcultures, which adversely affects fruiting body formation and metabolite biosynthesis. Moreover, genetic instability during long-term culture, contamination risks in large-scale production, and the lack of standardized cultivation and extraction protocols often result in variable product quality. The absence of efficient genetic transformation systems and the low success rate of genome editing approaches further complicate efforts in molecular strain improvement. This review provides a comprehensive overview of the principal bioactive compounds produced by C. militaris and critically evaluates the current challenges and limitations associated with both conventional and advanced strain improvement strategies. These include conventional approaches such as mutagenesis and protoplast fusion, as well as genome-editing technologies like CRISPR/Cas9, which are employed to enhance the biosynthesis of target metabolites. Moreover, the integration of metabolic engineering frameworks offers significant potential for rational strain design, optimization of bioprocesses, and the discovery of novel therapeutic agents.},
}
@article {pmid41032013,
year = {2025},
author = {Huang, X and Li, H and Du, J and Xie, W and Liu, Y and Lin, Y and Xing, H},
title = {HUH endonuclease-mediated DNA-protein conjugates: sequence-specific tools and cellular applications.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc04628a},
pmid = {41032013},
issn = {1364-548X},
abstract = {This highlight review article summarizes recent advances in employing HUH endonucleases as self-labeling protein tags for the sequence-specific covalent conjugation of unmodified ssDNA and examines their applications in cellular studies via engineered DNA-protein conjugates. We outline the structural basis and catalytic mechanism of the conserved HUH and Y motifs, which enable high selectivity, bioorthogonality, and robust conjugation under physiological conditions. Recent applications demonstrate the versatility of HUH-based DNA-protein conjugates in programmable cellular interface engineering, targeted therapeutic delivery, and enhancement of genome editing systems such as CRISPR-Cas. In the perspective section, we further highlight two emerging directions: computational tools such as the HUHgle platform for predictive substrate design, and directed evolution strategies extending HUH reactivity toward RNA substrates. Together, these advancements establish HUH endonucleases as powerful, programmable tools for generating DNA-protein conjugates that enable innovations in chemical biology, synthetic biology, and therapeutics.},
}
@article {pmid41030550,
year = {2025},
author = {Yang, ZQ and Li, MJ and Ahmad, F and Jin, CZ and Li, T and Jin, FJ and Shin, KS and Jin, L},
title = {Application of the transposon-associated TnpB system of CRISPR-Cas in bacteria: Deinococcus.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1604032},
pmid = {41030550},
issn = {1664-302X},
abstract = {Deinococcus radiodurans is one of the most radioresistant organisms found on Earth to date, showing extreme resistance to damage factors such as UV, drought, and mutagens, and is of great interest to scientists around the world. It was determined that the TnpB protein from D. radiodurans ISDra2 functions as an RNA-guided endonuclease, serving as a functional ancestor for the widely used CRISPR-Cas endonucleases. The CRISPR-Cas system is an "acquired immune system" found in most Bacteria and Archaea, and used in a wide range of biological and medical research fields. Cas12f is the smallest RNA-directed nuclease that is currently known and possesses unique characteristics. There has been extensive research conducted on the origin, classification, and mechanism of action of CRISPR-Cas12f, as well as its application in the field of gene editing. TnpB, as the protein closest to Cas12f in the evolutionary tree, has the potential to be used as a new micro-editing tool. Systematic studies have been conducted on it to develop smaller volumes of precision gene editing and treatment tools. In this review, the research progress, mechanism, and application of TnpB protein in D. radiodurans were reviewed. In addition, the classification of CRISPR-Cas system and the application and function of CRISPR-Cas12f in gene editing are also introduced and summarized.},
}
@article {pmid41028589,
year = {2026},
author = {Bhargava, CN and Karuppannasamy, A and Ramasamy, A},
title = {CRISPR/Cas9-Mediated Genome Editing in the Management of Oriental Fruit Fly, Bactrocera dorsalis (Hendel) (Tephritidae: Diptera).},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2966},
number = {},
pages = {259-270},
pmid = {41028589},
issn = {1940-6029},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Tephritidae/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Insect ; Pest Control, Biological/methods ; },
abstract = {The oriental fruit fly, Bactrocera dorsalis (Hendel), is a highly invasive polyphagous pest that causes significant damage to horticultural crops of global importance. Traditional management practices have not been effective in controlling this pest, and therefore, there is a need for alternative management strategies. CRISPR/Cas9-driven genome editing has been successfully used in a wide range of insects to induce site-specific, off-target minimized mutations that result in loss of function. This technique can be used to develop precision-guided sterile insect technique (pgSIT) and gene drive programs, which can be used for area-wide suppression of the pest. This chapter provides a brief overview of the workflow for RNP-based genome editing, which can be used to validate and establish gene function for large-scale gene drive programs aimed at combating this pest. The RNP, or ribonucleoprotein complex, comprises the sgRNA and Cas9 protein, which are microinjected into the G0 stage embryos for heritable editing of the target gene(s).},
}
@article {pmid41028581,
year = {2026},
author = {Ashokkumar, S and Ponnurangan, V and Krish, KK and Loganathan, A and Eswaran, K and Vaikuntavasan, P and Duraialagaraja, S and Shanmugam, V},
title = {CRISPR-Mediated Gene Editing for Inducing Thermosensitive Genic Male Sterility and Sheath Blight Resistance in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2966},
number = {},
pages = {127-140},
pmid = {41028581},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Oryza/genetics/microbiology ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Infertility/genetics ; *Plant Diseases/genetics/microbiology ; Plants, Genetically Modified/genetics ; },
abstract = {Recent advances in genome editing enable the researchers to focus more and more on the ability to manipulate genomes at specific sites. Efficient methods for genome editing further promote gene discovery and functional gene analyses in model plants as well as the introduction of novel desired agricultural traits in important species. CRISPR/Cas9 technology enables precise genetic modification through the creation of double-strand breaks in a target region and the generation of desired alterations during the repair process. In this chapter, we describe the cloning strategy, transformation protocols, triparental mating procedure, and characterization of genome-edited genetic male sterile mutants and sheath blight disease-resistant mutant plants.},
}
@article {pmid41028476,
year = {2026},
author = {Raschmanová, H and Weninger, A and Kovar, K},
title = {Engineering Pichia pastoris Strains Using CRISPR/Cas9 Technologies: The Basic Protocol.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2697},
number = {},
pages = {361-371},
pmid = {41028476},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Saccharomycetales/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Metabolic Engineering/methods ; *Pichia/genetics ; Genome, Fungal ; },
abstract = {The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats, CRISPR-associated protein 9) system has become a commonly used tool for genome editing and metabolic engineering. For Komagataella phaffii, commercialized as Pichia pastoris, the CRISPR/Cas9 protocol for genome editing was established in 2016 and since then has been employed to facilitate genetic modifications such as markerless gene disruptions and deletions as well as to enhance the efficiency of homologous recombination.In this chapter, we describe a robust basic protocol for CRISPR-based genome editing, demonstrating near 100% targeting efficiency for gene inactivation via a frameshift mutation. As described in other chapters of this volume, CRISPR/Cas9 technologies for use in P. pastoris have been further optimized for various specific purposes.},
}
@article {pmid41028475,
year = {2026},
author = {Smirnov, K and Rieder, L and Glieder, A},
title = {High-Throughput Generation of Pichia pastoris Knock-Out Strains by Using CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2697},
number = {},
pages = {345-360},
pmid = {41028475},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plasmids/genetics ; *Gene Knockout Techniques/methods ; *Saccharomycetales/genetics ; Genetic Vectors/genetics ; },
abstract = {The CRISPR/Cas9 (CRISPR is an acronym for clustered regularly interspaced short palindromic repeats) system is a powerful molecular biological tool simplifying the process of genome engineering. Originally demonstrated to function in human and mouse cells, the portfolio of organisms that can be engineered by the new and groundbreaking technology was soon expanded. In the past years, CRISPR/Cas9 tools for use in Komagataella phaffii were reported to allow the generation of K. phaffii mutant strains in less than 2 weeks. In addition, the K. phaffii tailored system uses episomal vectors for the expression of the CRISPR/Cas9 elements, which allows the recycling of the plasmid after the CRISPR editing to obtain empty mutant strains. This means that the engineered strains do not carry the expression cassette of the resistance marker and CRISPR/Cas9 plasmid in their genome and are therefore a superb starting point for further investigations.In this chapter, we describe a pipeline for the high-throughput generation of K. phaffii mutant strains with interrupted open reading frames of genes, by using the CRISPR/Cas9 system in combination with error-prone repair of the double-strand break by NHEJ. The pipeline we developed consists of four steps: (a) CRISPR/Cas9 plasmids assembly, (b) transformation of K. phaffii, (c) screening for mutant strains, and (d) plasmid elimination and is due to the detailed description of every step being easily reproducible. To intensify and simplify the research work, most of the described procedures can be performed in a 96-well format.},
}
@article {pmid41028463,
year = {2026},
author = {Pichler, C and Weiss, F and Glieder, A},
title = {Autonomously Replicating Sequence-Bearing Plasmids Utilized in Pichia pastoris.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2697},
number = {},
pages = {191-203},
pmid = {41028463},
issn = {1940-6029},
mesh = {*Plasmids/genetics ; *Saccharomycetales/genetics ; *DNA Replication ; Transformation, Genetic ; Saccharomyces cerevisiae/genetics ; },
abstract = {Plasmids are a common tool in biotechnology to deliver recombinant DNA into microbial cells for the production of enzymes, pharmaceutical proteins, chemicals, or metabolites. Therefore, a stable plasmid system that provides reliable gene expression over generations is essential for the successful utilization of single-cell organisms in research and production applications. Most Komagataella phaffii expression clones are generated by the integration of linear plasmids into the genome, as circular episomal plasmids are not stable under non-selective conditions. The low rate of homology-directed specific integration and the large variation among transformants of random integration limit the organism's application in enzyme engineering approaches or comparative studies where high transformation rates and uniform expression levels are desired. In the yeast Saccharomyces cerevisiae, the problem of circular plasmid stability and partition to the daughter cells during mitosis has been solved by combining centromeric sequences or elements of the 2-micron plasmid with an autonomously replicating sequence (ARS) that serves as an origin of replication. Similar attempts have not yet been successful or widely adapted in K. phaffii; hence, permanent selection pressure is required to maintain episomal plasmids in K. phaffii. There are no reports so far about functional 2-micron plasmids for P. pastoris, and CEN/ARS plasmids for P. pastoris are usually rather large and do not provide the high transformation rates as known for episomal plasmids of S. cerevisiae expression systems. However, the availability of a broad set of resistance, auxotrophic, and carbon source utilization markers facilitates reliable plasmid selection in small-scale screening applications and recently also proved to be successful for bioreactor-scale expression. This allows the combined advantages of high transformation rates and low clonal variability of ARS plasmids to be exploited. This article describes the successful utilization of ARS1-containing plasmids in K. phaffii, including antibiotic-free selection, complementation of knockout strains, or even for the application of CRISPR/Cas by transient gRNA and CAS9 gene expression in K. phaffii.},
}
@article {pmid41028441,
year = {2025},
author = {Kannan, S and Tennyson, J},
title = {Regeneration of Transgenic Nicotiana benthamiana Raised from the Genome-Edited Protoplast.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2973},
number = {},
pages = {261-274},
pmid = {41028441},
issn = {1940-6029},
mesh = {*Nicotiana/genetics/growth &amp; development ; *Protoplasts/metabolism ; *Gene Editing/methods ; *Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; *Genome, Plant ; Transfection/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Regeneration/genetics ; },
abstract = {Plant genome editing is an emerging technique that has revolutionized plant genome engineering which helps to edit the plant genome precisely for the development of traits in many crops. Specifically, with clustered regularly interspaced short palindromic sequence (CRISPR)-CRISPR-associated protein (Cas) system, a progressive improvement in genome editing has been achieved with protoplast. Though protoplast isolation, transfection, and regeneration are available for many plants, regeneration of protoplast for many plants remains major challenge. In this methodology chapter, we outlined the construction of sgRNA for genome editing, transfection, and regeneration of transgenic N. benthamiana from the genome-edited protoplast and assay for gene targeting.},
}
@article {pmid41028420,
year = {2025},
author = {Andrew-Peter-Leon, MT and Pillai, MA and Kumar, KK and Sumithra, V},
title = {Agrobacterium-Mediated Genetic Transformation and Genome Editing Using CRISPR-Cas9 Constructs in Rice.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2973},
number = {},
pages = {27-49},
pmid = {41028420},
issn = {1940-6029},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Transformation, Genetic ; *Agrobacterium/genetics ; Plants, Genetically Modified/genetics ; *Genome, Plant ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)9 has become an essential tool in every molecular plant breeding laboratory. CRISPR-Cas9 causes highly precise knock-out mutations in plants that can be exploited in crop improvement programmes. However, poor response to Agrobacterium-mediated genetic transformation in recalcitrant rice genotypes is a major limiting factor. This protocol describes a detailed procedure for genome editing with CRISPR-Cas9 in recalcitrant rice genotypes that otherwise show a poor response to tissue culture. With this method, high transformation efficiency can be achieved in relatively a short period.},
}
@article {pmid41028383,
year = {2026},
author = {Yang, H and Bao, A and Tran, LP and Cao, D},
title = {CRISPR/Cas9-Based Gene Editing in Soybean.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2977},
number = {},
pages = {251-267},
pmid = {41028383},
issn = {1940-6029},
mesh = {*Glycine max/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Agrobacterium/genetics ; Transformation, Genetic ; },
abstract = {CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated Cas9)-based gene editing is a robust tool for functional genomics research and breeding programs in various crops. In soybean (Glycine max), a number of laboratories have obtained mutants by the CRISPR/Cas9 system; however, there has not yet been a detailed method for the CRISPR/Cas9-based gene editing in soybean. Here, we describe the procedures for constructing the CRISPR/Cas9 plasmid suitable for soybean gene editing and the modified protocols for Agrobacterium-mediated soybean transformation and regeneration from cotyledonary node explants containing the Cas9/sgRNA (single-guide RNA) transgenes.},
}
@article {pmid41028112,
year = {2025},
author = {Najar, IN and Sharma, P and Das, R and Mondal, K and Singh, AK and Radha, A and Sharma, V and Sharma, S and Thakur, N and Gandhi, SG and Kumar, V},
title = {Unveiling the probiotic potential of the genus Geobacillus through comparative genomics and in silico analysis.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {33748},
pmid = {41028112},
issn = {2045-2322},
mesh = {*Probiotics ; *Geobacillus/genetics ; *Genome, Bacterial ; *Genomics/methods ; Computer Simulation ; CRISPR-Cas Systems ; Humans ; Prophages/genetics ; },
abstract = {Pursuing new probiotic targets has surged, driven by next-generation sequencing, facilitating a thorough exploration of bacterial traits. The genus Geobacillus stands out as a promising candidate for probiotics. The study explored the genetic attributes of the genus Geobacillus for their resilience to gastrointestinal conditions, nutrient production, and immunomodulatory compound creation, revealing potential probiotic traits. Predictive analyses of genomic elements like prophages, CRISPR-Cas systems, insertion sequences, genomic islands, antibiotic resistance genes, and CAZymes were conducted to assess safety. Comparative genomic analysis was performed using 18 published Geobacillus genomes and a few Lactobacillus and Bifidobacterium genomes as controls. Genes associated with probiotic traits, such as adhesion, stress tolerance (acid/bile, osmotic, oxidative), immune modulation, and molecular chaperones, were uniformly detected in Geobacillus. Mobile genetic elements (such as plasmids, prophages, and insertion sequences), virulence factors, toxins, and antibiotic resistance genes were absent, while CRISPR-Cas systems and CAZymes were present. The pan-genome comprised 25,284 protein-coding genes. Comparative genomic analysis revealed an open pan-genome for Geobacillus. Pan-genome exhibited variability, particularly in genes linked to environmental interaction and secondary metabolite synthesis. Geobacillus appears potentially safe and well-suited for the gut habitat. However, further in vitro studies are essential to confirm its probiotic potential.},
}
@article {pmid41027434,
year = {2025},
author = {Jin, S and Zhu, Z and Li, Y and Zhang, S and Liu, Y and Li, D and Li, Y and Luo, Y and Cheng, Z and Zhao, KT and Gao, Q and Yang, G and Li, H and Liang, R and Zhang, R and Qiu, JL and Zhang, YE and Liu, JG and Gao, C},
title = {Functional RNA splitting drove the evolutionary emergence of type V CRISPR-Cas systems from transposons.},
journal = {Cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.cell.2025.09.004},
pmid = {41027434},
issn = {1097-4172},
abstract = {Transposon-encoded TnpB nucleases gave rise to type V CRISPR-Cas12 effectors through multiple independent domestication events. These systems use different RNA molecules as guides for DNA targeting: transposon-derived right-end RNAs (reRNAs or omega RNAs) for TnpB and CRISPR RNAs for type V CRISPR-Cas systems. However, the molecular mechanisms bridging transposon activity and CRISPR immunity remain unclear. We identify TranCs (transposon-CRISPR intermediates) derived from distinct IS605- or IS607-TnpB lineages. TranCs utilize both CRISPR RNAs and reRNAs to direct DNA cleavage. The cryoelectron microscopy (cryo-EM) structure of LaTranC from Lawsonibacter sp. closely resembles that of the ISDra2 TnpB complex; however, unlike a single-molecule reRNA, the LaTranC guide RNA is functionally split into a tracrRNA and crRNA. An engineered RNA split of ISDra2 TnpB enabled activity with a CRISPR array. These findings indicate that functional RNA splitting was the primary molecular event driving the emergence of diverse type V CRISPR-Cas systems from transposons.},
}
@article {pmid41026506,
year = {2025},
author = {Demissie, HA and Das, S and Thompson, JR and Lucks, JB},
title = {An Integrated Nucleic Acid Sequence-Based Amplification (NASBA) and CRISPR-Cas13a-Based Platform for Accurate and Sensitive Detection of Cucumber Mosaic Virus.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00406},
pmid = {41026506},
issn = {2161-5063},
abstract = {Cucumber mosaic virus (CMV) is a highly prevalent ssRNA viral crop pathogen that contributes to substantial losses in agricultural productivity worldwide. The first step in managing the impact of this pathogen is an accurate and timely diagnosis. However, current sensing strategies are hampered by several limitations, including insufficient sensitivity, off-target effects, and the need for complex instrumentation. To address these challenges, we refined a highly specific and sensitive system that pairs nucleic acid sequence-based amplification (NASBA) with clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a to selectively amplify and detect crop pathogens. To configure this system for CMV biosensing, we first screened guide RNAs and successfully validated designs that detect attomolar concentrations of purified CMV fragments. We then developed a simplified reaction assembly workflow toward optimizing the system for downstream point-of-use utility. Using this workflow, we demonstrated minimal matrix effects when detecting purified CMV fragments in a range of plant lysate backgrounds and showed high test specificity to CMV in the presence of common nontarget viral crop pathogens. We also showed that the NASBA-Cas13a system effectively detects the viral target in infected plant samples, as validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Finally, we optimized the system for lyophilization and long-term storage, toward preparing it for point-of-use settings. This work expands the suite of CMV diagnostic tools, offering a sensitive, specific, and user-friendly biosensing strategy. Through modular design, this assay has the potential to be reconfigured for the detection of a range of crop viruses, enhancing viral surveillance and improving infection management.},
}
@article {pmid41026406,
year = {2025},
author = {Rahman, MU and Shah, JA and Khan, MN and Bilal, H and Zhu, D and Du, Z and Mu, DS},
title = {Innovative Approaches to Combat Antimicrobial Resistance: A Review of Emerging Therapies and Technologies.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41026406},
issn = {1867-1314},
abstract = {The threat of antimicrobial resistance (AMR) presents a challenge in infectious diseases, leading to higher illness and deaths worldwide. No new antibiotic has been introduced, leaving healthcare systems vulnerable to resistant pathogens. Researchers are exploring innovative approaches to overcome this growing resistance crisis. One promising strategy is synergistic therapy using combined drugs to enhance efficacy and reduce resistance. Other approaches focus on targeting the specific enzymes or proteins responsible for resistance mechanisms, thereby neutralizing the defense strategies of microorganisms. Advances in drug delivery systems have also shown promise in improving the effectiveness of existing antimicrobial agents. Biotechnological breakthroughs, such as bacteriophages and antibodies, have seen partial clinical implementation, while newer approaches like antimicrobial peptides (AMPs), lysins, and probiotics are still under development. Emerging technologies such as CRISPR-Cas and engineered phages demonstrate significant potential in preclinical studies, offering precision targeting of resistance genes and pathogen-specific lysis, respectively. However, their translational success hinges on overcoming delivery challenges, scalability, and regulatory hurdles. Additionally, physicochemical methods that disrupt microbial activity are being explored as alternative treatments. While innovative therapies like phage-derived lysins and CRISPR-Cas systems show promise in preclinical models, their clinical impact remains to be validated through large-scale trials. Their integration into mainstream medicine will depend on addressing practical challenges such as manufacturing consistency, cost considerations, and real-world efficacy assessments. These efforts are crucial for addressing the growing threat of AMR and advancing more effective, sustainable infection control strategies in clinical settings.},
}
@article {pmid41025805,
year = {2025},
author = {Nadar, S and Brown, JC and Coe, LSY and Koukoulidis, NM and Czyż, EM and Czyż, DM},
title = {Antimicrobial resistance and One Health in the high school biology curriculum.},
journal = {Journal of microbiology &amp; biology education},
volume = {},
number = {},
pages = {e0014525},
doi = {10.1128/jmbe.00145-25},
pmid = {41025805},
issn = {1935-7877},
abstract = {Antimicrobial resistance (AMR) is the ability of a microbial organism to resist treatment designed to kill it. It poses a significant global threat to public health, affecting humans, animals, and the environment, in a concept collectively referred to as One Health. While one of the major mitigators of this pressing issue is education, the high school curriculum in the United States does not cover any aspects of AMR. As such, to address this challenge, we developed and delivered a one-week-long unit on AMR within a One Health framework into a high school biology curriculum. The unit aimed to enhance students' understanding of AMR and its implications across the One Health sectors. A survey was designed and administered to measure current knowledge, awareness, interest, and motivation. Through a combination of lectures developed using Universal Design of Learning principles, interactive discussions using team-based learning (TBL) with the help of content experts, hands-on laboratory exercise, and poster presentations, biology students explored the mechanisms of resistance and novel mitigation strategies. Pre- and post-assessments revealed a marked improvement in students' knowledge and comprehension of AMR and therapeutic strategies, such as silver nanoparticles, bacteriocins, bacteriophages, CRISPR-Cas, and immunotherapy. This research study provides a detailed overview of the curriculum design, instructional strategies, and assessment outcomes, offering a replicable model for broadly integrating AMR education into high school curricula. We found that the AMR mitigation strategies lesson, delivered through TBL, significantly enhanced students' understanding of novel therapeutic strategies and fostered high levels of engagement throughout the AMR and One Health unit.},
}
@article {pmid40961329,
year = {2025},
author = {Joseph, RA and Haley, RM and Padilla, MS and Ricciardi, AS and Yamagata, HM and Mitchell, MJ},
title = {Cas9 Protein Outperforms mRNA in Lipid Nanoparticle-Mediated CFTR Repair.},
journal = {Nano letters},
volume = {25},
number = {39},
pages = {14348-14355},
doi = {10.1021/acs.nanolett.5c03548},
pmid = {40961329},
issn = {1530-6992},
mesh = {*Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Cystic Fibrosis/genetics/therapy ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics ; Humans ; *Nanoparticles/chemistry ; Animals ; *RNA, Messenger/genetics/chemistry/administration &amp; dosage ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mice ; *Lipids/chemistry ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are currently one of the most clinically advanced delivery systems for nucleic acid cargo and hold great potential for clinical applications in gene editing and the treatment of genetic diseases. LNP-mediated delivery of Cas9 with single guide RNA (sgRNA) and homology-directed repair DNA template (ssDNA) enables efficient and precise editing in vitro and in vivo. Comparative analysis of LNP delivery of Cas9 as protein or mRNA for relevant clinical targets, such as cystic fibrosis (CF), which is caused by mutations in the CFTR gene, is imperative in the design of corrective therapeutics for genetic diseases. Here, we show that delivery of Cas9 protein LNPs outperforms Cas9 mRNA LNPs when evaluated for in vivo lung editing as well as corrective CRISPR/Cas9 editing and functional recovery of the CFTR protein. These results demonstrate the ability to optimize the use of CRISPR/Cas9 LNPs for cystic fibrosis applications.},
}
@article {pmid40938323,
year = {2025},
author = {Martin-Vicente, A and Nywening, AV and Xie, J and Thorn, HI and Guruceaga, X and Fortwendel, JR},
title = {Genetic analysis of common triazole resistance mechanisms in a collection of Aspergillus lentulus clinical isolates from the United States.},
journal = {Antimicrobial agents and chemotherapy},
volume = {69},
number = {10},
pages = {e0069025},
doi = {10.1128/aac.00690-25},
pmid = {40938323},
issn = {1098-6596},
support = {R01AI143197/NH/NIH HHS/United States ; R01AI158442/NH/NIH HHS/United States ; R21AI178048/NH/NIH HHS/United States ; },
mesh = {*Triazoles/pharmacology ; *Antifungal Agents/pharmacology ; *Aspergillus/drug effects/genetics/isolation &amp; purification ; *Drug Resistance, Fungal/genetics ; Microbial Sensitivity Tests ; United States ; Fungal Proteins/genetics/metabolism ; Aspergillosis/microbiology/drug therapy ; Humans ; Cytochrome P-450 Enzyme System/genetics/metabolism ; Aspergillus fumigatus/drug effects/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Aspergillus fumigatus continues to be the leading cause of invasive aspergillosis. However, the number of cases by drug-resistant cryptic species has increased in recent years. Aspergillus lentulus is a sibling species of Aspergillus section Fumigati that can only be distinguished from A. fumigatus by molecular methods. The clinical importance of this species resides in its low susceptibility to triazoles and intrinsic resistance to amphotericin B, making invasive aspergillosis treatments extremely challenging and producing high mortality rates. In this study, we investigate known molecular mechanisms important for triazole resistance in A. fumigatus in a collection of 25 clinical A. lentulus isolates from the United States. Using CRISPR-Cas9 gene editing technology, we performed cyp51A and hmg1 allele replacements between susceptible and resistant isolates. Phenotypic characterization of the resulting mutants, together with mRNA expression analyzes of cyp51A, cyp51B, and the putative ABC efflux pump, abcC, suggests that triazole resistance in our A. lentulus isolates is independent of the mechanisms studied.},
}
@article {pmid40692527,
year = {2025},
author = {Kohso, A and Inaba, H and Kanemaki, MT and Gabazza, EC and Toyoda, H and Hirayama, M and Goto, H},
title = {Aurora-A Promotes Cell-Cycle Progression From Quiescence Through Primary Cilia Disassembly.},
journal = {Cancer science},
volume = {116},
number = {10},
pages = {2763-2773},
doi = {10.1111/cas.70153},
pmid = {40692527},
issn = {1349-7006},
support = {JP22K06219//Japan Society for the Promotion of Science/ ; JP23H04925//Japan Society for the Promotion of Science/ ; JP23K06674//Japan Society for the Promotion of Science/ ; JP23K07651//Japan Society for the Promotion of Science/ ; //Takeda Science Foundation/ ; },
mesh = {Humans ; *Cilia/metabolism ; HCT116 Cells ; *Aurora Kinase A/metabolism/genetics ; *Cell Cycle ; CRISPR-Cas Systems ; Apoptosis/genetics ; Cell Proliferation ; },
abstract = {Aurora-A (AurA) is a member of the mitotic kinase family and is highly expressed in various tumors. Inhibition of AurA generally leads to fetal mitotic errors. We previously reported that AurA inhibition induces G0/G1 cell cycle arrest in noncancerous cells by promoting the reassembly of primary cilia. However, the mechanisms by which AurA regulates cell cycle progression beyond mitosis remain largely unknown. In this study, we generated noncancerous RPE1 and cancerous HCT116 cell lines expressing endogenous AurA tagged with a minimal auxin-inducible degron (mAID) using CRISPR/Cas9-based gene editing. This system enabled specific and rapid depletion of endogenous AurA protein. By combining this approach with cell synchronization in RPE1 cells, we investigated AurA function specifically in the transition from quiescence to the proliferative cell cycle. Targeted degradation of AurA not only delayed cell cycle progression but also impaired the disassembly of primary cilia at the G0/G1 transition in RPE1 cells. Since this delay in cell cycle progression was rescued by forced deciliation via the knockout of IFT20, AurA facilitates deciliation, which in turn accelerates the transition from quiescence to the proliferative phase of the cell cycle in RPE1 cells. AurA depletion for 4 days increased apoptotic markers in HCT116 cells but not in RPE1 cells. Notably, forced deciliation in RPE1 cells partially enhanced apoptosis induced by AurA depletion. These results suggest that the ability to assemble primary cilia may serve as a protective mechanism against cell death following AurA inhibition.},
}
@article {pmid40675820,
year = {2025},
author = {Turn, RE and Hilgendorf, KI and Johnson, CT and Han, K and Aziz-Zanjani, MO and Swails Bollinger, S and Domizi, P and Cheng, R and Rabiee, A and Zhu, Y and Jiang, Z and Asthana, A and Demeter, J and Svensson, KJ and Bassik, MC and Jackson, PK},
title = {A genome-wide, CRISPR-based screen reveals new requirements for translation initiation and ubiquitination in driving adipogenic fate change.},
journal = {Genes &amp; development},
volume = {39},
number = {19-20},
pages = {1241-1264},
doi = {10.1101/gad.352779.125},
pmid = {40675820},
issn = {1549-5477},
support = {P30 DK116074/DK/NIDDK NIH HHS/United States ; R01 DK125260/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Ubiquitination/genetics ; *Adipogenesis/genetics ; 3T3-L1 Cells ; Lipogenesis/genetics ; Adipocytes/cytology/metabolism ; *Peptide Chain Initiation, Translational/genetics ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mitosis/genetics ; },
abstract = {In response to excess nutrients, white adipose tissue expands by both generating new adipocytes and upregulating lipogenesis in existing adipocytes. Here, we performed a genome-wide functional CRISPR screen to identify regulators of adipogenesis in the mouse 3T3-L1 preadipocyte model. In this pooled screening strategy, we used FACS to isolate populations based on lipid content, gating for fluorescence intensity of lipophilic fluorescent BODIPY dye. Additionally, we categorized whether the gene functions primarily during mitotic clonal expansion, lipogenesis, or both. We found that translation initiation and ubiquitin-dependent protein stability regulators drive both adipogenic fate change and lipogenesis. We further supported these findings with proteomics, demonstrating that essential changes in protein reprogramming can drive or inhibit 3T3-L1 adipogenesis independent of transcription. Furthermore, we demonstrated that specific branches of the hypusination pathway, a conserved regulator of translation initiation, are critical for translating adipogenic inducers of mitotic clonal expansion and that the neddylation/ubiquitin pathway modulates insulin sensitivity during lipogenesis.},
}
@article {pmid40671314,
year = {2025},
author = {Kumar, P and Bisht, NC},
title = {High-level production of health-beneficial glucoraphanin by multiplex editing of AOP2 gene family in mustard.},
journal = {Plant biotechnology journal},
volume = {23},
number = {10},
pages = {4668-4680},
doi = {10.1111/pbi.70171},
pmid = {40671314},
issn = {1467-7652},
support = {BT/PR23893/GET/119/81/2017//Department of Biotechnology, Ministry of Science and Technology, India/ ; BT/PR53959/PBN/18/17/2024//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {Sulfoxides ; *Glucosinolates/metabolism/biosynthesis ; Oximes ; *Imidoesters/metabolism ; *Mustard Plant/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems ; Plants, Genetically Modified/metabolism ; *Plant Proteins/genetics/metabolism ; *Isothiocyanates/metabolism ; },
abstract = {Intake of glucosinolates through the consumption of cruciferous vegetables has been associated with numerous health benefits. In recent decades, glucosinolate glucoraphanin has gained a lot of attention, as its hydrolysis product (sulforaphane) is known to possess numerous health-promoting benefits, including anti-cancer and chemopreventive activities. However, due to the low availability of glucoraphanin in most of the cultivated Brassica crops (except broccoli), there is an increasing interest in many laboratories around the world to manipulate the glucosinolate profile for human benefit. Here, we report the high-level production of health-beneficial glucoraphanin by CRISPR/Cas9 editing of the ALKENYL HYDROXALKYL PRODUCING 2 (BjuAOP2) gene family, displaying distinct expression profiles in the allotetraploid mustard, Brassica juncea. Multiplex editing of five BjuAOP2 homologues, using four gRNAs, provided glucoraphanin accumulation up to 41.60, 75.10, 59.21 and 27.64 μmoles/g dry weight in sprouts, microgreens, seeds and leaves, respectively, of the transgene-free BjuAOP2-edited lines, while providing a significant reduction of the anti-nutritional and goitrogenic alkenyl glucosinolates including progoitrin. The glucoraphanin enhancement in BjuAOP2-edited lines was found to be dose-dependent, wherein loss-of-function mutations in BjuAOP2.A09 and BjuAOP2.B01 homologues had a more prominent effect. The transgene-free BjuAOP2-edited lines were stable for high glucoraphanin and performed at par with the wild-type plants for various seed quality and yield parameters when tested under containment conditions in the field. The development of high-glucoraphanin mustard will help its adoption as a global superfood with health-promoting benefits and as a bioactive source of high-value sulforaphane for industrial production.},
}
@article {pmid40652545,
year = {2025},
author = {Zhu, M and Yan, L and Zhan, Z and Chen, H and Wang, D and Xu, M and Zheng, Z and Zhang, Y and Yang, N and Wu, J and Zhan, H and Tian, Y and Xiong, L and He, Y},
title = {Transgene-Killer-CRISPR version 2 (TKC2) eliminates occasional transgene escape by coupling with a RUBY reporter.},
journal = {Plant biotechnology journal},
volume = {23},
number = {10},
pages = {4621-4632},
doi = {10.1111/pbi.70257},
pmid = {40652545},
issn = {1467-7652},
support = {32200335//National Natural Science Foundation of China/ ; U21A20207//National Natural Science Foundation of China/ ; Y2023QC39//Youth innovation Program of Chinese Academy of Agricultural Sciences/ ; CARS-01//Earmarked Fund for China Agriculture Research System/ ; YBXM2405//Nanfan special project of CAAS/ ; YBXM2446//Nanfan special project of CAAS/ ; B23CJ0208//Hainan Seed Industry Laboratory/ ; },
mesh = {*Transgenes/genetics ; Plants, Genetically Modified/genetics ; *Gene Editing/methods ; Genes, Reporter/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Oryza/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {A critical step in generating gene-edited plants is the removal of CRISPR-related transgenes from T0 plants and their progenies, a process that is generally time-consuming and labour-intensive. We previously reported a Transgene Killer CRISPR (TKC) technology that enables self-elimination of transgenes after the targeted gene has been edited. However, we observed that a small number of T1 plants generated by TKC still retained the transgenes. Herein, we first integrated Cas9 and guide RNA (gRNA) with the RUBY reporter gene (RUBY-CRISPR) to monitor the Cas9/sgRNA expression and track the presence or absence of transgenes in the T0 generation and its progenies. We then combined the RUBY-CRISPR unit with several TKC variants to develop four RUBY-TKC (TKC2) systems including TKC2.1, TKC2.2, TKC2.3 and TKC2.4, to facilitate the elimination of escaped transgene plants. Compared to non-TKC, TKC alone and RUBY-CRISPR, our TKC2s were much more efficient in the generation of transgene-free edited progenies by up to 100% in the T0 generation. TKC2s not only omit the need for screening of the plants with transgenes in the T0 generation, but also enable visualisation of the escape of plants with transgenes in the following progenies. The TKC2 systems developed here provide straightforward yet highly effective approaches for the generation of transgene-free edited plants for either rice functional genomics or genetic improvement, with potential applications in gene editing of other crops.},
}
@article {pmid39970341,
year = {2025},
author = {Fan, F and Wu, MY and Zhang, HQ and Li, G and Luo, C},
title = {Rapid and Simple Detection of Anilinopyrimidine Resistance in Botrytis cinerea by Combining Recombinase Polymerase Amplification with the CRISPR/Cas12a Assay.},
journal = {Plant disease},
volume = {109},
number = {9},
pages = {1831-1838},
doi = {10.1094/PDIS-11-24-2346-SR},
pmid = {39970341},
issn = {0191-2917},
mesh = {*Botrytis/drug effects/genetics ; *Drug Resistance, Fungal/genetics ; *CRISPR-Cas Systems/genetics ; *Fungicides, Industrial/pharmacology ; *Nucleic Acid Amplification Techniques/methods ; Plant Diseases/microbiology ; *Pyrimidines/pharmacology ; Mutation ; Recombinases/genetics/metabolism ; },
abstract = {Anilinopyrimidine (AP) fungicides have been widely adopted to control Botrytis cinerea since the 1990s. As a high-risk pathogen for the development of fungicide resistance, B. cinerea developed resistance to AP fungicides soon after their application. To ensure the proper use of these fungicides, it is necessary to establish a rapid and simple method for resistance detection. Our previous study demonstrated that the E407K mutation in Bcmdl1 was the major mutation conferring AP resistance in China. Based on the combination of recombinase polymerase amplification (RPA) and CRISPR/Cas12a nucleic acid detection assay (RPA/Cas12a detection assay), a simple method for the rapid detection of AP resistance was established by specifically identifying this resistance-related mutation. The new detection assay could precisely identify the E407K mutants from other mutants and wild-type isolates within 50 min, relying solely on a water/metal bath and a UV flashlight. Moreover, this assay could detect genomic DNA at a concentration as low as 1.8 × 10[6] fg/μl, which is comparable with conventional PCR, indicating its high sensitivity. High specificity among different species was also observed in this assay. Above all, this assay was compatible with a 2-min DNA extraction method, implying its feasibility for field application. In conclusion, the RPA/Cas12a detection assay developed in this study is rapid and simple, making it an ideal method for AP resistance detection in local agencies and other points of care. Instant information on resistance monitoring can provide important guidance on resistance management.},
}
@article {pmid41025245,
year = {2025},
author = {Shin, K and Kim, ET},
title = {Efficient CRISPR-based genome editing for inducible degron systems to enable temporal control of protein function in large double-stranded DNA virus genomes.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {9},
pages = {e2504008},
doi = {10.71150/jm.2504008},
pmid = {41025245},
issn = {1976-3794},
support = {RS-2024-00352590//Ministry of Science and ICT/ ; RS-2023-00270936//Ministry of Education/ ; //Korea Health Industry Development Institute/ ; RS-2022-KH129726//Ministry of Health and Welfare/ ; RS-2024-00438990//Ministry of Health and Welfare/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Cytomegalovirus/genetics ; *Genome, Viral ; *Viral Proteins/genetics/metabolism ; Indoleacetic Acids/metabolism/pharmacology ; Green Fluorescent Proteins/genetics ; Recombinational DNA Repair ; Degrons ; },
abstract = {CRISPR-Cas9-based gene editing enables precise genetic modifications. However, its application to human cytomegalovirus (HCMV) remains challenging due to the large size of the viral genome and the essential roles of key regulatory genes. Here, we establish an optimized CRISPR-Cas9 system for precise labeling and functional analysis of HCMV immediate early (IE) genes. By integrating a multifunctional cassette encoding an auxin-inducible degron (AID), a self-cleaving peptide (P2A), and GFP into the viral genome via homology-directed repair (HDR), we achieved efficient knock-ins without reliance on bacterial artificial chromosome (BAC) cloning, a labor-intensive and time-consuming approach. We optimized delivery strategies, donor template designs, and component ratios to enhance HDR efficiency, significantly improving knock-in success rates. This system enables real-time fluorescent tracking and inducible protein degradation, allowing temporal control of essential viral proteins through auxin-mediated depletion. Our approach provides a powerful tool for dissecting the dynamic roles of viral proteins throughout the HCMV life cycle, facilitating a deeper understanding of viral pathogenesis and potential therapeutic targets.},
}
@article {pmid41025215,
year = {2025},
author = {Park, J and Sipe, GO and Tang, X and Ojha, P and Fernandes, G and Leow, YN and Zhang, C and Osako, Y and Natesan, A and Drummond, GT and Jaenisch, R and Sur, M},
title = {Astrocytic modulation of population encoding in mouse visual cortex via GABA transporter 3 revealed by multiplexed CRISPR/Cas9 gene editing.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
doi = {10.7554/eLife.107298},
pmid = {41025215},
issn = {2050-084X},
support = {R01DA049005/NH/NIH HHS/United States ; R01MH126351/NH/NIH HHS/United States ; R01NS130361/NH/NIH HHS/United States ; R01MH133066/NH/NIH HHS/United States ; Multidisciplinary University Research Initiative W911NF2110328//United States Department of Defense/ ; F32EY022264/NH/NIH HHS/United States ; Autism Research Initiative Bridge to Independence award//Simons Foundation/ ; Picower Postdoctoral Fellowship//JPB Foundation/ ; },
mesh = {Animals ; *Visual Cortex/physiology ; *Astrocytes/physiology/metabolism ; *GABA Plasma Membrane Transport Proteins/metabolism/genetics ; Mice ; *CRISPR-Cas Systems ; *Gene Editing ; Mice, Knockout ; Neurons/physiology ; },
abstract = {Astrocytes, which are increasingly recognized as pivotal constituents of brain circuits governing a wide range of functions, express GABA transporter 3 (Gat3), an astrocyte-specific GABA transporter responsible for maintenance of extra-synaptic GABA levels. Here, we examined the functional role of Gat3 in astrocyte-mediated modulation of neuronal activity and information encoding. First, we developed a multiplexed CRISPR construct applicable for effective genetic ablation of Gat3 in the visual cortex of adult mice. Using in vivo two-photon calcium imaging of visual cortex neurons in Gat3 knockout mice, we observed changes in spontaneous and visually driven single neuronal response properties such as response magnitudes and trial-to-trial variability. Gat3 knockout exerted a pronounced influence on population-level neuronal activity, altering the response dynamics of neuronal populations and impairing their ability to accurately represent stimulus information. These findings demonstrate that Gat3 in astrocytes profoundly shapes the sensory information encoding capacity of neurons and networks within the visual cortex.},
}
@article {pmid41024337,
year = {2025},
author = {Zhang, L and Fu, J and Long, T and Zhang, C and Fan, F and Lang, Z and Zhu, JK},
title = {A Modular and Customizable CRISPR/Cas Toolkit for Epigenome Editing of Cis-regulatory Modules.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e03917},
doi = {10.1002/advs.202503917},
pmid = {41024337},
issn = {2198-3844},
support = {KQTD20240729102038044//Shenzhen Science and Technology Program/ ; 32188102//National Natural Science Foundation of China/ ; },
abstract = {Epigenome and cis-regulome, comprising cis-regulatory elements (CREs) and modules (CRMs), jointly define the architecture of gene regulation. However, the causal mechanisms by which epigenetic marks influence CRM function remain elusive. To address this, modular epigenome editing frameworks, exemplified by dead Cas9-coupled DNA demethylation (dCd) and DNA methylation (dCm) platforms, are developed for programmable dissection and engineering of CRM activity. The dCd system modulates methylation levels and transcriptional output at CRMs in situ or ex situ, in accordance with CRM-specific methylation responsiveness, and alters co-transcriptional RNA processing to yield predictable phenotypic outcomes in plants. These findings underscore the reliability of targeted DNA demethylation. In parallel, the dCm system reconstitutes methylation-dependent and -sensitive CRMs of diverse origins in Saccharomyces cerevisiae, a species devoid of native DNA methylation, enabling causal dissection of epigenetic regulation and revealing cross-species portability. This system further uncovers crosstalk between DNA methylation and chromatin modifications, and enables logic-gated control of endogenous genes through CRM engineering. Incorporation of optogenetic and temperature-sensitive anti-CRISPR inhibitors confers tunable, reversible regulation, proposing dCm as a foundation for input-responsive synthetic epigenome editors. Together, these frameworks provide a versatile platform to decode and reprogram cis-regulatory epigenetic logic, with broad applications in trait design and synthetic biology.},
}
@article {pmid41023727,
year = {2025},
author = {Zhao, XY and Gao, C and Zhao, WW and Zhou, ZH and Zhuang, TC and Guo, C and Ji, MH},
title = {Development of a single-tube, dual-target CRISPR Cas12a/Cas13a system for rapid screening of coinfection with respiratory syncytial virus and rhinovirus.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {311},
pmid = {41023727},
issn = {1743-422X},
support = {Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Jiangsu Education Department (2023) No.11//"Nursing Science" Funded by the 4th Priority Discipline Development Program of Jiangsu Higher Education Institutions/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; Ym2023064//Jiangsu Provincial Health Commission Public Health Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; NJYFKT202409//Nanjing Preventive Medicine Research Project/ ; },
mesh = {Humans ; *Coinfection/diagnosis/virology ; *Respiratory Syncytial Virus Infections/diagnosis/virology ; *Rhinovirus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Respiratory Syncytial Virus, Human/genetics/isolation &amp; purification ; *Picornaviridae Infections/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; *Respiratory Tract Infections/diagnosis/virology ; CRISPR-Associated Proteins/genetics ; Infant ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND: Respiratory syncytial virus (RSV) and human rhinovirus (HRV) are leading causes of respiratory infections in children, with increasing reports of coinfections leading to severe complications. Current CRISPR-based detection systems, such as Cas12a and Cas13a, are limited in multiplex detection due to the lack of specific reporter cleavage mechanisms. This study aims to develop a rapid, sensitive, and single-tube dual-gene detection method for RSV and HRV using the orthogonal trans-cleavage activities of CRISPR-Cas12a/13a combined with reverse transcription-recombinase polymerase amplification (RT-RPA).<br><br>METHODS: We designed a novel detection system leveraging RT-RPA for amplification and the distinct cleavage activities of Cas12a and Cas13a for simultaneous dual-gene detection.<br><br>RESULTS: The reaction components were optimized to complete detection within 30&#xa0;min, achieving sensitivities of 10 copies/&#xb5;L for RSV and 10[2] copies/&#xb5;L for HRV. Clinical validation was performed on 543 respiratory infection samples, confirming high accuracy and specificity.<br><br>CONCLUSIONS: The RT-RPA-CRISPR-Cas12a/13a system provides a rapid, sensitive, and efficient solution for RSV and HRV coinfection detection. This method supports early diagnosis and improved clinical management, offering significant potential for public health applications in preventing severe respiratory complications in children.},
}
@article {pmid41023259,
year = {2025},
author = {Ravenel, K and Poirier, W and Razafimandimby, B and Bouchara, JP and Gastebois, A and Giraud, S},
title = {Optimization of the Genome Editing CRISPR-Cas9 Technology in Scedosporium apiospermum.},
journal = {Mycopathologia},
volume = {190},
number = {6},
pages = {94},
pmid = {41023259},
issn = {1573-0832},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Scedosporium/genetics ; },
abstract = {Scedosporium species are opportunistic pathogens causing a large variety of human infections. To date, there is limited information on the pathogenic mechanisms of these fungi, partly because of the limited number of genetic tools available. Here, the CRISPR-Cas9 technology, which provided promising results for functional genomic studies in filamentous fungi, was optimized for Scedosporium species using in vitro assembled Cas9 ribonucleoprotein (RNP) complexes. In these fungi, functional genomic studies are particularly complex in a wild-type strain, because of the high frequency of non-homologous recombination. Prior disruption of the KU70 gene encoding one of the components of the non-homologous end joining system is required, which necessitates the use of a first selection marker. The cleavage of the target gene at each end using a dual RNA-guided Cas9 complex, followed by recombination with a repair template containing the hygromycin resistance gene, allowed disruption of the target gene in the ΔKU70 mutant. Four genes encoding dioxygenases, catalyzing the critical ring-opening step in aromatic hydrocarbons, were successfully disrupted, and the optimum efficiency was observed using 5 μg of the HygR repair cassette. Alternatively, in the wild-type strain, the exclusive use of two Cas9 RNP complexes was enough to achieve an efficient deletion method; one dioxygenase gene was successfully deleted in up to 20% of the obtained colonies. These last experimental conditions path the way to multiple gene deletions and complementation experiments, which cannot be reached using our first procedure since only two selection markers are available for Scedosporium species.},
}
@article {pmid41022734,
year = {2025},
author = {Fu, C and Saddawi-Konefka, R and Chinai, JM and Kim, SY and Kammula, AV and Perera, JJ and Jiang, A and Tiwari, P and Kistler, EN and Tang, S and Luna, SM and Colvin, KJ and Dubrot, J and Anderson, S and Fetterman, RA and Chuong, CL and Lane-Reticker, SK and Cheruiyot, CK and Muscato, AJ and Alipour, Z and Adkins, DR and Griffin, GK and Bernstein, BE and Egloff, AM and Yates, KB and Chernock, RD and Gutkind, JS and Uppaluri, R and Manguso, RT},
title = {In vivo CRISPR screening in head and neck cancer reveals Uchl5 as an immunotherapy target.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8572},
pmid = {41022734},
issn = {2041-1723},
support = {U01DE029188//U.S. Department of Health &amp; Human Services | NIH | National Institute of Dental and Craniofacial Research (NIDCR)/ ; },
mesh = {Animals ; Humans ; *Ubiquitin Thiolesterase/genetics/metabolism/immunology ; Mice ; *Head and Neck Neoplasms/genetics/immunology/therapy/pathology ; *Immunotherapy/methods ; *Squamous Cell Carcinoma of Head and Neck/genetics/immunology/therapy/pathology ; Epithelial-Mesenchymal Transition/genetics/immunology ; Cell Line, Tumor ; CD8-Positive T-Lymphocytes/immunology ; CRISPR-Cas Systems ; Extracellular Matrix/metabolism ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; Tumor Escape/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation, Neoplastic ; Female ; },
abstract = {Recurrent/metastatic head and neck squamous cell carcinoma (HNSCC) is an aggressive malignancy with a significant unmet need for enhancing immunotherapy response given current modest efficacy. Here, we perform an in vivo CRISPR screen in an HNSCC mouse model to identify immune evasion genes. We identify several regulators of immune checkpoint blockade (ICB) response, including the ubiquitin C-terminal hydrolase 5 (UCHL5). Loss of Uchl5 in tumors increases CD8[+] T cell infiltration and improved ICB responses. Uchl5 deficiency attenuates extracellular matrix (ECM) production and epithelial-mesenchymal-transition (EMT) transcriptional programs, which contribute to stromal desmoplasia, a histologic finding we describe as associated with reduced anti-PD1 response in human HNSCCs. COL17A1, a collagen highly and specifically expressed in HNSCC, mediates in part Uchl5-mediated immune evasion. Our findings suggest an unappreciated role for UCHL5 in promoting EMT in HNSCC and highlight ECM modulation as a strategy to improve immunotherapy responses.},
}
@article {pmid41022133,
year = {2025},
author = {Jin, X and Ouyang, C and Sun, T and Li, C and Gu, J and An, B and Wang, Z},
title = {A CRISPR/Cas9 mutant resource for OsSm RNA-binding genes in rice.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70475},
doi = {10.1111/tpj.70475},
pmid = {41022133},
issn = {1365-313X},
support = {2021CX02N173//Guangdong Pearl River Talents Program/ ; 2025B03J0025//Guangzhou Science and Technology Plan Project/ ; 2024KJ31//the Provincial Rural Revitalization Strategy Special Project of Guangdong in 2024/ ; ZDYF2024XDNY179//Hainan Province Science and Technology Special Fund/ ; 32100252//National Natural Science Foundation of China/ ; 32100294//National Natural Science Foundation of China/ ; 32171292//National Natural Science Foundation of China/ ; 211207157080997//Zhanjiang plan for navigation/ ; QT2024-017//Young Talent Support Project of Guangzhou Association for Science and Technology/ ; 2023A1515010428//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {*Oryza/genetics/growth &amp; development/metabolism ; *CRISPR-Cas Systems/genetics ; *Plant Proteins/genetics/metabolism ; Mutation ; Gene Expression Regulation, Plant ; *RNA-Binding Proteins/genetics/metabolism ; Genes, Plant ; RNA Splicing ; },
abstract = {Pre-mRNA, produced by eukaryotic DNA transcription, undergoes splicing by the spliceosome, which removes introns and joins exons to form mRNA. The spliceosome is a large and highly dynamic molecular machine. Its core components include five small nuclear ribonucleoproteins (snRNPs) and the various spliceosome-related proteins. The conserved Smith (Sm) complex and the Sm-like proteins (LSm) serve as primary components of the snRNPs. Sm proteins are involved in processes such as pre-mRNA splicing and mRNA degradation, which can regulate gene expression, thereby influencing plant growth, development, and stress responses. While 25 Sm proteins have been identified in rice, their specific roles in regulating rice growth and development remain unclear. In this study, we employed the CRISPR/Cas9 system to edit 15 OsSm genes, and 13 mutants were obtained, with mutation rates ranging from 20.83 to 83.87%. In comparison to the wild type (WT), the mutants exhibited dwarfism, reduced tiller numbers, lower seed-setting rates or sterility, and increased susceptibility to diseases. One Sm mutant, ossmf-2, exhibited dwarfism, delayed flowering, and small grains. Through transcriptome analysis, three target genes, OsMRG702, OsRGG2, and OsLA1, were identified. Mutations of the OsSmF protein may lead to the abnormal splicing of these genes and finally lead to the inhibition of growth and development. Our study first edited the OsSm genes and generated a mutant library in rice. Most of the mutants exhibited abnormal growth and development, underscoring the essential roles of OsSm proteins in rice physiology. Furthermore, this work addresses a critical gap in the functional characterization of Sm proteins in rice. The resulting mutant collection offers valuable germplasm resources and lays a theoretical foundation for elucidating the molecular regulatory networks involving spliceosomal components and their target genes in the control of crop growth, development, and reproduction.},
}
@article {pmid41021290,
year = {2025},
author = {Johnson, KA and Cooper, C and Philippe, C and Catchpole, RJ and Mitchell, S and Terns, MP},
title = {A Phage Variable Region Encodes Anti-CRISPR Proteins Inhibiting All Streptococcus thermophilus CRISPR Immune Systems.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {},
doi = {10.1177/25731599251369720},
pmid = {41021290},
issn = {2573-1602},
abstract = {Bacteria and archaea utilize CRISPR-Cas systems to defend against invading mobile genetic elements (MGEs) such as phages and plasmids. In turn, MGEs have evolved anti-CRISPR (Acr) proteins to counteract these defenses. While several type II-A Acrs have been identified in Streptococcus thermophilus (Sth) phages, a more comprehensive understanding of Acr diversity in Sth phages has yet to be explored. Guided by the genomic context of known Acrs, we systematically screened uncharacterized phage proteins and identified several novel Acrs that inhibit type I-E, type II-A or type III-A Sth CRISPR-Cas systems. These acr genes are clustered within a variable phage genomic region, indicating a hotspot for anti-defense activity. We also identified neighboring proteins with predicted enzymatic or structural domains that may modulate phage-host interactions through Acr-independent mechanisms. Together, our findings expand the known repertoire of Sth Acrs and highlight the phage variable region as a key reservoir of immune-modulating factors.},
}
@article {pmid41021273,
year = {2025},
author = {Sgodda, M and Gebel, E and Dignas, L and Alfken, S and Eggenschwiler, R and Stalke, A and Dröge, C and Pfister, ED and Baumann, U and Luedde, T and Esposito, I and Keitel, V and Cantz, T},
title = {iPSC-based hepatic organoids reveal a heterozygous MYO5B variant as driver of intrahepatic cholestasis.},
journal = {Hepatology communications},
volume = {9},
number = {10},
pages = {},
doi = {10.1097/HC9.0000000000000812},
pmid = {41021273},
issn = {2471-254X},
mesh = {Humans ; *Cholestasis, Intrahepatic/genetics/metabolism ; *Organoids/metabolism ; ATP Binding Cassette Transporter, Subfamily B, Member 11/genetics/metabolism ; *Induced Pluripotent Stem Cells ; Heterozygote ; *Myosin Type V/genetics ; *Myosin Heavy Chains/genetics ; ATP Binding Cassette Transporter, Subfamily B/genetics/metabolism ; Liver/metabolism ; Multidrug Resistance-Associated Protein 2 ; },
abstract = {BACKGROUND: Hereditary intrahepatic cholestasis is caused by variants of various genes involved in enterohepatic bile circulation, metabolization, and conjugation. Originally classified into 3 groups, the number of contributing genes is still increasing, underlining the need for a deeper understanding of the molecular interaction during intrahepatic cholestasis.<br><br>METHODS: In the present study, we investigate the interplay of heterozygous variants in 3 cholestasis-associated genes (ABCB11, ABCB4, and MYO5B) by exploiting iPSC-based hepatic organoids from a patient suffering from recurrent intrahepatic cholestasis.<br><br>RESULTS: Functional characterization of MRP2-mediated cholyl-lysyl-fluorescein (CLF) and BSEP-mediated Tauro-nor-THCA-24-DBD transport demonstrated a marked reduction of transport in MYO5B-deficient organoids, in comparison to unaffected control organoids. Moreover, iPSC-based organoids derived from the patient carrying 3 heterozygous variants in ABCB11, ABCB4, and MYO5B also exhibited absence of BSEP-mediated Tauro-nor-THCA-24-DBD transport, but functional MRP2-mediated CLF-transport. Interestingly, CRISPR/Cas9-mediated correction of the mutated ABCB11 allele could not restore the impaired BSEP function, suggesting the heterozygous MYO5B variant as the main driver of the transport deficiency. In fact, CRISPR/Cas-mediated correction of the MYO5B variant finally resulted in a restoration of the BSEP-mediated Tauro-nor-THCA-24-DBD transport.<br><br>CONCLUSIONS: iPSC-based organoids serve as an authentic model for functional assessment of the hepatobiliary transport with fluorescent substrates. This allows the characterization of variants of uncertain significance and other variants in cholestasis-associated genes and revealed that a heterozygous MYO5B variant increases the susceptibility to defective hepatobiliary BSEP-mediated transport.},
}
@article {pmid41021067,
year = {2025},
author = {Zou, S and Chen, W and Cao, Y and Liu, X and Wang, J and Wang, Y and Zhou, S},
title = {Lethal endotoxin (ccdB) based counterselection improved the efficiency of sequential gene editing in Escherichia coli.},
journal = {Biotechnology letters},
volume = {47},
number = {5},
pages = {118},
pmid = {41021067},
issn = {1573-6776},
support = {Guike AA24206048//Science and Technology Major Project of Guangxi/ ; AA24206050//Science and Technology Major Project of Guangxi/ ; 4301/00960//Hubei University of Technology High-Level Talent Research Startup Fund Program/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; Plasmids/genetics ; CRISPR-Cas Systems ; *Endotoxins/genetics ; *Escherichia coli Proteins/genetics ; },
abstract = {The CRISPR/Cas9-based technology has been used for sequential gene editing in E. coli. The plasmids carrying the sgRNA and/or Cas9 genes need to be cured after each round of editing. Curing of these plasmids, particularly the sgRNA plasmid, limits the efficiency of sequential gene editing. In this study, a lethal endotoxin (ccdB) based counterselection was established for improving the overall efficiency of sequential gene editing in E. coli. This approach was validated for sequential editing (deletion) of cstA and ppsA genes in HBUT-P2 strain (W derivative). The experimental results showed that the transformation efficiency of sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20) reached 10[8]-10[9] cfu/μg-DNA, resulting in a 100% and 93.75% recombination rate for cstA and ppsA gene, respectively. Upon completion of cstA gene editing, the sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20 (cstA)) was effectively cured through ccdB based counterselection at 42 °C, with a 43.75% efficiency. At the end of sequential editing of ppsA gene, both Cas9 (25A) and sgRNA (pTargetF-tcr-PL-ccdB-N20 (ppsA)) plasmids were cured simultaneously through the sacB and ccdB based counterselections by incubating the cells on LB-sucrose (5%) plate at 42 °C, achieving a curing rate of 100% for Cas9 plasmid (25A), 37.5% for sgRNA plasmid (pTargetF-tcr-PL-ccdB-N20 (ppsA)), and 37.5% for both Cas9 and sgRNA plasmids. Moreover, this approach was further validated through efficient site-specific insertion of the csc operon into the slmA gene in DH5α (K12 derivative) and S322 (C derivative) strains. These results demonstrated that the endotoxin (ccdB) based counterselection improved the transformation efficiency of sgRNA plasmid, the recombination rate of the editing target gene, the curing rate of sgRNA plasmid, and the overall efficiency of sequential gene editing.},
}
@article {pmid41020501,
year = {2025},
author = {Yu, L and Jin, Y and Chen, J and Zhu, Z and Su, S and Wilkerson, EM and Gongora, J and Cloer, EW and Major, MB and Liu, P},
title = {Stable Cas9 expression regulates cell growth by facilitating mTORC2 activation.},
journal = {Nucleic acids research},
volume = {53},
number = {18},
pages = {},
pmid = {41020501},
issn = {1362-4962},
support = {//Mary Kay Ash Foundation/ ; //University of North Carolina/ ; //Chapel Hill University Cancer Research/ ; },
mesh = {Humans ; Ribosomal Proteins/metabolism/genetics ; *Mechanistic Target of Rapamycin Complex 2/metabolism/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Cell Line, Tumor ; *Cell Proliferation/genetics ; Signal Transduction ; *CRISPR-Cas Systems ; Adaptor Proteins, Signal Transducing/metabolism/genetics ; Repressor Proteins/metabolism/genetics ; Gene Editing ; Phosphatidylinositol 3-Kinases/metabolism ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR), widely used for gene editing, relies on bacterial endonucleases like Cas9 to study gene functions and develop therapies. However, its potential effects on mammalian cellular behavior remain unclear. Here, we systematically profiled effects of stable Cas9 expression on growth of 32 cell lines spanning 9 cancer types and non-cancerous cells, finding growth alterations in a subset. To investigate mechanisms, we established the SpCas9 interactome in DU145 and MDA-MB-231 cells, both showing Cas9-enhanced growth, and identified ribosomal proteins as the top shared interactors. RNA-seq analysis revealed that Cas9 expression in DU145 cells activated PI3K signaling. Mechanistic studies showed that ribosomal proteins, including RPL26 and RPL23a, bind to Sin1, a core mTORC2 component, leading to mTORC2 activation. Notably, SpCas9 interacts with both RPL26/RPL23a and Sin1, acting as a scaffold to stabilize their association and enhance mTORC2 activation, even in the absence of growth factors. Our study systematically characterizes Cas9's effects on cell growth regulation and uncovers a novel Cas9-ribosome-mTORC2 signaling axis that promotes cell growth. These findings underscore the need to consider unintended cellular effects in CRISPR applications and highlight the importance of engineering safer Cas9 variants for biomedical research and clinical therapies.},
}
@article {pmid41020266,
year = {2025},
author = {Back, F and Sandoval, A and Vu, LM and Hong, VM and Bhaskara, A and Rodriguez, SR and O'Brien, JT and Kolber, BJ and Kroener, S and Ploski, JE},
title = {Adeno-associated viral vector resource for the RNA-targeting Cas13d: A comparison of high-fidelity variants, DjCas13d and hfCas13d.},
journal = {Molecular therapy. Methods &amp; clinical development},
volume = {33},
number = {4},
pages = {101565},
pmid = {41020266},
issn = {2329-0501},
abstract = {RNA-targeting CRISPR-Cas systems have emerged as alternatives to RNA-interference technology to knock down specific RNA transcripts. In particular, Cas13d derived from Ruminococcus flavefaciens (CasRx, RfxCas13d) has generated interest due to its superior knockdown efficiencies; however, accumulating evidence indicates that CasRx is prone to inducing transcriptome alterations due to its tendency to cleave bystander RNAs. High-fidelity Cas13d (hfCas13d) derived from CasRx and DjCas13d, an ortholog of Cas13d derived from Ruminococcus sp. UBA7013 (gut metagenome), are two recently identified variants that are superior to CasRx, as they both show a reduced tendency to cleave bystander RNAs. In this study, we created a resource of adeno-associated viral (AAV) vectors designed to deliver Cas13d, including hfCas13d and DjCas13d. We directly compared hfCas13d and DjCas13d for their on- and off-target potential in 293FT and neuro 2A cells. Specifically, we examined their ability to knockdown several endogenous and ectopically expressed transcripts using several different guide RNAs (gRNAs), and we examined knockdown specificity using a combination of reporter assays, RNA integrity analysis, and RNA sequencing (RNA-seq). We report that while both of these enzymes exhibit generally similar levels of knockdown potential, with DjCas13d sometimes outperforming hfCas13d, hfCas13d consistently caused significantly fewer transcriptome alterations when targeting highly expressed genes compared to DjCas13d.},
}
@article {pmid40974028,
year = {2025},
author = {Teng, J and Chen, Y and Zhang, W and Xu, H and Ke, L and Xu, H and Wang, J},
title = {An RCA-CRISPR-Enhanced SERS Platform for Ultrasensitive and Single-Nucleotide-Resolved Detection of Exosomal miRNA-21 in Early Lung Cancer.},
journal = {Analytical chemistry},
volume = {97},
number = {38},
pages = {21098-21105},
doi = {10.1021/acs.analchem.5c04448},
pmid = {40974028},
issn = {1520-6882},
mesh = {*Lung Neoplasms/diagnosis/genetics ; Humans ; *MicroRNAs/genetics/analysis/blood ; *Exosomes/genetics/chemistry ; *Spectrum Analysis, Raman/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; CRISPR-Cas Systems ; Biomarkers, Tumor/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Exosomal miRNA-21 has emerged as a promising biomarker for early-stage lung cancer due to its close association with tumor progression and its stability in circulation. However, its low abundance, short sequence length, and high-sequence similarity present significant detection challenges. To address this, we developed an ultrasensitive surface-enhanced Raman scattering (SERS) platform that integrates rolling circle amplification (RCA) with clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 12a (Cas12a) for the detection of exosomal miRNA-21. RCA provides target-dependent amplification with stringent sequence discrimination via padlock probe ligation, while the CRISPR/Cas12a system facilitates robust signal generation through trans-cleavage activity. The final SERS readout enables molecular-level sensitivity by detecting nanotag-labeled cleavage events. The assay achieved a limit of detection as low as 0.62 aM and effectively discriminated miRNA-21 from multiple single- and multinucleotide variants. As a proof of concept, we applied this method to the detection of exosomal miRNA-21 extracted from the serum of 20 early-stage lung cancer patients and 20 healthy controls, achieving 100% sensitivity and 100% specificity (AUC = 1.0) in this preliminary cohort. These findings demonstrate the strong potential of the RCA-CRISPR-SERS platform for noninvasive early-stage lung cancer diagnosis based on exosomal miRNA-21 detection.},
}
@article {pmid40955638,
year = {2025},
author = {Sun, F and Mao, R and Li, J and Wang, X and Hou, P and Zhou, H},
title = {Organic Photoelectrochemical Transistor/Visual Sensing Platform Based on CS/MCS Schottky Heterojunction and CRISPR/Cas12a-Driven Triple-Modal Synergistic Signal Amplification.},
journal = {Analytical chemistry},
volume = {97},
number = {38},
pages = {21079-21088},
doi = {10.1021/acs.analchem.5c04321},
pmid = {40955638},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; *Transistors, Electronic ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; DNA, Catalytic/chemistry/metabolism ; Photochemical Processes ; G-Quadruplexes ; Colorimetry ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Developing novel signal amplification and transduction technologies is the key to overcoming the bottlenecks of high-sensitivity and on-site detection in nucleic acid analysis. In this study, a dual-mode sensing platform based on organic electrochemical transistors (OPECT) and colorimetry was established to achieve ultrasensitive detection of miRNA-21. 1D/3D Co9S8/Mn0.3Cd0.7S Schottky heterojunction was synthesized as the photoactive material, which significantly enhanced the photoelectric conversion efficiency. The sensing and detection system cleverly integrated a quadruple signal amplification mechanism. The target triggered the catalytic hairpin assembly (CHA) reaction, generating H1 and H2 long chains. These chains activated the CRISPR/Cas12a system, which carried out nondiscriminatory cleavage to block the tandem strand displacement reaction (TSDR). This triggered the hybrid chain reaction (HCR) and formation of G-quadruplex/hemin DNAzyme (GQH DNAzyme), realizing cascade signal amplification. Under the catalysis of GQH DNAzyme, the detection had dual-signal outputs. It catalyzed the oxidation of 4-CN to form a deposition layer, inhibiting electron transport and achieving cascade signal amplification for OPECT. It catalyzed the H2O2-mediated TMB colorimetric reaction to complete the visual colorimetric analysis. Through triple-modal synergistic signal amplification of biological, chemical, and electronic modalities, this biosensing platform reduced the detection limits to as low as 36.5 aM and 3.8 fM, respectively. It provided a new solution for the accurate analysis of miRNA markers in the early diagnosis of cancer.},
}
@article {pmid40916664,
year = {2025},
author = {Barker, EN and Ashiri, M and Saville, JT and Hemming, R and Furletti, N and Dhume, SH and Yu, S and Anjos, E and Wu, X and Fresnoza, A and Merz, DC and Jackson, M and Del Bigio, MR and Siddiqui, TJ and Fuller, M and Mark, BL and Triggs-Raine, B},
title = {Generation of mice with combined Hexa Gly269Ser KI or KO and Neu3 KO alleles to create new models of GM2 gangliosidoses.},
journal = {Biology open},
volume = {14},
number = {9},
pages = {},
doi = {10.1242/bio.062045},
pmid = {40916664},
issn = {2046-6390},
support = {/CAPMC/CIHR/Canada ; //Research Manitoba/ ; /AS/Autism Speaks/United States ; //Natural Sciences and Engineering Council of Canada/ ; },
mesh = {Animals ; *Disease Models, Animal ; Mice ; *Alleles ; Mice, Knockout ; *Gangliosidoses, GM2/genetics/metabolism/pathology ; Phenotype ; CRISPR-Cas Systems ; *beta-Hexosaminidase alpha Chain/genetics ; Gene Knock-In Techniques ; Mutation ; Humans ; N-Acetylgalactosaminyltransferases ; },
abstract = {The GM2 gangliosidoses are lysosomal storage disorders exhibiting a spectrum of neurological phenotypes ranging from childhood death to debilitating adult-onset neurological impairment. To date, no mouse model harbouring a specific human mutation causing GM2 gangliosidosis has been created. We used CRISPR/Cas9 to generate knockin (KI) mice with the common adult-onset Hexa Gly269Ser variant as well as knockout (KO) mice with Hexa mutations expected to cause complete HexA deficiency. We also created Neu3 KO alleles that combined with Hexa KO or KI alleles were expected to create acute and chronic models of GM2 gangliosidosis, respectively. However, both models accumulated GM2 ganglioside throughout the brain when compared to controls (CON), and exhibited progressive loss of reflexes, gait abnormalities, and premature death by 24 weeks of age. Although survival and behavioural phenotypes did not differ between KO and KI models, the KI model had substantial Hexa mRNA and evidence of GM2 turnover. This KI model will be useful for developing gene editing to correct the variant causing the Gly269Ser substitution and its novel biochemical phenotype suggests it may be suitable for testing therapies that treat partial β-hexosaminidase A deficiency.},
}
@article {pmid39433694,
year = {2025},
author = {Farrokhi, S and Eslahi, A and Alizadeh, F and Kerachian, MA and Mojarrad, M},
title = {Assessment the Efficacy of the CRISPR System for Inducing Mutations in the AIMP2 Gene to Create a Cell Line Model of HLD17 Disease.},
journal = {Molecular biotechnology},
volume = {67},
number = {10},
pages = {3922-3929},
pmid = {39433694},
issn = {1559-0305},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *Gene Editing/methods ; *Mutation ; Plasmids/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Hypomyelinating leukodystrophy-17 is a neurodevelopmental disorder caused by autosomal recessive mutations in the AIMP2 gene, resulting in a lack of myelin deposition during brain development, leading to variable neurological symptoms. Research on brain function in these disorders is challenging due to the lack of access to brain tissue. To overcome this problem, researchers have utilized different cell and animal models. The CRISPR-Cas9 system is considered the most optimal and effective method for genetic modification and developing cell models. We studied the efficacy of the CRISPR-Cas9 technology in inducing mutations in the AIMP2 gene in HEK293 cell lines. The study involved transfecting HEK293 cells with recombinant PX458 plasmids targeting spCas-9 and AIMP2 sgRNA. The cells were evaluated using fluorescent microscopy and enriched using serial dilution. The CRISPR/Cas9 plasmids were validated through PCR and Sanger sequencing. After serial dilution, AS-PCR, Sanger sequencing, and TIDE program analysis showed the construct successfully induces an indel mutation in HEK cells. Our findings demonstrated the great efficacy of the CRISPR system and produced a construct for inducing mutations in the AIMP2 gene, which can be utilized to edit the AIMP2 gene in nerve cells and create a cellular model of the HLD17 disease.},
}
@article {pmid39377911,
year = {2025},
author = {Bhoomika, S and Salunkhe, SR and Sakthi, AR and Saraswathi, T and Manonmani, S and Raveendran, M and Sudha, M},
title = {CRISPR-Cas9: Unraveling Genetic Secrets to Enhance Floral and Fruit Traits in Tomato.},
journal = {Molecular biotechnology},
volume = {67},
number = {10},
pages = {3786-3799},
pmid = {39377911},
issn = {1559-0305},
mesh = {*Solanum lycopersicum/genetics/growth &amp; development ; *Fruit/genetics/growth &amp; development ; *CRISPR-Cas Systems ; *Flowers/genetics/growth &amp; development ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; Plant Breeding/methods ; },
abstract = {Tomato, a globally consumed vegetable, possesses vast genetic diversity, making it suitable for genetic manipulation using various genetic improvement techniques. Tomatoes are grown extensively for their market value and health benefits, primarily contributed by enhanced yield and nutritional value respectively, influenced by floral and fruit traits. Floral morphology is maintained by genes involved in meristem size control, regulation of inflorescence transition, and pollen development. SP (SELF-PRUNING) and SP5G (SELF-PRUNING 5G) determine growth habit and flowering time. RIN (RIPENING INHIBITOR) and PG (POLYGALACTURONASE) are responsible for the shelf life of fruits. In addition to this, nutrition-enriched tomatoes have been developed in recent times. In this review, we comprehensively discuss the major genes influencing floral morphology, flowering time, fruit size, fruit shape, shelf life, and nutritional value, ultimately resulting in enhanced yield. Additionally, we address the advances in CRISPR/Cas9 applied for the genetic improvement of tomatoes along with prospects of areas in which research development in terms of tomato genetic improvement has to be advanced.},
}
@article {pmid41020188,
year = {2025},
author = {Shalaby, KE and Hmila, I and Uddin, SMN and Zawia, NH and El-Agnaf, OMA and Aouida, M},
title = {Enhanced Cellular Uptake of Compact Cas Proteins: A Comparative Study of Cas12f and Cas9 in Human Cells.},
journal = {Engineering in life sciences},
volume = {25},
number = {9},
pages = {e70042},
pmid = {41020188},
issn = {1618-0240},
abstract = {The clinical translation of CRISPR genome-editing therapies is often hindered by inefficient delivery of the CRISPR-Cas RNA-protein complex into target cells. The most widely used CRISPR-Cas9 system poses a significant challenge for efficient delivery into cells due to its large size (∼1.4 kDa). Recently reported compact Cas proteins, such as Cas12f (552 Da), Cas12k (639 Da), and Cas12m (596 Da) represent attractive alternatives as cargoes for delivery. In this brief research report, we employ efficient delivery vectors to evaluate the efficiency of cellular uptake of a compact Cas protein (Cas12f) compared to the widely used larger Cas9 in human cells. Our findings demonstrate that compact Cas proteins may facilitate more efficient cellular penetration and delivery, making them a promising alternative for the development of CRISPR-based therapies. Practical Application: Our study demonstrates that compact Cas proteins significantly enhance cellular uptake compared to larger Cas proteins. This improved uptake efficiency suggests that compact Cas proteins could be more effective for clinical application, where size constraints and delivery efficiency are critical challenges. Combined with the optimization and refinement of the editing efficiencies of compact Cas systems, our study provokes further exploration of compact Cas proteins in various therapeutic contexts to advance the development of more efficient CRISPR-based therapies.},
}
@article {pmid41019300,
year = {2025},
author = {Bilger, R and Drepper, F and Knapp, B and Berndt, T and Landerer, H and Putzer, H and Huesgen, PF and Hess, WR},
title = {Involvement of RNase J in CRISPR RNA maturation in the cyanobacterium Synechocystis sp. PCC 6803.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf022},
pmid = {41019300},
issn = {2633-6693},
abstract = {Many bacteria and archaea use CRISPR-Cas systems, which provide RNA-based, adaptive, and inheritable immune defenses against invading viruses and other foreign genetic elements. The proper processing of CRISPR guide RNAs (crRNAs) is a crucial step in the maturation of the defense complexes and is frequently performed by specialized ribonucleases encoded by cas genes. However, some systems employ enzymes associated with degradosome or housekeeping functions, such as RNase III or the endoribonuclease RNase E. Here, the endo- and 5´-exoribonuclease RNase J was identified as an additional enzyme involved in crRNA maturation, acting jointly with RNase E in the crRNA maturation of a type III-Bv CRISPR-Cas system, and possibly together with a further RNase in the cyanobacterium Synechocystis sp. PCC 6803. Co-IP experiments revealed a small set of proteins that were co-enriched with RNase J, among them the exoribonuclease polyribonucleotide nucleotidyltransferase (PNPase). Despite a measured, strong 3' exonucleolytic activity of the recombinant enzyme, PNPase was not confirmed to contribute to crRNA maturation. However, the co-IP results indicate that PNPase in Synechocystis is an enzyme that can recruit either RNase E or RNase J, together with additional proteins.},
}
@article {pmid41018953,
year = {2025},
author = {Ajmal, H and Nandi, S and Kebabci, N and Ryan, CJ},
title = {Benchmarking genetic interaction scoring methods for identifying synthetic lethality from combinatorial CRISPR screens.},
journal = {NAR genomics and bioinformatics},
volume = {7},
number = {3},
pages = {lqaf129},
pmid = {41018953},
issn = {2631-9268},
mesh = {*Synthetic Lethal Mutations ; Benchmarking ; Humans ; Algorithms ; *CRISPR-Cas Systems ; *Epistasis, Genetic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Synthetic lethality (SL) is an extreme form of negative genetic interaction, where simultaneous disruption of two non-essential genes causes cell death. SL can be exploited to develop cancer therapies that target tumour cells with specific mutations, potentially limiting toxicity. Pooled combinatorial CRISPR screens, where two genes are simultaneously perturbed and the resulting impacts on fitness estimated, are now widely used for the identification of SL targets in cancer. Various scoring methods have been developed to infer SL genetic interactions from these screens, but there has been no systematic comparison of these approaches. Here, we performed a comprehensive analysis of five scoring methods for SL detection using five combinatorial CRISPR datasets. We assessed the performance of each algorithm on each screen dataset using two different benchmarks of paralog SL. We find that no single method performs best across all screens but identify two methods that perform well across most datasets. Of these two scores, Gemini-Sensitive has an available R package that can be applied to most screen designs, making it a reasonable first choice.},
}
@article {pmid41017815,
year = {2025},
author = {Alipanahi, R and Safari, L and Khanteymoori, A},
title = {Advancing CRISPR with deep learning: A comprehensive review of models and databases.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102691},
pmid = {41017815},
issn = {2162-2531},
abstract = {CRISPR is considered a powerful tool for targeted genome editing. However, off-target effects remain a significant challenge in the CRISPR field, hindering its broader clinical application. To enhance the development of gene-editing therapies, it is essential to predict the efficiency of CRISPR-based genome editing experiments, before trying them on clinical cases. Machine learning (ML) and deep learning (DL) tools are projected to become the leading methods for predicting CRISPR on-target and off-target activity. Current prediction accuracy is limited by the amount of available training data. As more sequence features are identified and incorporated in DL tools, predictions of them are expected to better align with experimental results. Hence, the increasing focus on ML/DL approaches to predict off-target sites necessitates large and easily searchable databases. In this review, we will take a closer look at available CRISPR databases.},
}
@article {pmid41017813,
year = {2025},
author = {Sharma, S and Pokharel, YR},
title = {Measles and rubella: From global health challenges to advancements in molecular diagnostics in the elimination era.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102698},
pmid = {41017813},
issn = {2162-2531},
abstract = {Measles and rubella are highly contagious viral infections with significant public health implications, particularly in low- and middle-income countries. Despite the availability of effective vaccines, these diseases continue to cause periodic outbreaks, contributing to substantial global morbidity, mortality, and economic burden. Immunization programs have drastically abridged disease incidence; however, gaps in vaccination coverage and surveillance systems deter complete elimination. The economic impact of outbreaks includes direct healthcare costs and indirect societal losses, emphasizing the need for robust disease control strategies. Accurate and timely diagnosis is pivotal to measles and rubella elimination efforts. Current diagnostic approaches range from conventional RT-PCR (including multiplex and real-time formats), ELISA, and plaque reduction neutralization test (PRNT), to emerging methods such as isothermal amplification loop-mediated isothermal amplification, recombinase polymerase amplification), CRISPR-Cas systems, next-generation sequencing (NGS), microfluidics, and lateral flow assays. Despite their sensitivity, many of these methods require complex infrastructure and skilled personnel, limiting their utility in field settings. To bridge diagnostic gaps, there is an urgent need for rapid, affordable, and field-deployable nucleic acid-based diagnostics that are simple to use with minimal training. Innovations like CRISPR-Cas and microfluidic platforms hold promise for decentralized testing and real-time surveillance, potentially transforming global measles and rubella elimination programs for the future.},
}
@article {pmid41017535,
year = {2025},
author = {Han, B and Xie, X and Zhao, Y and Zhang, J and Yang, X and Jiang, Y and Zhang, W and Zhang, X},
title = {Recent development and applications of emerging biosensing technologies and on-site analytical devices for food adulteration detection: a critical review.},
journal = {Critical reviews in food science and nutrition},
volume = {},
number = {},
pages = {1-20},
doi = {10.1080/10408398.2025.2564216},
pmid = {41017535},
issn = {1549-7852},
abstract = {The increasing incidence of food adulteration poses a significant challenge to global health and food safety. Although current detection methods can effectively complete food adulteration detection, they usually require complex pre-preparation processes and professional technicians to some extent. Therefore, the development of rapid and on-site detection technologies for food adulteration is imperative. Recently, biosensing technologies and portable devices have been developed for efficient and precise food adulteration detection. In this review, the strengths and weaknesses of conventional food adulteration detection methods were compared. The recent development of emerging biosensing technologies (i.e., antibody-based biosensors, aptamer-based biosensors, molecular imprinted polymers (MIPs)-based biosensors, and clustered regularly interspaced short palindromic repeats-associated proteins (CRISPR/Cas) systems-based biosensors) and portable analytical devices (e.g., lateral flow assays (LFAs), microfluidic devices, handheld Raman, and nanopore-based devices) for food adulteration detection has been comprehensively summarized and discussed. Remarkably, the challenges and opportunities in this field have been proposed.},
}
@article {pmid41016988,
year = {2025},
author = {Zeng, J and Luo, J and Zeng, Y},
title = {Cancer gene therapy: historical perspectives, current applications, and future directions.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {200},
pmid = {41016988},
issn = {1438-7948},
support = {S202413705064//Innovation and Entrepreneurship Training Program for College Students/ ; 82104084//National Natural Science Foundation of China/ ; 2024qnGzn12 and 2024kjTzn08//CMC Excellent-talent Program of Chengdu Medical College/ ; },
mesh = {Humans ; *Genetic Therapy/methods/trends/history ; *Neoplasms/therapy/genetics ; Gene Editing ; CRISPR-Cas Systems ; Genetic Vectors/genetics ; Animals ; Oncolytic Virotherapy ; },
abstract = {Gene therapy has emerged as a transformative approach in cancer treatment, leveraging genetic modifications to target malignancies with enhanced precision. Early efforts faced challenges such as inefficient vector delivery (< 5% tumor transduction rates with first-generation adenoviruses), immune responses (neutralizing antibodies in ~ 30% of patients), and limited clinical efficacy (< 10% objective response rates in 1990s trials). However, advancements in viral and non-viral vectors (e.g., AAVs achieving > 50% transduction efficiency in solid tumors), alongside CRISPR-Cas9 (90% target gene knockout rates in preclinical models) and RNA interference technologies, have revolutionized the field. Presently, gene therapy strategies, including tumor suppressor gene restoration, oncogene silencing, and immune modulation, demonstrate promising clinical outcomes. Despite persistent hurdles like off-target effects and high costs, emerging innovations in personalized gene editing, oncolytic viruses, and combination therapies signal a paradigm shift in oncology. This review explores the evolution of gene therapy for cancer, highlighting key milestones, current applications, and future directions that could unlock its full therapeutic potential.},
}
@article {pmid41016806,
year = {2025},
author = {Watanabe, T},
title = {[Exploration and Functional Analysis of Epstein-Barr Virus Pathogenic Factors Using a Multidimensional Approach].},
journal = {Uirusu},
volume = {75},
number = {1},
pages = {73-86},
doi = {10.2222/jsv.75.73},
pmid = {41016806},
issn = {0042-6857},
mesh = {*Herpesvirus 4, Human/genetics/pathogenicity/physiology ; Humans ; Animals ; Mice ; *Epstein-Barr Virus Infections/virology ; Genome, Viral/genetics ; CRISPR-Cas Systems ; Disease Models, Animal ; Chromosomes, Artificial, Bacterial ; Gene Editing ; Virus Latency/genetics ; },
abstract = {Epstein-Barr virus (EBV), a member of the herpesvirus family, infects more than 90% of adults and establishes a lifelong latent infection. In addition to its involvement in a wide range of malignancies such as lymphomas, nasopharyngeal carcinoma, and gastric cancer, recent evidence has shown its potential association with autoimmune diseases, positioning EBV as an interdisciplinary research model linking virology, oncology, and immunology. Historically, EBV research has been hindered by technical limitations in viral culture systems and animal models. However, recent advances-including whole-genome cloning using bacterial artificial chromosomes (BACs), gene editing via CRISPR/Cas9, and the development of in vivo models such as humanized mice-have accelerated the elucidation of EBV' s unique life cycle and tumorigenic mechanisms. In this review, we discuss the evolution of techniques for generating recombinant EBVs and in vivo modeling, both essential for functional genetic analysis, and highlight our contributions to the advancement of these tools and their application in researching EBV-associated tumorigenesis.},
}
@article {pmid41016566,
year = {2025},
author = {Hussen, BM and Abdullah, SR and Hidayat, HJ and Glassy, MC and Safarzadeh, A and Komaki, A and Samsami, M and Taheri, M},
title = {CRISPR/Cas as a Tool to Overcome Drug Resistance in Cancer: From Challenge to Opportunity.},
journal = {Molecular and cellular probes},
volume = {},
number = {},
pages = {102052},
doi = {10.1016/j.mcp.2025.102052},
pmid = {41016566},
issn = {1096-1194},
abstract = {Drug resistance remains a significant challenge in cancer therapy, often resulting in treatment failure, tumor progression, and metastasis. The underlying resistance mechanisms-including genetic mutations, epigenetic alterations, and modifications in drug efflux pathways-are complex and not yet fully understood. This review explores the application of CRISPR-Cas gene editing technology in understanding and overcoming drug resistance in cancer. It focuses on how CRISPR can identify and target resistance-associated genes to restore drug sensitivity. CRISPR-based approaches enable precise genetic modifications that offer new insights into the molecular basis of drug resistance. The technology has shown promise in dissecting resistance mechanisms and developing targeted therapeutic strategies. Nevertheless, key limitations such as inefficient delivery systems, off-target effects, and limited specificity hinder clinical translation. Current efforts focus on improving guide RNA design, creating more effective delivery vectors, and integrating CRISPR with existing treatments. CRISPR-Cas technology holds significant potential to address drug resistance in cancer by enabling targeted genetic interventions. Continued advancements are required to enhance its safety, specificity, and delivery, paving the way for its integration into future clinical applications.},
}
@article {pmid41015553,
year = {2025},
author = {Zhang, Y and Shi, Q and Xie, H and Xie, B and Li, L and Wu, W and Xie, H and Xiao, Z and Xie, D and Lai, R},
title = {Construction and phenotypic analysis of p2rx2 knockout zebrafish lines.},
journal = {Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences},
volume = {50},
number = {6},
pages = {919-930},
pmid = {41015553},
issn = {1672-7347},
support = {2023JJ30753//the Natural Science Foundation of Hunan Province/ ; 2023SK4030//the Innovative Construction Foundation of Hunan Province/ ; kq2208326//the Natural Science Foundation of Changsha/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Receptors, Purinergic P2X2/genetics/deficiency ; CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques ; Phenotype ; *Zebrafish Proteins/genetics ; Disease Models, Animal ; },
abstract = {OBJECTIVES: The purinergic receptor P2X2 (P2RX2) encodes an ATP-gated ion channel permeable to Na[+], K[+], and especially Ca[2+]. Loss-of-function mutations in P2RX2 are known to cause autosomal dominant nonsyndromic deafness 41 (DFNA41), which manifests as high-frequency hearing loss, accelerated presbycusis, and increased susceptibility to noise-induced damage. Zebrafish, owing to their small size, rapid development, high fecundity, transparent embryos, and high gene conservation with humans, provide an ideal model for studying human diseases and developmental mechanisms. This study aims to generate a p2rx2 knockout zebrafish model using CRISPR/Cas9 gene editing system to investigate the effect of p2rx2 deficiency on the auditory system, providing a basis for understanding P2RX2-related hearing loss and developing gene therapy strategies.<br><br>METHODS: Two CRISPR targets (sgRNA1 and sgRNA2) spaced 47 bp apart were designed within the zebrafish p2rx2 gene. Synthesized sgRNAs and Cas9 protein were microinjected into single-cell stage T&#xfc;bingen (TU)-strain zebrafish embryos. PCR and gel electrophoresis verified editing efficiency at 36 hours post-fertilization (hpf). Surviving embryos were raised to adulthood (F0), tail-clipped, genotyped, and screened for positive mosaics. F1 heterozygotes were generated by outcrossing, and F2 homozygous mutants were obtained by intercrossing. Polymerase chain reaction (PCR) combined with sequencing verified mutation type and heritability. At 5 days post-fertilization (dpf), YO-PRO-1 staining was used to examine hair cell morphology and count in lateral line neuromasts and the otolith region. Auditory evoked potential (AEP) thresholds at 600, 800, 1 000, and 2 000 Hz were measured in nine 4-month-old wild type and mutant zebrafish per group.<br><br>RESULTS: A stable p2rx2 knockout zebrafish line was successfully established. Sequencing revealed a 66 bp insertion at the first target site introducing a premature stop codon (TAA), leading to early termination of protein translation and loss of function. Embryos developed normally with no gross malformations. At 5 dpf, mutants exhibited significantly reduced hair cell density in the otolith region compared with wild type, although lateral line neuromasts were unaffected. AEP testing showed significantly elevated auditory thresholds at all 4 frequencies in homozygous mutants compared with wild type (all P<0.001), indicating reduced hearing sensitivity.<br><br>CONCLUSIONS: We successfully generated a p2rx2 loss-of-function zebrafish model using CRISPR/Cas9 technology. p2rx2 deficiency caused hair cell defects in the otolith region and increased auditory thresholds across frequencies, indicating its key role in maintaining zebrafish auditory hair cell function and hearing perception. The phenotype's restriction to the otolith region suggests tissue-specific roles of p2rx2 in sensory organs. This model provides a valuable tool for elucidating the molecular mechanisms of P2RX2-related hearing loss and for screening otoprotective drugs and developing gene therapies.},
}
@article {pmid41013258,
year = {2025},
author = {Shelenkov, A and Slavokhotova, A and Yunusova, M and Kulikov, V and Mikhaylova, Y and Akimkin, V},
title = {Genomic typing, antimicrobial resistance gene, virulence factor and plasmid replicon database for the important pathogenic bacteria Staphylococcus aureus.},
journal = {BMC genomic data},
volume = {26},
number = {1},
pages = {65},
pmid = {41013258},
issn = {2730-6844},
mesh = {*Virulence Factors/genetics ; *Plasmids/genetics ; *Staphylococcus aureus/genetics/classification/pathogenicity/drug effects ; *Replicon ; *Drug Resistance, Bacterial/genetics ; Genome, Bacterial ; *Databases, Genetic ; Humans ; Genomics ; Staphylococcal Infections/microbiology ; Anti-Bacterial Agents/pharmacology ; },
abstract = {BACKGROUND: Bacterial infections pose a global health threat across clinical and community settings. Over the past decade, the alarming expansion of antimicrobial resistance (AMR) has progressively narrowed therapeutic options, particularly for healthcare-associated infections. This critical situation has been formally recognized by the World Health Organization as a major public health concern. Epidemiological studies have demonstrated that the dissemination of AMR is frequently mediated by specific high-risk bacterial lineages, often designated as "global clones" or "clonal complexes." Consequently, surveillance of these epidemic clones and elucidation of their pathogenic mechanisms and AMR acquisition pathways have become essential research priorities. The advent of whole genome sequencing has revolutionized these investigations, enabling comprehensive epidemiological tracking and detailed analysis of mobile genetic elements responsible for resistance gene transfer. However, despite the exponential increase in available bacterial genome sequences, significant challenges persist. Current genomic datasets often suffer from uneven representation of clinically relevant strains and inconsistent availability of accompanying metadata. These limitations create substantial obstacles for large-scale comparative studies and hinder effective surveillance efforts.<br><br>DESCRIPTION: This database represents a comprehensive genomic analysis of 98,950&#xa0;Staphylococcus aureus&#xa0;isolates, a high-priority bacterial pathogen of global clinical significance. We provide detailed isolate characterization through several established typing schemes including multilocus sequence typing (MLST), clonal complex (CC) assignments,&#xa0;spa&#xa0;typing results, and core genome MLST (cgMLST) profiles. The dataset also documents the presence of CRISPR-Cas systems in these isolates. Beyond fundamental typing data, our resource incorporates the distribution of antimicrobial resistance determinants, virulence factors, and plasmid replicons. These systematically curated genomic features offer researchers valuable insights into isolate epidemiology, resistance mechanisms, and horizontal gene transfer patterns in this highly concerning pathogen.<br><br>CONCLUSION: This database is freely available under CC BY-NC-SA at https://doi.org/10.5281/zenodo.14833440 . The data provided enables researchers to identify optimal reference isolates for various genomic studies, supporting critical investigations into&#xa0;S. aureus&#xa0;epidemiology and antimicrobial resistance evolution. This resource will ultimately inform the development of more effective prevention and control measures against this high-priority pathogen.},
}
@article {pmid41013219,
year = {2025},
author = {Zhao, C and Xia, J and Liang, B and Lin, S and Song, Y and Hong, D and Gu, J},
title = {Large-scale screening of genes responsible for silique length and seed size in Brassica Napus via pooled CRISPR library.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {829},
pmid = {41013219},
issn = {1471-2164},
support = {2022ZD04008//Biological Breeding-National Science and Technology Major Project/ ; 2022ZD04008//Biological Breeding-National Science and Technology Major Project/ ; XGKJ2024020003//Natural Science Foundation of Xiaogan Municipality/ ; },
mesh = {*Brassica napus/genetics/growth &amp; development ; *Seeds/genetics/growth &amp; development/anatomy &amp; histology ; *CRISPR-Cas Systems ; Gene Editing ; Gene Library ; *Genes, Plant ; Phenotype ; Mutation ; Gene Expression Regulation, Plant ; },
abstract = {BACKGROUND: Enhancing rapeseed (Brassica napus, B. napus) yield is critical for ensuring global vegetable oil security. However, yield is heavily influenced by silique development and seed size, the enhancement of which is limited by scarce genetic resources. The CRISPR/Cas9 system has emerged as a powerful tool for constructing genome-wide mutant libraries, even in polyploid crops with complex genomes.<br><br>RESULTS: The transcriptome-wide association study (TWAS) data, tissue-specific expression profiles data and reported genes were integrated to identify candidate genes regulating silique development and seed size. We constructed a sgRNA library targeting these genes and generated a CRISPR/Cas9 editing mutant library through genetic transformation. Specifically, 6124 sgRNAs were designed for 1739 candidate genes with &#x2266;&#x2009;4 orthologues. 681 T0 plants were obtained through genetic transformation, which harbor 453 sgRNAs. Of 408 T0 plants analyzed, 151 (37.00%) exhibited successful gene editing events, targeting 84 candidate genes. Ten homozygous mutant plants were isolated and preliminary phenotypic analysis was performed in mutants targeting the BnaHRDs. The results suggest that mutations in BnaHRD.A03 and BnaHRD.C03 may modulate plant height (PH), main inflorescence length (MIL), silique length (SL), effective silique number per plant (ENS), seed number per silique (SNPS), and thousand-seed weight (TSW).<br><br>CONCLUSIONS: This study harnessed the CRISPR/Cas9 technology to establish a preliminary library of gene-edited mutants in B. napus, thereby laying a robust foundation for the future screening of candidate genes pertaining to silique development and seed size. Furthermore, this study provides a methodological framework for rapid functional gene discovery in B. napus through CRISPR-based approaches.},
}
@article {pmid41012695,
year = {2025},
author = {Madirov, A and Iksat, N and Masalimov, Z},
title = {Tomato Bushy Stunt Virus (TBSV): From a Plant Pathogen to a Multifunctional Biotechnology Platform.},
journal = {Viruses},
volume = {17},
number = {9},
pages = {},
pmid = {41012695},
issn = {1999-4915},
support = {No. BR21882269//Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {*Biotechnology/methods ; *Tombusvirus/genetics/physiology ; Genetic Vectors ; Gene Editing ; *Plant Diseases/virology ; CRISPR-Cas Systems ; Gene Silencing ; Nanotechnology ; },
abstract = {Plant viruses have evolved from being viewed exclusively as pathogens into versatile and powerful tools for modern biotechnology. Among them, Tomato bushy stunt virus (TBSV) holds a special place due to its well-studied molecular biology and unique structural properties. This review systematizes the knowledge on TBSV's dual role as a multifunctional platform. On one hand, we cover its application as a viral vector for the highly efficient expression of recombinant proteins in plants, as well as a tool for functional genomics, including Virus-Induced Gene Silencing (VIGS) and the delivery of CRISPR/Cas9 gene-editing components. On the other hand, we provide a detailed analysis of the use of the stable and monodisperse TBSV virion in nanobiotechnology. Its capsid serves as an ideal scaffold for creating next-generation vaccine candidates, platforms for targeted drug delivery to tumor cells, and as a building block for the programmable self-assembly of complex nanoarchitectures. In conclusion, key challenges limiting the widespread adoption of the platform are discussed, including the genetic instability of vectors and difficulties in scalable purification, along with promising strategies to overcome them.},
}
@article {pmid41012691,
year = {2025},
author = {Zhao, X and Jiang, G and Ruan, Q and Qu, Y and Yang, X and Shi, Y and Wang, D and Zhou, J and Liu, J and Hou, L},
title = {Rapid Visual Detection of Senecavirus A Based on RPA-CRISPR/Cas12a System with Canonical or Suboptimal PAM.},
journal = {Viruses},
volume = {17},
number = {9},
pages = {},
pmid = {41012691},
issn = {1999-4915},
support = {National Key Research and Development Program of China (2023YFD1800501)//Lei Hou/ ; Introduction Program of High-Level Innovation and Entrepreneurship Talents in Jiangsu Province//Jue Liu/ ; the 111 Project D18007 (D18007)//Jue Liu/ ; Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)//Jue Liu/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Swine ; *Picornaviridae/genetics/isolation &amp; purification ; *Swine Diseases/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Picornaviridae Infections/diagnosis/veterinary/virology ; Recombinases/metabolism/genetics ; },
abstract = {Senecavirus A (SVA) is an emerging pathogen responsible for vesicular lesions and neonatal mortality in swine. In the absence of effective vaccines or therapeutics, early and accurate diagnosis is essential for controlling SVA outbreaks. Although nucleic acid-based detection methods are commonly employed, there remains a pressing need for rapid, convenient, highly sensitive, and specific diagnostic tools. Here, we developed a two-pot assay combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a containing crRNA targeting canonical protospacer adjacent motifs (PAMs) for simple, rapid, and visual identification of SVA in clinical samples. Subsequently, we successfully streamlined this system into a one-pot assay by selecting a specially designed crRNA targeting suboptimal PAM and integrating RPA amplification reagents and CRISPR/Cas12a detection components into a single reaction system in one tube. The developed methods exhibited diagnostic specificity, showing no cross-reactivity with four major swine viruses, while showing remarkable sensitivity with a lower detection limit of just two copies. Clinical validation in field samples using these two methods revealed perfect agreement (100% concordance) with conventional quantitative PCR (qPCR) results (sample size, n = 28), with both assays completing detection within 30 min. These results demonstrate that both the one-pot and two-pot RPA-CRISPR/Cas12a assays offer a reliable and efficient method for detecting SVA in this pilot study. Despite the limited sample size, the assays combine rapid reaction time with high sensitivity and specificity, showing great potential for future diagnostic applications.},
}
@article {pmid41012605,
year = {2025},
author = {Lee, MF and Tham, SK and Poh, CL},
title = {Antiviral Strategies Targeting Enteroviruses: Current Advances and Future Directions.},
journal = {Viruses},
volume = {17},
number = {9},
pages = {},
doi = {10.3390/v17091178},
pmid = {41012605},
issn = {1999-4915},
mesh = {*Antiviral Agents/pharmacology/therapeutic use ; Humans ; *Enterovirus Infections/drug therapy/virology ; *Enterovirus/drug effects/genetics ; Animals ; Virus Replication/drug effects ; },
abstract = {Enteroviruses, a diverse genus within the Picornaviridae family, are responsible for a wide range of human infections, including hand, foot, and mouth disease, respiratory disease, aseptic meningitis, encephalitis, myocarditis, and acute flaccid paralysis. Despite their substantial global health burden and the frequent emergence of outbreaks, no specific antiviral therapies are currently approved for clinical use against non-polio enteroviruses. This review provides a comprehensive overview of the current landscape of antiviral strategies targeting enteroviruses, including direct-acting antivirals such as capsid binders, protease inhibitors, and viral RNA polymerase inhibitors. We also examine the potential of host-targeting agents that interfere with virus-host interactions essential for replication. Emerging strategies such as immunotherapeutic approaches, RNA interference, CRISPR-based antivirals, and peptide-based antivirals are also explored. Furthermore, we address key challenges, including viral diversity, drug resistance, and limitations in preclinical models. By highlighting recent advances and ongoing efforts in antiviral development, this review aims to guide future research and accelerate the discovery of effective therapies against enterovirus infections.},
}
@article {pmid41011478,
year = {2025},
author = {Cortés, M and Olate, P and Rodriguez, R and Diaz, R and Martínez, A and Hernández, G and Sepulveda, N and Paz, EA and Quiñones, J},
title = {Human Microbiome as an Immunoregulatory Axis: Mechanisms, Dysbiosis, and Therapeutic Modulation.},
journal = {Microorganisms},
volume = {13},
number = {9},
pages = {},
doi = {10.3390/microorganisms13092147},
pmid = {41011478},
issn = {2076-2607},
support = {N&#xb0; 21231033//Agencia Nacional de Investigaci&#xf3;n y Desarrollo/ ; },
abstract = {The human microbiome plays a central role in modulating the immune system and maintaining immunophysiological homeostasis, contributing to the prevention of immune-mediated diseases. In particular, the gut microbiota is a key ecosystem for immune system maturation, especially in early life. This review aimed to analyze the molecular and cellular mechanisms linking the microbiome to immune and neuronal functions, as well as the impact of dysbiosis and emerging therapeutic strategies targeting the microbiome. The analysis was based on scientific databases, prioritizing studies published since 2000, with special emphasis on the past decade. The microbiome influences immune signaling through microorganism-associated molecular patterns (MAMPs) and pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). Additionally, microbial metabolites-such as short-chain fatty acids (SCFAs), tryptophan derivatives, and secondary bile acids-exert significant immunomodulatory effects. The intestinal epithelial barrier is also described as an active immunological interface contributing to systemic regulation. The literature highlights innovative therapies, including fecal microbiota transplantation (FMT), probiotics, and microbiome editing with CRISPR-Cas technologies. These strategies aim to restore microbial balance and improve immune outcomes. The growing body of evidence positions the microbiome as a valuable clinical and diagnostic target, with significant potential for application in personalized medicine.},
}
@article {pmid41011338,
year = {2025},
author = {Meng, S and Zhao, Z and Huang, L and Peng, X and Chen, H and Tang, X},
title = {CRISPR/Cas Technology for the Diagnosis of Animal Infectious Diseases.},
journal = {Microorganisms},
volume = {13},
number = {9},
pages = {},
doi = {10.3390/microorganisms13092006},
pmid = {41011338},
issn = {2076-2607},
support = {2023174004//Beihai Science and Technology Program/ ; AB241484035//Guangxi Key R&amp;D Program Project/ ; YCSW2024140//Innovation Project of Guangxi Graduate Education/ ; },
abstract = {Increasingly complex epidemics of animal infectious diseases have emerged as a major risk to livestock production and human health. However, current detection methods for animal infectious diseases suffer from shortcomings such as insufficient sensitivity, complicated operation, and reliance on skilled personnel, highlighting the urgent need for novel sensing platforms. CRISPR/Cas systems are adaptive immune systems found in many prokaryotes. Owing to their ability to precisely and reliably target and cleave nucleic acids, the CRISPR/Cas-based nucleic acid detection technology is considered a promising new detection method. When leveraged with a pre-amplification step and established readout methods, CRISPR/Cas-based sensing platforms can achieve a high sensitivity of single-base resolution or attomolar levels on-site. In this review, we first outline the history, working principles, and nucleic acid detection platforms derived from various CRISPR/Cas systems. Next, we evaluate the advantages and limitations of different nucleic acid pre-amplification methods integrated with CRISPR/Cas systems, followed by a discussion of readout methods employed in CRISPR/Cas-based sensing platforms. Additionally, we highlight recent applications of CRISPR/Cas-based sensing platforms in identifying animal infectious diseases. Finally, we address the challenges and prospects of CRISPR/Cas-based sensing platforms for the early and accurate diagnosis of animal infectious diseases.},
}
@article {pmid41010035,
year = {2025},
author = {Angelis, KJ and Holá, M and Vágnerová, R and Vaculíková, J and Paleček, JJ},
title = {The Phenotype of Physcomitrium patens SMC6 Mutant with Interrupted Hinge Interactions.},
journal = {Genes},
volume = {16},
number = {9},
pages = {},
doi = {10.3390/genes16091091},
pmid = {41010035},
issn = {2073-4425},
support = {GA20-05095S//Czech Science Foundation/ ; GA23-05284S)//Czech Science Foundation/ ; },
mesh = {*Bryopsida/genetics/metabolism ; *Cell Cycle Proteins/genetics/metabolism ; *Plant Proteins/genetics/metabolism/chemistry ; Phenotype ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; Mutation ; *Chromosomal Proteins, Non-Histone/genetics ; },
abstract = {Background/Objectives: The Structural Maintenance of Chromosomes (SMC) proteins form essential heterocomplexes for the preservation of DNA structure and its functions, and hence cell viability. The SMC5/6 dimer is assembled by direct interactions of ATP heads via the kleisin NSE4 bridge and by SMC hinges. The structure might be interrupted by a single point mutation within a conserved motif of the SMC6-hinge. We describe the phenomena associated with the impairment of the SMC5/6 complex with morphology, repair of DNA double strand breaks (DSB), mutagenesis, recombination and gene targeting (GT) in the moss Physcomitrium patens (P. patens). Methods: Using CRISPR/Cas9-directed oligonucleotide replacement, we have introduced two close G to R point mutations in the hinge domain of SMC6 of P. patens and show that both mutations are not toxic and allow viability of mutant lines. Results: The G514R mutation fully prevents the interaction of SMC6 not only with SMC5, but also with NSE5 and NSE6, while the mutation at G517R has no effect. The Ppsmc6_G514R line has aberrant morphology, spontaneous and bleomycin-induced mutagenesis, and maintenance of the number of rDNA copies. The most unique feature is the interference with gene targeting (GT), which is completely abolished. In contrast, the Ppsmc6_G517R line is close to WT in many aspects. Surprisingly, both mutations have no direct effect on the rate of DSB repair in dividing and differentiated cells. Conclusions: Abolished interactions of SMC6 with SMC5 and NSE5,6 partners, which allow DSB repair, but impair other repair and recombination functions, suggests also regulatory role for SMC6.},
}
@article {pmid41009844,
year = {2025},
author = {Haval, M and Unakal, C and Ghagane, SC and Pandit, BR and Daniel, E and Siewdass, P and Ekimeri, K and Rajamanickam, V and Justiz-Vaillant, A and Lootawan, KA and Oliveira, FM and Bashetti, N and Naqvi, TA and Shettar, A and Bhasme, P},
title = {Biofilms Exposed: Innovative Imaging and Therapeutic Platforms for Persistent Infections.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {14},
number = {9},
pages = {},
doi = {10.3390/antibiotics14090865},
pmid = {41009844},
issn = {2079-6382},
abstract = {Biofilms constitute a significant challenge in the therapy of infectious diseases, offering remarkable resistance to both pharmacological treatments and immunological elimination. This resilience is orchestrated through the regulation of extracellular polymeric molecules, metabolic dormancy, and quorum sensing, enabling biofilms to persist in both clinical and industrial environments. The resulting resistance exacerbates chronic infections and contributes to mounting economic burdens. This review examines the molecular and structural complexities that drive biofilm persistence and critically outlines the limitations of conventional diagnostic and therapeutic approaches. We emphasize advanced technologies such as super-resolution microscopy, microfluidics, and AI-driven modeling that are reshaping our understanding of biofilm dynamics and heterogeneity. Further, we highlight recent progress in biofilm-targeted therapies, including CRISPR-Cas-modified bacteriophages, quorum-sensing antagonists, enzyme-functionalized nanocarriers, and intelligent drug-delivery systems responsive to biofilm-specific cues. We also explore the utility of in vivo and ex vivo models that replicate clinical biofilm complexity and promote translational applicability. Finally, we discuss emerging interventions grounded in synthetic biology, such as engineered probiotic gene circuits and self-regulating microbial consortia, which offer innovative alternatives to conventional antimicrobials. Collectively, these interdisciplinary strategies mark a paradigm shift from reactive antibiotic therapy to precision-guided biofilm management. By integrating cutting-edge technologies with systems biology principles, this review proposes a comprehensive framework for disrupting biofilm architecture and redefining infection treatment in the post-antibiotic era.},
}
@article {pmid41009658,
year = {2025},
author = {Zhang, H and Teng, C and Lyu, S and Fan, Y},
title = {High-Frequency Generation of Homozygous/Biallelic Mutants via CRISPR/Cas9 Driven by AtKu70/80 Promoters.},
journal = {International journal of molecular sciences},
volume = {26},
number = {18},
pages = {},
doi = {10.3390/ijms26189094},
pmid = {41009658},
issn = {1422-0067},
support = {ZR2023MC070//Natural Science Foundation of Shandong province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Promoter Regions, Genetic ; *Arabidopsis/genetics ; *Gene Editing/methods ; *Ku Autoantigen/genetics ; Homozygote ; *Arabidopsis Proteins/genetics ; Plants, Genetically Modified/genetics ; *Mutation ; Glycine max/genetics ; Alleles ; DNA End-Joining Repair ; },
abstract = {CRISPR/Cas9 gene editing technology is widely used in plant gene editing to verify gene function or improve agronomic traits. In the CRISPR/Cas9 system, Cas9 expression hinges on promoter choice, and CRISPR/Cas9 driven by a strong promoter or cell division-specific promoter has a higher editing efficiency. The CRISPR/Cas9 mechanism involves the CAS9 enzyme, which, directed by guide RNA, cleaves target double-stranded DNA and subsequently induces insertions or deletions (InDels) through the non-homologous end joining (NHEJ) repair pathway. The Ku protein plays a central role in the NHEJ repair process. It remains unclear whether driving Cas9 with promoters of AtKu70 and AtKu80, which are subunits of the Ku protein, will enhance gene editing efficiency. In this study, the promoters of AtKu70 and AtKu80 were cloned and used to drive Cas9 in the CRISPR/Cas9 system. Four different genes, GmRj7, GmNNL1, AtPDS3, and AtBRI1, were designed for soybean hairy root transformation and Arabidopsis transformation. The results showed that the CRISPR/Cas9 systems driven by the promoters of AtKu70 and AtKu80 achieved higher homozygous/biallelic mutation efficiencies than the CRISPR/Cas9 system driven by the 35S promoter in hairy root transformation by Rhizobium rhizogenes and stable genetic transformation with Rhizobium tumefaciens.},
}
@article {pmid41006228,
year = {2025},
author = {Maric, M and Segura-Bayona, S and Kuthethur, R and Takaki, T and Borel, V and Stanage, TH and Ivanov, MP and Parnandi, N and Hewitt, G and Millar, R and Fonseca, CS and Patel, H and Llorian, M and Warchal, S and Howell, M and Chaudhuri, AR and Kotsantis, P and Boulton, SJ},
title = {EXO1 as a therapeutic target for Fanconi Anaemia, ZRSR2 and BRCA1-A complex deficient cancers.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8476},
pmid = {41006228},
issn = {2041-1723},
mesh = {*Exodeoxyribonucleases/genetics/metabolism ; Humans ; *BRCA1 Protein/genetics/metabolism/deficiency ; *Fanconi Anemia/genetics/metabolism ; DNA Repair/genetics ; *Neoplasms/genetics/metabolism ; DNA Replication/genetics ; *DNA Repair Enzymes/genetics/metabolism ; DNA Damage ; Cell Line, Tumor ; Cisplatin/pharmacology ; Synthetic Lethal Mutations ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; CRISPR-Cas Systems ; },
abstract = {Exonuclease EXO1 performs multiple roles in DNA replication and DNA damage repair (DDR). However, EXO1 loss is well-tolerated, suggesting the existence of compensatory mechanisms that could be exploited in DDR-deficient cancers. Using CRISPR screening, we find EXO1 loss as synthetic lethal with many DDR genes somatically inactivated in cancers, including Fanconi Anaemia (FA) pathway and BRCA1-A complex genes. We also identify the spliceosome factor and tumour suppressor ZRSR2 as synthetic lethal with loss of EXO1 and show that ZRSR2-deficient cells are attenuated for FA pathway activation, exhibiting cisplatin sensitivity and radial chromosome formation. Furthermore, FA or ZRSR2 deficiencies depend on EXO1 nuclease activity and can be potentiated in combination with PARP inhibitors or ionizing radiation. Finally, we uncover dysregulated replication-coupled repair as the driver of synthetic lethality between EXO1 and FA pathway attributable to defective fork reversal, elevated replication fork speeds, post-replicative single stranded DNA exposure and DNA damage. These findings implicate EXO1 as a synthetic lethal vulnerability and promising drug target in a broad spectrum of DDR-deficient cancers unaddressed by current therapies.},
}
@article {pmid41006213,
year = {2025},
author = {Chen, PR and Qin, PP and Wang, YN and Liu, PF and Zhang, XY and Qian, T and Ye, BC and Yin, BC},
title = {De novo design of hypercompact transcript degraders by engineering substrate-specific toxins and Cas6-CBS system.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8446},
pmid = {41006213},
issn = {2041-1723},
mesh = {Escherichia coli/genetics/metabolism ; Humans ; *Escherichia coli Proteins/genetics/metabolism ; *Endoribonucleases/metabolism/genetics ; CRISPR-Cas Systems ; RNA Stability ; Protein Engineering/methods ; Binding Sites ; HEK293 Cells ; Bacterial Toxins/genetics/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics ; DNA-Binding Proteins ; },
abstract = {Artificial assembly of small functional proteins provides effective strategies for development of compact RNA degradation systems, which overcome the challenges associated with delivery. Here, we excavate and evolve three small toxin endoribonucleases with simple RNA cleavage motifs (barnase, MqsR, and MaZF), and integrate catalytically dead Cas6 (dCas6) along with its cognate stem-loop RNA (Cas6 binding site, termed CBS) from Escherichia coli (E. coli) to create hypercompact transcript degraders (317 ~ 430 amino acids), named STAR (small toxin- and dEcCas6-CBS-based RNA degraders). We experimentally find that CBS can be fine-tuned for EcCas6 processing but exhibits high conservatism in EcCas6 and dEcCas6 binding, laying a foundation for the design of CBS guides to effectively recruit dEcCas6-toxins. STAR exhibits high-efficiency knockdown of both cytoplasmic and nuclear transcripts in the tested mammalian cells, with significantly reduced off-target activities compared to established CRISPR and RNA interference (RNAi) technologies. Moreover, the small size of STAR enables delivery via a single adeno-associated virus (AAV) for ease of multiplex RNA knockdown, including effective silencing of the oncogenic RNA MYC in human cancer cells. Together, STAR unlocks new territory for employing toxin to design miniature, efficacious and safer RNA degraders.},
}
@article {pmid41005481,
year = {2025},
author = {Gong, Z and Lu, T and Ruan, Z and Zhang, R and Zhu, S and Xia, Z and Zhong, J and Wang, G and Li, Y and He, Q and Liu, R and Che, J},
title = {A multiplexed TSA/CRISPR-mediated one-pot system for rapid detection of high-risk animal-derived infectious diseases.},
journal = {Journal of microbiological methods},
volume = {238},
number = {},
pages = {107277},
doi = {10.1016/j.mimet.2025.107277},
pmid = {41005481},
issn = {1872-8359},
abstract = {The importance of rapid and convenient pathogen detection has been emphasized by the alarming threat of the Coronavirus Disease 2019 (COVID-19) pandemic since 2019. Point-of-care testing (POCT) provides rapid diagnostic results directly at the sampling site. However, isothermal amplification-based POCT faces technical challenges including primer design complexity and false-positive rates. To address these limitations, we developed the Thermostatic Step Amplification (TSA)/Clustered regularly interspaced short palindromic repeats (CRISPR) One-Pot System (TCOPS). This sensitive, rapid, and efficient platform specifically detects Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Mpox virus (MPXV) and Rabíes virus (RV) through integrated amplification and CRISPR-based detection. Our integrated TCOPS overcomes the technical challenges through single-tube reactions combining thermostatic amplification and CRISPR detection, reducing contamination while maintaining high accuracy for field applications. TCOPS enables single-tube CRISPR detection of high-risk viruses, with 10 copies/μL sensitivity shown using cloned DNA template for RV. In evaluations against Quantitative Polymerase Chain Reaction (qPCR) using 50 clinical samples, TCOPS incorporating freeze-dried reagents and a newly developed miniature fluorescence system (Q max) demonstrated >90 % sensitivity and 100 % specificity. Combined with the portable Q max device and its lyophilized reagent kit, TCOPS enables simple, rapid detection of multiple zoonotic viruses (SARS-CoV-2, MPXV, and RV) at the point of care. This integrated system achieves high sensitivity and specificity while establishing a practical, field-deployable prototype for next-generation POCT applications in resource-limited settings.},
}
@article {pmid41004579,
year = {2025},
author = {Gao, Q and Gao, Y and Cao, Y and Xu, H and Ma, Y and Zu, M and Yang, Q and Yang, K and Zhu, Z and Liu, C and Shi, X and Reis, RL and Kundu, SC and Ma, S and Han, H and Xiao, B},
title = {Reinforced plant-derived lipid nanoparticles for oral precise epigenome editing in colonic diseases.},
journal = {Science advances},
volume = {11},
number = {39},
pages = {eadw9275},
pmid = {41004579},
issn = {2375-2548},
mesh = {*Nanoparticles/chemistry/administration &amp; dosage ; *Gene Editing/methods ; Animals ; Administration, Oral ; Humans ; Mice ; *Lipids/chemistry ; CRISPR-Cas Systems ; *Epigenome ; *Colonic Diseases/genetics/therapy ; Ribonucleoproteins/genetics ; Colitis/genetics ; *Epigenesis, Genetic ; Histone Demethylases/genetics ; Epigenome Editing ; Liposomes ; },
abstract = {The clinical application of CRISPR-Cas9 remains limited by delivery challenges, particularly for oral administration. Lysine-specific demethylase 1 (Lsd1) plays a key role in colonic inflammation and tumorigenesis. Here, we developed an oral genome-editing platform (TPGS-RNP@LNP), where Lsd1-targeting ribonucleoproteins (RNPs) were encapsulated in mulberry leaf lipid nanoparticles (LNPs) and formulated with d-α-tocopherol polyethylene glycol succinate (TPGS). TPGS reinforced the lipid bilayer of LNPs, enhanced gastrointestinal stability, and facilitated colonic mucus penetration. Upon the galactose receptor-mediated endocytosis of TPGS-RNP@LNPs by macrophages, their fusion with the endosomal membrane and the presence of nuclear localization signals ensured the nuclear delivery of RNPs. TPGS-RNP@LNPs achieved 59.7% Lsd1 editing efficiency in macrophages, surpassing the commercial CRISPRMAX (43.0%). Oral TPGS-RNP@LNPs promoted H3K4 methylation to modulate epigenetic states, achieving inflammation mitigation, epithelial barrier restoration, and retardation of colitis and its associated tumorigenesis. As an LNP-based oral RNP delivery system, TPGS-RNP@LNPs provide a promising platform for precise treatment of colorectal diseases.},
}
@article {pmid41004518,
year = {2025},
author = {Wilbie, D and Eising, S and Amo-Addae, V and Walther, J and Bosman, E and Lei, Z and de Jong, OG and Molenaar, JJ and Mastrobattista, E},
title = {Anti-cancer compound screening identifies Aurora Kinase A inhibition as a means to favor CRISPR/Cas9 gene correction over knock-out.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0332617},
pmid = {41004518},
issn = {1932-6203},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Aurora Kinase A/antagonists &amp; inhibitors/genetics ; HEK293 Cells ; *Antineoplastic Agents/pharmacology ; Gene Knockout Techniques ; *Protein Kinase Inhibitors/pharmacology ; Gene Editing ; Pyrimidines/pharmacology ; Drug Screening Assays, Antitumor ; Recombinational DNA Repair/drug effects ; Cell Line, Tumor ; Azepines ; },
abstract = {CRISPR gene therapy holds the potential to cure a variety of genetic diseases by causing a targeted DNA break, which is repaired by host DNA damage responses. One option to introduce precise gene corrections is via the homology-directed repair (HDR) pathway. The problem in utilizing this pathway is that CRISPR-induced double stranded DNA breaks are more likely to be erroneously repaired by the non-homologous end joining (NHEJ) pathway, which may introduce random insertions or deletions at the cut site. We screened a small library of oncological drug compounds to steer the DNA repair process towards preferential HDR activation. We included forty compounds in the screen based on their mechanism of action. After optimizing the toxicity and adding these compounds during gene editing, nine showed a potential benefit for HDR activation. Three were shown to be beneficial after validation: rucaparib, belinostat and alisertib. The Aurora Kinase A inhibitor alisertib in particular led to an over 4-fold increase in preferential gene correction over gene knock-out in two cell models (HEK293T and Hepa 1-6) at sub-micromolar dosages on the eGFP locus, prompting further validation. On the long term this pathway did show cytotoxicity especially in the HEK293T cells, indicating further mechanistic investigation is needed, but this toxicity was less pronounced in primary hepatocytes.},
}
@article {pmid41004222,
year = {2025},
author = {Taweechai, S and Totañes, FIG and Westhead, D and Herrera-Arozamena, C and Foster, R and McConkey, GA},
title = {Validated antimalarial drug target discovery using genome-scale metabolic modeling.},
journal = {Antimicrobial agents and chemotherapy},
volume = {},
number = {},
pages = {e0045925},
doi = {10.1128/aac.00459-25},
pmid = {41004222},
issn = {1098-6596},
abstract = {Given the rapid resistance of Plasmodium falciparum to antimalarial drugs, there is a continual need for new treatments. A genome-scale metabolic (GSM) model was developed with integrated metabolomics and constraint-based, experimental flux-balance data to predict genes essential for P. falciparum growth as drug targets. We selected the highly ranked P. falciparum UMP-CMP kinase (UCK) to test its necessity and the ability to inhibit parasite growth in the presence of inhibitors. Conditional deletion mutants using the DiCre recombinase system, generated by CRISPR-Cas genome editing, exhibited defective asexual growth and stage-specific developmental arrest. Based on in silico and in vitro screening, inhibitors were identified that are selective for P. falciparum UCK and exhibit antiparasitic activity. This study, for the first time, shows assertions from a GSM model identifying novel, validated "druggable" targets. These findings show a role for GSM models in antimalarial drug discovery and identify P. falciparum UCK as a novel, valid malaria drug target.},
}
@article {pmid41003754,
year = {2025},
author = {Zubair, A and Ali, M and Ahmad, F and Althobaiti, SA},
title = {Unlocking the role of transcription activator-like effector nuclease (TALENs) and zinc finger nuclease (ZFN) in the treatment of HIV.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {948},
pmid = {41003754},
issn = {1573-4978},
mesh = {Humans ; *Transcription Activator-Like Effector Nucleases/genetics/metabolism/therapeutic use ; *Zinc Finger Nucleases/genetics/metabolism/therapeutic use ; *HIV Infections/therapy/genetics ; Gene Editing/methods ; HIV-1/genetics ; CRISPR-Cas Systems ; Receptors, CCR5/genetics ; Zinc Fingers ; },
abstract = {Some nucleases may be programmed to break just certain portions of DNA; examples of such enzymes include zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Insertions and deletions are used by cellular machinery to repair damaged DNA. By specifically targeting long terminal repeats (LTRs), zinc-finger nucleases (ZFNs) efficiently and accurately remove HIV-1 proviral DNA from inactive human T cells, offering a new and different way to eradicate HIV-1 infections. This paper examines the potential, evaluates the current situation, and draws attention to the challenges surrounding the use of TALENs and ZFNs as therapeutic tools for the treatment of HIV infection, to mitigate the adverse off-target effects that result from their extended expression. There is less off-target editing and higher success in targeting HIV escape mutations using TALENs and ZFNs than with CRISPR/Cas-9. The use of ZFNs and TALEN has resulted in changes to many host genes. These include the entrance receptors CCR5 and CXCR4, as well as the proviral integration protein LEDGF/p75. One of the viral targets is the big terminal repeats of proviral DNA. The advancement of gene therapy from the laboratory to the clinic is hindered by the need to reduce immunogenicity, cytotoxicity, and off-target editing while simultaneously enhancing cleavage efficiency and dispersion. However, TALENs technology and breakthroughs in ZFNs are making cleavage more efficient and selective. The strategy for treating HIV might be drastically changed, and maybe even eradicated, by the creation of synthetic nucleases like ZFNs and TALENs. This review explores the current developments about ZFNs and TALENs for the treatment of HIV.},
}
@article {pmid41003718,
year = {2025},
author = {Behera, B and Singh, R and Sharma, K and Rai, A and Singh, S and Balan, B},
title = {A comprehensive review of advanced strategies to combat antimicrobial resistance.},
journal = {Archives of microbiology},
volume = {207},
number = {11},
pages = {281},
pmid = {41003718},
issn = {1432-072X},
mesh = {Humans ; *Anti-Bacterial Agents/pharmacology/therapeutic use ; Gene Editing ; *Drug Resistance, Bacterial ; *Bacteria/drug effects/genetics ; CRISPR-Cas Systems ; Antimicrobial Peptides/pharmacology/therapeutic use ; Phage Therapy ; *Bacterial Infections/drug therapy/microbiology/therapy ; },
abstract = {Antimicrobial Resistance (AMR) is a growing global issue, as many first-line antibiotics are becoming less effective due to their overuse and misuse. Recent advances in novel antibiotic derivatives reveal mechanisms designed to counteract AMR. Even though conventional antimicrobial therapy has failed, no new antibiotic class has been developed in the past decade. Consequently, various innovative alternative tactics have been discovered to counteract drug-resistant pathogens. The article reviews novel approaches in combating AMR, which include antimicrobial peptides, phage therapy, CRISPR-Cas gene editing, nanomaterial-based antimicrobials, immunomodulatory agents, innovative physicochemical strategies, and combination therapy. Collectively, these approaches utilize cutting edge technologies that mark a shift from the traditional paradigm of antibiotics to integrated next-generation therapeutics. AMR remains a serious issue despite all of the noted advancements, and hence, a collaborative and multidisciplinary action involving researchers, healthcare professionals, policymakers, and pharmaceutical sector is urgently required. The emergence and burden of AMR can be better tackled by inventiveness, cooperation, and proactive approaches.},
}
@article {pmid41002431,
year = {2025},
author = {Vivarelli, R and Vantaggiato, C and Bassi, MT and Santorelli, FM and Marchese, M},
title = {Wings of Discovery: Using Drosophila to Decode Hereditary Spastic Paraplegia and Ataxias.},
journal = {Cells},
volume = {14},
number = {18},
pages = {},
doi = {10.3390/cells14181466},
pmid = {41002431},
issn = {2073-4409},
support = {GJC21131//Telethon Foundation/ ; Ricerca Corrente 2024-2025//Italian Minister of Health/ ; },
mesh = {Animals ; *Spastic Paraplegia, Hereditary/genetics/pathology ; Disease Models, Animal ; Humans ; *Drosophila melanogaster/genetics ; CRISPR-Cas Systems ; },
abstract = {Hereditary spastic paraplegia (HSP) and hereditary ataxias (HA) are clinically and genetically heterogeneous neurodegenerative disorders that primarily affect motor coordination and neural integrity. Despite distinct pathological features, such as pyramidal tract degeneration in HSP and spinocerebellar pathway involvement in HA, these conditions share overlapping genetic pathways and mechanisms. The fruit fly Drosophila melanogaster has emerged as a powerful model organism for investigating the molecular basis of rare diseases, including HSP and HA. Its genetic tractability, rapid life cycle, and high degree of gene conservation with humans make it a cost-effective and ethically viable platform for disease modelling. In this review, we provide a comprehensive overview of Drosophila-based models for HSP and HA. We highlight the use of advanced genetic tools, including RNA interference, CRISPR/Cas9, and the GAL4/UAS system, as well as behavioral and neuroanatomical assays to model disease features. Furthermore, we discuss the application of genetic "avatars" and high-throughput drug screening platforms to test therapeutic candidates. Collectively, these models have deepened our understanding of the pathophysiology of HSP and HA, offering valuable insights for the development of targeted therapies and approaches to personalized medicine.},
}
@article {pmid41002335,
year = {2025},
author = {Jayakumar, S and Vengadassalapathy, S and Venkadassalapathy, S and Durairajan, S and Vijayaraj, R and Govindan, L},
title = {Advancements and Applications of Split Technology in CRISPR/Cas12a: Transforming Molecular Diagnostics and Biosensing.},
journal = {Biosensors},
volume = {15},
number = {9},
pages = {},
doi = {10.3390/bios15090595},
pmid = {41002335},
issn = {2079-6374},
mesh = {*Biosensing Techniques ; *CRISPR-Cas Systems ; Humans ; *Pathology, Molecular ; *Molecular Diagnostic Techniques ; },
abstract = {The rapid evolution of CRISPR technology has revolutionized molecular biology, and among the various systems, CRISPR/Cas12a stands out for its high specificity and efficient collateral cleavage activity. This review article focuses on the recent advancements and applications of split technology within the CRISPR/Cas12a framework, highlighting its transformative role in molecular diagnostics and biosensing. Split technology innovatively divides functional nucleic acid components into modular segments that are activated by specific targets, significantly enhancing the specificity and sensitivity of biosensors. This design addresses the inherent limitations of traditional sensor systems, enabling the direct detection of ultrashort nucleic acids and improved discrimination of single-nucleotide variants, thereby facilitating the simultaneous detection of multiple biomolecules. The versatility of split-enabled biosensors extends beyond genetic testing, making them valuable tools in diagnostics, therapeutics, and environmental science. Despite challenges such as crRNA degradation and reassembly kinetics, ongoing research and engineering solutions continue to enhance the stability and performance of these systems. This review synthesizes the foundational principles, recent advancements, and potential applications of split technology while also identifying challenges and opportunities for future exploration. Ultimately, our insights provide a comprehensive resource to leverage the full potential of CRISPR/Cas12a-based split technology in advancing biosensing methodologies and clinical applications.},
}
@article {pmid41002137,
year = {2025},
author = {Zhang, S and Ji, Z and Cheng, X and Ma, Y and Feng, M and Cai, D and Bai, T},
title = {TMTP1-Modified Small Extracellular Vesicles Target BRAF Mutation in Anaplastic Thyroid Cancer Reversing Vemurafenib Resistance With CRISPR/Cas9 Delivery.},
journal = {Journal of extracellular vesicles},
volume = {14},
number = {9},
pages = {e70170},
doi = {10.1002/jev2.70170},
pmid = {41002137},
issn = {2001-3078},
mesh = {Humans ; *Thyroid Carcinoma, Anaplastic/genetics/drug therapy/metabolism ; *Vemurafenib/pharmacology ; *Extracellular Vesicles/metabolism ; *CRISPR-Cas Systems ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Proto-Oncogene Proteins B-raf/genetics ; Cell Line, Tumor ; Animals ; *Thyroid Neoplasms/genetics/drug therapy ; Mutation ; Mice ; Gene Editing ; Reactive Oxygen Species/metabolism ; Drug Delivery Systems ; },
abstract = {This study investigates a novel approach to overcome Vemurafenib resistance in BRAF-mutant Anaplastic thyroid carcinoma (ATC) using CRISPR/Cas9 gene editing and TMTP1-modified extracellular vesicles (TMTP1-sgBRAF-EVs). By knocking out the BRAF gene, the study elucidates Vemurafenib-induced ferroptosis mechanisms involving lipid peroxidation and reactive oxygen species (ROS) generation in ATC cells. The developed TMTP1-sgBRAF-EVs system demonstrates superior tumour-targeting and drug delivery capabilities, significantly enhancing Vemurafenib efficacy in both in vitro and in vivo models. This innovative combination of gene editing technology with a nanoparticle delivery system shows promising potential as a therapeutic strategy for treating aggressive BRAF-mutant ATC.},
}
@article {pmid41000010,
year = {2025},
author = {Wang, Z and Wang, Q and Zhang, J and Li, B and Li, Y and Chen, Z and Guo, D and Feng, S},
title = {CRISPR-driven diagnostics: Molecular mechanisms, clinical efficacy and translational challenges.},
journal = {Clinical and translational medicine},
volume = {15},
number = {10},
pages = {e70482},
doi = {10.1002/ctm2.70482},
pmid = {41000010},
issn = {2001-1326},
support = {23ZX005//the Basic Research Project of the Key Research Program of Colleges and Universities in Henan Province/ ; 2023M731023//China Postdoctoral Science Foundation/ ; //Joint Funds of Science and Technology Research and Development Plan of Henan Province/ ; 232300421164//Natural Science Foundation of Henan Province/ ; //Basic Research Project of the Key Research Program of Colleges and Universities in Henan Province/ ; 82402600//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Translational Research, Biomedical ; },
abstract = {BACKGROUND: In the realm of public health, among the primary perils menacing human well-being, the issue of pathogen infection persists as a significant concern. Precise and timely diagnosis of diseases constitutes the bedrock for effective therapeutic interventions and epidemiological monitoring. Hence, it is crucial to develop quick, sensitive, and highly effective methods for identifying pathogen and their variants.<br><br>MATERIAL AND METHODS: This article reviews the recent research progress in the CRISPR/Cas system for detecting nucleic acids, with an emphasis on CRISPR/Cas9, CRISPR/Cas12, and CRISPR/Cas13. Initially, we provided a concise overview of the nucleic acid detection mechanism utilizing the CRISPR/Cas system. Subsequently, we dissect the molecular mechanisms of CRISPR tools, compare their clinical efficacy against traditional methods, and explore frontier innovations such as amplification-free detection and AI integration.<br><br>CONCLUSION: Ultimately, we argue that CRISPR diagnostics must evolve beyond technical optimization to embrace ecological adaptability, ensuring that precision medicine serves as a bridge-rather than a barrier-to global health equity.<br><br>KEY POINTS: Core Mechanism: Explains the molecular basis of CRISPR-Cas (Cas9, Cas12, Cas13) for nucleic acid detection, leveraging crRNA-guided targeting and trans-cleavage activity for ultra-sensitive (aM level) and specific pathogen identification. Superior Performance: Outperforms traditional methods in speed, sensitivity, and cost, making it ideal for point-of-care use in resource-limited settings. Cutting-Edge Innovations: Covers key advances like amplification-free detection, portable device integration, and multiplex platforms. Translation Challenges: Discusses hurdles in clinical adoption, including inhibitor interference in complex samples, scalability limitations, the need for multi-center clinical data, and varying regional regulations. Future Outlook: Highlights emerging directions such as integrated "sample-to-result" systems and AI integration, while also addressing associated biosafety and ethical concerns, calling for robust regulatory frameworks.},
}
@article {pmid40998781,
year = {2025},
author = {Porter, DF and Meyers, RM and Miao, W and Reynolds, DL and Hong, AW and Yang, X and Srinivasan, S and Mondal, S and Siprashvili, Z and Fabo, T and Zhou, R and Nguyen, T and Ducoli, L and Meyers, JM and Nguyen, DT and Ko, LA and Kellman, LN and Elfaki, I and Guo, M and Winge, MC and Jackrazi, LV and Lopez-Pajares, V and Liu, BB and Qu, Y and Porter, IE and Kim, SH and Kim, G and Tao, S and Engreitz, JM and Khavari, PA},
title = {Disease-linked regulatory DNA variants and homeostatic transcription factors in epidermis.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8387},
pmid = {40998781},
issn = {2041-1723},
support = {AR076965, AR045192//U.S. Department of Health &amp; Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; HG010856//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {Humans ; *Transcription Factors/metabolism/genetics ; *Epidermis/metabolism/pathology ; *Polymorphism, Single Nucleotide ; Homeostasis/genetics ; *Skin Diseases/genetics ; Genetic Predisposition to Disease ; *DNA/metabolism/genetics ; Multifactorial Inheritance/genetics ; Promoter Regions, Genetic ; Alleles ; CRISPR-Cas Systems ; },
abstract = {Identifying noncoding single nucleotide variants (SNVs) in regulatory DNA linked to polygenic disease risk, the transcription factors (TFs) they bind, and the genes they dysregulate is a goal in polygenic disease research. Here, we use massively parallel reporter analysis of 3451 SNVs linked to risk for polygenic skin diseases with disrupted epidermal homeostasis to identify 355 differentially active SNVs (daSNVs). daSNV target gene analysis, combined with daSNV editing, underscored dysregulated epidermal differentiation as a shared pathomechanism. CRISPR knockout screens of 1772 human TFs revealed 123 TFs essential for epidermal homeostasis, highlighting ZNF217 and CXXC1. Population sampling CUT&amp;RUN of 27 homeostatic TFs identified allele-specific DNA binding (ASB) differences at daSNVs enriched near epidermal homeostasis and monogenic skin disease genes, with notable representation of SP/KLF and AP-1/2 TFs. High TF-occupancy promoters were "buffered" against ASB. This resource implicates dysregulated binding of specific homeostatic TF families in risk for diverse polygenic skin diseases.},
}
@article {pmid40997767,
year = {2025},
author = {Shoeva, OY and Zedgenizova, VD and Egorova, AA and Gerasimova, SV and Kukoeva, TV and Vasiliev, GV and Kovaleva, ON and Zakhrabekova, S and Hansson, M and Hertig, CW and Hoffie, I and Kumlehn, J and Khlestkina, E},
title = {Analysis of Anthocyanin-Less 2 Diversity in Barley Reveals a Specific Allele to Cause Purple-Colored Grains.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c05032},
pmid = {40997767},
issn = {1520-5118},
abstract = {The purple color of the barley (Hordeum vulgare L.) grain is attributed to anthocyanins which are beneficial for human health. Synthesis of these pigments in both grain and vegetative tissues is controlled by ANT1 and ANT2 that belong to the families R2R3-MYB and bHLH of transcription factors, respectively. Here, we investigated the role of the Ant2 gene in pigmentation of grains compared to vegetative tissue. After screening of 504 barley accessions, six relevant alleles of Ant2 were identified. These involve distinct insertions in the promoter and/or in intron 6. Allele Ant2.l was found in all purple-grained barley accessions and was more strongly expressed compared to the alleles present in the other lines. The crucial role of Ant2.l in grain pigmentation was validated by targeted mutagenesis using RNA-guided endonuclease Cas9. It is further shown that the other Ant2 alleles do not affect grain pigmentation while being essential for pigmentation of vegetative tissues.},
}
@article {pmid40996619,
year = {2025},
author = {Parkinson, JE and Baldwin, GE and Papotto, PH and Humphreys, NE and Day, AJ and Adamson, AD and Allen, JE and Sutherland, TE},
title = {Generation of a Ym1 deficient mouse utilising CRISPR-Cas9 in CB6 embryos.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {44},
pmid = {40996619},
issn = {1573-9368},
support = {MR/K01207X/2/MRC_/Medical Research Council/United Kingdom ; MR/K01207X/2/MRC_/Medical Research Council/United Kingdom ; MR/K01207X/2/MRC_/Medical Research Council/United Kingdom ; MR/K01207X/2/MRC_/Medical Research Council/United Kingdom ; MRY0036831/MRC_/Medical Research Council/United Kingdom ; 097820/Z/11/B/WT_/Wellcome Trust/United Kingdom ; 203128/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; 203128/Z/16/Z/WT_/Wellcome Trust/United Kingdom ; MRFAUK-2015-302//Asthma and Lung UK/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Mice, Inbred C57BL ; *Chitinase-3-Like Protein 1/genetics/deficiency ; Mice, Inbred BALB C ; Mice, Transgenic ; Female ; Embryo, Mammalian/metabolism ; },
abstract = {Chitinase-like proteins (CLPs) are of wide interest due to their significant roles during both homeostatic and pathological processes. Human CLPs such as YKL-40 have been proposed as biomarkers of disease severity in many conditions. Murine CLPs Brp39, Ym1, and Ym2 are similarly upregulated in multiple mouse models of pathology. Investigation of Ym1 and Ym2 is hampered by recent gene duplication events on the C57BL/6, but not BALB/c, background leading to complexity in the genomic locus. Here, we have generated a Ym1 deficient mouse using a novel CRISPR-Cas9 targeting approach involving CB6 (C57BL/6 X BALB/c) mixed background embryos. Validation using flow cytometry, ELISA, and immunofluorescence confirmed no expression of mature Ym1 protein. Additionally, expression of related genes including Chia, Chil1, and Chil4 were not altered in Ym1-deficent animals. This new transgenic mouse line will be key for future investigations of CLP functions and the utilised approach to genetic manipulation may provide a useful strategy for other genes which show differences in copy number between inbred mouse strains.},
}
@article {pmid40996539,
year = {2025},
author = {Nie, H and Wang, Z and Lin, Z and Gao, Y and Zhang, Y and Zheng, J and Cheng, Y},
title = {Enhancing lipase activity in Aspergillus niger through CRISPR/Cas9-mediated protease gene knockout and fermentation optimization.},
journal = {Biotechnology letters},
volume = {47},
number = {5},
pages = {114},
pmid = {40996539},
issn = {1573-6776},
support = {cstc2021jscx-jbgsX0002//Application Development Special Key Project of Chongqing/ ; 2022R01015//Leader-type Innovation and Entrepreneurship Team of Zhejiang/ ; },
mesh = {*Aspergillus niger/genetics/enzymology/metabolism ; *Lipase/metabolism/genetics ; *CRISPR-Cas Systems ; Fermentation ; *Gene Knockout Techniques/methods ; Fungal Proteins/genetics/metabolism ; *Peptide Hydrolases/genetics ; Metabolic Engineering/methods ; },
abstract = {The engineered Aspergillus niger strain AnCALB005 was selected as the research strain, which is a high-yield strain of Candida antarctica B lipase constructed in our laboratory. CRISPR/Cas9-mediated gene knockout was employed to construct the multiple protease-deficient strains targeting five genes (pepA, pepB, pepC, pepE and pepF) in the A. niger AnCALB005. Among the engineered variants, a triple-knockout strain lacking pepA, pepB, and pepF demonstrated 56% enhanced hydrolytic lipase activity relative to the parental strain. Fermentation culture conditions were initially screened through single-factor experiments. Building on these results, critical parameters were statistically determined via Plackett-Burman (PB) design. This was followed by a steepest ascent method combined with Box-Behnken (BB) response surface methodology. Key factors influencing lipase production (identified as maltose concentration, corn steep concentration, and shaking speed) were optimized. The final optimized fermentation conditions comprised: maltose (52 g/L), corn steep (52 g/L), K2HPO4 (5 g/L), soybean cake flour (30 g/L), initial pH 6.5, inoculation amount 10% (v/v), and shaking speed 220 rpm. Under the optimized fermentation conditions, Shake-flask validation of the engineered A. niger yielded a lipase activity of 46.66 U/mL, representing an increase of 92.01%. Scale-up fermentation in a 5 L bioreactor applying these optimized conditions over 120 h of cultivation achieved a lipase activity of 79.31 U/mL.},
}
@article {pmid40996239,
year = {2025},
author = {Calvert, RW and Knott, GJ},
title = {And… cut! - how conformational regulation of CRISPR-Cas effectors directs nuclease activity.},
journal = {The Biochemical journal},
volume = {482},
number = {19},
pages = {},
doi = {10.1042/BCJ20240481},
pmid = {40996239},
issn = {1470-8728},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; Protein Conformation ; *Bacterial Proteins/metabolism/chemistry/genetics ; Nucleic Acid Conformation ; },
abstract = {Controlling the conformation of dynamic protein, RNA and DNA molecules underpins many biological processes, from the activation of enzymes and induction of signalling cascades to cellular replication. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) effectors are enzymes tightly controlled by conformational steps that gate activation of nuclease domains core to their function in bacterial adaptive immunity. These precise conformational checkpoints combined with programmable activation specified by RNA guides have driven the success of CRISPR-Cas tools in biotechnology, medicine and beyond. To illustrate the importance of conformation in controlling CRISPR-Cas activity, we review the discrete conformational checkpoints at play in class 2 CRISPR-Cas systems. Using Cas9, Cas12a and Cas13a as examples, we describe how protein and nucleic acid conformations precisely control the loading of guide RNA, the selection of target nucleic acids and the activation of nuclease domains. Much like a director controls the timing of transitions between scenes in a movie, CRISPR effectors use conformational checkpoints to precisely direct their enzymatic activity.},
}
@article {pmid40957828,
year = {2025},
author = {Qiu, M and Zhang, X and Zhang, J and Li, Y and Jiang, Y and Zhao, Q and Man, C and Zhang, X},
title = {CRISPR/Cas12a-Responsive Hydrogel Biosensing Platform Based on an Aggregation-induced Emission Metal-Organic Framework Nanozyme for Sensitive and Reliable Detection of Foodborne Pathogens.},
journal = {ACS sensors},
volume = {10},
number = {9},
pages = {6775-6787},
doi = {10.1021/acssensors.5c01523},
pmid = {40957828},
issn = {2379-3694},
mesh = {*Metal-Organic Frameworks/chemistry ; *Biosensing Techniques/methods ; *Hydrogels/chemistry ; *CRISPR-Cas Systems ; Colorimetry ; *Salmonella enterica/isolation &amp; purification ; Limit of Detection ; Food Microbiology ; },
abstract = {Programmable CRISPR/Cas12a has emerged as a promising and powerful tool for the construction of biosensors, yet the challenges persist regarding the signal transduction efficiency, sensitivity, and multisignal outputs. In this work, a metal-organic framework (MOF) nanozyme with an aggregation-induced emission (AIE) property and peroxidase (POD)-like activity was synthesized by encapsulating hemin into a Zr-based MOF using an in situ encapsulation strategy, named AIE MOFzyme. The prepared AIE MOFzyme showed an enhanced fluorescent property via the restriction of the intramolecular motion effect for boosting the significant fluorescent response and a highly efficient POD-like nanozyme activity for activating visual colorimetric response. Benefiting from these merits, AIE MOFzyme, as a difunctional signal probe, was embedded into a DNA hydrogel for the construction of a CRISPR-responsive AIE MOFzyme hydrogel platform (abbreviated as Cas12a-H-AIE). In this platform, the activated CRISPR/Cas12a system led to DNA hydrogel phase transitions to release AIE MOFzymes, triggering highly efficient fluorescent and colorimetric signal amplification. Importantly, the CRISPR-responsive AIE MOFzyme hydrogel platform for the detection of Salmonella enterica (S. enterica) showed a high sensitivity, which was superior to that of numerous existing methods. This work provided new insight for the design of multifunctional and programmable nanozyme hydrogels, which may also offer guidance for the development of novel CRISPR/Cas12a-based biosensors.},
}
@article {pmid40936456,
year = {2025},
author = {Huang, M and Shang, K and Ying, L and Han, Y and Hong, N and Yao, YF},
title = {CRISPR/Cas12-Driven Exponential Amplification Combined with a Lateral Flow Biosensor Enabling Rapid and Highly Sensitive DNA Detection.},
journal = {ACS sensors},
volume = {10},
number = {9},
pages = {6553-6563},
doi = {10.1021/acssensors.5c00944},
pmid = {40936456},
issn = {2379-3694},
mesh = {*Biosensing Techniques/methods ; Animals ; *CRISPR-Cas Systems ; Rabbits ; *Nucleic Acid Amplification Techniques/methods ; *DNA, Viral/analysis/genetics ; Humans ; Herpesvirus 1, Human/genetics/isolation &amp; purification ; Limit of Detection ; Point-of-Care Systems ; Tears/virology/chemistry ; *DNA/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and precise detection of specific DNA is valuable for biological research and clinical disease diagnosis. Clustered regularly interspaced short palindromic repeat (CRISPR) technology can enhance existing DNA testing, making DNA detection faster, more portable, and more accurate. This study presents a new Cas12-driven exponential amplification-based lateral flow biosensor (CADEX-LF) for rapid and highly sensitive DNA detection. CADEX-LF takes full advantage of the highly specific target-loading-dependent trans-cleavage activity of Cas12 and the extremely high efficiency of nicking endonuclease-mediated exponential amplification. The adoption of lateral flow readout enables CADEX-LF for point-of-care (POC) use without requiring complicated supplementary equipment. CADEX-LF was shown to achieve a detection sensitivity of 2 × 10[-15] M within 45 min of measurement time and displayed outstanding specificity with double-base resolution. Furthermore, CADEX-LF could identify herpes simplex virus 1 (HSV-1) DNA in tears of rabbits and clinical patients with HSV-1 keratitis, exhibiting its practical application potential in clinical diagnosis. The proposed CADEX-LF biosensor may have great promise for point-of-care disease diagnosis in resource-limited environments.},
}
@article {pmid40921247,
year = {2025},
author = {Huerta, M&#xc1; and Codony, X and Ruiz-Cantero, MC and Porras, M and Tejada, M&#xc1; and Rickert-Llàcer, A and Artacho-Cordón, A and Zamanillo, D and Cobos, EJ and Nieto, FR},
title = {Generation and phenotypic characterization of a sigma-1 receptor knockout rat.},
journal = {Life sciences},
volume = {380},
number = {},
pages = {123953},
doi = {10.1016/j.lfs.2025.123953},
pmid = {40921247},
issn = {1879-0631},
mesh = {Animals ; *Receptors, sigma/genetics/metabolism ; Sigma-1 Receptor ; Rats ; Male ; Phenotype ; Gene Knockout Techniques ; Behavior, Animal ; Rats, Sprague-Dawley ; Neuralgia/genetics ; CRISPR-Cas Systems ; },
abstract = {The sigma-1 receptor (σ1R) is a chaperone involved in multiple physiological and pathological processes, including pain modulation, neuroprotection, and neurodegenerative diseases. Despite its functional significance, its precise roles remain unclear due to the lack of suitable models for detailed mechanistic studies. In this work, we describe the generation and phenotypic characterization of a novel σ1R knockout (σ1R KO) rat model. Using CRISPR/Cas9 technology, we introduced a specific 218-base-pair deletion into the σ1R gene, resulting in a complete loss of receptor expression, as confirmed by Western blot, immunohistochemistry, and binding assays. Comprehensive phenotypic analyses revealed no major developmental or behavioral abnormalities in σ1R KO rats under baseline conditions, suggesting that σ1R is not essential for development or survival. Additionally, no genotype-related differences were observed in cellular or biochemical blood parameters. Motor function tests (rotarod, grip strength, and wheel running) showed no deficits; however, σ1R KO rats displayed reduced exploratory behavior in actimetry and markedly diminished burrowing behavior. By contrast, no anxiodepressive-like behaviors were observed in the open field, startle, or forced swim tests. Sensory testing of naive rats revealed no significant genotype-related differences in responses to mechanical, heat, or cold stimuli, or in the formalin test (chemical-induced pain). However, σ1R KO rats displayed attenuated neuropathic pain after traumatic nerve injury (spared nerve injury), highlighting the role of σ1R in pain sensitization pathways. This study establishes the σ1R KO rat as a valuable tool for investigating σ1R-mediated mechanisms and for developing therapeutic strategies targeting σ1R for chronic pain, neurodegeneration, and psychiatric disorders.},
}
@article {pmid40676485,
year = {2025},
author = {Ran, F and Huang, H and Shang, B and Peng, W and Wu, L and Ling, K and Xie, X},
title = {An ultrasensitive sensing strategy based on CRISPR/Cas13a and T7 RNA polymerase amplification for detection of extracellular vesicles.},
journal = {Analytical sciences : the international journal of the Japan Society for Analytical Chemistry},
volume = {41},
number = {10},
pages = {1627-1636},
pmid = {40676485},
issn = {1348-2246},
support = {82272960//the National Science Foundation of China/ ; HBCH2024005//the Open Project of Hubei Clinical Research Center of Hypertension/ ; 24Y142//and the Science and Technology Key Program of Shiyan/ ; 24Y154//and the Science and Technology Key Program of Shiyan/ ; 24Y161//and the Science and Technology Key Program of Shiyan/ ; Y202522//the Young Talent Project of Affiliated Dongfeng Hospital/ ; Y202512//the Young Talent Project of Affiliated Dongfeng Hospital/ ; Y202518//the Young Talent Project of Affiliated Dongfeng Hospital/ ; },
mesh = {*Extracellular Vesicles/chemistry/metabolism ; *DNA-Directed RNA Polymerases/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Viral Proteins/metabolism/genetics ; *Nucleic Acid Amplification Techniques ; *Biosensing Techniques/methods ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; },
abstract = {Extracellular vesicles (EVs) are important biomarkers for an early diagnosis of lung cancer. Herein, we proposed an ultrasensitive fluorescent sensing platform for EVs detection, which involves aptamer and streptavidin-modified magnetic nanoparticles (SA-MB) magnetic separation technology as well as T7 RNA polymerase-assisted CRISPR/Cas13a system, which can achieve target recycling signal amplification. In this detection method, biotin-modified CD63 aptamer hybridizes first with the aptamer Blocker (T7 promoter) and then binds to SA-MB. When adding EVs, the CD63 aptamer in CD63 aptamer/Blocker/SA-MB complex captures EVs causing the release of Blocker single chain. Subsequently, large amounts of ssRNAs, which are generated with the assistance of Blocker-initiated T7 RNA polymerase, were recognized by CRISPR/Cas13a and trigger its trans-cleavage report probe (F-Q). Eventually, the report probe labeled with fluorescent dye (FAM) and quench group (BHQ) at both ends was cut to produce fluorescent signal. The designed sensor combined this with a signal amplification strategy based on T7 RNA polymerase and CRISPR/Cas13a to significantly enhance the sensitivity and specificity of EVs detection. The use of magnetic separation technology eliminates interference from complex matrices and improves EVs detection efficiency, while the introduction of T7 RNA polymerase and CRISPR/Cas13a enables multiple amplifications of the sensor signals, and enhancing the accuracy and sensitivity of the method. Ultimately, the combination of multiple amplification reactions resulted in a detection limit (LOD) for EVs as low as 60 particles/mL (approximately 1 zmol/L). In addition, this detection method can specifically distinguish EVs from other confounding substances and efficiently detect plasma EVs from lung cancer and healthy individuals in actual samples. Indicating this sensing platform is a valuable tool for early lung cancer detection.},
}
@article {pmid40557518,
year = {2025},
author = {Li, K and Liu, C and Wan, G and Zhang, L and Fan, R and Zhang, X and Su, Y and He, J and Liu, N and Guan, F and Dong, W and Gao, S and Kong, W and Qi, X and Zhang, X and Ma, Y},
title = {Comparative analysis of two newly established Cre rat lines, NeuN-Cre and Thy1-Cre, for neurological research.},
journal = {Animal models and experimental medicine},
volume = {8},
number = {8},
pages = {1468-1479},
doi = {10.1002/ame2.70051},
pmid = {40557518},
issn = {2576-2095},
support = {Program CBYI202102//Research Project of China Baoyuan Investment Co., Ltd/ ; HH24KYZX0007//Haihe Laboratory of Cell Ecosystem Innovation Fund/ ; 2021-I2M-1-024//CAMS Innovation Fund for Medical Sciences/ ; 2021-I2M-1-034//CAMS Innovation Fund for Medical Sciences/ ; 2023-I2M-2-001//CAMS Innovation Fund for Medical Sciences/ ; 3332022040//Fundamental Research Funds for the Central Universities/ ; 3332023164//Fundamental Research Funds for the Central Universities/ ; 202411/WT_/Wellcome Trust/United Kingdom ; 2060204//State Key Laboratory Special Fund/ ; 2023-PT180-01.//the Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences/ ; 202411/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; Rats ; *Integrases/genetics/metabolism ; *Thy-1 Antigens/genetics/metabolism ; *Neurons/metabolism ; CRISPR-Cas Systems ; Male ; *Nerve Tissue Proteins/genetics ; Rats, Sprague-Dawley ; Rats, Transgenic ; },
abstract = {BACKGROUND: The Cre/loxP system is most popular in mice, but its application in rats has largely lagged far behind. The rat is vital laboratory animal, especially in toxicological and neurological studies. Generating genetic tools to manipulate neurons in rats could benefit neurological research.<br><br>METHODS: Using the CRISPR/Cas9 system, we inserted a Cre cassette into endogenous Thy1 and NeuN loci. Thy1-Cre rats featured a downstream P2A-linked insertion, while NeuN-Cre was inserted at the transcriptional start site. The Cre activity was assessed by crossing with a Cre reporter (Rosa26[imCherry]) rat and through analyzing mCherry expression patterns. The specificity of cell type was further confirmed by immunofluorescence with NeuN antibody. Phenotypic consequences were assessed by crossing with ND1[LSL] rats to deplete ND1, followed by monitoring weight/survival and conducting motor function tests.<br><br>RESULTS: We generated two neuron-specific rats (Thy1-Cre and NeuN-Cre), which exhibited high neuron-specific Cre expression in brain and spinal cord with minor leakage in other tissues. Thy1-Cre showed minor leakage in spleen, lung and kidney while NeuN-Cre showed minor leakage in spleen and kidney. ND1[Thy1-Cre] and ND1[NeuN-Cre] rats both showed decreased body weights and survival times. The ND1[NeuN-Cre] rats died within two weeks, while ND1[Thy1-Cre] rats lived longer with impaired motor function.<br><br>CONCLUSIONS: We successfully generated two neuron-specific NeuN-Cre and Thy1-Cre rats, and systemically analyzed their expression pattern.},
}
@article {pmid40175512,
year = {2025},
author = {Duan, M and Gao, P and Zhang, YZ and Hu, YL and Zhou, L and Xu, ZC and Qiu, HY and Tong, XH and Ji, RJ and Lei, XL and Yin, H and Guo, CL and Zhang, Y},
title = {TOPO-seq reveals DNA topology-induced off-target activity by Cas9 and base editors.},
journal = {Nature chemical biology},
volume = {21},
number = {10},
pages = {1554-1564},
pmid = {40175512},
issn = {1552-4469},
support = {82450105//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *DNA/chemistry/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; High-Throughput Nucleotide Sequencing ; },
abstract = {With the increasing use of CRISPR-Cas9, detecting off-target events is essential for safety. Current methods primarily focus on guide RNA (gRNA) sequence mismatches, often overlooking the impact of DNA topology in regulating off-target activity. Here we present TOPO-seq, a high-throughput and sensitive method that identifies genome-wide off-target effects of Cas9 and base editors while accounting for DNA topology. TOPO-seq revealed that topology-induced off-target sites frequently harbor higher mismatches than the relaxed DNA sequence, with over 50% of off-target sites containing six mismatches, which are usually overlooked using previous methods. Applying TOPO-seq to three therapeutic gRNAs in hematopoietic stem cells identified 47 bona fide off-target loci, six of which are specifically induced by DNA topology. These findings highlight DNA topology as a regulator of off-target editing rates, establish TOPO-seq as a robust method for capturing DNA topology-induced off-target events and underscore its importance in off-target detection for developing safe genome-editing therapies.},
}
@article {pmid40995763,
year = {2025},
author = {Pramanik, D and Wang, K and Lee, K},
title = {CRISPR/Cas9-Mediated Gene Knockout in Cereal Crops.},
journal = {Current protocols},
volume = {5},
number = {9},
pages = {e70210},
doi = {10.1002/cpz1.70210},
pmid = {40995763},
issn = {2691-1299},
mesh = {*CRISPR-Cas Systems/genetics ; *Edible Grain/genetics ; *Gene Editing/methods ; *Crops, Agricultural/genetics ; *Gene Knockout Techniques/methods ; Plants, Genetically Modified/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Plant ; },
abstract = {High-precision genome editing tools, such as programmable nucleases, are poised to transform crop breeding and significantly impact fundamental plant research. Among these tools, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated 9) system is a programmable, RNA-guided nuclease that introduces targeted, site-specific double-stranded breaks in the target DNA loci. When these breaks are repaired, it often results in a frame-shift mutation via short insertion/deletion (indel), leading to gene knockout. Since its first successful use in plants, CRISPR/Cas9 has been widely adopted for targeting genes of agronomic and scientific importance in multiple crops, including rice, maize, wheat, and sorghum. These cereal crops ensure global food security, provide essential nutrition, and support economic stability. Additionally, such crops support biofuel production, livestock feed, and sustainable farming practices through crop rotation. This article outlines the strategies for implementing CRISPR/Cas9 genome editing in plants, including a step-by-step process of guide RNA target selection, oligonucleotide design, construct development, assembly, and analysis of genome edits. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: CRISPR/Cas9 guide RNA target selection Support Protocol 1: Genomic DNA extraction in-house protocol Basic Protocol 2: Construction of a binary plasmid vector Support Protocol 2: Agrobacterium transformation with a binary vector construct and stability check Support Protocol 3: Plant transformation Basic Protocol 3: Genotyping of edited events.},
}
@article {pmid40994338,
year = {2025},
author = {Zhang, T and Wang, Z and Song, Y and Wang, J and Guo, F and Zhang, Y and Lu, F and Li, M},
title = {[Establishment and optimization of a genetic manipulation system for Staphylococcus pasteuri].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {9},
pages = {3604-3616},
doi = {10.13345/j.cjb.250167},
pmid = {40994338},
issn = {1872-2075},
mesh = {*Staphylococcus/genetics/metabolism/drug effects ; *Gene Editing/methods ; Electroporation/methods ; Plasmids/genetics ; CRISPR-Cas Systems ; Genetic Engineering/methods ; },
abstract = {One of the technical bottlenecks limiting the high yield of 1,4-butanediamine is the insufficient tolerance of strains to 1,4-butanediamine. Enhancing the tolerance of strains to 1,4-butanediamine is therefore a primary challenge that needs to be addressed for the construction of strains with high yields of 1,4-butanediamine. Staphylococcus pasteuri 326180 exhibits exceptional tolerance to high-concentration 1,4-butanediamine, serving as both an ideal model for studying the mechanism underlying the 1,4-butanediamine tolerance and a novel host for constructing strains capable of efficiently producing 1,4-butanediamine. However, for both the research on the tolerance mechanism and the modification of chassis strains, gene editing of S. pasteuri needs to be carried out at the molecular level. The research objective of this paper is to establish a genetic manipulation system for S. pasteuri, laying foundation for subsequent studies on tolerance mechanism and the modification of chassis strains. This study systematically optimized the electroporation conditions, including key parameters such as the growth phase of cells, electric field strength, electroporation buffer, and recovery medium, successfully establishing an electroporation method for S. pasteuri. Additionally, we constructed the gene editing plasmid pCpfOA by replacing the resistance expression cassette, optimized the selection markers for gene editing, and finally established a CRISPR/Cpf1-based gene editing technology for S. pasteuri, achieving an editing efficiency of 90%. The genetic manipulation system of S. pasteuri established in this study provides technical support for research into the tolerance mechanism of this bacterium and the genetic modification of chassis strains.},
}
@article {pmid40994249,
year = {2025},
author = {Zaheer, U and Munir, F and Qiao, Q and Salum, YM and Abbas, AN and Tariq, M and Huang, S and Zheng, C and Yang, G and He, W},
title = {Functional Role of the PxGRHPR2 Gene in the Host Plant Adaptation of Diamondback Moth (Plutella xylostella).},
journal = {Archives of insect biochemistry and physiology},
volume = {120},
number = {1},
pages = {e70100},
doi = {10.1002/arch.70100},
pmid = {40994249},
issn = {1520-6327},
support = {//This study was supported by the National Natural Science Foundation of China (32472659 and 32172503), Natural Science Foundation of Fujian Province in China (2023J01069), Open Research Project of the Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests (MIMCP-202402), and Innovation Fund of FAFU (KFB23014A)./ ; },
mesh = {Animals ; *Moths/genetics/growth &amp; development/physiology/enzymology ; Larva/genetics/growth &amp; development/physiology ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Raphanus ; Adaptation, Physiological/genetics ; },
abstract = {The diamondback moth (Plutella xylostella), a major lepidopteran pest with a wide host range, presents persistent challenges to sustainable agriculture due to its high adaptability to cruciferous host plants. Although glyoxylate/hydroxypyruvate reductases (GRHPRs) have been well-characterized in plants and humans, their functional role in insects, particularly in host plant adaptation, remains largely unexplored. In this study, we characterized PxGRHPR2, a member of the GRHPR gene family, using a bioinformatics analysis, expression profiling, and CRISPR/Cas9-mediated gene knockout. RT-qPCR analysis showed that PxGRHPR2 was predominantly expressed in larval stage, with the highest transcript levels observed in the second instar and larval midgut tissues. Three homozygous PxGRHPR2 knockout strains were successfully generated using CRISPR/Cas9 system. Mutation of PxGRHPR2 led to significant reductions in larval weight, survival, and eclosion rates when larvae were fed on radish seedlings, whereas no such effects were observed under artificial diet conditions. These findings suggest that PxGRHPR2 plays a critical role in detoxification and metabolic regulation, thereby facilitating host plant adaptability in P. xylostella. Overall, this study provides new insights into insect-plant interactions and identifies PxGRHPR2 as a potential molecular target for developing sustainable pest management strategies.},
}
@article {pmid40994005,
year = {2025},
author = {Wang, S and Hu, Z},
title = {The marine diatom Phaeodactylum tricornutum as a versatile bioproduction chassis: Current progress, challenges and perspectives.},
journal = {Plant communications},
volume = {},
number = {},
pages = {101519},
doi = {10.1016/j.xplc.2025.101519},
pmid = {40994005},
issn = {2590-3462},
abstract = {Beyond its importance in diatom studies, the marine model diatom Phaeodactylum tricornutum has emerged as a versatile photosynthetic chassis for sustainable bioproduction, leveraging both native bioactive metabolites and engineered heterologous compounds through synthetic biology. Over the past three decades, transformative advances in genetic tool development, including transgenic element optimization, CRISPR/Cas genome editing and high-efficiency transformation systems, have driven strain engineering for elevated fucoxanthin, fatty acid, triacylglycerol yields and successful synthesis of diverse heterologous products, from terpenoids and therapeutic peptides to sustainable materials. Concurrently, advances in molecular toolkits have refined chassis optimization by elucidating fundamental biological mechanisms underlying nutrient uptake, environmental stress adaptation, stimuli sensing and cell development. Despite the progress, critical challenges persist, particularly suboptimal product yield, biomass limitations as well as a prohibitive production cost which hinder industrial translation. This review examines emerging strategies, such as chloroplastic gene expression, DNA site-specific integration and trophic alteration, promising for species improvement, while addressing other scale-up considerations including cultivation strategies, techno-economic analysis and regulatory policies. The integrative efforts could accelerate the transition of P. tricornutum from a model diatom to a scalable, eco-friendly biomanufacturing platform.},
}
@article {pmid40992924,
year = {2025},
author = {Warshauer, EM and Maier, PA and Runfeldt, G and Fuentes, I and Escamez, MJ and Valinotto, L and Natale, M and Manzur, G and Illera, N and Garcia, M and Del Rio, M and Mencia, A and Holguin, A and Larcher, F and Hellenthal, G and Brown, AR and Consuegra, L and Rivera, C and Nogueiro, I and Tang, J and Oro, A and Marinkovich, P and Palisson, F and Titeux, M and Hovnanian, AA and Sprecher, E and Skorecki, K and Norris, D and Bruckner, A and Kogut, I and Bilousova, G and Roop, D},
title = {Sephardic origins revealed for rare skin disorder, recessive dystrophic epidermolysis bullosa, in individuals carrying the unique c.6527insC mutation.},
journal = {Journal of medical genetics},
volume = {},
number = {},
pages = {},
doi = {10.1136/jmg-2025-110967},
pmid = {40992924},
issn = {1468-6244},
abstract = {BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and severe blistering skin disorder caused by loss-of-function mutations in the type VII collagen gene (COL7A1). The COL7A1 c.6527insC mutation is curiously prevalent among individuals with RDEB and is found worldwide in Europe and the Americas. Previous research has suggested the possibility of a Sephardic Jewish origin of the mutation; however, individuals with RDEB are not known to have predominant Jewish ancestry.<br><br>METHODS: In this study, a global cohort of individuals with RDEB with the c.6527insC founder mutation from Spain, France, Argentina, Chile, Colombia and the USA were investigated by autosomal genotyping, pairwise identical-by-descent matching and a local ancestry analysis. Age estimation analysis was performed to determine when Jewish founders introduced the c.6527insC mutation into Iberian and Native American populations (~900&#x2009;CE and 1492 CE, respectively).<br><br>RESULTS: Sephardic ancestry was identified at the haplotype spanning the c.6527insC mutation in 85% of the individuals, despite mixed ancestry elsewhere in the genome and no known recent Sephardic ancestry. Identical-by-descent matching between this RDEB subpopulation and a known crypto-Jewish community in Belmonte, Portugal was also ascertained, providing support for crypto-Jewish ancestry in this RDEB subpopulation.<br><br>CONCLUSION: The identification of this unique RDEB subpopulation unified by the single most prevalent c.6527insC mutation holds great potential to facilitate promising new RDEB therapies using CRISPR Cas 9 gene and base editing. The identification of a single guide RNA allowing efficient and safe editing of this variant would represent a unique drug to treat a large cohort of patients with the same founder mutation.},
}
@article {pmid40991439,
year = {2025},
author = {Yang, W and Wang, S and Ji, S and Wang, J and Lian, S and Li, Z and Jansen, RA and Wu, W and Niu, K and Sun, Z and Jia, Q and Zheng, J and Zhu, H and Deng, X and Wang, L and Fan, Z and Shi, Y and Lieftink, C and Guan, M and Beijersbergen, RL and Qin, W and Gao, Q and Bernards, R and Jin, H},
title = {CRISPR screens identify the ATPase VCP as a druggable therapeutic vulnerability in cholangiocarcinoma.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {39},
pages = {e2519568122},
doi = {10.1073/pnas.2519568122},
pmid = {40991439},
issn = {1091-6490},
support = {W2411079//MOST | National Natural Science Foundation of China (NSFC)/ ; 82222047//MOST | National Natural Science Foundation of China (NSFC)/ ; 82403226//MOST | National Natural Science Foundation of China (NSFC)/ ; 82303081//MOST | National Natural Science Foundation of China (NSFC)/ ; 22XD1423100//Science and Technology Commission of Shanghai Municipality (STCSM)/ ; 23YF1443500//Science and Technology Commission of Shanghai Municipality (STCSM)/ ; 2022XD057//Shanghai Municipal Health Commission ()/ ; 12539//KWF Kankerbestrijding (DCS)/ ; },
mesh = {Humans ; *Valosin Containing Protein/genetics/antagonists &amp; inhibitors/metabolism ; *Cholangiocarcinoma/drug therapy/genetics/pathology/metabolism ; Animals ; CRISPR-Cas Systems ; Mice ; *Bile Duct Neoplasms/drug therapy/genetics/pathology/metabolism ; Cellular Senescence/drug effects ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Xenograft Model Antitumor Assays ; Antineoplastic Agents/pharmacology ; },
abstract = {Cholangiocarcinoma (CCA) remains a lethal malignancy with limited therapeutic options. Through genome-wide CRISPR-Cas9 screening, we identified the adenosine triphosphatase (ATPase) valosin-containing protein (VCP) as a critical dependency in CCA. Compound screens revealed that the VCP inhibitor CB-5339 potently suppresses CCA proliferation in a panel of patient-derived organoids by inducing cellular senescence. It is known that senescent cells persist, and this can contribute to therapy resistance. To address this, we combined CB-5339 with senolytic agents (ABT-263 and conatumumab), which selectively eliminate senescent CCA cells, resulting in enhanced tumor suppression both in vitro and in vivo. Clinical analysis showed that VCP overexpression in CCA patients correlates with poor prognosis. Our study unveils a "one-two punch" strategy, targeting VCP-mediated senescence followed by senolytic clearance, offering a promising therapeutic approach for CCA.},
}
@article {pmid40991036,
year = {2025},
author = {Nikam, T and Rana, A and Saraf, SA and Awasthi, S},
title = {Micro- and nanoscale biosensing technologies for early diagnosis of Parkinson's disease.},
journal = {Mikrochimica acta},
volume = {192},
number = {10},
pages = {691},
pmid = {40991036},
issn = {1436-5073},
support = {BT/RLF/Re-Entry/40/2021//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {*Parkinson Disease/diagnosis ; Humans ; *Biosensing Techniques/methods ; Early Diagnosis ; Biomarkers/analysis ; Electrochemical Techniques ; },
abstract = {Parkinson's disease (PD), the second most prevalent neurodegenerative disorder, remains challenging to diagnose at its earliest stages due to the absence of definitive biomarkers and overlapping clinical features with other synucleinopathies, thereby delaying therapeutic intervention and effective disease management. This review provides an integrative evaluation of established and emerging approaches for detecting PD-specific biomarkers in biofluids and tissues with high sensitivity and specificity. Conventional assays such as seed amplification techniques, proximity ligation and extension methods, bead-based microarrays, and immunoassays including ELISA, electrochemiluminescence, and SIMOA are examined alongside their performance metrics and inherent limitations. We then highlight next-generation micro- and nanoscale biosensing platforms, including nanopore-based resistive pulse sensing, surface plasmon resonance (SPR), surface-enhanced Raman spectroscopy (SERS), field-effect transistors (FETs), electrochemical sensors, and lateral flow assays (LFAs), which are capable of ultrasensitive detection at nano- to attomolar concentrations. Particular emphasis is given to nucleic acid-based technologies such as aptasensors, genosensors, and CRISPR/Cas systems for their exceptional molecular recognition, programmable signal outputs, and portability. The potential of artificial intelligence and machine learning tools (e.g., SVM, RF, DNN) to improve biomarker interpretation, enable multiplexed analysis, and facilitate real-time monitoring is also discussed. Finally, we outline key translational challenges, including assay standardization, clinical validation, scalability, integration into wearable and point-of-care devices, and regulatory hurdles towards the development of robust, clinically deployable diagnostic platforms for early PD detection and monitoring.},
}
@article {pmid40988386,
year = {2025},
author = {Zuo, N and Zuo, F and Liu, Y and Xiang, B},
title = {Genome Editing Using the Endogenous Type I-E CRISPR-Cas System in Lactobacillus paracasei ATCC334.},
journal = {Biotechnology and applied biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1002/bab.70056},
pmid = {40988386},
issn = {1470-8744},
abstract = {Lactobacillus paracasei ATCC334 is a well-known beneficial strain that plays a crucial role in food industry and promotion of human health. However, despite its significance, our understanding of its gene functions remains limited due to obstacles in gene editing techniques. This gap hinders the full utilization and development of this beneficial bacterium. In this study, we targeted L. paracasei ATCC334 as editing chassis. Initially, bioinformatics tools were used to explore a type I-E endogenous clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system within L. paracasei ATCC334. We further analyzed its repeat sequences, spacer sequences, and leader sequence predicted the protospacer adjacent motif (PAM) recognized by this system. To validate our findings, we assessed the accuracy of potential PAM, evaluated the cutting activity of the endogenous CRISPR-Cas system, and studied the impact of the artificial mini-CRISPR array through plasmid interference and genome interference experiments. These results helped us to achieve successful gene knockout and gene integration. Finally, we engineered a strain capable of nicotine degradation. Our study provides valuable insights for the broader development and application of lactobacilli.},
}
@article {pmid40988083,
year = {2025},
author = {Zhu, R and Ren, C and Bao, Z},
title = {Fueling chromosomal gene diversification and artificial evolution with CRISPR.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {297},
pmid = {40988083},
issn = {1474-760X},
support = {2023YFF1204500//National Key Research and Development Program of China/ ; 22308316//National Natural Science Foundation of China/ ; 226-2022-00214//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*CRISPR-Cas Systems ; *Directed Molecular Evolution/methods ; *Gene Editing/methods ; *Chromosomes/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; *Genetic Variation ; Animals ; Evolution, Molecular ; },
abstract = {Gene diversification is an effective approach to massively dissecting variant functions and evolving sequences when paired with an appropriate assay. In vitro mutagenesis and ectopic gene expression, however, fail to simulate the endogenous regulatory environment of the variants. The development of clustered, regularly interspaced short palindromic repeats (CRISPR) systems has greatly boosted the efficiency of targeted gene diversification in various species. Here, we review recent CRISPR-assisted methods for chromosomal gene diversification and artificial evolution, focusing on the advantages and limitations of each approach, and propose possible strategies to overcome current limitations and directions in future technology development.},
}
@article {pmid40986369,
year = {2025},
author = {Ratna, TA and Sharon, BM and Barros Velin, CA and Palmer, K},
title = {Factors affecting CRISPR-Cas defense against antibiotic resistance plasmids harboured by Enterococcus faecalis laboratory model strains and clinical isolates.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {9},
pages = {},
doi = {10.1099/mic.0.001601},
pmid = {40986369},
issn = {1465-2080},
mesh = {*Enterococcus faecalis/genetics/drug effects/isolation &amp; purification ; *Plasmids/genetics ; *CRISPR-Cas Systems/genetics ; Gene Transfer, Horizontal ; Conjugation, Genetic ; Humans ; Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Multiple, Bacterial/genetics ; Gram-Positive Bacterial Infections/microbiology ; },
abstract = {Enterococcus faecalis is a Gram-positive bacterium and opportunistic pathogen that acquires resistance to a wide range of antibiotics by horizontal gene transfer (HGT). The rapid increase of multidrug-resistant (MDR) bacteria including MDR E. faecalis necessitates the development of alternative therapies and a deeper understanding of the factors that impact HGT. CRISPR-Cas systems provide sequence-specific defense against HGT. From previous studies, we know that E. faecalis CRISPR-Cas provides sequence-specific anti-plasmid defense during agar plate biofilm mating and in the murine intestine. Those studies were mainly conducted using laboratory model strains with a single, CRISPR-targeted plasmid in the donor. MDR E. faecalis typically possess multiple plasmids that are diverse in sequence and may interact with each other to impact plasmid transfer and CRISPR-Cas efficacy. Here, we altered multiple parameters of our standard in vitro conjugation assays to assess CRISPR-Cas efficacy, including the number and genotype of plasmids in the donor, and laboratory model strains as donor versus recent human isolates as donor during conjugation. We found that the plasmids pTEF2 and pCF10, which are not targeted by CRISPR-Cas in our recipient, enhance the conjugative transfer of the CRISPR-targeted plasmid pTEF1 into both WT and CRISPR-Cas-deficient (via deletion of cas9) recipient cells. However, the effect of pTEF2 on pTEF1 transfer is much more pronounced, with a striking 6-log increase in pTEF1 conjugation frequency when pTEF2 is also present in the donor and recipients are deficient for CRISPR-Cas (compared with 4-log for pCF10). Overall, this study provides insight about the interplay between plasmids and CRISPR-Cas defence, opening avenues for developing novel therapeutic strategies to curb HGT among bacterial pathogens and highlighting pTEF2 as a plasmid for additional mechanistic study.},
}
@article {pmid40986360,
year = {2025},
author = {Ren, J and Duan, Y and Li, R and Zhang, X and Shi, Y and Zhou, S and Xie, K and Wu, X and Irish, VF and Deng, X and Zhang, F and Guo, W},
title = {Transcriptional regulation of thorn tip sclerification in plants.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {39},
pages = {e2510775122},
doi = {10.1073/pnas.2510775122},
pmid = {40986360},
issn = {1091-6490},
support = {2022YFF1003100//MOST | National Key Research and Development Program of China (NKPs)/ ; 2024YFD1200501//MOST | National Key Research and Development Program of China (NKPs)/ ; 32425048//MOST | NSFC | National Science Fund for Distinguished Young Scholars (National Science Foundation for Distinguished Young Scholars)/ ; HBZY2023B00501//Department of Agriculture &amp; Rural Affairs of Hubei Province/ ; CARS-26//MOA | Earmarked Fund for China Agriculture Research System/ ; 2306142//National Science Foundation (NSF)/ ; },
mesh = {*Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; *Citrus/genetics/growth &amp; development/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Transcription Factors/genetics/metabolism ; Cell Wall/metabolism/genetics ; Plant Stems/genetics/growth &amp; development ; },
abstract = {A common characteristic of thorns, prickles, and spines is a hardened and sharp tip. This feature complicates cultivation and postharvest processing for many crops, yet the molecular mechanisms governing this specific sclerification process remain unclear. By genome editing screening, we identified a MYB family gene named SHORT and SOFT THORN 1 (SST1) that specifically promotes the development of sclerenchyma cells at Citrus thorn tips. CRISPR-Cas9 editing of SST1 results in the formation of short and soft thorns with significantly reduced secondary wall thickening at the tips, while leaving stem growth unaffected. SST1 directly activates NST1 and SND1, driving a hierarchical transcriptional regulatory network for secondary wall biosynthesis. Moreover, SST1 homologs in various species are specifically expressed in thorns, suggesting a potential conserved role. Our results uncover the molecular basis for hard plant protective structures, and modulation of this pathway could be an effective strategy for optimizing cultivation practices.},
}
@article {pmid40986203,
year = {2025},
author = {Sood, A and Sharma, P and Sharma, A and Chaudhary, DR and Manisha, },
title = {Genome editing in vegetable crops: a new era of sustainable agriculture.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {944},
pmid = {40986203},
issn = {1573-4978},
}
@article {pmid40985903,
year = {2025},
author = {Palma-Cobo, M and Toribio, V and Morales, J and López-Martín, S and Enrich, C and Lu, A and Yáñez-Mó, M},
title = {Genome-Wide CRISPR/Cas9 Screening Identifies the COMMANDER Recycling Complex as a Key Player in EV Uptake.},
journal = {Journal of extracellular vesicles},
volume = {14},
number = {9},
pages = {e70166},
pmid = {40985903},
issn = {2001-3078},
support = {//Ministerio Espa&#xf1;ol de Ciencia e Innovaci&#xf3;n/ ; //Universidad Aut&#xf3;noma de Madrid/ ; PIF2023/SAL-GL-29726//FPI-UAM/ ; //GEIVEX/ ; //Comunidad de Madrid/ ; //PIPF-2023/SAL-GL- 29726/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; K562 Cells ; *Extracellular Vesicles/metabolism/genetics ; HeLa Cells ; Cell Line, Tumor ; Genome, Human ; Melanoma/metabolism ; },
abstract = {Extracellular vesicles (EVs) hold immense potential in therapeutic delivery, warranting a comprehensive investigation of the mechanisms that regulate their uptake by target cells. To identify key molecular regulators of EV internalization, we conducted a genome-wide CRISPR (GWC) screen aimed to pinpoint candidate genes that influence EV uptake. We employed a GWC library spanning the entire human genome in K562 cells. 3.6 × 10[12] EVs isolated from the SKMEL147 human melanoma cell line were labelled with Alexa633-C5-Maleimide and incubated for 2 h with 500 × 10[6] K562 cells, providing a 2000× coverage of the library. The top 5% of high and low fluorescence populations were sorted. Next-generation sequencing (NGS) was performed to quantify sgRNA enrichment in the sorted populations compared to the unsorted control. Remarkably, among other genes, several members of the COMMANDER complex emerged as significant hits in our screen. We validated the hits in knockout (KO) cell lines of both K562 and HeLa cells using EVs derived either from melanoma or breast cancer cell lines. Kinetic follow-up of EV cargo, including surface or luminal proteins, suggests that the COMMANDER complex plays a pivotal role in the early stages of EV uptake but also in the final fate of EV components in the target cell.},
}
@article {pmid40938694,
year = {2025},
author = {Wei, Z and Zhang, L and Wang, Y and Xu, X and Cao, L and Lin, H and Sui, J and Wang, K and Wang, X},
title = {Development of a Label-Free Colorimetric and Fluorescent Diagnostic Platform for Foodborne Salmonella Based on RPA-CRISPR/Cas12 Assay in a Single Tube.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {38},
pages = {24447-24456},
doi = {10.1021/acs.jafc.5c06045},
pmid = {40938694},
issn = {1520-5118},
mesh = {*Salmonella/genetics/isolation &amp; purification ; *Colorimetry/methods/instrumentation ; CRISPR-Cas Systems ; Food Contamination/analysis ; Milk/microbiology ; Animals ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; Fishes/microbiology ; Bacterial Proteins/genetics/metabolism ; Food Microbiology ; *Endodeoxyribonucleases/genetics/metabolism ; Recombinases/genetics/metabolism/chemistry ; Fluorescence ; CRISPR-Associated Proteins ; },
abstract = {Foodborne pathogen Salmonella poses a significant threat to public health, and therefore, it is important to establish accurate, convenient, and rapid detection methods. Herein, a label-free colorimetric and fluorescent diagnostic platform for foodborne Salmonella was developed, integrating recombinase polymerase amplification (RPA), CRISPR/Cas12, and water-soluble cationic conjugated polythiophene (PMNT) in a single-tube system. Upon recognition of Salmonella-specific gene invA targets, RPA products can stimulate the cis- and trans-cleavage activity of Cas12a in the presence of crRNA. This enzymatic activity degrades single-stranded DNA (ssDNA), leading to the release of PMNT from PMNT-ssDNA complexes, which, in turn, produces a detectable fluorescence increase along with a visible color transition from pink to yellow. The one-tube strategy enables sensitive detection of 1.9 × 10[1] copies/μL invA target and could detect as low as 10[3] CFU/mL of Salmonella in artificially spiked milk and fish samples without enrichment, while the detection limit improved to 10[0] CFU/mL after 8 h enrichment. Importantly, the assay demonstrated high specificity with no cross-reactivity with other bacteria with and without complex food matrices. This one-tube, dual-signal assay provides a rapid, reliable, and equipment-minimal solution for on-site detection of Salmonella, with a reduced risk of aerosol contamination.},
}
@article {pmid40985777,
year = {2025},
author = {Nguyen, GT and Raju, A and Schelling, MA and Sashital, DG},
title = {Rapid CRISPR-Cas9 target-strand nicking can provide phage resistance by reducing DNA abundance.},
journal = {Nucleic acids research},
volume = {53},
number = {18},
pages = {},
doi = {10.1093/nar/gkaf900},
pmid = {40985777},
issn = {1362-4962},
support = {1652661//National Science Foundation/ ; GM140876/NH/NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; Magnesium/metabolism ; *Bacteriophages/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; *DNA, Viral/metabolism/genetics ; DNA Breaks, Double-Stranded ; },
abstract = {Cas9 is an RNA-guided immune endonuclease that provides bacterial defense against bacteriophages. Cas9 relies on divalent metal ions for cleavage catalysis by two domains, HNH and RuvC, and to facilitate conformational changes that are required for cleavage activation. While Cas9 typically produces double-strand breaks (DSBs) in DNA targets, we observed that reduced, physiologically relevant Mg2+ concentrations can result in a slow rate of non-target strand cleavage by RuvC. This raised the question of whether rapid target-strand nicking by the Cas9 HNH domain is sufficient to provide protection against phage. To address this, we tested phage protection by Cas9 nickases, in which only the HNH or RuvC domain is catalytically active. We find that nicking by HNH, but not RuvC, can be sufficient to provide immunity. Target-strand nicking prevents phage DNA accumulation and can reduce the susceptibility of Cas9 to viral escape. Cleavage by RuvC is strongly impaired in the presence of other biomolecules that can compete for binding of free Mg2+, preventing formation of a DSB. Overall, our results suggest that HNH cleavage may occur more rapidly than RuvC cleavage under physiological conditions, resulting in an initial target-strand nick that may be sufficient to provide CRISPR-mediated immunity.},
}
@article {pmid40985776,
year = {2025},
author = {Park, SG and Park, JU and Dodero-Rojas, E and Bryant, JA and Sankaranarayanan, G and Kellogg, EH},
title = {Comprehensive profiling of activity and specificity of RNA-guided transposons reveals opportunities to engineer improved variants.},
journal = {Nucleic acids research},
volume = {53},
number = {18},
pages = {},
doi = {10.1093/nar/gkaf917},
pmid = {40985776},
issn = {1362-4962},
support = {//NIH/ ; 5R01GM144566-02/GM/NIGMS NIH HHS/United States ; //Cystic Fibrosis Foundation/ ; //Jane Coffin Childs Memorial Fund/ ; //Korea Health Industry Development Institute/ ; HI19C1095//Ministry of Health and Welfare/ ; //Hartwell Center for Bioinformatics &amp; Biotechnology/ ; //ALSAC/ ; P30 CA021765/CA/NCI NIH HHS/United States ; },
mesh = {*DNA Transposable Elements/genetics ; CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; Mutation ; Gene Editing/methods ; },
abstract = {Recently discovered CRISPR-associated transposons (CASTs) are natural RNA-guided DNA transposition systems capable of single-step genomic integration of large DNA cargo. Wild-type CASTs exhibit low integration activity in heterologous systems; therefore, engineering efforts are required to develop therapeutically relevant tools. Here we developed a high-throughput dual genetic screen capable of accurately quantifying the relative activity and specificity of a large pool of CAST variants. Under the conditions of our screen, we discovered that the wild-type V-K CAST system can consistently achieve between 88% and 95% on-site targeting specificity. We used site-saturation mutagenesis of the conserved core transposition machinery (TnsB, TnsC, and TniQ) to reveal novel mechanistic insights into the function of these transposon proteins. Furthermore, we found that different components have varying trade-offs between activity and specificity, a critical aspect overlooked in conventional screening pipelines. These findings provide clear engineering principles for further optimization of CASTs. Finally, we identified several mutations that, together, enhance CAST activity up to four-fold while minimally impacting targeting specificity. These methods are a powerful tool to characterize the sequence-function landscape across multiple functional parameters while also providing a robust platform for developing enhanced genome-editing tools.},
}
@article {pmid40985758,
year = {2025},
author = {Kippnich, J and Benz, F and Uecker, H and Baumdicker, F},
title = {Effectiveness of CRISPR-Cas in Sensitizing Bacterial Populations with Plasmid-Encoded Antimicrobial Resistance.},
journal = {Genetics},
volume = {},
number = {},
pages = {},
doi = {10.1093/genetics/iyaf192},
pmid = {40985758},
issn = {1943-2631},
abstract = {The spread of bacteria resistant to antibiotics poses a serious threat to human health. Genes that encode antibiotic resistance are often harbored on plasmids, extra-chromosomal DNA molecules found in bacteria. The emergence of multiresistance plasmids is particularly problematic and demands the development of new antibiotics and alternative strategies. CRISPR-Cas derived tools with their sequence specificity offer a promising new approach to combating antibiotic resistance. By introducing CRISPR-Cas encoding plasmids that %specifically target antibiotic resistance genes on plasmids, the susceptibility of bacteria to conventional antibiotics can be restored. However, genetic variation within bacterial populations can hinder the effectiveness of such CRISPR-Cas tools by allowing some mutant plasmids to evade CRISPR-mediated cleaving or gene silencing. In this study, we develop a model to test the effectiveness of CRISPR-Cas in sensitizing bacterial populations carrying resistance on non-transmissible plasmids and assess the success probability of a subsequent treatment with conventional antibiotics. We evaluate this probability according to the target interference mechanism, the copy number of the resistance-encoding plasmid, and its compatibility with the CRISPR-Cas encoding plasmid. Our results identify promising approaches to revert antibiotic resistance with CRISPR-Cas encoding plasmids: A DNA-cleaving CRISPR-Cas system on a plasmid incompatible with the targeted plasmid is most effective for low copy numbers, while for resistance plasmids with higher copy numbers gene silencing by CRISPR-Cas systems encoded on compatible plasmids is the superior solution.},
}
@article {pmid40983443,
year = {2025},
author = {Belizário, JE and Occhiucci, JM and Garay-Malpartida, M and Cunha da Silva, JR},
title = {Phageptosis: A bacterial cell death program induced by crispr-cas systems.},
journal = {Progress in molecular biology and translational science},
volume = {217},
number = {},
pages = {211-231},
doi = {10.1016/bs.pmbts.2025.07.003},
pmid = {40983443},
issn = {1878-0814},
mesh = {*CRISPR-Cas Systems/genetics ; *Bacteriophages/physiology ; *Bacteria/virology/cytology/genetics ; Cell Death ; Apoptosis ; Humans ; },
abstract = {Programmed cell death (PCD) is a fundamental mechanism that has evolved across both unicellular and multicellular organisms for species preservation and self-protection. In certain contexts, genetically regulated cell death can enable surviving cells to thrive, safeguarding the genotype from extinction. Recent research on bacteria and archaea has revealed an ancient defense mechanism involving CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR-associated) proteins. These systems identify and eliminate invading genetic elements, such as bacteriophages, transposons, and plasmids, using sequence-specific RNA-guided targeting. A protease complex called Craspase, activated by non-self RNA, regulates Cas nuclease activity, facilitating this primitive form of immunity. Interestingly, this pathway shows structural and mechanistic similarities to apoptosis, the first recognized form of programmed mammalian cell death, characterized by chromatin condensation, nuclear fragmentation, and membrane blebbing. Other regulated cell death pathways, including necroptosis and pyroptosis, also share overlapping features. Comparative genomic studies reveal a conserved molecular framework underpinning these diverse death pathways across life forms. In this article, we explore the emerging parallels and distinctions between apoptosis and CRISPR-Cas-mediated cell death, a process we refer to as "phageptosis," highlighting evolutionary links and their implications for understanding cell death mechanisms.},
}
@article {pmid40983435,
year = {2025},
author = {Zhang, H and Bai, X and Wang, C and Pang, W and Zhang, J and Song, J and Zhou, H and Gao, Z and Zheng, B},
title = {Bimetallic-satellite enhanced SERS reporter integrated with CRISPR-Cas12a for ultrasensitive biological rhythm monitoring.},
journal = {Analytica chimica acta},
volume = {1374},
number = {},
pages = {344544},
doi = {10.1016/j.aca.2025.344544},
pmid = {40983435},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; *Spectrum Analysis, Raman/methods ; Gold/chemistry ; *Biosensing Techniques/methods ; *Metal Nanoparticles/chemistry ; Silver/chemistry ; Humans ; RNA, Messenger/analysis/genetics ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Biorhythmic played an important role in physical health. Biorhythmic disorders led to various health issue, including cardiovascular diseases and metabolic disorders. Early diagnosis of biorhythmic disorders was crucial in slowing the progression of disease. Therefore, it was important to develop technologies for detecting biorhythmic and their disorder markers. This study selected HMGB1(High mobility group box 1), BMAL1 (Brain and Muscle Aryl hydrocarbon receptor nuclear translocator-like 1), and MICU (mitochondrial calcium uptake 1) mRNA for detection.<br><br>RESULTS: CRISPR/Cas12a/SERS (surface-enhanced Raman spectroscopy) ultrasensitive biosensing platform was developed for the detection of biorhythmic markers. The biosensor was partly an ultrabright Raman signal reporter (on) of a novel satellite structure formed by gold/silver bimetallic nanoparticles. This novel satellite structure utilizes the potential difference between gold and silver nanoparticles to promote the transfer of electrons from the core structure to the AuNPs and MBA (4-mercaptobenzoic acid) molecules, while enhancing the electromagnetic field distribution. Compared with the Raman signal of the satellite structure composed of Au/Au bimetallic, the Raman signal of the satellite structure in this study is increased by about 10 times. CRISPR/Cas12a recognized the target molecule and activated trans-cleavage activity, cleaving the DNA/RNA hairpin structure and releasing RNA2&#x2217;. RNA2&#x2217; caused the satellite structure to disperse, significantly attenuating the ultra-strong Raman signal (off), thereby enabling quantitative detection of the target nucleic acid. The LOD for three targets were of less than 1&#xa0;fM.<br><br>SIGNIFICANCE: In summary, the CRISPR/Cas12a/SERS biosensing platform enables mRNA detection without preamplification. This biosensing platform can detect target molecules at the fM level, achieving ultra-sensitive detection. In addition, the satellite structure exhibits long-term stability, providing a theoretical basis for the practical application of this biosensing platform.},
}
@article {pmid40981773,
year = {2025},
author = {Eloiflin, R and Pérez-Antón, E and Camara, A and Dujeancourt-Henry, A and Boiro, S and Djetchi, MN and Traoré, MB and Koffi, M and Kaba, D and Le Pennec, Y and Doukouré, B and Camara, AD and Kagbadouno, M and Campagne, P and Camara, M and Jamonneau, V and Thévenon, S and Bart, JM and Glover, L and Rotureau, B},
title = {A SHERLOCK toolbox for eco-epidemiological surveillance of African trypanosomes in domestic pigs from Western Africa.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {40981773},
issn = {2050-084X},
support = {ANR-21-CE17-0022-01 SherPa//Agence Nationale de la Recherche/ ; ANR-10-LABX-62-IBEID//Agence Nationale de la Recherche/ ; ANR-11-LABX-0024-PARAFRAP//Agence Nationale de la Recherche/ ; Bourse Calmette-Yersin//Institut Pasteur/ ; },
mesh = {Animals ; *Trypanosomiasis, African/epidemiology/veterinary/parasitology/diagnosis ; *Trypanosoma/isolation &amp; purification/genetics/classification ; Swine ; *Swine Diseases/epidemiology/parasitology/diagnosis ; *Epidemiological Monitoring/veterinary ; Africa, Western/epidemiology ; CRISPR-Cas Systems ; Cote d'Ivoire/epidemiology ; },
abstract = {Animal African trypanosomosis (AAT), caused by protist parasites of the genus Trypanosoma, puts upward of a million head of livestock at risk across 37 countries in Africa. The economic impact of AAT and the presence of human-infectious trypanosomes in animals place a clear importance on improving diagnostics for animal trypanosomes to map the distribution of the veterinary parasites and identify reservoirs of human-infectious trypanosomes. We have adapted the CRISPR-based detection toolkit SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for trypanosomatid parasites responsible for AAT (SHERLOCK4AAT) including Pan-trypanosomatid, Trypanozoon, T. vivax, T. congolense, T. theileri, T. simiae, and T. suis assays. To test the applicability of this technique in the field, we analysed dried blood spots collected from 200 farm and 224 free-ranging pigs in endemic and historical human African trypanosomiasis foci in Guinea and Côte d'Ivoire, respectively. The results revealed that SHERLOCK4AAT can detect and discriminate between trypanosome species involved in multiple infections with a high sensitivity. 62.7% [58.1, 67.3] of pigs were found infected with at least one trypanosome species. T. brucei gambiense, a human-infectious trypanosome, was found in one animal at both sites, highlighting the risk that these animals may act as persistent reservoirs. These data suggest that, due to their proximity to humans and their attractiveness to tsetse flies, pigs could act as sentinels to monitor T. b. gambiense circulation using the SHERLOCK4AAT toolbox.},
}
@article {pmid40980906,
year = {2025},
author = {Croteau, FR and Tran, J and Hynes, AP},
title = {CRISPR adaptation in Streptococcus thermophilus benefits from phage environmental DNA.},
journal = {mSphere},
volume = {},
number = {},
pages = {e0045325},
doi = {10.1128/msphere.00453-25},
pmid = {40980906},
issn = {2379-5042},
abstract = {The CRISPR-Cas system is a bacterial adaptive immune system that protects against infection by phages: viruses that infect bacteria. To develop immunity, bacteria integrate spacers-fragments of the invading nucleic acids-into their CRISPR array to serve as the basis for sequence-targeted DNA cleavage. However, upon infection, a phage quickly takes over the metabolism of the bacterium, leaving little time for the bacterium to acquire new spacers, transcribe them, and use them to cut the invading DNA. To develop CRISPR immunity, bacteria must be safely exposed to phage DNA. Phage infection releases environmental DNA (eDNA) which could be involved in the development of CRISPR immunity. Using Streptococcus thermophilus and phages 2972 and 858 as a model for CRISPR immunity, we show that eDNA is involved in CRISPR immunity, as generation of phage-immune bacterial colonies decreases with eDNA digestion. Furthermore, it is phage eDNA specifically that impacts CRISPR immunity since only its addition increases the generation of phage-immune colonies. We also show that the effect of eDNA is phage-specific, sequence-specific, and can even be traced to a region of the genome covering the early-expressed genes, which differ between phages 2972 and 858. However, we also show that eDNA is not used as a source of genetic information for spacer acquisition. These results link eDNA to the CRISPR-Cas system, providing a better understanding of the context of the emergence of CRISPR immunity and could inform our understanding of the mechanisms through which bacteria detect phage infection.IMPORTANCEHow can a bacterial adaptive immune system (the CRISPR-Cas system) exist at all, when exposure to a virulent phage is so consistently lethal? We proposed that bacteria might actively sample their genetic environment for phage DNA through natural competence. In testing this hypothesis, we revealed that free phage DNA is important to CRISPR immunity-but not as the source of the immunological memory.},
}
@article {pmid40977958,
year = {2025},
author = {Alsallameh, SMS and Salman, HA and Al-Khafaji, K and Kuzukiran, O},
title = {Comprehensive Genomic Characterization of a Drug-Resistant Klebsiella pneumoniae Clinical Isolate in Iraq Using Whole Genome Sequencing.},
journal = {Iranian journal of medical sciences},
volume = {50},
number = {9},
pages = {638-648},
pmid = {40977958},
issn = {1735-3688},
mesh = {*Klebsiella pneumoniae/genetics/pathogenicity/isolation &amp; purification/drug effects ; Whole Genome Sequencing/methods ; Humans ; Iraq/epidemiology ; *Klebsiella Infections/microbiology/drug therapy/epidemiology ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Drug Resistance, Multiple, Bacterial/genetics ; Genome, Bacterial ; Genomics/methods ; Microbial Sensitivity Tests ; },
abstract = {BACKGROUND: Klebsiella pneumoniae is a Gram-negative encapsulated opportunistic pathogen, which presents a major threat to public health due to its ability for multi-antibiotic drug resistance. It is responsible for 30% of Gram-negative bacterial infections, including nosocomial infections, pneumonia, septicemia, and urinary tract infections. The study aimed to analyze the key phenotypic and genetic features of clinical K. pneumoniae isolates.<br><br>METHODS: Between 2022 and 2023, a total of 91 strains of Klebsiella pneumoniae were collected from Al-Imamian Al-Kadhimiyain Medical City (IKMC) and characterized using the VITEK-2 technique. Whole-genome sequencing (WGS) was employed to characterize the extreme drug-resistant strain. The whole genome was extracted and sequenced using the Next Generation Sequencing (NGS) technique. The genome of our bacterial isolate was analyzed using different bioinformatics tools such as Galaxy workflow, SPAdes, PROKKA, and Staramr.<br><br>RESULTS: The analysis identified Klebsiella pneumoniae serotype K36:O2a and sequencing type ST-437, containing 15 different plasmids carrying 54 resistance genes and more than 100 virulence genes with one region of CRISPR and no Cas. The sample obtained four intact bacteriophages and two questionable ones. Seven insertion sequences were revealed in the analysis as part of Other Mobile Genetic Elements (OMG). Additionally, the 16SrRNA phylogenetic tree identified a higher relationship of the bacteria to the strains from the USA and India than from Iraq.<br><br>CONCLUSION: It is the first study in Iraq to utilize WGS to comprehensively characterize an opportunistic pathogen. The study emphasizes the need for WGS to track the development of resistance and virulence patterns in clinical strains of K. pneumoniae.},
}
@article {pmid40977706,
year = {2025},
author = {Wang, JW and Feng, Q and Liu, JH and Xun, JJ},
title = {Opportunities, challenges, and future perspectives of oncolytic virus therapy for malignant melanoma.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1653683},
pmid = {40977706},
issn = {1664-3224},
mesh = {Humans ; *Oncolytic Virotherapy/methods/adverse effects ; *Melanoma/therapy/immunology ; *Oncolytic Viruses/immunology ; Animals ; Tumor Microenvironment/immunology ; Immunotherapy/methods ; Dendritic Cells/immunology ; *Skin Neoplasms/therapy/immunology ; },
abstract = {Malignant melanoma is characterized by high heterogeneity, aggressive metastatic potential, and a profoundly immunosuppressive "cold" tumor microenvironment, contributing to broad therapeutic resistance and suboptimal responses to immunotherapy. Conventional PD-1 inhibitors yield an ORR of only 38%. As an emerging class of immunotherapeutic agents, oncolytic viruses (OV) induce ICD, promoting the release of DAMPs and activating innate immune pathways such as cGAS-STING, thereby transforming "cold" tumors into "hot" phenotypes and eliciting robust anti-tumor responses. Mechanistically, OV therapy increases the proportion of CD103[+] dendritic cells (DCs) in lymph nodes from 5% to 25% and enhances DC-tumor synapse formation by 300%, facilitating efficient cross-presentation of tumor antigens and T-cell priming. Clinically, T-VEC combined with pembrolizumab achieves a 48.6% ORR with grade ≥3 AEs occurring in <20% of patients-superior to either monotherapy or conventional chemoradiotherapy. Nonetheless, OV therapy faces challenges including tumor heterogeneity, core mechanistic limitations, viral shedding risks, and regulatory hurdles. Over the next 5-10 years, single-cell RNA sequencing is expected to unravel molecular heterogeneity in melanoma, while CRISPR/Cas systems may enable the design of tailored OV to overcome resistance. Additional strategies such as serotype switching, JAK/STAT inhibition, and arming OV with hyaluronidase or STING agonists are under investigation to overcome immune and stromal barriers. Integration of artificial intelligence with biomarkers-such as neutralizing antibody titers, ISG expression, and STING methylation-may further enable personalized OV-based therapies. This review discusses OV therapy's mechanisms, clinical impact, and future prospects in melanoma treatment.},
}
@article {pmid40947068,
year = {2025},
author = {Das, LJ and Venkatesan, G and Krishnaswamy, N and Shekhawat, I and Vijayapillai, U and Priyanka, M and Dechamma, HJ},
title = {Effect of CRISPR-Cas9 mediated knockout of IRF3 gene in BHK-21 cells on immune gene expression and foot-and-mouth disease virus replication.},
journal = {Microbial pathogenesis},
volume = {208},
number = {},
pages = {108022},
doi = {10.1016/j.micpath.2025.108022},
pmid = {40947068},
issn = {1096-1208},
mesh = {Animals ; *Foot-and-Mouth Disease Virus/physiology/immunology ; *Interferon Regulatory Factor-3/genetics ; *CRISPR-Cas Systems ; *Virus Replication/genetics ; Cell Line ; *Gene Knockout Techniques ; Foot-and-Mouth Disease/immunology/virology ; Cricetinae ; },
abstract = {Foot-and-mouth disease (FMD) is an acute highly contagious viral disease of cloven-hoofed animals. Currently, FMD vaccine production and research mainly depend on the BHK-21 cell line. BHK-21 is highly sensitive to FMD virus, however, still a lot of room for improvement that can result in higher antigen yield in vaccine production facilities. IRF3 (Interferon regulatory factor 3) is a key transcription regulatory factor involved in the interferon (IFN) pathway, the immediate antiviral response of the cells. In this study, IRF3 knock-out (KO) BHK-21 cells were established using the CRISPR-Cas9 method. The KO cell line was stable in growth and morphological characteristics, unveiled in growth curve analysis. After infection with FMDV, the viral copy number, virus titer, and plaque forming units (PFU) were significantly increased in KO cells than those in parental BHK-21 (NC) cells. The relative gene expression of type I IFNs and ISGs such as IRF3, IRF7, viperin, Mx1 and ISG15 upon FMDV infection was severely reduced in KO cells. Results preliminarily reveal the role of IRF3 in cellular antiviral immune response, and the IRF3-KO cell line could also serve as a useful tool for FMDV research and vaccine production.},
}
@article {pmid40944915,
year = {2025},
author = {Daliri, K and Clement, K},
title = {Protocol for permanent gene repression by CRISPR-adenine base editing of promoter CCAAT motifs.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {104075},
doi = {10.1016/j.xpro.2025.104075},
pmid = {40944915},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; *Promoter Regions, Genetic/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mice ; Humans ; NIH 3T3 Cells ; Adenine ; Transfection ; },
abstract = {Here, we present a protocol to achieve permanent downregulation of gene expression by editing the CCAAT box in promoter regions using CRISPR-adenine base editors (ABEs). We outline steps for guide RNA (gRNA) design, transfection, genomic DNA extraction, Sanger sequencing, and gene expression quantification. The protocol is optimized for mammalian cell lines (e.g., NIH3T3). It allows for precise disruption of transcription factor binding site without double-strand breaks and offers a novel alternative to RNAi or CRISPR interference (CRISPRi). For complete details on the use and execution of this protocol, please refer to Daliri et al.[1].},
}
@article {pmid40938663,
year = {2025},
author = {Liu, WJ and Liu, J and Ma, F and Zhang, CY},
title = {CRISPR/Cas13a trans-Cleavage-Driven Programmable Autonomous Synthesis of a Functional G-Quadruplex for Ultrasensitive Detection of Circular RNAs with an Improved Signal-to-Background Ratio.},
journal = {Analytical chemistry},
volume = {97},
number = {37},
pages = {20323-20332},
doi = {10.1021/acs.analchem.5c03464},
pmid = {40938663},
issn = {1520-6882},
mesh = {*G-Quadruplexes ; *RNA, Circular/analysis/genetics ; Humans ; *CRISPR-Cas Systems ; Limit of Detection ; },
abstract = {Circular RNAs (circRNAs) are a prevalent class of endogenous RNAs that regulate gene expression in various biological pathways, and their dysregulation is closely linked to tumorigenesis. Herein, we demonstrate the CRISPR/Cas13a trans-cleavage-driven programmable autonomous synthesis of a functional G-quadruplex for ultrasensitive detection of circRNAs in living cells and clinical tissues. Upon specific binding to target circRNA, the activated Cas13a/crRNA enables collateral cleavage of a uracil ribonucleotide (rU)-containing substrate probe, releasing a trigger sequence with a 2',3'-cyclic phosphate at its 3'-end. Subsequently, the resultant trigger sequence initiates primer exchange reaction (PER) cascades after its 3'-end is repaired by T4 polynucleotide kinase (T4 PNK), generating numerous long G-quadruplex sequences with different lengths. The long G-quadruplex sequences can be lighted up by thioflavin T (ThT) to obtain a dramatically amplified fluorescence signal. Owing to the exceptional specificity of high-fidelity CRISPR/Cas13a, high amplification efficiency of PER cascades, and high signal-to-background ratio of the G-quadruplex/ThT complex, this method can achieve ultrasensitive detection of circRNA down to 0.83 aM and discriminate target circRNA from its mismatched variants with single-base resolution. Moreover, it can profile endogenous circRNA in single cancer cell and differentiate circRNA expression in breast cancer tissues and their healthy counterparts. Notably, this assay can be accomplished in a one-step and single-tube manner at physiological temperature with the characteristics of easy design, simplified operation, rapid turnaround, and cost-effectiveness, suitable for in-field molecular diagnostics with limited resources.},
}
@article {pmid40924991,
year = {2025},
author = {Zhao, J and Sui, Z and Wang, Y and Dong, H and Chen, B and Xu, J},
title = {Programmable Dual-Phase Electrochemical Biosensor Combines Homogeneous CRISPR/Cas12a Activation with Interfacial Poly-G Signaling for miRNA-21 Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {37},
pages = {20515-20526},
doi = {10.1021/acs.analchem.5c04010},
pmid = {40924991},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics/blood ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; DNA/chemistry ; Gold/chemistry ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; Limit of Detection ; Electrodes ; Bacterial Proteins ; },
abstract = {Despite the promise of electrochemical biosensors in amplified nucleic acid diagnostics, existing high-sensitivity platforms often rely on a multilayer surface assembly and cascade amplification confined to the electrode interface. These stepwise strategies suffer from inefficient enzyme activity, poor mass transport, and inconsistent probe orientation, which compromise the amplification efficiency, reproducibility, and practical applicability. To address these limitations, we report a programmable dual-phase electrochemical biosensing system that decouples amplification from signal transduction. In the homogeneous phase, a palindromic allosteric hairpin probe undergoes target-triggered polymerization and bidirectional strand extension, generating double-stranded DNA (dsDNA) amplicons. These dsDNAs activate CRISPR/Cas12a complexes, which, in turn, cleave thiolated reporter DNA immobilized on a gold electrode. The exposed 3'-hydroxyl termini then initiate terminal deoxynucleotidyl transferase (TdT)-mediated polyguanine (poly-G) extension in the presence of dGTP. Methylene blue, selectively binding to G-rich sequences, produces a strong voltammetric signal proportional to the original miRNA-21 concentration. By integration of homogeneous amplification with localized electrochemical signal generation, this dual-phase design circumvents the drawbacks of interface-bound cascades while leveraging their sensitivity advantages. The system achieves a detection limit of 25 attomolar for miRNA-21, excellent sequence specificity, and reliable performance in human blood samples. This approach provides a robust and generalizable platform for nucleic acid diagnostics with high sensitivity, modularity, and operational simplicity.},
}
@article {pmid40924964,
year = {2025},
author = {Guo, XM and Fu, SY and Wang, YQ and Chen, Y and Li, H and Zhang, YJ and Lin, JS and Li, JF},
title = {Background-Free Rolling Circle Amplification for SERS Bioassay Using a Chimeric Hairpin-Integrated CRISPR/Cas12a System.},
journal = {Analytical chemistry},
volume = {97},
number = {37},
pages = {20557-20566},
doi = {10.1021/acs.analchem.5c04250},
pmid = {40924964},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *African Swine Fever Virus/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Spectrum Analysis, Raman/methods ; Animals ; Swine ; DNA, Single-Stranded ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rolling circle amplification (RCA) has revolutionized nucleic acid detection owing to its isothermal simplicity. However, over two decades of clinical application have been hampered by off-target amplification and incompatibility with double-stranded DNA (dsDNA). Herein, a strategy, specifically cleavage of rationally designed DNA/RNA chimeric hairpin preprimer by dsDNA-targeted CRISPR/Cas12a to release ssRNA for initiating RCA (SCOPE-RCA), is proposed for nucleic acid identification of African swine fever virus (ASFV). Leveraging the transcleavage capability of CRISPR/Cas12a, the SCOPE-RCA system achieves 99% single-stranded DNA (ssDNA) cleavage within 20 min, thus largely suppressing nonspecific amplification. Simultaneously, dsDNA-to-ssRNA conversion is realized using a DNA/RNA chimeric hairpin primer, where RNA functions as an RCA activator, enabling dsDNA-compatible RCA. Eventually, cascaded with catalytic hairpin assembly (CHA) coupled with surface-enhanced Raman scattering (SERS) signal amplification, the SCOPE-RCA system can detect ASFV in mock-infected samples at levels as low as 59 copies/mL. With its ease of use and ultrasensitive detection capability, the SCOPE-RCA system demonstrates significant potential for nucleic acid research and clinical applications.},
}
@article {pmid40899816,
year = {2025},
author = {Fujii, C and Wang, D},
title = {An SRR1 domain-containing protein is required for efficient Orsay virus replication in Caenorhabditis elegans.},
journal = {Journal of virology},
volume = {99},
number = {9},
pages = {e0052125},
doi = {10.1128/jvi.00521-25},
pmid = {40899816},
issn = {1098-5514},
mesh = {Animals ; *Caenorhabditis elegans/virology/genetics/metabolism ; *Virus Replication ; *Caenorhabditis elegans Proteins/genetics/metabolism ; *Nodaviridae/physiology/genetics ; Protein Domains ; Host-Pathogen Interactions ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Viruses depend on their hosts for completing their life cycle, and a better understanding of virus replication can inform therapeutic strategies. Using the Orsay virus-Caenorhabditis elegans experimental platform, we identified by a forward genetic screen the host gene Y55F3BL.4 (renamed viro-9) as a novel host factor critical for Orsay virus replication. Three distinct mutations of viro-9 each resulted in a >1,000-fold reduction in Orsay viral load, demonstrating a pro-viral function of viro-9. viro-9 had no previously described function in C. elegans, but in the absence of viral infection, deletion of the viro-9 locus by CRISPR/Cas9 led to a reduction in brood size and a shortened lifespan. VIRO-9 contains a sensitivity to red light reduced (SRR1) protein domain. While SRR1 domains are present in diverse organisms, including plants, yeast, and mammals, little is known about their function. The Caenorhabditis briggsae ortholog of viro-9, CBG23913, can functionally complement the C. elegans viro-9 defect, demonstrating that the pro-viral function of the SRR1 domain is conserved over at least 80 million years of evolution. Furthermore, we identified three conserved amino acid residues within the SRR1 domain that are required for Orsay virus infection. This study provides the first insights into amino acids necessary for functionality of the SRR1 domain and demonstrates the essential role of viro-9 in virus infection.IMPORTANCEHost factors required for viral replication could serve as therapeutic targets for various viral species. The Caenorhabditis elegans-Orsay virus experimental system offers a platform for identifying genes important for virus infection in nematodes that may also be important for human-infecting viruses. We determined that viro-9, a previously uncharacterized gene in C. elegans containing the SRR1 domain, is required for Orsay virus replication. The related gene in Caenorhabditis briggsae, a relative of C. elegans that diverged about 80 million years ago, can substitute for viro-9, demonstrating that this protein's ability to promote virus replication is functionally conserved. Because SRR1 domain-containing proteins exist in nematodes, fungi, Drosophila, plants, and mammals, including humans, these proteins could be important for facilitating virus infection in other organisms as well.},
}
@article {pmid40879383,
year = {2025},
author = {Wang, Y and Peng, D and Li, M and Yao, M and Li, T and Li, S and Qiu, H-J and Li, L-F},
title = {Organoids: physiologically relevant ex vivo models for viral disease research.},
journal = {Journal of virology},
volume = {99},
number = {9},
pages = {e0113225},
doi = {10.1128/jvi.01132-25},
pmid = {40879383},
issn = {1098-5514},
support = {32372983//National Natural Science Foundation of China/ ; Y2025YC117//Central Public-interest Scientific Institution Basal Research Fund/ ; },
mesh = {*Organoids/virology ; Humans ; *Virus Diseases/virology/drug therapy ; Gene Editing ; Animals ; Antiviral Agents/pharmacology ; CRISPR-Cas Systems ; SARS-CoV-2 ; Models, Biological ; Viruses ; },
abstract = {Viral diseases pose serious threats to human health, resulting in substantial economic losses. However, traditional disease models often fail to capture the full complexity of viral pathogenesis. Pluripotent and tissue stem cell-derived organoids help bridge this gap by closely mimicking the structure and function of native organs, thereby enabling new breakthroughs in studying viral pathogenesis. This review discusses the diverse applications of organoid models in virology, including infection modeling, host-virus interaction studies, CRISPR/Cas9-based gene editing, antiviral drug screening, and vaccine development. Here, we focus on human organoid models used to investigate viral infections, covering systemic viral infections (exemplified by viruses such as SARS-CoV-2, Zika virus, influenza virus, and monkeypox virus) as well as localized viral infections (exemplified by viruses including respiratory syncytial virus, herpes simplex virus 1, rotavirus, norovirus, hepatobiliary viruses, and cytomegalovirus). By advancing mechanistic insights and accelerating therapeutic discovery, organoid technology shows significant potential as a complementary tool for combating viral diseases.},
}
@article {pmid40829261,
year = {2025},
author = {Rauff, R and Dong, C and Ayyar, S and Liang, FS},
title = {Transcript and temporal-specific RNA nucleotide editing technologies.},
journal = {Bioorganic &amp; medicinal chemistry},
volume = {130},
number = {},
pages = {118346},
doi = {10.1016/j.bmc.2025.118346},
pmid = {40829261},
issn = {1464-3391},
mesh = {*RNA Editing ; Humans ; *RNA/genetics/metabolism ; Oligonucleotides, Antisense/genetics/chemistry/metabolism ; Animals ; CRISPR-Cas Systems ; *Nucleotides/genetics/metabolism ; },
abstract = {Modified RNA nucleotides have emerged as a rapidly growing field due to their critical roles in post-transcriptional gene regulation. Early approaches investigating epitranscriptomic regulation primarily involved altering the expression of RNA nucleotide editing enzymes. However, the global perturbation of RNA nucleotide modifications complicates the investigation of site-specific effects of individual modifications on transcripts. To address this limitation, researchers have developed novel RNA modification editing tools that use CRISPR, antisense oligonucleotides (ASOs), chemical ligands, and light to selectively target RNA editing enzymes to chosen RNA sites with precise temporal control. In this review, we provide a comprehensive overview of current spatiotemporal controlled RNA nucleotide editing technologies.},
}
@article {pmid40773349,
year = {2025},
author = {Sharma, D and Sinha, R and Lesch, BJ and Cromer, MK},
title = {Protocol for efficient CRISPR/AAV-mediated genome editing and erythroid differentiation of human hematopoietic stem and progenitor cells.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {104018},
pmid = {40773349},
issn = {2666-1667},
mesh = {Humans ; *Gene Editing/methods ; *Hematopoietic Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; *Dependovirus/genetics ; Cell Differentiation/genetics ; *Erythroid Cells/cytology/metabolism ; },
abstract = {Here, we present a protocol for genome editing in human hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9 ribonucleoproteins and adeno-associated virus (AAV)-mediated homology-directed repair. We describe steps for AAV production, purification, and titration; HSPC thawing and culture; genome editing; and quantification of editing frequencies. We then detail procedures for erythroid differentiation assays. This protocol ensures high editing efficiency while maintaining cell viability and engraftment potential. For complete details on the use and execution of this protocol, please refer to Chu et al.[1].},
}
@article {pmid40768339,
year = {2025},
author = {Adarska, P and Fox, E and Heyza, J and Barnaba, C and Schmidt, J and Bottanelli, F},
title = {Protocol for fast antibiotic resistance-based gene editing of mammalian cells with CRISPR-Cas9.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {103949},
pmid = {40768339},
issn = {2666-1667},
support = {DP2 GM142307/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; HeLa Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Drug Resistance, Microbial/genetics ; Plasmids/genetics ; Transfection ; Recombinational DNA Repair ; },
abstract = {Protein tagging with CRISPR-Cas9 enables the investigation of protein function in its native environment but is limited by low homology-directed repair (HDR) efficiency. Here, we present a protocol for fast antibiotic resistance-based gene editing with CRISPR-Cas9 (FAB-CRISPR), which streamlines N/C-terminal tagging using an antibiotic resistance cassette for rapid selection and enrichment of gene-edited cells. We describe in detail guide RNA and HDR donor plasmid cloning, transfection of editing reagents into HeLa cells, and subsequent enrichment and verification of gene-edited cells. For complete details on the use and execution of this protocol, please refer to Wong-Dilworth et al.,[1] Stockhammer et al.,[2] Stockhammer et al.,[3] Heyza et al.,[4] and Broadbent et al.[5].},
}
@article {pmid40762955,
year = {2025},
author = {Arnson, B and Ilich, E and von Beck, T and Li, S and Brooks, ED and Gheorghiu, D and He, G and Weinrub, M and Chan, SY and Kang, HR and Courtney, D and Everitt, JI and Cullen, BR and Koeberl, DD},
title = {Efficacious genome editing in infant mice with glycogen storage disease type Ia.},
journal = {JCI insight},
volume = {10},
number = {18},
pages = {},
doi = {10.1172/jci.insight.181760},
pmid = {40762955},
issn = {2379-3708},
mesh = {Animals ; *Gene Editing/methods ; *Glycogen Storage Disease Type I/therapy/genetics ; Mice ; *Genetic Therapy/methods ; Genetic Vectors ; Dependovirus/genetics ; *Glucose-6-Phosphatase/genetics ; Disease Models, Animal ; Humans ; Liver/pathology/metabolism ; CRISPR-Cas Systems ; Hypoglycemia/prevention &amp; control ; Animals, Newborn ; Male ; },
abstract = {Glycogen storage disease type Ia (GSD Ia) is caused by a deficiency of glucose-6-phosphatase (G6Pase) in the liver leading to lethal hypoglycemia. Gene therapy with adeno-associated virus (AAV) vectors encoding G6Pase fails to stably treat GSD Ia early in life. We evaluated genome editing in 12-day-old infant mice with GSD Ia using 2 AAV vectors, one containing Cas9 from Streptococcus pyogenes and a second Donor vector that expresses a guide RNA and a G6PC transgene. Gene therapy with the Donor vector only was compared with genome editing using both Donor and CRISPR vectors. Treatment with genome editing (total vector dose 0.2 × 1013 to 2 × 1013 vector genomes/kg) and bezafibrate (to stimulate autophagy) was efficacious, as assessed by hypoglycemia prevention and the frequency of transgene integration, which correlated with improved survival. This therapy achieved 5.9% chromosomal transgene integration through homology-directed repair, which surpassed a threshold to prevent long-term hepatic complications. No integration was detected in the absence of the CRISPR vector. Importantly for safety, CRISPR vector genomes were depleted, and no intact, integrated CRISPR genomes were detected by long-read sequencing. Thus, genome editing warrants further development as a potentially stable treatment for human infants with GSD Ia.},
}
@article {pmid40700012,
year = {2025},
author = {Parra-Rivas, LA and Sharma, R and Rust, TE and Bazick, HO and Carlson-Stevermer, J and Zylka, MJ and Ogawa, Y and Roy, S},
title = {Protocol for CRISPR-based manipulation and visualization of endogenous α-synuclein in cultured mouse hippocampal neurons.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {103945},
pmid = {40700012},
issn = {2666-1667},
mesh = {Animals ; *alpha-Synuclein/genetics/metabolism ; *Hippocampus/cytology/metabolism ; Mice ; *Neurons/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cells, Cultured ; Gene Editing/methods ; },
abstract = {CRISPR-Cas9 technology enables acute gene knockdown and endogenous tagging to study single-synapse function. Here, we present a protocol for depleting alpha-synuclein (α-syn) or visualizing native α-syn with an endogenously inserted fluorescent tag in cultured mouse hippocampal neurons. We describe detailed steps, including CRISPR design, virus packaging/transduction (delivery), and validation of on-/off-target editing. This protocol should be useful for assigning precise function to contentious synaptic proteins and for visualizing protein trafficking without overexpression in cultured hippocampal neurons-an established model system for synaptic biology. For complete details on the use and execution of this protocol, please refer to Parra-Rivas et al.[1].},
}
@article {pmid40700010,
year = {2025},
author = {Shelake, RM and Kim, JY},
title = {Protocol for assessment of CRISPR base editors and their components in Escherichia coli.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {103973},
pmid = {40700010},
issn = {2666-1667},
mesh = {*Escherichia coli/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Base editing (BE) is a CRISPR technique that allows precise nucleobase conversions. However, high expression of BE components is often toxic in Escherichia coli. Here, we present a protocol for analyzing BE at single or multiple target sites using promoter-terminators for single guide RNA (sgRNA) and BE component expression. We describe steps for designing and cloning sgRNA, synthetic target, and BE biomodules. We then detail procedures for BE module assemblage, E. coli transformation, and testing base editors and components. For complete details on the use and execution of this protocol, please refer to Shelake and Kim,[1] Shelake et al.,[2] and Shelake et al.[3].},
}
@article {pmid40674218,
year = {2025},
author = {Wilbie, D and Mastrobattista, E and de Jong, OG},
title = {Protocol to rapidly screen CRISPR-Cas9 gene editing outcomes in a cell population by mutating eGFP to a blue or non-fluorescent phenotype.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {103950},
pmid = {40674218},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Green Fluorescent Proteins/genetics ; Humans ; Mutation/genetics ; Phenotype ; },
abstract = {When designing genome editing therapy, it is crucial to measure outcomes of DNA damage repair. Here, we present a protocol to distinguish the outcome of targeted DNA damage repair from the bottom up, through a previously established readout of enhanced green fluorescent protein (eGFP) to blue fluorescent protein (BFP) mutations. We describe steps for producing eGFP-positive cells and differentiating between non-homologous end joining-induced gene knockout and homology-directed repair-induced-directed mutation in these cells. This protocol has potential for high-throughput and scalable assessment of gene editing techniques. For complete details on the use and execution of this protocol, please refer to Walther et al.[1] and Wilbie et al.[2].},
}
@article {pmid40618370,
year = {2025},
author = {Zhang, Y and Yang, H and Yang, Y and Lu, Z and Cheng, L and Tan, H and Zhang, JZ},
title = {Protocol for high-efficiency generation of iPSCs stably expressing Cas9-EGFP using the selection by essential gene exon knockin method.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {103928},
pmid = {40618370},
issn = {2666-1667},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Exons/genetics ; *Gene Knock-In Techniques/methods ; *Green Fluorescent Proteins/genetics/metabolism ; *Gene Editing/methods ; Humans ; *Genes, Essential/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Animals ; },
abstract = {CRISPR-Cas9 is widely used for genome editing. However, Cas9 silencing occurs during the directed differentiation of induced pluripotent stem cells (iPSCs), even when it is inserted into the safe harbor locus. Here, we generate iPSC-Cas9-EGFP using selection by essential gene exon knockin technology. We describe steps for inserting the Cas9-EGFP into exon 9 of GAPDH, bypassing Cas9 silencing. We then detail procedures for Cas9 function validation. For complete details on the use and execution of this protocol, please refer to Zhang et al.[1].},
}
@article {pmid40569759,
year = {2025},
author = {Doorley, LA and Meza-Perez, V and Jones, SJ and Rybak, JM},
title = {A Candidozyma (Candida) auris-Optimized Episomal Plasmid-Induced Cas9-Editing System Reveals the Direct Impact of the S639F-Encoding FKS1 Mutation.},
journal = {The Journal of infectious diseases},
volume = {232},
number = {3},
pages = {e529-e536},
doi = {10.1093/infdis/jiaf285},
pmid = {40569759},
issn = {1537-6613},
support = {//St Jude Children's Research Hospital Children's Infection Defense Center/ ; //Society of Infectious Diseases Pharmacists Young Investigator Research/ ; //Hartwell Center for Bioinformatics and Biotechnology/ ; //St Jude Center for Advanced Genome Engineering/ ; //American Lebanese Syrian Associated Charities/ ; P30 CA021765/CA/NCI NIH HHS/United States ; P30 CA021765/CA/NCI NIH HHS/United States ; },
mesh = {*Glucosyltransferases/genetics ; Drug Resistance, Fungal/genetics ; *Plasmids/genetics ; Antifungal Agents/pharmacology ; *Gene Editing/methods ; Echinocandins/pharmacology ; Mutation ; *Fungal Proteins/genetics ; *Candida auris/genetics/drug effects ; Microbial Sensitivity Tests ; CRISPR-Cas Systems ; Humans ; beta-Glucans ; },
abstract = {BACKGROUND: Mutations in the Candidozyma (Candida) auris β-glucan synthase gene (FKS1) altering S639 are frequently associated with clinical echinocandin resistance. We have developed a novel C auris-optimized episomal plasmid-induced Cas9 (EPIC) gene-editing system capable of recyclable precision editing and demonstrate the contribution of FKS1S639F mutation to echinocandin resistance.<br><br>METHODS: The EPIC gene-editing system was generated for optimized use in C auris, and ADE2 modification was evaluated in 5 C auris clades. Mutations leading to Fks1S639F and Fks1WT were placed into echinocandin-susceptible and echinocandin-resistant isolates from clades III and I, respectively. Echinocandin susceptibility was determined by Clinical and Laboratory Standards Institute methods. Cell wall abundance of chitin and &#x3b2;-glucan was assessed by staining with calcofluor white and aniline blue.<br><br>RESULTS: The EPIC system was capable of targeted ADE2 editing in all tested C auris isolates and precise editing confirmed by sequencing. A single-nucleotide polymorphism (SNP) in FKS1 resulting in either the S639F substitution or a synonymous mutation was introduced in an echinocandin-susceptible clade III isolate. Precision FKS1 editing by the EPIC system was confirmed by whole genome sequencing. Introduction of the Fks1S639F allele increased echinocandin resistance, while correction of the Fks1S639F to the Fks1WT sequence in an echinocandin-resistant clade I isolate restored echinocandin sensitivity. Evaluation of cell wall composition showed strains harboring Fks1S639F to contain significantly elevated &#x3b2;-glucan and chitin content.<br><br>CONCLUSIONS: These data demonstrate the potential of our EPIC system in its ability to introduce SNPs in multiple C auris clade backgrounds while revealing the direct impact of the S639F-encoding FKS1 mutation on echinocandin resistance.},
}
@article {pmid40517388,
year = {2025},
author = {Jo, MJ and Nam, HJ},
title = {Protocol to screen chemicals to enhance homology-directed repair in CRISPR-Cas9 gene editing.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {103896},
doi = {10.1016/j.xpro.2025.103896},
pmid = {40517388},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Recombinational DNA Repair/drug effects/genetics ; *High-Throughput Screening Assays/methods ; Humans ; },
abstract = {CRISPR-Cas9-based gene editing via homology-directed repair (HDR) enables precise modifications, though its efficiency is limited by the prevalence of non-homologous end joining (NHEJ). Here, we present a protocol for enhancing HDR efficiency by identifying chemicals using high-throughput screening (HTS). We describe steps for designing 96-well plates, executing HTS, and performing data analysis. We then detail procedures for identifying small molecules that improve HDR-associated gene editing. This protocol has potential application in HTS analysis focused on discovering reliable HDR enhancers. For complete details on the use and execution of this protocol, please refer to Jang et al.[1].},
}
@article {pmid40974457,
year = {2025},
author = {Ma, D and Cai, F and Zhang, T},
title = {Advances in the detection of Drug-Resistant bacteria: current trends and innovations.},
journal = {European journal of clinical microbiology &amp; infectious diseases : official publication of the European Society of Clinical Microbiology},
volume = {},
number = {},
pages = {},
pmid = {40974457},
issn = {1435-4373},
abstract = {Facing challenges in the fields of microbial detection and antimicrobial resistance (AMR) monitoring, the scientific community is opening new research avenues with the help of cutting-edge technologies such as molecular biology, genomics, proteomics, nanotechnology, and bioinformatics. In this review, we comprehensively collate and elaborate on revolutionary detection methods and AMR surveillance strategies that go beyond traditional microbial culture techniques. These innovative methods have not only improved the sensitivity and speed of detection but also broadened our understanding of the microbial world, providing new weapons in the fight against drug-resistant microorganisms. Through the integration and innovation of interdisciplinary approaches, we are gradually constructing a more precise, efficient, and comprehensive new paradigm for microbial detection and AMR testing.},
}
@article {pmid40973454,
year = {2025},
author = {Cortolezzis, Y and Othman, Z and Agostini, F and Ibrahim, I and Picco, R and Salgado, GF and Di Giorgio, E and Xodo, LE},
title = {Post-transcriptional control of KRAS: functional roles of 5'UTR RNA G-quadruplexes, long noncoding RNA, and hnRNPA1.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
pmid = {40973454},
issn = {1362-4962},
support = {IG 2017//Associazione Italiana per la Ricerca sul Cancro/ ; 19898//Associazione Italiana per la Ricerca sul Cancro/ ; //Italian Ministry of Education/ ; //SD-Cancer Contributions of physics, chemistry and engineering sciences to oncology/ ; //Minist&#xe8;re de l'Europe et des Affaires &#xc9;trang&#xe8;res/ ; },
mesh = {Humans ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *5' Untranslated Regions/genetics ; *G-Quadruplexes ; *RNA, Long Noncoding/genetics/metabolism ; *Heterogeneous Nuclear Ribonucleoprotein A1/metabolism/genetics ; RNA, Messenger/genetics/metabolism ; RNA Stability ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; *RNA Processing, Post-Transcriptional ; Cell Line, Tumor ; },
abstract = {Previous studies have shown that human KRAS expression is regulated at the transcriptional level by G-quadruplex DNA structures within its promoter. Here we show an additional level of regulation involving a post-transcriptional mechanism centred on the 5'-untranslated region (5'UTR) of the messenger RNA (mRNA) characterized by G4 structures (rG4s). Long noncoding RNAs (lncRNAs) and the protein hnRNPA1 are also involved in this mechanism. RIP-seq confirmed the presence of rG4s in the 5'UTR. Deletion of the rG4 region using CRISPR/Cas9 resulted in a significant increase in KRAS mRNA levels, indicating the role of the 5'UTR in controlling mRNA levels. RIP shows that hnRNPA1 is recruited to the 5'UTR, where it unfolds the rG4 structures and potentially affects mRNA stability. In addition, lncRNAs transcribed from the LINC01750 locus can hybridize to the rG4 region of 5'UTR and form RNA duplexes leading to RNase III-assisted degradation of the targeted mRNA. Activation of the LINC01750 locus with dCas9-VP64 resulted in downregulation of KRAS mRNA, whereas its suppression with dCas9-KRAB led to upregulation of both KRAS mRNA and protein. Since lncRNA-mediated regulation of mRNA appears to be a crucial aspect of cellular homeostasis and its disruption contributes to various diseases, understanding these mechanisms may reveal promising new therapeutic targets.},
}
@article {pmid40973278,
year = {2025},
author = {Safarzadeh, S and Naghib, SM and Takdehghan, G and Forooshani, RS and Roozbahani, MH and Sharifianjazi, F and Tavamaishvili, K},
title = {Multimodal chitosan-based materials for combination immunotherapy in cancers: Structural engineering, immune regulatory mechanisms and synergistic therapeutic applications.},
journal = {Carbohydrate polymers},
volume = {369},
number = {},
pages = {124245},
doi = {10.1016/j.carbpol.2025.124245},
pmid = {40973278},
issn = {1879-1344},
mesh = {Humans ; *Immunotherapy/methods ; *Neoplasms/therapy/immunology ; *Chitosan/chemistry/therapeutic use ; Animals ; Combined Modality Therapy ; },
abstract = {Chitosan (CS)-based materials have gained considerable attention due to their intrinsic biodegradability, biocompatibility, and non-immunogenicity, establishing them as versatile candidates for state-of-the-art cancer immunotherapy. By harnessing the unique physicochemical and immunomodulatory properties of this polysaccharide, CS-based systems enable precise targeting, controlled release, and enhanced immune activation, thereby improving therapeutic efficacy while minimizing off-target effects. This review begins by outlining the cancer-immunity cycle and elucidating the structural and functional attributes of CS including cationic charge, chemical functionalization, biodistribution, targeting efficiency, and immune regulation that underpin its performance. The strategic roles of advanced CS derivatives, such as trimethyl CS (TMC), carboxymethyl CS (CMC), fluorinated CS (FCS), glycated CS (GC), hydroxypropyl CS (HPC), and thiolated CS (TC), are examined in optimizing these parameters. Cutting edge CS-based platforms, encompassing vaccines, immune checkpoint inhibitors, and nanostructures, are critically evaluated alongside their integration with synergistic modalities such as chemotherapy, radiotherapy (RT), phototherapy, sonodynamic therapy (SDT), gene therapy, and CRISPR-Cas mediated genome editing. The clinical and translational landscape is also addressed, highlighting emerging trials, manufacturing scalability, and regulatory considerations. Collectively, recent advances consolidate CS-based immunotherapy as a potent, adaptable, and clinically relevant approach capable of driving durable and tumor specific immune responses, with substantial promise for future cancer treatment.},
}
@article {pmid40973142,
year = {2025},
author = {Gong, M and Ding, Y and Jin, Y and Zhao, S and Jiang, X and Shao, S and Chen, S},
title = {CRISPR-based gene knockout in the model haloarchaeon Haloferax mediterranei.},
journal = {FEMS microbiology letters},
volume = {},
number = {},
pages = {},
doi = {10.1093/femsle/fnaf096},
pmid = {40973142},
issn = {1574-6968},
abstract = {Halophilic archaea, a specialized group of extremophiles that inhabit hypersaline environments, exhibit distinctive physiological and metabolic features. Traditional genetic manipulation of these organisms, predominantly reliant on homologous recombination techniques, suffers from limitations such as complex procedures and extended timelines, which hinder functional genomics research and the development of practical applications. This study established a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-mediated gene knockout system in the model halophilic archaeon Haloferax mediterranei. A polyethylene glycol (PEG)-mediated transformation method was used to deliver a plasmid carrying a mini-CRISPR array into H. mediterranei. The crtB gene, involved in pigment synthesis, was successfully knocked out, demonstrating the feasibility of CRISPR-based editing in H. mediterranei. To further validate the reliability and targeting accuracy of the system, the hlyR4 gene, encoding an extracellular serine protease, was also disrupted. The CRISPR-mediated gene knockout efficiency for hlyR4 reached 27%, significantly higher than the approximately 3% efficiency achieved with conventional homologous recombination. The establishment of this CRISPR-based gene knockout system provides a more efficient genetic tool for H. mediterranei and lays a new experimental foundation for exploiting microbial resources from extreme environments.},
}
@article {pmid40904316,
year = {2025},
author = {Li, Z and Song, Y and Huang, H and Chen, R and Liu, M and Yang, X and Luo, Z and Liu, BM and Wang, J},
title = {Highly stable Cas9 promotes HBV genome destruction by antagonizing HSC70-mediated degradation.},
journal = {Emerging microbes &amp; infections},
volume = {14},
number = {1},
pages = {2556728},
doi = {10.1080/22221751.2025.2556728},
pmid = {40904316},
issn = {2222-1751},
mesh = {Humans ; *HSC70 Heat-Shock Proteins/metabolism/genetics ; *Hepatitis B virus/genetics/physiology ; *CRISPR-Cas Systems ; *Genome, Viral ; Autophagy ; *CRISPR-Associated Protein 9/metabolism/genetics ; Proteolysis ; *Hepatitis B/virology ; Lysosomes/metabolism ; },
abstract = {As a naturally existing adaptive immune system of prokaryotes against phages and foreign genetic materials, the CRISPR/Cas9 system has been widely used to combat various viral infections. However, its ability to destroy the constantly replicating viral genome and subsequently clear viral infections still needs further improvement. This study found that Cas9 protein was mainly degraded through the chaperone-mediated autophagy (CMA)-lysosome pathway in human cells, which was mediated by the binding between heat shock cognate protein 70 (HSC70) and Cas9 protein. HRS could stabilize Cas9 protein by competing with HSC70 to bind to Cas9 and subsequently inhibiting its degradation via the CMA-lysosome pathway. The stability of Cas9 protein with mutant KFERQ-like motifs located at aa 670-674 and aa 894-898 was significantly increased by antagonizing the HSC70-mediated CMA degradation, thus this Cas9 mutant was referred to as a highly stable Cas9 (HSCas9). The enhanced ability of HSCas9 to destroy the constantly replicating hepatitis B virus (HBV) genome promoted the CRISPR/Cas9 system to clear HBV infection without exhibiting cytotoxicity or increasing off-target effects. In summary, this study uncovers the degradation mechanism of Cas9 protein in human cells and provides a strategy to enhance the ability of the CRISPR/Cas9 system to clear HBV infection.Abbreviations: ALP: autophagy-lysosome pathways; AR7: 7-Chloro-3-(4-methylphenyl)-2H-1,4-benzoxazine; cccDNA: covalently closed circular DNA; CMA: chaperone-mediated autophagy; CRISPR/Cas9: clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9); gRNA: single-guide RNA; HBV: hepatitis B virus; HRS: hepatocyte growth factor-regulated tyrosine kinase substrate; HSC70: heat shock cognate protein 70; HSCas9: highly stable Cas9; rcDNA: relaxed circular DNA; SNP: single nucleotide polymorphism; UPS: ubiquitin-proteasome system.},
}
@article {pmid40972923,
year = {2025},
author = {Sun, H and Bu, S and Wang, J and Zhang, Z and Li, C and Wan, Y and Zhao, Y and Zhou, H and Hao, Z and Feng, X and Li, C and Wan, J},
title = {PAM-free CRISPR/Cas12a biosensor for PNAs-assisted isothermal amplification detection of S. typhimurium.},
journal = {International journal of biological macromolecules},
volume = {328},
number = {Pt 2},
pages = {147721},
doi = {10.1016/j.ijbiomac.2025.147721},
pmid = {40972923},
issn = {1879-0003},
abstract = {CRISPR/Cas12a (Cpf1) have been generally used for detecting pathogenic microorganisms. However, rules for a protospacer adjacent motif (PAM) on target double-stranded DNA (dsDNA) hindered their application. To overcome this shortcoming, we developed a novel isothermal amplification scheme, peptide nucleic acids (PNAs)-assisted self-folding isothermal amplification for a PAM-free CRISPR/Cas12a biosensor (PSCas) to detect salmonella typhimurium (S. typhimurium). The PSCas biosensor uses PNAs to precisely invade the target gene dsDNA to form single-stranded DNA (ssDNA), which in turn binds to a self-folding primer (SP). When the self-folding strand of SP is complementary to the target DNA, the hairpin structure is formed, which exposes the start-up site of the new SP continuous hybridization. Consequently, this amplification used only one primer to produce a large number of ssDNA amplifiers, and exhibited a remarkable diagnostic sensitivity of 3 CFU•mL[-1] S. typhimurium. This platform provided a novel approach for developing innovative PAM-free CRISPR/Cas biosensors for PNAs-assisted isothermal amplification.},
}
@article {pmid40769810,
year = {2025},
author = {Liu, W and Zhu, X and Xue, Y and Wang, X and Chang, L and Wang, S and Li, L and Huang, J and Zhang, Y and Jiang, C and Chi, H and Zhang, Y and Li, CZ and Liang, T},
title = {RNA target-independent non-canonical activation (RINCA) of Cas13 trans-nuclease activity.},
journal = {Science bulletin},
volume = {70},
number = {18},
pages = {3005-3018},
doi = {10.1016/j.scib.2025.07.015},
pmid = {40769810},
issn = {2095-9281},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; *RNA/metabolism/genetics ; Animals ; Aptamers, Nucleotide/genetics ; Ribonucleases/metabolism/genetics ; Mutation ; Mice ; },
abstract = {A thorough understanding of unintended Cas13 activity is critical for ensuring the safe in vivo application of CRISPR-Cas13. In this study, we uncover the RNA target-independent, non-canonical activation (RINCA) of Cas13 by crRNA alone and elucidate its structural basis. RINCA poses both challenges and opportunities for Cas13-based technologies. On one hand, it constrains Cas13's utility due to its uncontrolled RNase activity. To address this, we screened over 40 rationally designed Cas13a mutants to identify those with diminished RINCA potential while preserving target-dependent responses. These optimized Cas13a variants improve biosensing sensitivity and significantly reduce cytotoxicity in vivo. On the other hand, RINCA can be harnessed for novel applications. By integrating RNA aptamers into spacer regions, the RINCA-capable LwCas13a/ap-crRNA system was engineered to sense intracellular metabolite levels, drive systemic RNA degradation, and demonstrate anti-cancer efficacy across multiple models. This study not only discovers the RINCA model of Cas13 activity but also provides effective mitigation strategies and establishes novel RINCA-based applications.},
}
@article {pmid40972653,
year = {2025},
author = {Serpa, G and Gong, Q and De, M and Rana, PSJB and Montgomery, CP and Wozniak, DJ and Long, ME and Hemann, EA},
title = {Detection of pre-existing immunity to bacterial Cas9 proteins in people with cystic fibrosis.},
journal = {ImmunoHorizons},
volume = {9},
number = {10},
pages = {},
doi = {10.1093/immhor/vlaf041},
pmid = {40972653},
issn = {2573-7732},
support = {LONG19F5-CI//Cystic Fibrosis Foundation/ ; LONG21R3//Cystic Fibrosis Foundation/ ; },
mesh = {Humans ; *Cystic Fibrosis/immunology/genetics ; *CRISPR-Associated Protein 9/immunology/genetics ; CRISPR-Cas Systems/immunology ; Gene Editing ; Male ; Adult ; Female ; Genetic Therapy ; Staphylococcus aureus/immunology ; Streptococcus pyogenes/immunology ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; Immunoglobulin G/blood/immunology ; T-Lymphocytes/immunology ; Young Adult ; Adolescent ; Interferon-gamma/metabolism ; },
abstract = {Cystic fibrosis (CF) is caused by homozygous mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, resulting in multi-organ dysfunction and decreased lifespan and quality of life. A durable cure for CF will likely require a gene therapy approach to correct CFTR. Rapid advancements in genome editing technologies, including CRISPR/Cas9, have already resulted in Food and Drug Administration (FDA) approval for cell-based gene editing therapies, providing new therapeutic avenues for many rare diseases. However, immune responses to gene therapy delivery vectors and editing tools remain a challenge, especially for strategies targeting complex in vivo tissues such as the lung. Previous findings in non-CF healthy individuals reported pre-existing antibody and T cell responses to recombinant Cas9 proteins, suggesting potential additional obstacles for incorporation of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technologies in gene therapies. To determine whether pre-existing immunity to Cas9 from S. aureus or S. pyogenes was present or augmented in people with CF, anti-Cas9 IgG levels and Cas9-specific T cell responses were determined from peripheral blood samples of people with CF and non-CF healthy controls. Overall, non-CF control and CF samples displayed evidence of pre-existing antibody and T cell responses to both S. aureus and S. pyogenes Cas9, although there were no significant differences between these populations. However, we observed global changes in CF activation of Th1 and CD8 T cell responses as measured by interferon γ (IFN-γ) and tumor necrosis factor (TNF) that warrant further investigation and mechanistic understanding as this finding has implications not only for CRISPR/Cas9 gene therapy for people with CF but also for protection against infectious disease.},
}
@article {pmid40972525,
year = {2025},
author = {Tatarakis, A and Saini, H and Yu, J and Feng, W and Pinzon-Arteaga, CA and Moazed, D},
title = {Requirements for establishment and epigenetic stability of mammalian heterochromatin.},
journal = {Molecular cell},
volume = {85},
number = {18},
pages = {3388-3406.e12},
doi = {10.1016/j.molcel.2025.08.025},
pmid = {40972525},
issn = {1097-4164},
mesh = {*Heterochromatin/genetics/metabolism ; Animals ; Mice ; *Histones/metabolism/genetics ; DNA Methylation ; *Epigenesis, Genetic ; *Mouse Embryonic Stem Cells/metabolism ; Chromatin Assembly and Disassembly ; Cell Differentiation ; Histone-Lysine N-Methyltransferase/metabolism/genetics ; Methylation ; CRISPR-Cas Systems ; Histone Deacetylases/metabolism/genetics ; },
abstract = {Heterochromatic domains of DNA account for a large fraction of mammalian genomes and play critical roles in silencing transposons and genes, but the mechanisms that establish and maintain these domains are not fully understood. Here, we use a CRISPR-based genetic screen to investigate the requirements for establishment and maintenance of histone H3 lysine 9 trimethylation (H3K9me3) heterochromatin. In mouse embryonic stem cells (mESCs), we show that transiently induced H3K9me3 heterochromatin is inherited for a limited number of cell divisions, independently of sequence-dependent recruitment, but becomes stable upon differentiation, concomitant with downregulation of enzymes erasing H3K9me and DNA methylation. In addition, ordered and non-redundant activities of multiple H3K9 and DNA methyltransferases, together with histone deacetylases, chromatin remodeling complexes, and RNA processing factors, are required for heterochromatin maintenance. Our findings suggest that a newly acquired H3K9me3 domain can be maintained like an imprint but requires reinforcement by DNA methylation and other pathways.},
}
@article {pmid40970972,
year = {2025},
author = {Yang, C and Li, B and Yu, H and Wang, Y and An, Z and Chen, M and He, C},
title = {GmCDC7 is involved in coordinating seed size and quality in soybean.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {10},
pages = {253},
pmid = {40970972},
issn = {1432-2242},
support = {2023ZD0406904//Biological Breeding-National Science and Technology Major Project/ ; 2023ZD0406802//Biological Breeding-National Science and Technology Major Project/ ; 31930007//National Natural Science Foundation of China/ ; 31525003//National Natural Science Foundation of China/ ; XDA08010105//Chinese Academy of Sciences/ ; },
mesh = {*Glycine max/genetics/growth &amp; development ; *Seeds/genetics/growth &amp; development ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; *Cell Cycle Proteins/genetics/metabolism ; Phenotype ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {We revealed essential roles of GmCDC7 in modulating seed size/weight and seed protein/oil content in soybean, presenting potential new targets for improving yield and quality of soybean and other crops. Seed size/weight is a critical factor determining crop yield; however, a limited number of genes regulating this trait have been characterized in soybean. In this study, we identified a Glycine max CELL DIVISION CYCLE 7 (GmCDC7) and revealed its essential roles in seed development. The putative GmCDC7 was highly conserved in both sequences and structure across various species. GmCDC7 transcripts were detectable in multiple tissues, with peak expression occurring during early seed development, while the GmCDC7 proteins were predominantly localized within the nucleus. CRISPR/Cas9-mediated knockout of GmCDC7 led to a significant increase in seed size and 100-seed weight, while overexpression of this gene resulted in a reduction in both seed size and weight. Further cytological analysis demonstrated that GmCDC7 promoted cell expansion and inhibited cell proliferation in seeds. Notably, the gene-edited gmcdc7 mutants showed a substantial increase in protein content alongside a reduction in oil content in seeds. Correspondingly, transcriptomic analyses revealed that GmCDC7 may significantly influence multifaceted regulatory pathways related to cell cycle-related activities, storage protein accumulation, and lipid transport and metabolism during seed development. These findings suggest that GmCDC7 plays pivotal roles in modulating seed size/weight and quality, offering new gene resources and insights into biotechnological strategies for soybean breeding.},
}
@article {pmid40970094,
year = {2025},
author = {Shu, J and Xie, X and Wang, S and Du, Z and Huang, P and Chen, Y and He, Z},
title = {CRISPR/Cas-edited iPSCs and mesenchymal stem cells: a concise review of their potential in thalassemia therapy.},
journal = {Frontiers in cell and developmental biology},
volume = {13},
number = {},
pages = {1595897},
pmid = {40970094},
issn = {2296-634X},
abstract = {Thalassemia, a prevalent single-gene inherited disorder, relies on hematopoietic stem cell or bone marrow transplantation as its definitive treatment. However, the scarcity of suitable donors and the severe complications from anemia and iron overload pose significant challenges. An immediate need exists for a therapeutic method that addresses both the illness and its associated complications. Advancements in stem cell technology and gene-editing methods, such as clustered regularly interspaced short palindromic repeats along with its associated protein (CRISPR/Cas), offer encouraging prospects for a therapy that could liberate patients from the need for ongoing blood transfusions and iron chelation treatments. The potential of genetic reprogramming using induced pluripotent stem cells (iPSCs) to address thalassemia is highly promising. Furthermore, mesenchymal stem cells (MSCs), recognized for their capacity to self-renew and differentiate into multiple lineages that include bone, cartilage, adipose tissue, and liver, demonstrate potential in alleviating several complications faced by thalassemia patients, including osteoporosis, cirrhosis, heart conditions, respiratory issues, and immune-related disorders. In this review, we synthesize and summarize relevant studies to assess the therapeutic potential and predict the curative effects of these cellular approaches.},
}
@article {pmid40968372,
year = {2025},
author = {Harle, V and Offord, V and Gökbağ, B and Fotopoulos, L and Williams, T and Alexander, D and Mehta, I and Thompson, NA and Olvera-León, R and Peidli, S and Iyer, V and Gonçalves, E and Kebabci, N and De Kegel, B and van de Haar, J and Li, L and Ryan, CJ and Adams, DJ},
title = {A compendium of synthetic lethal gene pairs defined by extensive combinatorial pan-cancer CRISPR screening.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {284},
pmid = {40968372},
issn = {1474-760X},
support = {20/FFP-P/8641 and 18/CRT/6214//Health Research Charities Ireland/ ; },
mesh = {Animals ; Humans ; *CRISPR-Cas Systems ; Mice ; Cell Line, Tumor ; *Neoplasms/genetics ; *Synthetic Lethal Mutations ; Mice, Knockout ; *Genes, Lethal ; },
abstract = {BACKGROUND: Synthetic lethal interactions are attractive therapeutic candidates as they enable selective targeting of cancer cells in which somatic alterations have disrupted one member of a synthetic lethal gene pair while leaving normal tissues untouched, thus minimising off-target toxicity. Despite this potential, the number of well-established and validated synthetic lethal gene pairs is modest.<br><br>RESULTS: We generate a dual-guide CRISPR/Cas9 Library and analyse 472 predicted synthetic lethal pairs in 27 cancer cell Lines from melanoma, pancreatic and lung cancer Lineages. We report a robust collection of 117 genetic interactions within and across cancer types and explore their candidacy as therapeutic targets. We show that SLC25A28 is an attractive target since its synthetic lethal paralog partner SLC25A37 is homozygously deleted pan-cancer. We generate knockout mice for Slc25a28 revealing that, except for cataracts in some mice, these animals are normal; suggesting inhibition of SLC25A28 is unlikely to be associated with profound toxicity.<br><br>CONCLUSIONS: We provide and validate an extensive collection of synthetic lethal interactions across cancer types.},
}
@article {pmid40968144,
year = {2025},
author = {Wang, M and Zhang, Z and Wang, X and Zhang, L and Chen, X and Li, N and Sun, Q and Lu, Y and He, Z and Yang, H and Tan, F and Qi, J and Chai, R},
title = {Optimized in vivo base editing restores auditory function in a DFNA15 mouse model.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8322},
pmid = {40968144},
issn = {2041-1723},
support = {82030029//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; 82330033//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; 92468302//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; 92149304//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; BK20232007//Natural Science Foundation of Jiangsu Province (Jiangsu Provincial Natural Science Foundation)/ ; BK20241692//Natural Science Foundation of Jiangsu Province (Jiangsu Provincial Natural Science Foundation)/ ; BG2024037//Natural Science Foundation of Jiangsu Province (Jiangsu Provincial Natural Science Foundation)/ ; JCYJ20240813161801003//Shenzhen Science and Technology Innovation Commission/ ; 2024M750455//China Postdoctoral Science Foundation/ ; 2025T180661//China Postdoctoral Science Foundation/ ; 82401369//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82271183//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82222017//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82192864//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82371161//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82371162//National Natural Science Foundation of China (National Science Foundation of China)/ ; U23A200440//National Natural Science Foundation of China (National Science Foundation of China)/ ; RF1028623028//Southeast University (SEU)/ ; CZXM-GSP-RC04//Southeast University (SEU)/ ; tsqn202408320//Taishan Scholar Project of Shandong Province/ ; 7252089//Natural Science Foundation of Beijing Municipality (Beijing Natural Science Foundation)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Disease Models, Animal ; Mice ; CRISPR-Cas Systems ; *Transcription Factor Brn-3C/genetics ; *Deafness/genetics/therapy ; Genetic Therapy/methods ; Mutation ; Dependovirus/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; Female ; Male ; },
abstract = {Genetic mutations cause hereditary deafness, in which mutations in the POU4 transcription factor 3 gene (POU4F3) lead to autosomal dominant non-syndromic deafness 15 (DFNA15), for which no effective clinical treatment currently exists. Gene editing holds promise for precisely repairing mutated nucleotides, thus offering a potential cure for hereditary hearing loss. Here, we establish a Pou4f3[WT/Q113*] mutant mouse model mimicking DFNA15. We develop and screen adenine base editors (ABEs) targeting the Pou4f3[Q113*] allele by fusing diverse adenine deaminases to Cas9 we discovered before. SchABE8e accomplishes highly precise and efficient editing (up to 48.5%) at sgRNA3 in vitro. Neonatal Pou4f3[WT/Q113*] mice are treated via synthetic AAV (Anc80L65)-delivered SchABE8e-sgRNA3, resulting in near-complete hearing recovery, with the effect persisting for at least four months. Biosafety analyses further support the feasibility of base editing, providing a therapeutic strategy for DFNA15.},
}
@article {pmid40966844,
year = {2025},
author = {Sihamok, W and Islam, SI and Khang, LTP and Dangsawat, O and Sangsawad, P and Tu, TA and Thao, CP and Dinh-Hung, N and Permpoonpattana, P and Linh, NV},
title = {Genomic insights into Bacillus sp. KNSH11 from Litopenaeus vannamei intestine: Probiotic potential, safety, and aquaculture applications.},
journal = {Comparative biochemistry and physiology. Part D, Genomics &amp; proteomics},
volume = {56},
number = {},
pages = {101633},
doi = {10.1016/j.cbd.2025.101633},
pmid = {40966844},
issn = {1878-0407},
abstract = {In the context of sustainable aquaculture, probiotics represent a promising alternative to antibiotics for promoting shrimp health and disease resistance. In this study, Bacillus sp. KNSH11, a Gram-positive, rod-shaped bacterium isolated from the intestine of whiteleg shrimp (Litopenaeus vannamei), was characterized to assess its probiotic potential. The strain exhibited excellent sporulation efficiency (> 99 %), supporting its resilience under harsh environmental conditions. Functional assays demonstrated that KNSH11 retained high viability under various stressors, including acidic pH (2-4), bile salts, elevated temperatures (up to 95 °C), and lysozyme exposure, indicating robust tolerance to gastrointestinal and processing challenges. Metabolic profiling revealed substantial lactic acid production with minimal levels of acetate and propionate, distinguishing it from conventional lactic acid bacteria. The strain also exhibited strong antioxidant activity and moderate antibiofilm effects against pathogenic bacteria. Antibiotic susceptibility testing showed sensitivity to amoxicillin, chloramphenicol, kanamycin, and tetracycline (all at 30 μg/disc), while resistance was observed against ampicillin and penicillin (10 μg/disc each). Whole genome sequencing confirmed the absence of virulence genes and identified the presence of mobile genetic elements, a CRISPR/Cas system, and gene clusters potentially responsible for bacteriocin production. Collectively, these results indicate that Bacillus sp. KNSH11 exhibits key probiotic characteristics and genomic features consistent with a safe profile, supporting its potential application in sustainable shrimp aquaculture, pending further in vitro and in vivo validation.},
}
@article {pmid40966510,
year = {2025},
author = {Rananaware, SR and Shoemaker, GM and Pizzano, BLM and Vesco, EK and Sandoval, LSW and Lewis, JG and Bodin, AP and Flannery, SJ and Lange, IH and Pedada, D and Fang, A and Antal, SG and Aguilar, D and Rakestraw, NR and Karalkar, VN and Meister, KS and Nguyen, LT and Jain, PK},
title = {AsCas12a tolerates insertions in target DNA.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
doi = {10.1093/nar/gkaf887},
pmid = {40966510},
issn = {1362-4962},
support = {//University of Florida/ ; //UF Herbert Wertheim College of Engineering/ ; //Shah Foundation/ ; //Exxon Mobil Gator Alumni Faculty/ ; R21AI156321/GF/NIH HHS/United States ; R21AI168795/AI/NIAID NIH HHS/United States ; 5R61AI181016/AI/NIAID NIH HHS/United States ; R35GM147788/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *DNA/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; *Acidaminococcus/enzymology/genetics ; *Bacterial Proteins/metabolism/chemistry/genetics ; Gene Editing ; DNA Cleavage ; Models, Molecular ; },
abstract = {CRISPR-Cas12a enzymes are RNA-guided nucleases widely used for programmable genome editing and diagnostics. Perfect complementarity between guide RNA and target DNA is essential for efficient binding and cleavage by Cas12a. However, we report that a particular ortholog of Cas12a, Acidaminococcus sp. Cas12a (AsCas12a), shows an unexpected tolerance to noncomplementary insertions at various positions in its DNA target. AsCas12a remains functional despite DNA bubbles or loops in the CRISPR-RNA (crRNA)-target DNA duplex, displaying both cis- and trans-cleavage activities even when the target harbors insertions of lengths 1-20 nucleotides in the crRNA-binding region. This activity is sequence-independent and works for ssDNA and is observed on dsDNA in vitro for specific insertion lengths/positions and DNA topologies but is strongly diminished in cells. Among 12 Cas12a orthologs tested, only AsCas12a exhibits this tolerance, making it a unique member of the Cas12a family. Structural analysis suggests a distinctive α-helix in AsCas12a's WED domain is required for this flexibility. Upon deleting this α-helix, AsCas12a loses its ability to tolerate insertions. This discovery can be utilized to detect single-nucleotide polymorphisms and enable protospacer adjacent motif (PAM)-flexible DNA cleavage with Cas12a. Our findings expand our fundamental understanding of CRISPR-Cas12a systems. In conclusion, we uncover and characterize a unique property of AsCas12a to tolerate insertions in its target.},
}
@article {pmid40966507,
year = {2025},
author = {Obi, I and Sengupta, P and Sabouri, N},
title = {CRISPR-Cas9 targeting of G-Quadruplex DNA in ADH1 promoter highlights its role in transcriptome and metabolome regulation.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
doi = {10.1093/nar/gkaf853},
pmid = {40966507},
issn = {1362-4962},
support = {//Insamlingsstiftelsen at Ume&#xe5; University/ ; 22 2380 Pj 01 H//Swedish Cancer Society/ ; VR-MH 2021-02&#xa0;468//Swedish Research Council/ ; KAW 2021.0173//Knut and Alice Wallenberg Foundations/ ; UPD2020-0097//Wenner-Gren Foundations/ ; 24 0907 PT 01 H//Swedish Cancer Society/ ; },
mesh = {*G-Quadruplexes ; *Promoter Regions, Genetic ; *CRISPR-Cas Systems ; *Schizosaccharomyces/genetics/metabolism ; *Metabolome/genetics ; *Alcohol Dehydrogenase/genetics/metabolism ; *Transcriptome/genetics ; Gene Expression Regulation, Fungal ; Mutation ; *Schizosaccharomyces pombe Proteins/genetics/metabolism ; TATA Box ; NAD/metabolism ; },
abstract = {G-quadruplex (G4) structures are critical regulators of gene expression, yet the role of an individual G4 within its native chromatin remains underexplored, especially outside human systems. Here, we used CRISPR-Cas9 to introduce guanine-to-thymine mutations at a G4-forming motif within the adh1+ promoter in yeast Schizosaccharomyces pombe, creating two mutant strains: one with G4-only mutations and another with both G4 and TATA-box mutations. Chromatin immunoprecipitation using BG4 antibody confirmed reduced G4 enrichment in both mutants, validating G4 structure formation in the wild-type chromatin. Detailed characterizations demonstrated that the G4 mutations alter its dynamics without fully preventing its formation. These mutations significantly reduce adh1 transcript levels, with G4 TATA-box mutant causing the strongest transcriptional suppression. This indicates a positive regulatory role for the G4 structure in transcription. Furthermore, both mutants displayed altered transcriptomic profiles, particularly impacting the oxidoreductase pathway. Metabolomic analyses by mass spectrometry further highlighted substantial disruptions in NAD+/NADH metabolism, a key energy reservoir for metabolic regulation. These results highlight that tuning G4 dynamics, without abolishing the structure, can still profoundly affect gene expression and metabolism, unlike prior studies on the human MYC promoter that disrupted G4 formation. This represents the first such finding in yeast.},
}
@article {pmid40949944,
year = {2025},
author = {Guy, J and Hein, E and Alexander-Howden, B and von Bock Und Polach, T and Mathieson, T and Kleinstiver, BP and Zoghbi, HY and Bird, AP},
title = {Translational reading frame determines the pathogenicity of C-terminal frameshift deletions in MeCP2: an alternative therapeutic approach.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40949944},
issn = {2692-8205},
abstract = {Mutations in the MECP2 gene cause the severe neurological disorder Rett syndrome. A cluster of frameshift-causing C-terminal deletions (CTDs) lead to loss of ~100 amino acids at the C-terminus of the MeCP2 protein, and account for approximately 10% of RTT-causing mutations. The pathogenicity of C-terminal deletions (CTDs) is unexpected, as this C-terminal domain is non-essential in mice. Utilising databases of pathogenic and benign human MECP2 mutations, we find that some individuals with apparently typical CTDs do not exhibit Rett syndrome, confirming that C-terminal truncations are not intrinsically pathogenic. Using human DNA sequence data and mouse models, we demonstrate that pathogenicity results from a drastic reduction in MeCP2 levels and is determined by the presence of the short amino acid motif proline-proline-stop (-PPX) at the C-terminus, which results from a shift to the +2 reading frame. Individuals with CTDs that shift to the +1 frame avoid this motif and do not develop Rett syndrome. Mutating the stop codon of the PPX motif to tryptophan rescues MeCP2 expression and RTT-like phenotypes in a CTD mouse model. Finally, we demonstrate that an adenine base editor can efficiently introduce this tryptophan substitution in cultured cells. Overall, our findings uncover a simple and reliable prognostic distinction between benign and pathogenic CTDs and provide proof-of-concept for an editing strategy that potentially corrects all disease-causing CTD mutations.},
}
@article {pmid40928467,
year = {2025},
author = {Rijal, S and Standage-Beier, K and Zhang, R and Stone, A and Youssef, A and Wang, X and Tian, XJ},
title = {CRISPRi-Linked Multimodule Negative Feedback Loops to Address Winner-Take-All Resource Competition.},
journal = {ACS synthetic biology},
volume = {14},
number = {9},
pages = {3646-3654},
doi = {10.1021/acssynbio.5c00394},
pmid = {40928467},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Regulatory Networks/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Feedback, Physiological ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Synthetic Biology/methods ; },
abstract = {Cellular resource limitations create unintended interactions among synthetic gene circuit modules, compromising circuit modularity. This challenge is particularly pronounced in circuits with positive feedback, where uneven resource allocation can lead to Winner-Takes-All (WTA) behavior, favoring one module at the expense of others. In this study, we experimentally implemented a Negatively Competitive Regulatory (NCR) controller using CRISPR interference (CRISPRi) and evaluated its effectiveness in mitigating WTA behavior in two gene circuits: dual self-activation and cascading bistable switch. We chromosomally integrated a tunable dCas9 gene and designed module-specific gRNAs, with each module encoding its own gRNA to self-repress via competition for limited dCas9. This configuration introduces strong negative feedback to the more active module while reallocating resources to the less active one, promoting balanced module activation. Compared to the control group lacking dCas9-mediated repression, the NCR controller significantly increased module coactivation and suppressed WTA behavior. Our quantitative results demonstrate that NCR provides an effective strategy for regulating resource competition and improving the modularity of synthetic gene circuits.},
}
@article {pmid40824237,
year = {2025},
author = {Holland, KL and Blancher, I and McKesey, M and Silas, M and Gandhi, S and Nickerson, A and Jackson, K and Blazeck, J},
title = {RNA Polymerase III Promoters Compatible with CRISPR Gene Regulation in Saccharomyces cerevisiae.},
journal = {ACS synthetic biology},
volume = {14},
number = {9},
pages = {3387-3400},
doi = {10.1021/acssynbio.5c00122},
pmid = {40824237},
issn = {2161-5063},
mesh = {*RNA Polymerase III/genetics/metabolism ; *Saccharomyces cerevisiae/genetics ; *Promoter Regions, Genetic/genetics ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Fungal ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Kluyveromyces/genetics ; },
abstract = {Saccharomyces cerevisiaeis a model organism commonly used to study gene regulation and function recently via CRISPR-(d)Cas9 technologies. Modulating the expression of multiple gene targets simultaneously is often important for synthetic biology and metabolic engineering applications and is crucial for genetic interaction studies. CRISPR-based systems can be used to target multiple genetic loci via expression of multiple single-guide RNAs (sgRNAs) in a single cell. However, there are currently a limited number of well-characterized RNA polymerase III (Pol III) promoters (e.g., pSNR52) for sgRNA expression in S. cerevisiae. Herein, we characterize 20 RNA Pol III promoters from different yeast species, from S. cerevisiae itself or from mammals, for their utility toward effectively mediating CRISPR activation and repression in S. cerevisiae. We show that the Pol III promoter cross-species functionality is impacted by promoter architecture and inclusion of core sequence motifs and that scaffold-mediated recruitment of multiple effectors can rescue poor promoter function in some contexts. Also, we highlight two Kluyveromyces lactis Pol III promoters that mediate CRISPR function as well as the gold standard S. cerevisiae pSNR52 and previously described tRNA promoters. Finally, we show that these non-native promoters enable effective simultaneous CRISPR-mediated activation and repression of endogenous S. cerevisiae genes to enhance resistance to hydrogen peroxide. The Pol III promoters described here highlight the cross-species compatibility of genetic units in simple eukaryotes and will be useful for synthetic biology and phenotype engineering applications in yeast.},
}
@article {pmid40810600,
year = {2025},
author = {Zhang, H and Khoury, LR and Xu, P},
title = {Multiplexed Genome Editing and Transcriptional Knockdown in Yarrowia lipolytica by CRISPR-Cpf1 and an Orthogonal T7 System.},
journal = {ACS synthetic biology},
volume = {14},
number = {9},
pages = {3377-3386},
doi = {10.1021/acssynbio.5c00104},
pmid = {40810600},
issn = {2161-5063},
mesh = {*Yarrowia/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; *Gene Knockdown Techniques/methods ; Transcription, Genetic ; Bacteriophage T7/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Yarrowia lipolytica, a nonconventional yeast, has become an industrial workhorse to synthesize valuable compounds, including lipids, oleochemicals, and nutraceuticals. While the synthetic biology toolkits to engineer the genome and endogenous metabolic pathways are not as developed as Baker's yeast, it has emerged as a promising microbial host for industrial applications. In this study, we examined the multiplexed editing capability of the CRISPR-AsCpf1 coupled with gRNAs generated from either a yeast native promoter or an orthogonal T7 promoter, which yielded 73.3% editing efficiency for up to four target genes and 100% editing efficiency for two genes. We also attempted two strategies to enhance homology-directed recombination (HDR) efficiency; only minor improvements were observed. We further demonstrated that CRISPR-dAsCpf1 with T7-driven gRNA achieved significant gene knockdown compared to the CRISPR-RfxCas13d system. Its knockdown efficiency was comparable to that of an antisense T7 promoter system. Taken together, this work provides a facile toolkit that enables efficient and multiplexed genome editing and transcriptional knockdown of critical genes by combining CRISPR-Cpf1 with an orthogonal T7 transcription system in Y. lipolytica.},
}
@article {pmid40492356,
year = {2025},
author = {Sha, R and Qin, H and Yuan, A and Deng, Z and Liao, W and Qu, G and Wen, B and Xie, W and Peng, H},
title = {Cellular Inflammation-Induced Cleavage of Phosphorothioate DNA Locker Activates CRISPR/Cas9 Regulator for Gene Editing.},
journal = {Chemistry, an Asian journal},
volume = {20},
number = {17},
pages = {e01696},
doi = {10.1002/asia.202401696},
pmid = {40492356},
issn = {1861-471X},
support = {2023YFA0915102//National Key Research and Development Program of China/ ; XDB0750100//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; 22276199//National Natural Science Foundation of China/ ; 22206201//Youth Fund from National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; HeLa Cells ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Inflammation/metabolism ; *Phosphorothioate Oligonucleotides/chemistry/metabolism ; *DNA/chemistry/metabolism/genetics ; Green Fluorescent Proteins/genetics ; },
abstract = {The CRISPR technology is a highly promising strategy for developing a versatile toolbox to engineer genetic circuits. However, achieving precise and specific control over the activity of the CRISPR/Cas9 system in response to intracellular processes remains a challenging endeavor. In this study, we present a cellular inflammation-induced activation of an engineered CRISPR/Cas9 regulator for gene regulation. A phosphorothioate (PS)-modified DNA sequence, referred as the "locker," is employed to deactivate single guide RNA (sgRNA), whose locker sequence complements the spacer region of sgRNA. In the presence of myeloperoxidase during cellular inflammation, a halogenation process is triggered, leading to the generation of HClO, specifically cleaving the PS site of locker and activating CRISPR/Cas9 for gene editing. The target GFP gene has been successfully edited, downregulating the GFP protein expression in HeLa cells. This study provides valuable insights into the CRISPR-based gene regulation through specific endogenous processes.},
}
@article {pmid40394347,
year = {2025},
author = {Kumar, N},
title = {Genome Editing in Gynecological Oncology: The Emerging Role of CRISPR/Cas9 in Precision Cancer Therapy.},
journal = {Therapeutic innovation &amp; regulatory science},
volume = {59},
number = {5},
pages = {937-948},
pmid = {40394347},
issn = {2168-4804},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Female ; *Genital Neoplasms, Female/therapy/genetics ; *Precision Medicine/methods ; Genetic Therapy/methods ; },
abstract = {INTRODUCTION: Gynecological cancers, including cervical, ovarian, and endometrial cancers, represent a significant global health challenge due to their high prevalence and profound impact on mortality and quality of life. This narrative review explores the transformative capability of genome editing, specifically clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) technology, in advancing the management of these cancers. Genome editing offers great opportunities to develop targeted therapies by enabling precise modifications of genes involved in cancer initiation, progression, and chemoresistance.<br><br>METHODOLOGY: A comprehensive literature search was conducted from October 2004 to October 2024. Only peer-reviewed relevant English articles with substantial insights into the impact of genome editing on cancer therapies were considered using keywords such as "CRISPR/Cas9," "genome editing," "gynecological cancers," and specific cancer types like "cervical cancer," "ovarian cancer," and "endometrial cancer."<br><br>CONCLUSION: Genome editing, particularly CRISPR/Cas9, holds substantial capacity for revolutionizing the treatment landscape of gynecological cancers by enabling highly specific, gene-targeted therapies that can overcome conventional treatment limitations such as chemoresistance and tumor recurrence. Emerging preclinical studies demonstrate the feasibility of correcting oncogenic mutations and enhancing the sensitivity of tumors to existing therapies. However, before clinical translation can be realized, critical challenges-including off-target effects, delivery system optimization, and immune responses-must be systematically addressed through rigorous research and clinical trials. Advancing these solutions will be essential for safely integrating CRISPR-based interventions into personalized medicine approaches for gynecological malignancies.},
}
@article {pmid40966498,
year = {2025},
author = {Xiao, Y and Chen, J and Hou, X and Wang, H and Zhang, K and Xu, S and Jiang, T and Huo, Y and Zhang, F and Gu, L},
title = {A NanoLock-enabled, Craspase-based strategy for highly sensitive RNA detection.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
doi = {10.1093/nar/gkaf907},
pmid = {40966498},
issn = {1362-4962},
support = {2024YFC27007400//Key Technologies Research and Development Program/ ; 2020CXGC011305//Key Technology Research and Development Program of Shandong Province/ ; 32371276//National Natural Science Foundation of China/ ; 32400148//National Natural Science Foundation of China/ ; ts20220107//Instrument Improvement Funds of Shandong University Public Technology Platform/ ; XDB37010301//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
mesh = {Humans ; *SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *RNA, Viral/genetics/analysis ; *COVID-19/diagnosis/virology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Limit of Detection ; *Caspases/genetics/metabolism ; },
abstract = {Rapid and sensitive detection of RNA is important in fields such as biomedical research and clinical diagnostics. However, current methods typically involve an amplification process, require substantial time, and are susceptible to aerosol contamination. Herein, we introduce a NanoLock-powered, amplification-free assay based on the type III-E clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated system for rapid, highly sensitive, and specific RNA diagnostics. This innovative platform, designated CRISPR-guided caspase (Craspase)-NanoLock-Csx30 (CNC), harmoniously integrates the precise protease activity of Craspase with the remarkable luminescent sensitivity of NanoLock, creating a novel and streamlined approach for RNA detection. The CNC platform exhibited exceptional sensitivity in detecting severe acute respiratory syndrome coronavirus-2 N gene RNA through the integration of three guide RNAs, achieving a detection limit of 250 fM in just 10 min without amplification. Preliminary studies further revealed the platform's extended diagnostic potential for detecting influenza A virus and human immunodeficiency virus. These findings collectively establish the CNC platform as an appealing tool for infectious disease detection and significantly broaden the scope of CRISPR-based diagnostic applications.},
}
@article {pmid40966253,
year = {2025},
author = {Takahashi, M and Nashimoto, M},
title = {Cleavage of MALAT1 RNA by 14-nt sgRNA-guided tRNase ZL.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0318968},
doi = {10.1371/journal.pone.0318968},
pmid = {40966253},
issn = {1932-6203},
mesh = {*RNA, Long Noncoding/genetics/metabolism ; Humans ; *Endoribonucleases/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Silencing ; Cell Line, Tumor ; *RNA Cleavage ; },
abstract = {We have been developing a gene suppression technology, tRNase ZL-utilizing efficacious (TRUE) gene silencing, in which artificially designed small guide RNA (sgRNA) guides tRNase ZL to cleave cellular target RNA. In this study, we examined 14-nt linear-type sgRNAs, which are fully 2'-O-methylated and have full phosphorothioate linkages, for their ability to suppress a level of a nuclear-localized long non-coding RNA, Metastasis Associated Lung Adenocarcinoma Transcript 1 (MALAT1). The MALAT1 RNA is implied to be involved in stress responses and diseases including cancers. Specifically, we designed six 14-nt linear-type sgRNAs, sgRM1 - sgRM6 that target the human MALAT1 RNA. sgRM1, sgRM2 and sgRM6 suppressed the MALAT1 RNA level, while the other sgRNAs showed little effect. In order to demonstrate that the suppression effect of sgRM1, sgRM2 and sgRM6 on the MALAT1 RNA level is caused by TRUE gene silencing, we performed in vitro tRNase ZL cleavage assay, microscopic analysis for nuclear existence of sgRNA, and tRNase ZL knockdown experiment. For the in vitro tRNase ZL cleavage assay, three 30-nt MALAT1 RNA fragments, TM1, TM2 and TM6 were prepared, which were RNA targets for sgRM1, sgRM2 and sgRM6, respectively. All of the sgRNAs guided recombinant tRNase ZL in vitro to cleave their own targets, although the cleavage efficiency changed depending on target/sgRNA pairs. By fluorescence microscopy, a 14-nt 5'-Alexa568-labeled sgRNA released from liposome was observed to be distributed ubiquitously in A549 cells with higher density in the nucleus, where both the target MALAT1 RNA and tRNase ZL exist. Knockdown of tRNase ZL by siRNA attenuated the suppression effect of sgRM1, sgRM2 and sgRM6 on the MALAT1 RNA level. We also demonstrated that the effective sgRNAs sgRM1, sgRM2 and sgRM6 reduce A549 cell viability.},
}
@article {pmid40964963,
year = {2025},
author = {Shigeta, M and Inoue, KI and Shimada, N and Tobe, A and Abe, T and Kiyonari, H},
title = {Generation of Knock-In Syrian Hamsters via Zygote Microinjection Using CRISPR/Cas9 Genome Editing.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {63},
number = {5},
pages = {e70027},
doi = {10.1002/dvg.70027},
pmid = {40964963},
issn = {1526-968X},
support = {//RIKEN Intramural/ ; JP20H05767//JSPS KAKENHI/ ; JP23H04945//JSPS KAKENHI/ ; JP23H04939//JSPS KAKENHI/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Mesocricetus/genetics ; *Gene Editing/methods ; *Zygote/metabolism ; *Gene Knock-In Techniques/methods ; Microinjections/methods ; Cricetinae ; Female ; Male ; },
abstract = {Syrian hamsters (Mesocricetus auratus) have long served as valuable model organisms in diverse research fields such as oncology, immunology, and physiology owing to their unique biological and pathological characteristics. Although embryo manipulation techniques such as embryo collection, pronuclear microinjection, and embryo transfer have been established, gene knock-in (KI) hamsters have not yet been reported. Here, we report the successful generation of gene KI Syrian hamsters by microinjecting CRISPR/Cas9 components and plasmid DNA into pronuclear-stage zygotes. Targeted insertion of a DNA cassette up to 8 kb was achieved at the ROSA26 orthologous locus and other genomic sites. Importantly, we confirmed functional expression of a reporter cassette inserted at the ROSA26 site, providing evidence of transcriptional activity at this locus in Syrian hamsters. Furthermore, we demonstrated that frozen-thawed KI embryos could give rise to live offspring using a simplified freezing and thawing protocol originally developed for mice and rats. These results confirm the feasibility and applicability of advanced genome editing technologies in Syrian hamsters. These technological advancements enable the development of versatile KI models for applications such as gene expression monitoring and conditional mutagenesis, thereby expanding the utility of Syrian hamsters as model organisms, comparable to mice and rats.},
}
@article {pmid40963829,
year = {2025},
author = {Ton, LB and Qayyum, Z and Amas, J and Thomas, WJW and Edwards, D and Batley, J and Dolatabadian, A},
title = {Applications of CRISPR/Cas tools in improving stress tolerance in Brassica crops.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1616526},
pmid = {40963829},
issn = {1664-462X},
abstract = {Brassica species, which include economically important Brassica crops grown around the globe, are important as popular vegetables, forage, and oilseed crops, supplying food for humans and animals. Despite their importance, these crops face increasing challenges from biotic and abiotic stresses, exacerbated by climate change and the evolving threat of crop pathogens. Enhancing crop resilience against these stresses has become a key priority to ensure stable crop production. Recent advancements in genomic studies on Brassica crops and their pathogens have facilitated the deployment of CRISPR/Cas systems in breeding major Brassica crops. This review highlights recent progress in CRISPR/Cas-based gene editing technologies to improve resistance to pathogens and enhance tolerance to drought, salinity, and extreme temperatures. It also summarises the molecular mechanisms underlying crop responses to these stresses. Furthermore, the review discusses the workflow for employing the CRISPR/Cas system to boost stress tolerance and resistance, outlines the associated challenges, and explores prospects based on gene editing research in Brassica species.},
}
@article {pmid40963206,
year = {2025},
author = {Nie, YF and Yue, SJ and Huang, P and Hu, DK and Xu, Z and Aguilar-Vera, A and Carreri, JU and Zhang, XH and Hu, HB},
title = {Harnessing the Endogenous Type I-F CRISPR/Cas System for Efficient Genome Engineering and Gene Repression in Pseudomonas chlororaphis LX24.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00371},
pmid = {40963206},
issn = {2161-5063},
abstract = {Pseudomonas chlororaphis, a nonpathogenic plant growth-promoting rhizobacterium, holds immense potential for agricultural and industrial applications due to its ability to biosynthesize bioactive metabolites. However, the lack of efficient genetic tools has hindered its metabolic engineering. In this study, we first characterized an endogenous type I-F CRISPR/Cas system in P. chlororaphis LX24 and established a programmable genome editing toolkit based on this system. Concurrently, the plasmid transformation efficiency of P. chlororaphis LX24 was enhanced by identifying and deleting the restriction-modification systems. We further demonstrated the DNA interference capability with different PAM sequences of the type I-F CRISPR/Cas system, which also exhibited various editing efficiencies ranging from 22 to 87% in P. chlororaphis LX24. By introducing the λ-Red recombination system, the knockout efficiency of the phenazine cluster (8.3 kb) increased by over 9-fold. Next, introducing the sacB-based counterselection marker achieved a 100% plasmid curing success within 36 h. The optimized toolkit was further applied to single-step gene insertion and replacement with 100% success rates. Additionally, we established a CRISPR interference (CRISPRi) system for transcriptional repression in P. chlororaphis LX24 by knocking out nuclease Cas3. Through modulating the induction time and concentration of IPTG, the production of phenazines was reduced to 21-89% within 24 h in P. chlororaphis LX24. Overall, our work developed a convenient and precise genetic tool for the P. chlororaphis LX24, and the methods may also provide a reference for repurposing endogenous CRISPR systems in non-model prokaryotes.},
}
@article {pmid40962795,
year = {2025},
author = {Huang, Z and Song, Z and Zeng, J and Liu, X and Fang, M and Wu, Z and Zhao, Y and Chen, Y and Li, D and Huang, H and Fu, L and Xu, P and Ning, B and Chen, J and Guan, M and Sun, L and Lyon, CJ and Fan, XY and Lu, S and Hu, T},
title = {Sensitive pathogen DNA detection by a multi-guide RNA Cas12a assay favoring trans- versus cis-cleavage.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8257},
pmid = {40962795},
issn = {2041-1723},
support = {R01HD090927, R01HD103511//U.S. Department of Health &amp; Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; R01AI144168, R01AI175618, R01AI173021, R01AI174964, R01AI177986, R01AI179714, R21AI169582-01A1//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; W8IXWH1910026//U.S. Department of Defense (United States Department of Defense)/ ; Weatherhead Presidential Endowment fund//Tulane University/ ; 82302614//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *Mycobacterium tuberculosis/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Sensitivity and Specificity ; *DNA, Bacterial/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics ; *Molecular Diagnostic Techniques/methods ; Adult ; Feces/microbiology ; Tuberculosis, Meningeal/diagnosis/microbiology/cerebrospinal fluid ; Child ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Most CRISPR assays lack clinical utility due to their complex workflows and limited validation. Here we present a streamlined "one-pot" asymmetric CRISPR tuberculosis assay that attenuates amplicon degradation to achieve 5 copies/μL sensitivity within 60 min and detect positive patient samples within 15 min. This assay exhibited 93%, 83%, and 93% sensitivity with adult respiratory, pediatric stool, and adult cerebral spinal fluid specimens, and detected 64% of clinically diagnosed tuberculous meningitis cases, in a cohort of 603 clinical samples. This assay achieves complete specificity and greater sensitivity (74% vs. 56%) than the most sensitive reference test with prospectively collected tongue swabs, and exhibits similar performance when adapted to a lateral flow assay format and employed to analyze self-collected tongue swabs. These results demonstrate the utility of this approach across diverse specimen types, including those suitable for use in remote and resource-limited settings, to improve access to molecular diagnostics.},
}
@article {pmid40962474,
year = {2025},
author = {Gao, YH and Deng, JJ and Xiao, X and Pan, LY and He, MD and Zhang, YB},
title = {Role of ppp6r3 in zebrafish gonadal differentiation and gametogenesis.},
journal = {Yi chuan = Hereditas},
volume = {47},
number = {9},
pages = {1023-1031},
doi = {10.16288/j.yczz.25-093},
pmid = {40962474},
issn = {0253-9772},
mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Gametogenesis/genetics ; Male ; *Zebrafish Proteins/genetics/metabolism ; Female ; *Phosphoprotein Phosphatases/genetics/metabolism ; *Gonads/growth &amp; development/metabolism/cytology ; Cell Differentiation/genetics ; Mutation ; Spermatogenesis/genetics ; Testis/growth &amp; development/metabolism ; CRISPR-Cas Systems ; },
abstract = {The gonad differentiation and gametogenesis of fish is regulated by various factors. Protein phosphatase (PP) have the function of catalyzing the dephosphorylation of proteins in organisms. As a member of the protein phosphatase family, protein phosphatase type 6 (PP6) plays an important role in gonadal development and gametogenesis. The role of ppp6r3, which encodes the regulatory subunit 3 of protein phosphatase 6, in gonadal differentiation and gametogenesis is still unclear. In this study, two zebrafish ppp6r3 mutant lines were constructed using CRISPR/Cas9 technology. It was found that the absence of ppp6r3 leads to the development of only male zebrafish, and these mutants are incapable of fertilizing wild-type eggs. Further investigations revealed that in the testes of ppp6r3 mutants, the transition of spermatocytes to mature sperm was blocked, disrupting spermatogenesis. In summary, this study established a ppp6r3 mutant model, exhibiting defects in gonadal differentiation and gametogenesis, which provides a new model for further research on the mechanisms by which Ppp6r3 regulates germ cell proliferation, differentiation, and sex determination.},
}
@article {pmid40961787,
year = {2025},
author = {Zhang, H and Wu, Y and Liu, D and Feng, S and Xuan, X and Dong, G and Cheng, J and Qin, Y and Ngo, HH},
title = {Insights into microalgal biotechnology: Current applications, key challenges, and future prospects.},
journal = {Journal of environmental management},
volume = {394},
number = {},
pages = {127263},
doi = {10.1016/j.jenvman.2025.127263},
pmid = {40961787},
issn = {1095-8630},
abstract = {Microalgae have emerged as multifunctional biofactories capable of simultaneously supporting carbon capture, renewable energy production, environmental remediation, and the synthesis of high value bioproducts. Despite this promise, large-scale deployment remains limited by techno-economic barriers, particularly the high costs of biomass harvesting and dewatering. Recent advances including bioflocculation, magnetic separation, and solar-assisted drying are helping to reduce energy inputs and enhance feasibility. In parallel, breakthroughs in synthetic biology, such as CRISPR/Cas genome editing, are enabling the development of engineered strains with enhanced lipid, carbohydrate, and hydrogen productivity. Innovations in photobioreactor design have further improved light-use efficiency, reduced contamination risks, and supported high-density cultivation. Life cycle assessments indicate that integrating microalgal systems with flue gas utilization and wastewater treatment can substantially lower freshwater use and greenhouse gas emissions. To unlock the full potential of this technology, future efforts should prioritize modular biorefinery systems, intelligent process control, and supportive policy frameworks that incentivise negative-emission technologies. These integrated strategies can help position microalgae as a key enabler of a sustainable, circular bioeconomy.},
}
@article {pmid40959562,
year = {2025},
author = {Tang, S and Chen, X and Tong, X and Zhu, L},
title = {Overcoming the Delivery Challenges in CRISPR/Cas9 Gene Editing for Effective Cancer Treatment: A Review of Delivery Systems.},
journal = {International journal of medical sciences},
volume = {22},
number = {14},
pages = {3625-3649},
pmid = {40959562},
issn = {1449-1907},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Neoplasms/therapy/genetics ; *Genetic Therapy/methods ; Animals ; Genetic Vectors/genetics/administration &amp; dosage ; *Gene Transfer Techniques ; Extracellular Vesicles/genetics ; },
abstract = {Therapeutic strategies based on gene editing provide the ability to modify faulty genes contributing to the development of diseases such as cancer by directly altering the cellular machinery. The clustered regularly interspaced short palindromic repeats associated nuclease 9 (CRISPR/Cas9) system is currently the primary tool used for gene editing. Several effective Cas9 variants have already been established to address the complex genetic modifications that arise during diseases. Although gene-editing systems have made significant advancements, a primary obstacle that requires attention is the transportation of CRISPR/Cas to diverse target cells, both in vivo and in vitro, to render them suitable for clinical implementation. Various strategies can be utilized to facilitate the transportation of the CRISPR/Cas systems into mammalian cells. Herein, we reviewed contemporary research about delivery systems for gene-editing systems that interact effectively in biological systems. This review explores the benefits and drawbacks of using extracellular vesicles and viral vectors as vehicles for delivering the CRISPR/Cas system in the context of cancer treatment.},
}
@article {pmid40938937,
year = {2025},
author = {Hossain, MF and Popsuj, S and Vitrinel, B and Kaplan, NA and Stolfi, A and Christiaen, L and Ruggiu, M},
title = {Neuron-specific Agrin splicing by Nova RNA-binding proteins regulates conserved neuromuscular junction development in chordates.},
journal = {PLoS biology},
volume = {23},
number = {9},
pages = {e3003392},
doi = {10.1371/journal.pbio.3003392},
pmid = {40938937},
issn = {1545-7885},
support = {R01 HD096770/HD/NICHD NIH HHS/United States ; R01 GM096032/GM/NIGMS NIH HHS/United States ; R01 HD104825/HD/NICHD NIH HHS/United States ; R15 GM119099/GM/NIGMS NIH HHS/United States ; R01 HL108643/HL/NHLBI NIH HHS/United States ; R35 GM158421/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Neuromuscular Junction/metabolism/growth &amp; development ; *Agrin/genetics/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; Alternative Splicing ; Neuro-Oncological Ventral Antigen ; *Nerve Tissue Proteins/metabolism/genetics ; Motor Neurons/metabolism ; Receptors, Cholinergic/metabolism ; Neurons/metabolism ; Humans ; CRISPR-Cas Systems ; },
abstract = {In mammals, neuromuscular synapses rely on clustering of acetylcholine receptors (AChRs) in the muscle plasma membrane, ensuring optimal stimulation by motor neuron-released acetylcholine neurotransmitter. This clustering depends on a complex pathway based on alternative splicing of Agrin pre-mRNAs by the RNA-binding proteins Nova1/2. Neuron-specific expression of Nova1/2 ensures the inclusion of small "Z" exons in Agrin, resulting in a neural-specific form of this extracellular proteoglycan carrying a short peptide motif that is required for binding to Lrp4 receptors on the muscle side, which in turn stimulate AChR clustering. Here we show that this intricate pathway is remarkably conserved in Ciona robusta, a non-vertebrate chordate in the tunicate subphylum. We use in vivo tissue-specific CRISPR/Cas9-mediated mutagenesis and heterologous "minigene" alternative splicing assays in cultured mammalian cells to show that Ciona Nova is also necessary and sufficient for Agrin Z exon inclusion and downstream Lrp4-mediated AChR clustering. We present evidence that, although the overall pathway is well conserved, there are unexpected differences in Nova structure-function between Ciona and mammals. We further show that, in Ciona motor neurons, the transcription factor Ebf is a key activator of Nova expression, thus ultimately linking this RNA switch to a conserved, motor neuron-specific transcriptional regulatory network.},
}
@article {pmid40889206,
year = {2025},
author = {Li, T and Wang, Y and Zhang, X and Wu, Z and Zhang, L},
title = {CRISPR-SDA: an integrated isothermal amplification and CRISPR-Cas12a biosensing platform for sensitive detection of microRNA-21.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {36},
pages = {7266-7271},
doi = {10.1039/d5ay01227a},
pmid = {40889206},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis/blood ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Humans ; Limit of Detection ; Animals ; Cattle ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {MicroRNA-21 (miRNA-21), a critical oncogenic biomarker, poses detection challenges due to low abundance and limitations of conventional methods. Herein, we developed a novel CRISPR-SDA biosensing platform by integrating strand displacement amplification (SDA) with CRISPR-Cas12a, leveraging SDA's efficient isothermal amplification of miRNA-21 and Cas12a's precise target recognition and trans-cleavage activity for signal amplification. Optimized conditions achieved high sensitivity with a detection limit of 10.1 fM and segmented linear ranges of 0.05-25 pM and 25-500 pM. It showed excellent selectivity against other miRNAs and anti-interference in complex matrices (e.g., salmon sperm DNA). Spike recovery experiments in fetal bovine serum yielded recoveries of 92.0 ± 7.8% to 105.6 ± 3.3%, confirming its reliability in complex biological samples. This CRISPR-SDA platform overcomes the drawbacks of conventional methods, enabling rapid, sensitive, and equipment-friendly detection. It holds great potential for early cancer diagnosis and point-of-care testing and provides a versatile framework for detecting other disease-associated nucleic acids.},
}
@article {pmid40865701,
year = {2025},
author = {Gao, J and Li, W and Li, M and Ji, G and Liu, Z},
title = {Cd36 deficiency enhances the immune defence against Grass Carp Reovirus in zebrafish.},
journal = {Developmental and comparative immunology},
volume = {170},
number = {},
pages = {105446},
doi = {10.1016/j.dci.2025.105446},
pmid = {40865701},
issn = {1879-0089},
mesh = {Animals ; *Zebrafish/immunology/virology/genetics ; *Reoviridae/immunology/physiology ; *CD36 Antigens/genetics/metabolism ; *Reoviridae Infections/immunology ; *Fish Diseases/immunology/virology ; Immunity, Innate ; CRISPR-Cas Systems/genetics ; *Zebrafish Proteins/genetics/metabolism ; Carps/immunology ; Transcriptome ; Liver ; Gene Expression Profiling ; Viral Load ; },
abstract = {CD36, a single-chain transmembrane glycoprotein belonging to the class B scavenger receptor family, remains enigmatic in terms of its antiviral immune function, with some conflicting conclusions. This study aims to elucidate the role of Cd36 in antiviral immunity using zebrafish as a model organism. We generated cd36[-/-] zebrafish mutants using CRISPR/Cas9 technology and challenged them with Grass Carp Reovirus (GCRV), a dsRNA virus. Compared to wild-type (WT) zebrafish, cd36[-/-] zebrafish exhibited stronger antiviral immunity, reduced viral load, and lower mortality. Transcriptome sequencing analysis of liver tissue revealed that the differentially expressed genes (DEGs) between WT and cd36[-/-] zebrafish were primarily enriched in metabolic pathways, such as glycan biosynthesis and fatty acid metabolism. This suggests that Cd36 may modulate antiviral immune responses by influencing metabolic activities. Our findings provide a foundation for further investigations into the functional and molecular mechanisms of Cd36 in antiviral immunity in fish.},
}
@article {pmid40959550,
year = {2025},
author = {Mahto, RK and B S, C and Singh, RK and Kumar, A and Kumar, S and Yadav, R and Dey, D and Hamwieh, A and Kumar, R},
title = {Symbiotic nitrogen fixation for sustainable chickpea yield and prospects for genome editing in changing climatic situations.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1621191},
pmid = {40959550},
issn = {1664-462X},
abstract = {Chickpea (Cicer arietinum L.) is a vital/essential legume crop valued for its nutritional, agricultural, and economic importance, with a relatively large genome size of approximately 738 megabases. Chickpea roots establish symbiotic relationships with soil microorganisms, resulting in the formation of root nodules essential for biological nitrogen fixation. In this study, 20 chickpea genotypes were selected from a genome-wide association panel to assess nodulation traits under eight different treatment combinations involving biofertilizers (Rhizobium, vesicular-arbuscular mycorrhiza - VAM) and inorganic fertilizers (NPK) using a randomized block design with three replications. Pre-planting soil preparation included the application of fertilizers and biofertilizers. Comprehensive analyses including descriptive statistics, correlation, path analysis, principal component analysis, agglomerative hierarchical clustering, and gene expression studies were conducted. Among treatments, the NPK+Rhizobium combination significantly enhanced nodulation across genotypes, while the Rhizobium+VAM (T7) treatment identified ICC-9085 as a superior donor for the number of nodules, aiming for sustainable chickpea productivity. Gene expression profiling through qRT-PCR revealed that the RZ+VAM treatment notably upregulated several key genes, including CaNFP, GST, Leghemoglobin, Nodulin6, and CaLYK3, with CaNFP emerging as a pivotal regulator of nodulation. The marked upregulation of CaNFP underlines its potential as a target for enhancing symbiotic efficiency. The availability of the chickpea draft genome opens new avenues for employing genome editing tools such as CRISPR/Cas systems. Targeted editing of the CaNFP gene offers a promising strategy to improve nodule formation, nitrogen fixation, and overall plant vigor. Integrating CaNFP gene through genome editing with potential genotypes and use of microbial treatments can accelerate the development of elite chickpea cultivars, enhancing productivity while reducing reliance on chemical fertilizers and supporting sustainable agricultural practices.},
}
@article {pmid40959136,
year = {2025},
author = {Zhou, D and Yu, H and Shao, Y and Gao, C and Xia, C and Qi, Y},
title = {Rapid and accurate detection method for bluetongue virus based on CRISPR-Cas13a combined with RT-ERA.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1621012},
pmid = {40959136},
issn = {2235-2988},
mesh = {*Bluetongue virus/isolation &amp; purification/genetics ; Animals ; *Bluetongue/diagnosis/virology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sheep ; RNA, Viral/genetics ; },
abstract = {INTRODUCTION: Bluetongue virus (BTV), a vector-borne pathogen of domestic and wild ruminants, poses substantial global threats to livestock health and trade. Conventional detection methods, such as RT-qPCR, remain constrained by reliance on specialized equipment and prolonged turnaround times, limiting their utility in field settings.<br><br>METHODS: To overcome these challenges, we developed an integrated isothermal amplification-CRISPR detection platform-Reverse Transcription-Enzymatic Recombinase Amplification coupled with CRISPR-Cas13a (RT-ERA/CRISPR-Cas13a)-enabling rapid, sensitive, specific and visual pan-serotype detection of BTV.<br><br>RESULTS: The assay demonstrated a sensitivity of 20 RNA copies/reaction within 55 min using three readout modalities: fluorescence values, visual fluorescence signals, and lateral flow test strips. Specificity evaluation revealed no cross-reactivity with 9 non-target pathogens, including epidemiologically significant viruses such as EHDV, AKAV, and CHUV. Clinical validation using 263 field samples demonstrated that RT-ERA/CRISPR-Cas13a achieved clinical sensitivities of 100%, 100%, and 96% with fluorescence values, fluorescence signals, and lateral flow strips, respectively, while maintaining 100% clinical specificity via all modalities. Field adaptation using Nucleic Acid Release Reagent (NARR) simplified crude sample processing, delivering 97% clinical sensitivity and 100% clinical specificity even in the presence of inhibitors from unpurified samples.<br><br>CONCLUSION: This work represents the first CRISPR-Cas13a-based platform for pan-serotype BTV detection, combining portability, cost-efficiency, and detective accuracy suitable for point-of-care deployments. By bridging the gap between high laboratory sensitivity and practical field applicability, this system enables real-time BTV surveillance and facilitates timely outbreak containment in resource-constrained agricultural and veterinary settings.},
}
@article {pmid40957902,
year = {2025},
author = {Ekins, TG and Rybicki-Kler, C and Deng, T and Brooks, IAW and Jedrasiak-Cape, I and Donoho, E and Ahmed, OJ},
title = {Psychedelic neuroplasticity of cortical neurons lacking 5-HT2A receptors.},
journal = {Molecular psychiatry},
volume = {},
number = {},
pages = {},
pmid = {40957902},
issn = {1476-5578},
support = {R01MH129282//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R34NS127101//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; P50NS123067//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; AARG-NTF-21-846572//Alzheimer&apos;s Association/ ; T32DA007268//U.S. Department of Health &amp; Human Services | NIH | National Institute on Drug Abuse (NIDA)/ ; T32DC000011//U.S. Department of Health &amp; Human Services | NIH | National Institute on Deafness and Other Communication Disorders (NIDCD)/ ; T32DC000011//U.S. Department of Health &amp; Human Services | NIH | National Institute on Deafness and Other Communication Disorders (NIDCD)/ ; },
abstract = {Classical psychedelic drugs show promise as a treatment for major depressive disorder and related psychiatric disorders. This therapeutic efficacy stems from long-lasting psychedelic-induced neuroplasticity onto prefrontal cortical neurons and is thought to require the postsynaptic expression of serotonin 2A receptors (5-HT2AR). However, other cortical regions such as the granular retrosplenial cortex (RSG) - important for memory, spatial orientation, fear extinction, and imagining oneself in the future, but impaired in Alzheimer's disease - lack 5-HT2AR and are thus considered unlikely to benefit from psychedelic therapy. Here, we show that RSG pyramidal cells lacking postsynaptic 5-HT2A receptors still undergo long-lasting psychedelic-induced synaptic enhancement. A newly engineered CRISPR-Cas-based conditional knockout mouse line reveals that this form of psychedelic-induced retrosplenial plasticity requires presynaptic 5-HT2A receptors expressed on anterior thalamic axonal inputs to RSG. These results highlight a broader psychedelic therapeutic utility than currently appreciated, suggesting potential for augmenting RSG circuit function in Alzheimer's disease, post-traumatic stress disorder, and other neuropsychiatric conditions, despite the lack of postsynaptic 5-HT2A receptors.},
}
@article {pmid40956304,
year = {2025},
author = {Zhuang, Q and Xiao, C and Tang, S and Song, Y and Chen, F and Zhao, G},
title = {Single-Cas, Single-Reporter, and Time-Resolved CRISPR-Cas by Stoichiometry Coding of Multiplex crRNAs in a Single Tube for Brucella Species and ABO Genotype Identification.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c01034},
pmid = {40956304},
issn = {2379-3694},
abstract = {Genotyping is significant in identifying pathogen gene types and disease-related allele genes. Multiplex nucleic acid detection is convenient and useful for genotyping. Though CRISPR-Cas has great advantages in nucleic acid detection, multiplex detection strategies remain to be developed. Here, we propose a novel CRISPR-Cas strategy characterized by using stoichiometry coding of multiplex crRNAs. Different targets can be recognized by the corresponding crRNA with a certain amount, leading to distinguishable fluorescence difference. We employed this strategy to achieve Brucella species identification and human cell ABO blood genotyping. The detection can be conveniently visualized in a time-resolved way using a single tube, single Cas, and single reporter. The strategy can be employed in a PCR-coupled, RAA-coupled, or amplification-free way. The multiplex crRNA-coding CRISPR-Cas has potential application in differentiating gene types in various fields.},
}
@article {pmid40956103,
year = {2025},
author = {Babajanyan, SG and Garushyants, SK and Wolf, YI and Koonin, EV},
title = {Evolution of antivirus defense in prokaryotes, depending on the environmental virus prevalence and virome dynamics.},
journal = {mBio},
volume = {},
number = {},
pages = {e0240925},
doi = {10.1128/mbio.02409-25},
pmid = {40956103},
issn = {2150-7511},
abstract = {UNLABELLED: Prokaryotes can acquire antivirus immunity via two fundamentally distinct types of processes: direct interaction with the virus, as in clustered regularly interspaced short palindromic repeats (CRISPR)-Cas adaptive immunity systems, and horizontal gene transfer (HGT), which is the main route of transmission of innate immunity systems. These routes of defense evolution are not mutually exclusive and can operate concomitantly, but observations suggest that at least in some bacterial and archaeal species, one or the other route dominates the defense landscape. We hypothesized that the observed dichotomy stems from different life-history trade-offs characteristic of these organisms. To test this hypothesis, we analyzed a mathematical model of a well-mixed prokaryote population under a stochastically changing viral prevalence. Optimization of the long-term population growth rate reveals two contrasting modes of defense evolution. In stable, predictable environments, direct interaction with the virus is the optimal route of immunity acquisition. In fluctuating, unpredictable environments with a moderate viral prevalence, horizontal transfer of defense genes is preferred. In the HGT-dominant mode, we observed a universal distribution of the fraction of microbes with different immune repertoires. Under very low virus prevalence, the cost of immunity exceeds the benefits such that the optimal state of a prokaryote is complete absence of defense systems. By contrast, under very high virus prevalence, horizontal spread of defense systems dominates regardless of the stability of the virome. These findings might explain consistent but enigmatic patterns in the spread of antivirus defense systems among prokaryotes, such as the ubiquity of adaptive immunity in hyperthermophiles contrasting their patchy distribution among mesophiles.<br><br>IMPORTANCE: The virus-host arms race is a major component of the evolutionary process in all organisms that drove the evolution of a broad variety of immune mechanisms. In the last few years, over 200 distinct antivirus defense systems have been discovered in prokaryotes. There are two major modes of immunity acquisition: innate immune systems spread through microbial populations via HGT, whereas adaptive-type immune systems acquire immunity via direct interaction with the virus. We developed a mathematical model to explore the short-term evolution of prokaryotic immunity and showed that in stable environments with predictable viral repertoires, adaptive-type immunity is the optimal defense strategy, whereas in fluctuating environments with unpredictable virus composition, HGT dominates the immune landscape.},
}
@article {pmid40954158,
year = {2025},
author = {Gottschling, DC and Döring, F},
title = {The novel HRD motif kinase SPE-60 is required for sperm development and motility in Caenorhabditis elegans.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {32539},
pmid = {40954158},
issn = {2045-2322},
mesh = {*Caenorhabditis elegans/genetics/physiology/metabolism ; Male ; Animals ; *Sperm Motility/genetics ; *Caenorhabditis elegans Proteins/metabolism/genetics/chemistry ; *Spermatozoa/metabolism ; *Protein Serine-Threonine Kinases/metabolism/genetics ; *Spermatogenesis ; Amino Acid Motifs ; },
abstract = {Sexual reproduction requires sperm development, activation and motility. Many of these post-meiotic processes remain incompletely understood. We identified the gene spe-60, which encodes a tau-tubulin kinase with a conserved catalytic HRD motif. Localisation via immunostaining reveals that SPE-60 is present in sperm and dynamically colocalises with membranous organelles (MOs), that store factors essential for activation and fertility. spe-60 null mutants are sterile due to immotile sperm that undergo normal MO-plasma membrane fusion but fail to extend normal pseudopods essential for motility and fertilisation. Furthermore, mutant sperm exhibit arrested spermatocyte development. The functional loss observed in spe-60 null sperm is phenocopied by sperm carrying a CRISPR/Cas-mediated point mutation within the HRD motif. Genetic epistasis places SPE-60 downstream of the serine/threonine kinase SPE-6 in the canonical sperm activation pathway. Thus, SPE-60 is required for sperm development and motility, but not MO fusion. These findings suggest that the HRD-containing kinase SPE-60 fulfils a dual function as a key evolutionarily conserved factor.},
}
@article {pmid40938948,
year = {2025},
author = {He, W and Yang, Z and Wang, J and Yang, F and Li, N and Xu, R and Zeng, S and Xiao, L and Feng, Y and Guo, Y},
title = {Cryptosporidium parvum protease INS6 plays an important role in parasite proliferation and pathogenicity.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {9},
pages = {e0013532},
doi = {10.1371/journal.pntd.0013532},
pmid = {40938948},
issn = {1935-2735},
mesh = {*Cryptosporidium parvum/pathogenicity/genetics/enzymology/growth &amp; development ; Animals ; *Cryptosporidiosis/parasitology/pathology ; Mice ; *Protozoan Proteins/genetics/metabolism ; Humans ; Mice, Knockout ; Male ; Virulence ; *Peptide Hydrolases/genetics/metabolism ; CRISPR-Cas Systems ; Gene Deletion ; },
abstract = {BACKGROUND: Cryptosporidium parvum is a common protozoan pathogen responsible for moderate to severe diarrhea in humans and animals. Parasite invasion and egress cause damage to intestinal epithelial cells, which is mediated by a variety of secretory proteins from several unique organelles, such as micronemes. Previous spatial proteomic analysis has identified insulinase-like protease 6 (INS6) as a putative microneme protein in C. parvum. However, the functional contribution of INS6 to Cryptosporidium pathogenicity remains poorly characterized. In this study, we used genetic manipulation techniques to investigate the expression and biological functions of INS6 in C. parvum.<br><br>The INS6 gene was tagged and deleted in C. parvum using CRISPR/Cas9 technology. The expression of INS6 was determined by immunofluorescence analysis, ultrastructure-expansion microscopy, and immunoelectron microscopy. Endogenous labelling showed low levels of INS6 expression, which is found in C. parvum micronemes and is absent during the male gamont stage. The effect of INS6 deletion on parasite growth and pathogenicity was assessed in vitro using HCT-8 cultures and in vivo by infection of interferon-&#x3b3; knockout mice. Deletion of the INS6 gene impaired C. parvum proliferation in vitro and significantly reduced the parasite burden in infected mice. In addition, mice infected with the &#x394;ins6 strain showed a significant reduction in the intestinal villus-to-crypt ratio, attenuated body weight loss and increased survival rates, compared to those infected with the INS6-3HA strain.<br><br>CONCLUSIONS/SIGNIFICANCE: These results indicate that INS6 protein is involved in C. parvum proliferation and plays a critical role in modulating the pathogenicity of this zoonotic parasite. Deletion of this gene affects the invasion efficiency and pathogenicity of the parasite.},
}
@article {pmid40907495,
year = {2025},
author = {Bayraktar, E and Rodriguez-Aguayo, C and Stur, E and Kumar, S and Mangala, LS and Jennings, NB and Bayram, NN and Corvigno, S and Asare, A and Ivan, C and Kim, MS and Vu, TC and Hanjra, P and Kim, S and Ahumada, AL and Wu, W and Lee, S and Szymanowska, A and Oztatlici, H and Estecio, MR and Lee, JS and Jain, AK and Sahni, N and Hagan, JP and Baylin, S and Liu, J and Lopez-Berestein, G and Pradeep, S and Sood, AK},
title = {Epigenetic modulation of BARD1 to enhance anti-VEGF therapy.},
journal = {Cell reports. Medicine},
volume = {6},
number = {9},
pages = {102329},
doi = {10.1016/j.xcrm.2025.102329},
pmid = {40907495},
issn = {2666-3791},
mesh = {Humans ; *Epigenesis, Genetic/drug effects ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Animals ; *Vascular Endothelial Growth Factor A/antagonists &amp; inhibitors/metabolism ; Female ; *Tumor Suppressor Proteins/genetics/metabolism ; DNA Methylation/drug effects/genetics ; Cell Line, Tumor ; Mice ; *Ovarian Neoplasms/genetics/drug therapy/pathology ; Neovascularization, Pathologic/genetics/drug therapy ; Promoter Regions, Genetic ; CRISPR-Cas Systems/genetics ; Drug Resistance, Neoplasm/genetics/drug effects ; Mixed Function Oxygenases ; Proto-Oncogene Proteins ; },
abstract = {Despite the clinical use of anti-vascular endothelial growth factor (VEGF) antibodies (AVAs) in cancer therapy, resistance frequently develops, leading to disease progression. To address this, we identify a previously unknown role for breast cancer type 1 susceptibility protein (BRCA1)-associated RING domain 1 (BARD1) in modulating AVA sensitivity. Epigenetic modulation-via global and targeted DNA methylation-reveals BARD1 as a key regulator of angiogenesis. Sequential treatment with azacytidine overcomes AVA resistance in vivo. To enable precise epigenetic reactivation, we develop a liposomal CRISPR-deactivated Cas9 (dCas9)-TET1 system guided by BARD1-targeting single-guide RNAs (sgRNAs). This platform achieves CpG-specific demethylation of the BARD1 promoter, restores expression, and enhances AVA response. Additionally, BARD1 restoration, through either dCas9-TET1 or small interfering RNA (siRNA), significantly reduces tumor growth in combination with AVA in ovarian cancer models. These findings uncover a previously unrecognized function of BARD1 in tumor angiogenesis and demonstrate the potential of gene-specific epigenetic targeting to overcome AVA resistance.},
}
@article {pmid40905401,
year = {2025},
author = {Chen, G and Shan, Y and Wang, J and Zhang, Q and Yao, L and Chen, X},
title = {Multiple Lignocellulosic Inhibitor-Tolerant Saccharomyces cerevisiae Strains Developed by Evolutionary Engineering and CRISPR/Cas9 Gene Editing Technology.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {37},
pages = {23486-23497},
doi = {10.1021/acs.jafc.5c04039},
pmid = {40905401},
issn = {1520-5118},
mesh = {*Lignin/metabolism/antagonists &amp; inhibitors ; *Saccharomyces cerevisiae/genetics/metabolism/growth &amp; development/drug effects ; CRISPR-Cas Systems ; Gene Editing ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; Furaldehyde/metabolism/pharmacology ; Acetic Acid/metabolism/pharmacology ; Benzaldehydes/pharmacology/metabolism ; Ethanol/metabolism ; Fermentation ; },
abstract = {Through evolutionary engineering strategies, scientists have successfully cultivated multiple strains of Saccharomyces cerevisiae with enhanced tolerance, demonstrating significant potential in improving S. cerevisiae resistance. In this study, S. cerevisiae CEN.PK113-7D was continuously cultured for 80 days in a medium containing lignocellulosic inhibitors (furfural, acetic acid, and vanillin). The evolved strain, S. cerevisiae CEN.PK113-AL80-4, exhibited 12 h reduction in lag phase under multiple stress conditions and 17% increase in the ethanol conversion rate. The double mutant strain RG was constructed by mutating genes such as Rad18 and Gcn1 using CRISPR/Cas9 gene editing technology. Under the stress of 2 g/L furfural, 3 g/L acetic acid, and 1.5 g/L vanillin, ethanol yield reached 5.88 ± 0.28 g/L (the conversion rate was 0.29 ± 0.01 g/g). However, the original strain cannot grow. Mechanism studies have shown that Rad18 and Gcn1 significantly enhance stress tolerance by increasing the activities of catalase (CAT) (75%) and superoxide dismutase (SOD) (27.6%), increasing intracellular glycerol content, and strengthening carbon metabolism and oxidative stress responses. This study lays a solid theoretical foundation for developing more robust strains and advancing efficient utilization of lignocellulosic biomass.},
}
@article {pmid40897678,
year = {2025},
author = {Liu, S and Lun, J and Hu, L and Pan, L},
title = {Rapid and Field-Deployable Detection of the S59G Mutation in Glutamine Synthetase Endowing Glufosinate Resistance in Eleusine indica.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {37},
pages = {23292-23299},
doi = {10.1021/acs.jafc.5c08583},
pmid = {40897678},
issn = {1520-5118},
mesh = {*Glutamate-Ammonia Ligase/genetics/metabolism ; *Aminobutyrates/pharmacology ; *Herbicide Resistance ; *Herbicides/pharmacology ; *Plant Proteins/genetics/metabolism ; *Eleusine/genetics/drug effects/enzymology ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Glufosinate is a crucial nonselective herbicide used in both conventional and transgenic cropping systems. Its effectiveness is increasingly compromised by resistant weed species like Eleusine indica (L.) Gaertn. A single nucleotide mutation in the GS1-1 gene, specifically the S59G substitution in glutamine synthetase, is the only known target-site mutation conferring resistance to glufosinate in E. indica. This study presents a rapid and sensitive detection system for the GS1-1 S59G mutation, integrating recombinase-aided amplification (RAA) with CRISPR/Cas12a technology. We optimized key components to achieve robust performance, detecting target sequences at concentrations as low as approximately 10[4] aM. The system effectively differentiates between susceptible and S59G-mutated resistant E. indica using fluorescence readouts or lateral flow assays. This RAA-CRISPR/Cas12a detection system serves as an efficient molecular diagnostic tool for monitoring glufosinate resistance in E. indica, aiding in effective weed management strategies.},
}
@article {pmid40842431,
year = {2025},
author = {Erol, A and Celebi-Birand, D and Yilmaz, E and Polat, C and Kasap, OE and Alten, B and Duman, M},
title = {A novel CRISPR-Cas12a-based diagnostic for rapid and highly sensitive detection of West Nile virus.},
journal = {Journal of materials chemistry. B},
volume = {13},
number = {36},
pages = {11318-11328},
doi = {10.1039/d5tb01268f},
pmid = {40842431},
issn = {2050-7518},
mesh = {*West Nile virus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; *West Nile Fever/diagnosis/virology ; Animals ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism ; Nucleic Acid Amplification Techniques ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; },
abstract = {Climate change is increasing the global threat of vector-borne diseases, including West Nile Virus (WNV), a significant human and animal pathogen transmitted primarily by Culex mosquitoes. Current WNV diagnostic methods, while including sensitive techniques like RT-PCR, have limitations in early detection, practicality, and cost-effectiveness. There is an urgent need to develop novel and more efficient strategies to address these challenges and to facilitate the surveillance and management of WNV infections and their spread. This study presents a highly specific and sensitive CRISPR-Cas12a-based detection protocol for WNV detection. Through systematic analysis of key reaction parameters (time: 0-60 min; reporter concentration: 1-80 nM, Cas12a and crRNA concentration: 5.625-90 nM; and template amount: 10[-2]-10[5] pg) and integration with reverse transcriptase recombinase polymerase amplification to enhance sensitivity through an isothermal technique, this assay demonstrates a novel strategy for the rapid detection of WNV, achieving 10 femtomolar sensitivity within one hour. Moreover, the assay retained its efficacy at different temperatures (25 °C and 37 °C) and in biological matrices containing the host (fly or human) genetic material, which supports its applicability in resource-limited settings. Therefore, the method presented here has the potential for broad application in diverse point-of-care settings for rapid diagnosis of WNV.},
}
@article {pmid40757862,
year = {2025},
author = {Fernbach, J and Baggenstos, J and Svorjova, E-A and Riedo, J and McCallin, S and Loessner, MJ and Kilcher, S},
title = {CRISPR-Cas9 enables efficient genome engineering of the strictly lytic, broad-host-range staphylococcal bacteriophage K.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {9},
pages = {e0201424},
doi = {10.1128/aem.02014-24},
pmid = {40757862},
issn = {1098-5336},
support = {PZ00P3_174108//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung/ ; },
mesh = {*CRISPR-Cas Systems ; *Staphylococcus Phages/genetics/physiology ; *Staphylococcus aureus/virology ; Host Specificity ; *Genetic Engineering/methods ; Animals ; *Genome, Viral ; Staphylococcal Infections/microbiology/veterinary/diagnosis ; Cattle ; Humans ; },
abstract = {Staphylococcus aureus is a major opportunistic pathogen, increasingly difficult to treat due to rising resistance to methicillin, vancomycin, and other antimicrobials. Bacteriophages offer a promising alternative, particularly when conventional therapies fail and their efficacy can be enhanced through genetic engineering. Among S. aureus phages, the strictly lytic, broad-host-range members of the Twortvirinae subfamily are among the most promising therapeutic candidates. However, their large genome sizes make them notoriously difficult to engineer. In this study, we utilized Twortvirus K as a model to develop an efficient phage engineering platform, leveraging homologous recombination and CRISPR-Cas9-assisted counterselection. As proof of principle, this platform was utilized to construct a nanoluciferase (nluc)-encoding reporter phage (K::nluc) and tested as a bioluminescence-based approach for identifying viable Staphylococcus cells. Independent of their phage-resistance profile, 100% of tested clinical S. aureus isolates emitted bioluminescence upon K::nluc challenge. This diagnostic assay was further adapted to complex matrices such as human whole blood and bovine raw milk, simulating S. aureus detection scenarios in bacteremia and bovine mastitis. Beyond reporter phage-based diagnostics, our engineering technology opens avenues for the design and engineering of therapeutic Twortvirinae phages to combat drug-resistant S. aureus strains.IMPORTANCEPhage engineering, the process of modifying bacteriophages to enhance or customize their properties, offers significant potential for advancing precision antimicrobial therapies and diagnostics. While methods for engineering small Staphylococcus phage genomes are well-established, larger Staphylococcus phages have historically been challenging to modify. In this study, we present a novel method that enables the engineering of Twortvirinae, a subfamily of Staphylococcus phages known for their broad host range and strictly lytic lifestyle, making them highly relevant for diagnostic and therapeutic applications. Using this method, we successfully developed a phage-based diagnostic tool capable of rapid and sensitive detection of S. aureus cells across various matrices. This approach has the potential to extend beyond diagnostics, enabling applications such as phage-mediated delivery of antimicrobial effector proteins in the future.},
}
@article {pmid40752587,
year = {2025},
author = {Liang, G and Lu, B and Dai, S and Li, M and Yao, J and Liu, H and Liu, X and Liu, X and Wang, D},
title = {Creation of colorless transparent tilapia using CRISPR/Cas9 mediated multi-gene mutation.},
journal = {New biotechnology},
volume = {89},
number = {},
pages = {163-176},
doi = {10.1016/j.nbt.2025.07.009},
pmid = {40752587},
issn = {1876-4347},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Tilapia/genetics ; *Mutation/genetics ; *Gene Editing ; },
abstract = {Transparent mutant fish have been obtained from small-sized fish (medaka, zebrafish, and killifish), and are often used as experimental models in biological and medical research. However, transparent fish have never been created in medium- or large-sized fish through gene editing. In this study, mutants without xanthophores, erythrophores and pigmented melanophores were first obtained by crossing to form tyrb;csf1ra double mutant tilapia. Subsequently, single mutants lacking reflective platelets without visible iridophores were obtained by mutation of pnp4a and tfec using CRISPR/Cas9 gene editing, the recovery of iridophores were observed in pnp4a mutants but not in tfec mutants in the later stage. In addition, we also found that tfec mutation led to a decrease in number of melanophores and diameter of melanophores/erythrophores. Finally, transparent triple mutant homozygotes without visible pigment cells were obtained by triple mutation of tyrb;csf1ra;pnp4a (named amber) and tyrb;csf1ra;tfec (named ruby), through crossing of double and single mutants. The two mutant lines were no longer transparent at 60 dpf (days post fertilization) and 120 dpf, respectively, due to the recovery of iridophores in the former, and thickening of the body wall in the latter. In summary, this study created two transparent lines without visible pigment cells through aggregation of multiple gene mutation and crossing. The transparent fish is suitable for in vivo imaging, and is currently the only medium-sized transparent fish obtained through gene editing.},
}
@article {pmid40705967,
year = {2025},
author = {Uçar, SK and Yıldırım, C and Opladen, T},
title = {A CRISPR/Cas9 knockout model for AADC deficiency reveals structural loop3 instability as a key driver of catalytic failure.},
journal = {The FEBS journal},
volume = {292},
number = {18},
pages = {4804-4807},
doi = {10.1111/febs.70204},
pmid = {40705967},
issn = {1742-4658},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Aromatic-L-Amino-Acid Decarboxylases/genetics/deficiency/chemistry/metabolism ; *Amino Acid Metabolism, Inborn Errors/genetics/pathology/metabolism ; Gene Knockout Techniques ; },
abstract = {The CRISPR-Cas9 dopa decarboxylase (DDC) gene knockout SH-SY5Y model for aromatic L-amino acid decarboxylase (AADC) deficiency provides a valuable neuronal platform for functional and structural investigation of pathogenic variants. In their study, Bertoldi et al. successfully recapitulate the biochemical and metabolic hallmarks of AADC deficiency using the AADC catalytic variants R347Q and L353P. Their combined structural and cellular approach identifies loop3 dynamics as a critical determinant of enzymatic dysfunction. This model may pave the way for the development of precision therapies.},
}
@article {pmid40681150,
year = {2025},
author = {Xi, C and Chiu, S and Voje, WE and Carothers, JM and Moon, TS},
title = {Conditional guide RNA deactivation by mRNA and small molecule triggers in Saccharomyces cerevisiae.},
journal = {New biotechnology},
volume = {89},
number = {},
pages = {105-118},
pmid = {40681150},
issn = {1876-4347},
support = {R01 AT009741/AT/NCCIH NIH HHS/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *RNA, Messenger/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Fungal ; },
abstract = {CRISPR interference (CRISPRi) technologies have revolutionized bioengineering by providing precise tools for gene expression modulation, enabling targeted gene perturbation and metabolic pathway optimization. Despite these advances, achieving dynamic control over gene expression by CRISPR-based regulation remains a challenge due to its inherently static nature. Utilizing toehold-mediated strand displacement and ligand-responsive ribozymes (aptazymes), this study introduces switchable guide RNAs (gRNAs) that facilitate tunable gene expression mediated by mRNA or small molecule signals. We demonstrate complete silencing of gRNA via strategically designed 5' or 3' extensions that impede the gRNA spacer or the dCas9 handle, with subsequent restoration of function through sequestration or cleavage of the obstructive sequence. The resulting toehold-embedded or aptazyme-embedded gRNAs can be deactivated by specific signals, including two full-length translatable mRNAs and two small molecule triggers, thereby lifting CRISPRi repression on targeted genes. This modular approach allows for gRNA-based biocomputing through multi-layer or multi-input genetic logic gates in Saccharomyces cerevisiae. Offering a versatile strategy for post-CRISPR regulation in response to environmental signals or cellular states, this methodology expands the toolkit in eukaryotic systems for reversible control of gene expression.},
}
@article {pmid40318155,
year = {2025},
author = {Carmona-Carmona, CA and Bisello, G and Franchini, R and Lunardi, G and Galavotti, R and Perduca, M and Ribeiro, RP and Belviso, BD and Giorgetti, A and Caliandro, R and Lievens, PM and Bertoldi, M},
title = {The CRISPR-Cas9 knockout DDC SH-SY5Y in vitro model for AADC deficiency provides insight into the pathogenicity of R347Q and L353P variants: a cross-sectional structural and functional analysis.},
journal = {The FEBS journal},
volume = {292},
number = {18},
pages = {4833-4853},
doi = {10.1111/febs.70120},
pmid = {40318155},
issn = {1742-4658},
support = {PRIN2022 code 2022BTMTP8//Ministero dell'Universit&#xe0; e della Ricerca, National Recovery and Resilience Plan (NRRP)/ ; IIS154//PTC Therapeutics/ ; PE0000006//National Recovery and Resilience Plan, #NEXTGENERATIONEU (NGEU)/ ; 155311.10.2022//A Multiscale integrated approach to the study of the nervous system in health and disease/ ; },
mesh = {Humans ; *Aromatic-L-Amino-Acid Decarboxylases/genetics/deficiency/chemistry/metabolism ; *CRISPR-Cas Systems/genetics ; *Amino Acid Metabolism, Inborn Errors/genetics/pathology/metabolism ; Cell Line, Tumor ; Dopamine/metabolism ; Gene Knockout Techniques ; Crystallography, X-Ray ; Molecular Dynamics Simulation ; },
abstract = {Aromatic amino acid decarboxylase (AADC) deficiency is a severe inherited recessive neurotransmitter disorder caused by an impairment in dopamine synthesis due to the lack/modification of AADC, the enzyme converting l-dopa to dopamine. Patients exhibit severe movement disorders and neurodevelopmental delay, with a high risk of premature mortality. Given the lack of a reliable model for the disease, we developed a dopa decarboxylase knockout model using CRISPR/Cas9 technology in the SH-SY5Y neuroblastoma cell line. This model showed a deficiency in AADC protein and activity, with an altered dopamine metabolites profile (low homovanillic acid and high 3-O-methyldopa) and a modified expression of key enzymes, such as dopamine beta-hydroxylase and monoamine oxidases, which are involved in the catecholamine pathway. We then transfected the DDC-KO cells with two AADC catalytic variants, R347Q and L353P, which resulted in loss-of-function and an altered profile of dopamine metabolites. By combining several structural approaches (X-ray crystallography, molecular dynamics, small angle X-ray scattering, dynamic light scattering, and spectroscopy), we determined that both variants alter the flexibility of the structural element to which they belong, whose integrity is essential for catalysis. This change causes a mispositioning of essential residues at the active site, leading, in turn, to an unproductive external aldimine, identifying the molecular basis for the loss-of-function. Overall, the DDC-KO model recapitulates some key features of AADC deficiency, is useful to study the molecular basis of the disease, and represents an ideal system for small molecule screening regarding specific enzyme defects, paving the way for a precision therapeutic approach.},
}
@article {pmid39484758,
year = {2025},
author = {Sharma, B and Chauhan, I and Kumar, G and Bhardwaj, K and Tiwari, RK},
title = {Nanoparticle Carriers: A New Era of Precise CRISPR/Cas9 Gene Editing.},
journal = {MicroRNA (Shariqah, United Arab Emirates)},
volume = {14},
number = {2},
pages = {101-111},
pmid = {39484758},
issn = {2211-5374},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Nanoparticles/chemistry ; Animals ; Drug Delivery Systems/methods ; },
abstract = {The revolutionary CRISPR/Cas9 gene editing technology holds immense potential for treating genetic diseases and tackling conditions like cancer. However, efficient delivery remains a significant challenge. This is where nanoparticles come into play, emerging as powerful allies in the realm of drug delivery. Nanoparticles can accommodate larger insertion sizes, enabling the incorporation of larger Cas9 enzymes and complex guide RNAs, thus opening up the possibility of editing previously inaccessible genetic regions. Their relatively straightforward and scalable production processes make them cost-effective options for wider applications. Notably, nanoparticles excel in vivo, demonstrating efficient tissue penetration and targeted delivery, which are crucial for maximizing therapeutic impact while minimizing side effects. This review aims to explore the potential of nanoparticle-based delivery systems for CRISPR/Cas9, highlighting their advantages and challenges in gene editing applications. The diverse range of nanoparticles further bolsters their potential. Polymeric nanoparticles, for instance, offer tunable properties for customization and controlled release of the CRISPR cargo. Lipid-based nanoparticles facilitate efficient cellular uptake and endosomal escape, ensuring the CRISPR components reach the target DNA. Even gold nanoparticles, known for their unique biocompatibility and photothermal properties, hold promise in light-activated editing strategies. Non-viral delivery systems, particularly those based on nanoparticles, stand out due to their inherent advantages. Collectively, the evidence paints a promising picture: nanoparticles are not merely passive carriers but active participants in the CRISPR/Cas9 delivery landscape. Their versatility, efficiency, and safety position them as key enablers of a future where gene editing can revolutionize drug development, offering personalized and targeted therapies for a wide range of diseases.},
}
@article {pmid40954078,
year = {2025},
author = {Bexte, T and Wagner, DL},
title = {Next-generation multiplex-edited CAR-NK cells: more edits, more power?.},
journal = {Journal for immunotherapy of cancer},
volume = {13},
number = {9},
pages = {},
doi = {10.1136/jitc-2025-012841},
pmid = {40954078},
issn = {2051-1426},
mesh = {*Killer Cells, Natural/immunology/metabolism/transplantation ; Humans ; *Gene Editing/methods ; Animals ; *Receptors, Chimeric Antigen/genetics/metabolism ; Mice ; *Immunotherapy, Adoptive/methods ; CRISPR-Cas Systems ; },
abstract = {First clinical trials demonstrated the safety of adoptive cell transfer with allogeneic natural killer (NK) cell products from healthy donors, making them an attractive candidate for 'off-the-shelf' chimeric antigen receptor (CAR)-immune cell therapy. However, reduced persistence and inactivation of NK cells by immunosuppressive cues likely limit the performance of CAR-redirected NK cells. Wang and colleagues demonstrate that multiplex CRISPR base editing allows optimization of the intrinsic functionality of CAR-NK cells improving their therapeutic potential. In contrast to conventional CRISPR-Cas nucleases, base editing avoided most double-stranded DNA breaks while enabling highly efficient editing at up to six sites simultaneously. The study further demonstrates the feasibility of a non-viral approach to integrate CAR transgene and multiplex base editing of several immune checkpoints in NK cells using a single electroporation. CAR-NK cells harboring up to three base edits demonstrate improved potency over unedited counterparts in vitro. Xenograft mouse models confirmed increased potency, but also indicated signs of organ toxicity - a phenomenon that will require future studies prior to clinical translation. The study demonstrates that CRISPR base editing is a powerful tool to unleash the full cytotoxic potential of NK cells, but it also warrants the question: How many internal breaks can be removed without hurting CAR-NK cell therapy's impeccable safety record?},
}
@article {pmid40952971,
year = {2025},
author = {Leonova, EI and Chirinskaite, AV and Akhmarov, II and Luganskaya, PS and Kirillov, OA and Kandina, DA and Romanovich, AE and Shkodenko, LA and Fedotov, SA and Rubel, AA and Sopova, YV},
title = {Improved Genome Editing via Oviductal Nucleic Acids Delivery-based In Vivo Electroporation Technique for Knockout Mice Generation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {222},
pages = {},
doi = {10.3791/68704},
pmid = {40952971},
issn = {1940-087X},
mesh = {Animals ; Female ; *Electroporation/methods ; Mice ; Mice, Knockout ; Pregnancy ; *Gene Editing/methods ; *Fallopian Tubes ; Male ; CRISPR-Cas Systems ; *Nucleic Acids/administration &amp; dosage/genetics ; Oviducts ; RNA, Guide, CRISPR-Cas Systems/administration &amp; dosage/genetics ; *Gene Knockout Techniques/methods ; },
abstract = {Methods for creating knockout mice typically involve three main steps: (1) collecting embryos from donor females, (2) microinjecting genetic constructs into the zygotes ex vivo, and (3) surgically transferring them into the oviduct of pseudo-pregnant females. This process requires a significant number of animals, as it involves not only donor females but also vasectomized males and pseudo-pregnant females. Moreover, microinjections into the cytoplasm or pronucleus of mouse zygotes present challenges such as needle clogging, membrane permeability issues due to high elasticity, and potential embryo death. The development of advanced electroporators, such as the Nepa21, provides a unique opportunity to generate mice with targeted gene knockouts in a single step through a method known as Improved Genome Editing via Oviductal Nucleic Acids Delivery (I-GONAD). This technique involves microinjecting CRISPR-Cas components (Cas9 protein and guide RNA) into the oviducts of pregnant females at 0.7 days post-conception, followed by in vivo electroporation to deliver these components directly into the zygotes. Following the I-GONAD procedure, the pregnant mouse carries and gives birth to pups with the targeted gene knockout. This article provides a detailed, step-by-step protocol for implementing the I-GONAD method in mice, offering a more efficient and accessible alternative to traditional knockout mouse generation techniques.},
}
@article {pmid40952968,
year = {2025},
author = {Datta, A and Nelson, N and Orallo, GK and Krull, S},
title = {Targeted Corneal Sensory Nerve Depletion via Subconjunctival Injection: A Model for Investigating Bacterial Adhesion and Neuroimmune Interactions.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {222},
pages = {},
doi = {10.3791/68614},
pmid = {40952968},
issn = {1940-087X},
mesh = {Animals ; *Cornea/innervation/microbiology/immunology/drug effects ; *Bupivacaine/administration &amp; dosage/pharmacology ; *Bacterial Adhesion/drug effects/physiology ; Pseudomonas aeruginosa/physiology ; Conjunctiva/innervation/microbiology ; Staphylococcus aureus/physiology ; Staphylococcus epidermidis/physiology ; *Sensory Receptor Cells/drug effects/microbiology/immunology ; Mice ; },
abstract = {Corneal sensory nerves play a pivotal role in supporting ocular surface integrity and immune defense mechanisms. Loss of this innervation has been associated with increased vulnerability to microbial invasion, yet the precise contribution of nerve depletion to bacterial adhesion on the cornea remains insufficiently characterized. Here, we present a reproducible and temporally controlled method for selective corneal sensory nerve suppression using bupivacaine, a long-acting sodium channel blocker. By combining subconjunctival and topical delivery routes, this dual-application strategy achieves robust, sustained denervation, allowing for precise investigation of how altered sensory input influences corneal epithelial susceptibility to bacterial colonization. Using this model, we investigate how sensory denervation influences microbial adhesion dynamics for Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa three clinically relevant pathogens with distinct adhesion mechanisms. Standardized bacterial inoculation via the laboratory wipe blotting method ensures uniform deposition on the corneal surface, followed by quantitative assessment of bacterial adhesion. Bupivacaine-induced nerve depletion correlates with reduced corneal nerve density and increased bacterial adhesion, confirming a functional link between sensory depletion and microbial susceptibility. By simulating neuropathic conditions such as diabetic neuropathy and neurotrophic keratitis, this approach provides a novel framework for studying neuroimmune interactions in ocular infections. Beyond infection models, this subconjunctival injection strategy offers a versatile platform for investigating ocular drug pharmacokinetics, neuroprotective interventions, and immune modulation. Furthermore, it can be adapted for gene modification studies, including subconjunctival delivery of CRISPR/Cas constructs or viral vectors, broadening its applications in ophthalmic research and therapeutics.},
}
@article {pmid40951304,
year = {2025},
author = {Chen, J and Wang, Y and Aikebaier, R and Liu, H and Li, Y and Yang, L and Haiyilati, A and Wang, L and Fu, Q and Shi, H},
title = {RAA-CRISPR/Cas12a-based visual field detection system for rapid and sensitive diagnosis of major viral pathogens in calf diarrhea.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1616161},
pmid = {40951304},
issn = {2235-2988},
mesh = {Animals ; Cattle ; *Diarrhea/veterinary/virology/diagnosis ; Sensitivity and Specificity ; *Cattle Diseases/diagnosis/virology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; China ; *Nucleic Acid Amplification Techniques/methods ; Feces/virology ; *Virus Diseases/diagnosis/veterinary/virology ; Recombinases/metabolism ; *Viruses/genetics/isolation &amp; purification/classification ; },
abstract = {Calf diarrhea is a complex digestive disorder in cattle that imposes significant economic losses in terms of calf mortality, growth impairment, and treatment costs. Both infectious and non-infectious agents contribute to its aetiology; however, most of the infectious cases are caused by viruses, often accompanied by severe co-infections. To identify viral culprits, we performed viral metagenomic sequencing on three pooled samples from the 150 diarrheal samples from Xinjiang, China, which helped with identification of the following four predominant agents: bovine nepovirus (BNeV), bovine coronavirus (BCoV), bovine viral diarrhea virus (BVDV) and bovine enterovirus (BEV). Currently, the process of diagnosing these pathogens involves time-consuming workflows, limited sensitivity, poor portability, and lack of field applicability. Keeping these diagnostic shortcomings in mind, an integrated platform called RAA-CRISPR/Cas12a system was developed by combining recombinase-aided amplification (RAA) at 37°C with CRISPR/Cas12a-mediated fluorescence detection, which achieved 100-100,000 times higher sensitivity than conventional polymerase chain reaction (PCR) (detection limits: 1-10 copies/μL) and demonstrated 100% specificity against non-target pathogens. Clinical validation of sensitivity and specificity of 252 samples revealed 1.6-4.9 times higher detection rates (239 positives) than PCR (81 positives), which was consistent with PCR-confirmed cases. The assay's 40-min. workflow enables rapid on-site deployment without specialized instrumentation, as it requires only a portable heat block and blue LED transilluminator. Hence, with its laboratory accuracy and field applicability, this method helps in early identification of pathogens, outbreak containment and mitigation of economic loss in the global cattle industry.},
}
@article {pmid40950114,
year = {2025},
author = {Saito, A and Tankou, S and Ishii, K and Sakao-Suzuki, M and Oh, EC and Murdoch, H and Namkung, H and Adelakun, S and Furukori, K and Fujimuro, M and Salomoni, P and Maul, GG and Hayward, GS and Tang, Q and Yolken, RH and Houslay, MD and Katsanis, N and Kosugi, I and Yang, K and Kamiya, A and Ishizuka, K and Sawa, A},
title = {DISC1-PML protein interaction for congenital CMV infection-induced cortical neural progenitor deficit: perturbance of host signaling via viral IE1.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40950114},
issn = {2692-8205},
abstract = {Congenital CMV infection is the most common perinatal infection, affecting up to 0.5% of infants. This elicits long-term disabilities that include neuropsychiatric manifestations, such as intellectual disability, microcephaly. Despite its high prevalence, the underlying mechanism of how congenitally acquired CMV infection causes brain pathology remains unknown. Here we discovered the molecular interplay of key host (DISC1 and PML) and viral (IE1) proteins within the neural progenitor cells, which underlay an attenuated neural progenitor proliferation. Abolishing the viral IE1 protein by delivering IE1-targeting CRISPR/Cas9 to fetal brain rescued this progenitor cell deficit, a key pathology in congenital CMV infection. A selective targeting to a viral-specific protein by the CRISPR/Cas9 system is minimal in off-target effects. Therefore, we believe that a pivotal role of IE1 in an attenuated neural progenitor proliferation in the developing cortex through its interfering with interaction between host DISC1 and PML proteins.},
}
@article {pmid40947195,
year = {2025},
author = {Zhou, G and Ding, J and Li, Y and Xu, Q and Zhang, Y and Tang, H and Qi, P and Deng, M and Ma, J and Chen, G and Wang, J and Lin, N and Wei, Y and Jiang, Q},
title = {Ethylene-responsive factor HvERF72 regulates starch synthesis and B-type starch granules initiation in barley.},
journal = {Carbohydrate polymers},
volume = {368},
number = {Pt 2},
pages = {124167},
doi = {10.1016/j.carbpol.2025.124167},
pmid = {40947195},
issn = {1879-1344},
mesh = {*Hordeum/metabolism/genetics ; *Starch/biosynthesis ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Transcription Factors/metabolism/genetics ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; },
abstract = {Starch biosynthesis is a pivotal determinant of barley grain quality and yield, yet its regulatory mechanisms remain incompletely characterized. This study identifies HvERF72, an AP2-domain transcription factor, as a key regulator of starch biosynthesis and granule initiation in barley grains. Comparative analyses of CRISPR/Cas9-generated HvERF72 knockout mutants revealed enhanced B-type granule formation and elevated total starch content, whereas overexpression lines exhibited contrasting phenotypes, including reduced starch accumulation and suppressed B-type granule initiation. Transcriptional profiling at 15 DAF indicated significant upregulation of critical starch biosynthesis genes (HvAGPL1, HvAGPS1, HvSS2a, HvSBEI, HvSBEIIb, and HvGBSSI) in mutants, while overexpression lines showed downregulation of these genes. Mechanistic investigation demonstrated that HvERF72 directly binds to GCC-box motifs in the promoter regions of HvSS2a and HvSBEI, repressing their transcription. These findings establish HvERF72 as dual-function regulator that modulates starch biosynthesis and B-type granule initiation, providing novel molecular targets for optimizing starch yield and industrial quality in barley breeding programs.},
}
@article {pmid40943650,
year = {2025},
author = {Ulloa, D and Núñez, C and Matamala, R and San Martín, A and Páez-De Ávila, D and Mercado-Vides, J and Narváez, J and Aguirre, J and Effer, B and Iturrieta-González, I},
title = {CRISPR-Cas12 Application for the Detection of Pneumocystis jirovecii in Immunodepression Patients Through Fluorescent and Lateral Flow Colorimetric Assay.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943650},
issn = {1422-0067},
support = {21251211//ANID - Chile, Scholarship Programa de Doctorado Nacional 2025/ ; UDM010//FONCIENCIAS - Universidad del Magdalena/ ; },
mesh = {Humans ; *Pneumocystis carinii/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Colorimetry/methods ; *Pneumonia, Pneumocystis/diagnosis/microbiology/immunology ; *Immunocompromised Host ; Sensitivity and Specificity ; Tubulin/genetics ; },
abstract = {Pneumonia caused by Pneumocystis jirovecii poses a serious threat, particularly to immunocompromised patients such as those with HIV/AIDS, transplant recipients, or individuals undergoing chemotherapy. Its diagnosis is challenging because current methods, such as microscopy and certain molecular tests, have limitations in sensitivity and specificity, and require specialized equipment, which delays treatment initiation. In this context, CRISPR-Cas12-based methods offer a promising alternative: they are rapid, highly specific, sensitive, and low-cost, enabling more timely and accessible detection, even in resource-limited settings. We developed a simple and rapid detection platform based on the CRISPR-Cas12 coupled with lateral flow strips. A guide RNA was designed against DHPS, β-tubulin, and mtLSU rRNA genes. The guide corresponding to β-tubulin showed high sensitivity in the detection of P. jirovecii to produce a detectable fluorescence signal within the first 20-30 min. In addition, it demonstrated high specificity for P. jirovecii when DNA from other microorganisms was used. When coupled with lateral flow strips, high sensitivity and specificity were also observed for detecting positive samples, without the need for genetic amplification. CRISPR-Cas12 successfully detected P. jirovecii infection in an initial diagnostic application, demonstrating the potential of this method for integration into public health diagnostic systems, particularly in field, due to its adaptability, speed, and ease of use.},
}
@article {pmid40943640,
year = {2025},
author = {Yu, I and Jeong, J},
title = {Advancing Gene Therapy for Phenylketonuria: From Precision Editing to Clinical Translation.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943640},
issn = {1422-0067},
support = {RS-2023-00260529//National Research Foundation of Korea/ ; 2022R1A2C1002884//National Research Foundation of Korea/ ; 2024-0036//Seoul Women's University/ ; },
mesh = {*Phenylketonurias/therapy/genetics ; Humans ; *Genetic Therapy/methods ; *Gene Editing/methods ; Animals ; CRISPR-Cas Systems ; Phenylalanine Hydroxylase/genetics ; Gene Transfer Techniques ; Dependovirus/genetics ; Translational Research, Biomedical ; Nanoparticles/chemistry ; },
abstract = {Phenylketonuria (PKU) is an inherited disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene that result in the amino acid phenylalanine (Phe) building up in the blood. Current therapies suggest low-Phe dietary management and (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) therapy, which are limited in efficacy and require lifelong treatment. Recent advances in gene therapy, including gene editing and viral-mediated gene delivery, produce therapeutic effects. Advancements in gene editing technologies, notably adenine base editors (ABEs) and CRISPR-based systems, in conjunction with enhanced delivery methods such as lipid nanoparticles (LNPs) and recombinant viruses, have demonstrated substantial promise in preclinical studies. This review details the pathophysiology of PKU treatment, and progress in preclinical and clinical gene therapy strategies. Emphasis is on adenine base editing using LNPs, recombinant adeno-associated virus (rAAV)-mediated gene transfer, and the translational challenges associated with these technologies. We also discuss future directions for therapeutic reach and ensuring long-term safety and efficacy.},
}
@article {pmid40943604,
year = {2025},
author = {Yamaji, M and Nakahara, T and Nakanishi, T and Aoyama-Kikawa, S and Yamaguchi, K and Furukawa, Y and Nakamura, M and Okada, T and Tabata, H and Fuse, R and Shimizu, E and Kasajima, R and Imoto, S and Kukimoto, I and Saito, I and Kiyono, T},
title = {Disruption of Human Papillomavirus 16 E6/E7 Genes Using All-in-One Adenovirus Vectors Expressing Eight Double-Nicking Guide RNAs.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943604},
issn = {1422-0067},
support = {JP21fk0108106//Japan Agency for Medical Research and Development/ ; JP22fk0310523//Japan Agency for Medical Research and Development/ ; JP25K02541//Japan Society for the Promotion of Science/ ; JP19K0647//Japan Society for the Promotion of Science/ ; JP23H02402//Japan Society for the Promotion of Science/ ; (JP22ym0126804//Japan Agency for Medical Research and Development/ ; 20jk0210009h0001//Japan Agency for Medical Research and Development/ ; },
mesh = {Humans ; *Oncogene Proteins, Viral/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Papillomavirus E7 Proteins/genetics ; *Adenoviridae/genetics ; *Genetic Vectors/genetics ; CRISPR-Cas Systems ; *Human papillomavirus 16/genetics ; Gene Editing/methods ; *Repressor Proteins/genetics ; Animals ; Cell Line, Tumor ; Papillomavirus Infections/virology/therapy ; Mice ; Female ; Genetic Therapy ; },
abstract = {Human papillomavirus (HPV) is a prime target for genome-editing therapy as its E6 and E7 oncogenes are crucial for cancer development and maintenance. A key challenge in CRISPR/Cas9 therapy is the off-target effects. This study utilized a double-nicking technique to introduce DNA breaks in the E6 and E7 regions of HPV16. From 146 gRNA candidates, 16 double-nicking pairs were selected. Multiple combinations of double-nicking (DN)-gRNA pairs were delivered to HPV16-positive cells via lentiviruses, followed by Cas9 nickase (Cas9n) expression. Combinations of 3-4 DN-gRNA pairs effectively killed HPV16-positive cells while sparing HPV-negative cells. Off-target effects were reduced by nearly three orders of magnitude. An "all-in-one" adenovirus (AdV) system expressing four gRNA pairs and Cas9n showed promise in inhibiting tumor growth in HPV16-positive cancer models, demonstrating its potential as a safe and effective treatment for HPV-induced tumors.},
}
@article {pmid40943194,
year = {2025},
author = {Qi, Y and Jia, X and Lin, C and Qian, W and Chen, H and Fang, D and Han, C},
title = {CRISPR/Cas9-Mediated Overexpression of HGF Potentiates Tarim Red Deer Antler MSCs into Osteogenic Differentiation.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943194},
issn = {1422-0067},
mesh = {Animals ; *Osteogenesis/genetics ; *Deer/genetics ; *Antlers/cytology/metabolism ; *Mesenchymal Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems ; *Cell Differentiation/genetics ; *Hepatocyte Growth Factor/genetics/metabolism ; Signal Transduction ; Proto-Oncogene Proteins c-akt/metabolism ; Phosphatidylinositol 3-Kinases/metabolism ; Cells, Cultured ; },
abstract = {Previous studies conducted by our research groups have demonstrated that the HGF/c-Met signaling pathway promotes the proliferation and migration of MSCs in the antlers of Tarim red deer. However, the role and mechanism of this gene in the osteogenic differentiation of antler MSCs remain unclear. In this study, we used antler MSCs as experimental materials. CRISPR/Cas9 technology was employed to knock out the HGF gene, and lentivirus-mediated overexpression of the HGF gene was constructed in antler MSCs. Subsequently, antler MSCs were induced to undergo osteogenic differentiation in vitro. Alizarin Red staining was employed to identify calcium nodules, while the expression levels of various osteogenic differentiation marker genes were assessed using immunohistochemistry, RT-qPCR, and Western blotting techniques. The findings indicated that the HGF gene facilitates the osteogenic differentiation of antler MSCs. Analysis of genes associated with the PI3K/Akt and MEK/ERK signaling pathways demonstrated that in antler MSCs with HGF gene knockout, the expression levels of PI3K/Akt and MEK/ERK pathway genes were significantly downregulated on days 7 and 14 of osteogenic differentiation (p < 0.05). In contrast, antler MSCs with HGF gene overexpression exhibited a significant upregulation of the PI3K/Akt and MEK/ERK signaling pathways on days 4 and 6 of osteogenic differentiation (p < 0.01). These findings suggest that the HGF gene in antlers enhances the osteogenic differentiation of MSCs by activating the PI3K/Akt and MEK/ERK pathways.},
}
@article {pmid40943160,
year = {2025},
author = {Givi, S and Lohnes, BJ and Ebrahimi, S and Riedel, S and Khokhali, S and Khan, SA and Keller, M and Wölfel, C and Echchannaoui, H and Bockamp, E and Andre, MC and Abken, H and Theobald, M and Hartwig, UF},
title = {CRISPR/Cas9 TCR-Edited NKp30 CAR T Cells Exhibit Superior Anti-Tumor Immunity to B7H6-Expressing Leukemia and Melanoma.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943160},
issn = {1422-0067},
support = {2022.046.1//Wilhelm-Sander-Stiftung/ ; },
mesh = {Animals ; Humans ; *Melanoma/therapy/immunology/genetics ; Mice ; *CRISPR-Cas Systems ; *Immunotherapy, Adoptive/methods ; *B7 Antigens/genetics/metabolism/immunology ; *Natural Cytotoxicity Triggering Receptor 3/genetics/immunology/metabolism ; Cell Line, Tumor ; *Receptors, Chimeric Antigen/genetics/immunology ; *T-Lymphocytes/immunology/metabolism ; *Leukemia, Myeloid, Acute/therapy/immunology/genetics ; Xenograft Model Antitumor Assays ; *Receptors, Antigen, T-Cell/genetics ; },
abstract = {Chimeric antigen receptor (CAR) T-cell therapy directed to CD19 and B-cell maturation antigen has revolutionized treatment of B-cell leukemia and lymphoma, and multiple myeloma. However, identifying suitable targets for acute myeloid leukemia (AML) remains challenging due to concurrent expression of potential target antigens on normal hematopoietic stem cells or tissues. As the stress-induced B7H6 molecule is rarely found on normal tissues but expressed on many cancers including AML and melanoma, the NKp30-ligand B7H6 emerges as a promising target for NKp30-based CAR T therapy for these tumors. In this study, we report a comprehensive B7H6 expression analysis on primary AML and melanoma as well as on different tumor cell-lines examined by RT-qPCR and flow cytometry, and efficient anti-tumor reactivity of NKp30-CAR T cells to AML and melanoma. To overcome limitations of autologous CAR T-cell fitness-dependent efficacy and patient-tailored production, we generated CRISPR/Cas9-mediated TCR-knockout (TCR[KO]) NKp30-CAR T cells as an off-the-shelf approach for CAR T therapy. Functional studies comparing NKp30-CD28 CAR or NKp30-CD137 CAR TCR[+] and TCR[KO] T lymphocytes revealed superior anti-tumoral immunity of NKp30-CD28 CAR TCR[KO] T cells to AML and melanoma cell lines in vitro, and effective control of tumor burden in an NSG melanoma-xenograft mouse model. In conclusion, these findings highlight the therapeutic potential of NKp30 CAR TCR[KO] T cells for adoptive T-cell therapy to B7H6-expressing cancers, including melanoma and AML.},
}
@article {pmid40942109,
year = {2025},
author = {Ferrara, F and Sepe, A and Sguizzato, M and Marconi, P and Cortesi, R},
title = {A Pre-Formulation Study for Delivering Nucleic Acids as a Possible Gene Therapy Approach for Spinocerebellar Ataxia Disorders.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {17},
pages = {},
pmid = {40942109},
issn = {1420-3049},
mesh = {Humans ; *Genetic Therapy/methods ; Liposomes/chemistry ; *Spinocerebellar Ataxias/therapy/genetics ; Cell Line, Tumor ; Gene Transfer Techniques ; *Nucleic Acids/chemistry/genetics ; Transfection ; CRISPR-Cas Systems ; RNA, Messenger/genetics ; Green Fluorescent Proteins/genetics ; Plasmids/genetics ; DNA/genetics/chemistry ; },
abstract = {Liposomes are lipid bilayer vesicles that are highly biocompatible, able to interact with the cell membrane, and able to release their cargo easily. The improvement of the physicochemical properties of liposomes, such as surface charge, lipid composition, and functionalization, makes these vesicles eligible delivery nanosystems for the gene therapy of many pathological conditions. In the present study, pre-formulation analysis was conducted to develop liposomes that facilitate the delivery of nucleic acids to neuronal cells, with the aim of future delivery of a CRISPR/Cas9 system designed to silence genes responsible for autosomal dominant neurodegenerative disorders. To this aim, different nucleic acid cargo models, including λ phage DNA, plasmid DNA, and mRNA encoding GFP, were considered. Liposomes with varying lipid compositions were produced using the ethanol injection method and analyzed for their dimensional stability and ability to interact with DNA. The selected formulations were tested in vitro using a neuroblastoma cell line (SH-SY5Y) to evaluate their potential toxicity and the ability to transfect cells with a DNA encoding the green fluorescent protein (pCMV-GFP). Among all formulations, the one containing phosphatidylcholine, phosphatidylethanolamine, pegylated 1,2-distearoyl-sn-glycero-3-phosphethanolamine, cholesterol, and dioctadecyl-dimethyl ammonium chloride (in the molar ratio 1:2:4:2:2) demonstrated the highest efficiency in mRNA delivery. Although this study was designed with the goal of ultimately enabling the delivery of a CRISPR/Cas9 system for treating autosomal dominant neurodegenerative disorders such as polyglutamine spinocerebellar ataxias (SCAs), CRISPR/Cas9 components were not delivered in the present work, and their application remains the objective of future investigations.},
}
@article {pmid40940741,
year = {2025},
author = {Yao, X and Feng, M and Sun, C and Yang, S and Yuan, Z and Liu, X and Li, Q and Jiang, C and Weng, X and Song, J and Mu, Y},
title = {Establishment of a CRISPR/dCas9 Activation Library for Screening Transcription Factors Co-Regulating OCT4 with GATA4 in Pig Cells.},
journal = {Cells},
volume = {14},
number = {17},
pages = {},
pmid = {40940741},
issn = {2073-4409},
mesh = {Animals ; *GATA4 Transcription Factor/metabolism/genetics ; *Octamer Transcription Factor-3/metabolism/genetics ; Swine ; *CRISPR-Cas Systems/genetics ; *Transcription Factors/metabolism/genetics ; Cell Line ; Promoter Regions, Genetic/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; },
abstract = {OCT4 is a critical transcription factor for early embryonic development and pluripotency. Previous studies have shown that the regulation of OCT4 by the transcription factor GATA4 is species-specific in pigs. This study aimed to further investigate whether there are other transcription factors that co-regulate the transcription of OCT4 with GATA4 in pigs. A CRISPR activation (CRISPRa) sgRNA library was designed and constructed, containing 5056 sgRNAs targeting the promoter region of 1264 transcription factors in pigs. Then, a pig PK15 cell line was engineered with a single-copy OCT4 promoter-driven EGFP reporter at the ROSA26 locus, combined with the dCas9-SAM system for transcriptional activation. The CRISPRa sgRNA lentiviral library was used to screen for transcription factors, with or without GATA4 overexpression. Flow cytometry combined with high-throughput sequencing identified MYC, SOX2, and PRDM14 as activators and OTX2 and CDX2 as repressors of OCT4. In the presence of GATA4, transcription factors such as SALL4 and STAT3 showed synergistic activation. Functional validation confirmed that HOXD13 upregulates OCT4, while OTX2 inhibits it. GATA4 and SALL4 synergistically enhance OCT4 expression. These findings provide new insights into combinatorial mechanisms that control the transcriptional regulation of OCT4 in pigs.},
}
@article {pmid40940375,
year = {2025},
author = {Berger, T and Borisova, E and Gamerschlag, A and Terheyden-Keighley, D and Martins, S and Greber, B},
title = {Sequential factor delivery enables efficient workflow for universal gene editing in clinical grade iPS cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {32514},
pmid = {40940375},
issn = {2045-2322},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Workflow ; },
abstract = {Human induced pluripotent stem cells (iPSCs) are gaining momentum as a powerful starting material in cell therapy. To fully harness their potential, CRISPR technology permits endogenous gene modifications as well as the introduction of advanced features, to increase the immune compatibility of the cells or insert suicide genes for enhancing therapeutic safety, for instance. However, genetic manipulation of iPSCs, in particular the generation of knock-in lines, remains relatively inefficient. Conventional mitigation strategies, such as enriching for positive cells using antibiotic selection or complex instrumentation, may, however, cause conflicts with good manufacturing practice (GMP) requirements. To address this challenge, we have systematically optimized a basic gene editing procedure using both Cas9 and Cas12a-based ribonucleoprotein (RNP) complexes. Based on the sequential delivery of RNPs and donor plasmids as a critical hallmark, this virus-free approach permits knock-ins of full-length transgenes at above 30% efficiency, while readily identifying positive clones through random screening at small scale. We exemplify these advances by creating and characterizing homozygous iPSC lines depleted of HLA class I and carrying an inducible caspase-9 suicide gene. Isolated clones from independent GMP iPSC lines retained genomic integrity, differentiation capability, and functionality of the safety switch in the differentiated state. This improved methodology will form a flexible platform for custom gene editing universally applicable both in basic iPSC research and therapy.},
}
@article {pmid40897805,
year = {2025},
author = {Xu, N and Cho, HS and Hackland, JOS and Benito-Kwiecinski, S and Saurat, N and Harschnitz, O and Russo, MV and Garippa, R and Ciceri, G and Studer, L},
title = {Genome-wide CRISPR screen identifies Menin and SUZ12 as regulators of human developmental timing.},
journal = {Nature cell biology},
volume = {27},
number = {9},
pages = {1411-1421},
pmid = {40897805},
issn = {1476-4679},
support = {UM1 HG012654/HG/NHGRI NIH HHS/United States ; R01NS128087//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R01 NS128087/NS/NINDS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; UM1HG012654//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; P30 CA08748//U.S. Department of Health &amp; Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; },
mesh = {Humans ; Gene Expression Regulation, Developmental ; Cell Differentiation/genetics ; *Proto-Oncogene Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; *Polycomb Repressive Complex 2/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism/cytology ; Histones/metabolism/genetics ; PAX6 Transcription Factor/genetics/metabolism ; Promoter Regions, Genetic ; Epigenesis, Genetic ; Neurons/metabolism/cytology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Embryonic Development/genetics ; Neoplasm Proteins ; Transcription Factors ; },
abstract = {Embryonic development follows a conserved sequence of events across species, yet the pace of development is highly variable and particularly slow in humans. Species-specific developmental timing is largely recapitulated in stem cell models, suggesting a cell-intrinsic clock. Here we use directed differentiation of human embryonic stem cells into neuroectoderm to perform a whole-genome CRISPR-Cas9 knockout screen and show that the epigenetic factors Menin and SUZ12 modulate the speed of PAX6 expression during neural differentiation. Genetic and pharmacological loss-of-function of Menin or SUZ12 accelerate cell fate acquisition by shifting the balance of H3K4me3 and H3K27me3 at bivalent promoters, thereby priming key developmental genes for faster activation upon differentiation. We further reveal a synergistic interaction of Menin and SUZ12 in modulating differentiation speed. The acceleration effects were observed in definitive endoderm, cardiomyocyte and neuronal differentiation paradigms, pointing to chromatin bivalency as a general driver of timing across germ layers and developmental stages.},
}
@article {pmid40890563,
year = {2025},
author = {Li, X and Liu, C and Guo, G and Xu, Q and Ren, X and Tuerhongjiang, A and Liu, J and Zeng, J and Wen, CY},
title = {CRISPR-Driven Portable Piezoresistive Biosensor with Cascaded Signal Amplification for Ultrasensitive Osteocalcin Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {36},
pages = {19552-19559},
doi = {10.1021/acs.analchem.5c02678},
pmid = {40890563},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Osteocalcin/analysis/blood ; Limit of Detection ; Platinum/chemistry ; *CRISPR-Cas Systems ; Metal Nanoparticles/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Low-turnover osteoporosis diagnosis urgently requires sensitive detection of low-abundance osteocalcin (OC), yet conventional methods remain constrained by insufficient sensitivity, cumbersome instrumentation, and laborious operations. We devise a CRISPR-driven pressure bioassay that synergistically integrates molecular recognition, enzymatic amplification, and signal transduction for dual-amplification-enhanced OC quantification. The system features an engineered "locked-to-activated" molecular switch, where target binding liberates CRISPR-activating DNA strands, initiating Cas14a-catalyzed cleavage of ssDNA tethers on Fe3O4-ssDNA-Pt nanoassemblies. This cascade releases a multitude of platinum nanoparticles (the first amplification stage). Subsequently, the liberated platinum nanoparticles drive the catalytic decomposition of H2O2 within sealed microchambers, generating a massive flux of oxygen gas molecules (O2) (second amplification stage). Coupled with a laboratory-fabricated nanostructured piezoresistive sensor (20 Pa resolution), this two-stage amplification strategy achieves high sensitivity with a 7.31 pg/mL detection limit, 124-fold lower than commercial ELISA, while completing analysis within 60 min. The platform demonstrates remarkable specificity (spike recovery of 113%, 112%, and 110% in human serum), operational robustness across varying environmental temperatures (15-40 °C), and compatibility with miniaturized instrumentation. Clinical validation through serum matrix analysis reveals excellent correlation (R[2] = 0.982) with reference values. By integrating CRISPR programmability, nanozyme-amplified signaling, and portable piezoresistive sensing, this work provides a sensitive point-of-care osteoporosis screening method for resource-limited settings.},
}
@article {pmid40887819,
year = {2025},
author = {Pérez-Maroto, J and Sepp-Lorenzino, L and Castaño-Esteban, D and Palacios, D and Sot, B},
title = {Advancements in Nonviral Gene Editing Strategies for Rare Diseases.},
journal = {Human gene therapy},
volume = {36},
number = {17-18},
pages = {1118-1137},
doi = {10.1177/10430342251372056},
pmid = {40887819},
issn = {1557-7422},
mesh = {Humans ; *Gene Editing/methods ; *Rare Diseases/therapy/genetics ; *Genetic Therapy/methods ; Genetic Vectors/genetics ; Gene Transfer Techniques ; CRISPR-Cas Systems ; Animals ; Nanoparticles/chemistry ; Nanomedicine ; },
abstract = {Rare diseases are serious and often chronic conditions that affect a small number of individuals. However, with over 7,000 rare diseases identified, their cumulative global numbers and impact are substantial. A considerable proportion of these conditions is caused by genetic abnormalities. Among these, monogenic disorders are of particular relevance, as they are caused by mutations in specific genes. The development of gene therapy, and more specifically, gene editing, offers innovative approaches to treat these rare diseases. A significant challenge associated with the implementation of such strategies concerns the delivery of gene editing tools. Nonviral vectors based on nanomaterials have demonstrated considerable potential as promising alternatives to viral vectors, thereby overcoming their disadvantages. The biocompatibility and tunability of nanoparticles, along with their potential capacity to target diverse tissues, positions them as a promising therapeutic approach for the treatment of a wide range of organ-specific rare diseases. Here, we review current progress in the development and evaluation of novel nanomedicine strategies for gene editing in rare diseases, highlighting new gene editing approaches, delivery systems, and potential targets.},
}
@article {pmid40848918,
year = {2025},
author = {Coratella, E and Bohnert, R and Fischer, B and Lemmens, M and Alpert, T and Beibel, M and Oertli, M and Naumann, U and Elhajouji, A and Odermatt, A and Libertini, S},
title = {SACF and GILA assays on AML12 cells show limited predictive value for mouse liver genotoxicity.},
journal = {Toxicology and applied pharmacology},
volume = {504},
number = {},
pages = {117529},
doi = {10.1016/j.taap.2025.117529},
pmid = {40848918},
issn = {1096-0333},
mesh = {Animals ; Mice ; Gene Editing ; *Liver Neoplasms/genetics/pathology ; CRISPR-Cas Systems ; *Carcinoma, Hepatocellular/genetics/pathology ; Dependovirus/genetics ; *Liver/pathology/metabolism/drug effects ; Predictive Value of Tests ; Mutagenicity Tests/methods ; Humans ; },
abstract = {Hepatocellular carcinoma (HCC) has been observed in neonatal mice following the integration of recombinant Adeno-Associated Viruses (rAAV) into the Rian locus. rAAV-related oncogenic risk for patients remains unclear, and the lack of relevant in vitro methods hinders its proper assessment. The soft agar colony-forming (SACF) assay and the growth in low attachment assay (GILA) monitor anchorage-independent growth, a hallmark of transformed adherent cells, and have been previously proposed to assess the tumorigenicity of CRISPR/Cas9-edited human MCF10A cells. Here, we introduce murine versions of SACF and GILA as surrogate in vitro systems to evaluate the risk of HCC development following genome editing or rAAV induced insertional mutagenesis. Selected tumor suppressors linked to HCC onset in vivo were edited through CRISPR/Cas9 in the hepatic murine cell line AML12. The knockout of neurofibromin (Nf2) and the dual inactivation of tumor protein p53 (Tp53) and phosphatase and tensin homolog (Pten) induced anchorage-independence, while the editing of Axin1, Ctnnb1 (coding for β-catenin), and tuberous sclerosis complex 1 (Tsc1) did not promote growth in anchorage-free conditions. Additionally, we generated stable AML12 and MCF10A clones with the rAAV genome respectively integrated into Rian and MEG8, the human homolog of Rian; however, these clones did not show anchorage independence when seeded in SACF and GILA. Overall, the murine SACF and GILA exhibit low predictive value for HCC development, failing to detect rAAV- and tumor-suppressors-associated oncogenicity. While further optimization may improve assays performance, these results highlight the need for more appropriate in vitro methodologies to accurately evaluate rAAV genotoxicity.},
}
@article {pmid40845730,
year = {2025},
author = {Lai, B and Guo, H and Zhang, K and Wang, H and Qiu, X and Lin, L and Shi, R and Sedibe, M and Zou, Y and Norvienyeku, J},
title = {Putative SET domain-containing proteins play significant roles in regulating sporulation and pathogenicity in Phytophthora capsici.},
journal = {Microbiological research},
volume = {301},
number = {},
pages = {128316},
doi = {10.1016/j.micres.2025.128316},
pmid = {40845730},
issn = {1618-0623},
mesh = {*Phytophthora/pathogenicity/genetics/growth &amp; development ; *Plant Diseases/microbiology/parasitology ; Virulence/genetics ; Gene Expression Profiling ; Capsicum/microbiology ; CRISPR-Cas Systems ; *Fungal Proteins/genetics/metabolism ; },
abstract = {Phytophthora capsici is a filamentous oomycete responsible for root rot, fruit rot, leaf blight, and other economically destructive diseases in multiple plant species, including pepper (Capsicum annuum), tomato (Solanum lycopersicum), squash (Cucurbita pepo), eggplant (Solanum melongena), faba bean (Vicia faba), and lima bean (Phaseolus lunatus), among others. The pathogen causes significant yield losses in fruit and vegetable crops globally. Multiple molecular parameters, including effector proteins and epigenetic modulators, play vital roles in modulating the physio pathological development of P. capsici. Here, we identified 56 SET domain-containing proteins in P. capsici, with 35 predicted as potential effectors. Transcriptomic analysis revealed the upregulation of 13 candidate effector genes during infection, suggesting their roles in pathogenicity. We successfully deleted one effector, PcSET-C (DVH05_022087), and two non-effector proteins, PcSET-B (DVH05_004260), and PcSET-A (DVH05_000194), using CRISPR-Cas9 and homologous recombination strategies. Phenotypic analysis showed that targeted deletion of the PcSET-A, and PcSET-B gene significantly impaired vegetative growth, while ΔPcset-C strains displayed growth like wild-type strains. Additionally, we showed that targeted disruption of all three genes resulted in reduced asexual sporulation and almost completely abolished the pathogenicity or virulence of ΔPcset-A, ΔPcset-B, and ΔPcset-C strains on different P. capsici-susceptible plants. However, targeted replacement of the three SET domain-containing genes investigated in this study has no significant adverse effects on zoospore release in P. capsici. These findings provide insights into the crucial role of SET domain-containing proteins in both morphological and pathological development of P. capsici and underscore PcSETs as potential targets for disease control.},
}
@article {pmid40834922,
year = {2025},
author = {Zhao, JJ and Tian, SN and Peng, ZY and Ren, JX and Zhang, YY and Li, GH and Deng, DH and Zhang, JP and Zhang, XB},
title = {Biomembrane-inspired lipid nanoparticles enhance CRISPR-Cas9 editing for hemophilia A.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {386},
number = {},
pages = {114141},
doi = {10.1016/j.jconrel.2025.114141},
pmid = {40834922},
issn = {1873-4995},
mesh = {Animals ; *CRISPR-Cas Systems ; *Hemophilia A/therapy/genetics ; *Gene Editing/methods ; *Nanoparticles/administration &amp; dosage/chemistry ; Humans ; *Factor VIII/genetics ; Mice ; Mice, Inbred C57BL ; Genetic Therapy/methods ; Dependovirus/genetics ; Male ; *Lipids/chemistry/administration &amp; dosage ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) have substantially advanced RNA-based therapies; however, their use for CRISPR-Cas9 remains limited by sub-optimal endosomal escape, innate immune activation, transient nuclease expression, and restricted tissue specificity. Here, we engineered biomembrane-inspired LNPs containing sphingomyelin and C18-galactosyl ceramide (C18-GalCer) to improve liver-targeted CRISPR delivery. The optimized formulation increased in vivo editing efficiency 2.3-fold relative to a benchmark ALC-0315 LNP. Compared with adeno-associated virus (AAV) vectors, transient mRNA delivery reduced the theoretical risk of insertional mutagenesis, lowered innate immune readouts, and allowed dose titration. By pairing AAV-mediated delivery of a therapeutic Factor VIII donor with LNP-mediated CRISPR-Cas9, we achieved efficient and durable gene correction in a hemophilia A mouse model. A single LNP dose restored plasma Factor VIII activity to >50 % of wild-type levels and maintained this correction for >12 weeks, with low systemic cytokine induction, undetectable off-target insertions, and no overt toxicity. The LNPs retained physicochemical properties and editing potency after prolonged storage and multiple freeze-thaw cycles, supporting their translational potential. Collectively, our findings position biomembrane-inspired LNPs as a safe and efficient non-viral CRISPR platform with potential applicability to other gene-editing therapies.},
}
@article {pmid40749819,
year = {2026},
author = {Gaur, A and Bhakhri, H and Mishra, N and Sharma, S and Bansal, T and Kalaivani, M and Brijwal, M and Das, BK and Lodha, R and Sinha, S and Luthra, K},
title = {A rapid CRISPR/Cas12a-based assay for the detection of HIV-1 Clade C in infants.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {578},
number = {},
pages = {120518},
doi = {10.1016/j.cca.2025.120518},
pmid = {40749819},
issn = {1873-3492},
mesh = {Humans ; *HIV-1/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Infant ; *HIV Infections/diagnosis/virology ; Nucleic Acid Amplification Techniques ; },
abstract = {BACKGROUND: Early detection of HIV-1 infection is essential for initiating antiretroviral therapy (ART) to suppress viremia and prevent disease progression. Timely diagnosis, especially in infants, is critical as rapid antibody-based serology tests are ineffective due to the presence of maternal antibodies.<br><br>METHODS: We developed a CRISPR/Cas12a-based HIV-1 detection assay by optimizing components for coupled isothermal preamplification using recombinase polymerase amplification (RPA). The assay targeted the conserved region in the pol gene specific to HIV-1 with the designed CRISPR RNA (crRNA). CRISPR/Cas12a-mediated cleavage of viral cDNA was visualized through the collateral cleavage of a single-stranded DNA-FAM-BQ reporter, enabling rapid and visually detectable outcomes. The performance of the assay was evaluated using plasma from 41 HIV-1 Clade C (HIV-1C) seropositive individuals, including 28 HIV-1C infected infant samples, HIV-1 Indian Clade C and Clade B genome plasmids, viral disease control DNA/RNA samples (Influenza, RSV, Parvovirus, HPIV, CMV, and HBV), and 31 healthy donor plasma samples. Sensitivity and specificity were assessed, and detection was performed using fluorescence, visual readout, and lateral flow dipsticks.<br><br>RESULTS: The CRISPR/Cas12a-based HIV-1 Clade C detection assay achieved a sensitivity of 96&#xa0;% and a specificity of 92.65&#xa0;%. The assay successfully provided results through both fluorescence and visual readouts and was compatible with lateral flow dipstick formats, facilitating easy and rapid detection.<br><br>CONCLUSIONS: The developed CRISPR/Cas12a-based HIV-1C detection assay demonstrates high sensitivity and specificity for Clade C, indicating its potential as a robust point-of-care molecular diagnostic tool for HIV-1C. Additionally, it may serve as a rapid nucleic acid test alternative for detecting mother-to-child transmission of HIV-1C in infants under two years of age, where traditional antibody-based tests are ineffective. This assay holds promise for improving early HIV-1 diagnosis and timely initiation of ART, ultimately contributing to better disease management and outcomes.},
}
@article {pmid40709654,
year = {2025},
author = {Chang, MM},
title = {An Undergraduate Laboratory on Recombineering and CRISPR/Cas9-Assisted Gene Editing in Escherichia coli.},
journal = {Biochemistry and molecular biology education : a bimonthly publication of the International Union of Biochemistry and Molecular Biology},
volume = {53},
number = {5},
pages = {555-562},
doi = {10.1002/bmb.70002},
pmid = {40709654},
issn = {1539-3429},
mesh = {*Escherichia coli/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Laboratories ; Humans ; Students ; Universities ; *Recombination, Genetic ; *Genetic Engineering ; },
abstract = {Laboratory experience is vital to undergraduate science education. It allows students to observe and conduct engaging experiments to enhance their skills and literacy, helps them retain knowledge, and deepens their understanding of related content covered in lectures. This paper reports a 4-week undergraduate laboratory exercise on Escherichia coli gene editing by recombineering, recombination-mediated genetic engineering, with or without clustered regularly interspaced short palindromic repeats and their associated protein 9 (CRISPR/Cas9). Gene editing makes precise modifications to the DNA of living organisms that influence their development and functions. As technology evolves, recombineering and CRISPR/Cas9 have replaced methods that use restriction enzymes and DNA ligase and are applied to a wide variety of research and applications. It is necessary to introduce undergraduates to these two rapidly growing technologies. Student results obtained from the lab indicate that antisense single-stranded oligodeoxynucleotide (ssODN) has a 15-20 times higher recombineering efficiency than the sense strand. Treatment with a plasmid containing the crRNA target of CRISPR/Cas9 increased recombineering efficiency. Instructional assessments, based on student feedback, revealed that the lab had clear objectives, instructions, and explicit protocols, with sufficient time to complete them, and was found to be interesting and worthwhile. Student learning outcomes, assessed by comparing pre-lab questions and post-lab tests, suggested that they learned the underlying principles and detailed molecular mechanisms. Besides learning the technologies and acquiring basic laboratory skills, students practiced key components of scientific research, such as data collection, analysis, and scientific communication.},
}
@article {pmid40694540,
year = {2025},
author = {Knoll, N and Masser, S and Bordas, B and Ebright, RY and Li, G and Kesar, D and Destefanis, E and Kania, N and Rodriguez, DJ and Jen, J and Zagar, SE and Mensah, C and Chen, Z and Moffitt, SJ and Enakireru, EM and He, Y and Feng, B and Chokshi, MK and Jin, CY and Raghavan, S and Sellers, WR and Mulvaney, KM},
title = {CRISPR-Drug Combinatorial Screening Identifies Effective Combination Treatments for MTAP-Deleted Cancer.},
journal = {Cancer research},
volume = {85},
number = {18},
pages = {3518-3539},
doi = {10.1158/0008-5472.CAN-25-1464},
pmid = {40694540},
issn = {1538-7445},
support = {R35GM154987//National Institute of General Medical Sciences (NIGMS)/ ; 1R01CA233626//Center for Cancer Research (CCR)/ ; CA232543//Basic Research Laboratory (BRL)/ ; V Scholar Grant//V Foundation for Cancer Research (VFCR)/ ; Promos Scholarship//German Academic Exchange Service (DAAD)/ ; //Austrian Marshall Plan Scholarship/ ; Travel Scholarship//BioTechMed-Graz (BIOTECHMEDGRAZ)/ ; CA260442//Center for Cancer Research (CCR)/ ; //Hale Family Center for Pancreatic Cancer Research (Hale Family Research Center At Dana-Farber Cancer Institute)/ ; Claudia Adams Barr Program//Dana-Farber Cancer Institute (DFCI)/ ; },
mesh = {Humans ; *Purine-Nucleoside Phosphorylase/genetics ; Animals ; Mice ; *Protein-Arginine N-Methyltransferases/antagonists &amp; inhibitors/genetics ; *CRISPR-Cas Systems ; *Cyclin-Dependent Kinase Inhibitor p16/genetics ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; *Lung Neoplasms/genetics/drug therapy/pathology ; *Antineoplastic Combined Chemotherapy Protocols/pharmacology/therapeutic use ; },
abstract = {UNLABELLED: Cyclin-dependent kinase inhibitor 2A (CDKN2A)/methylthioadenosine phosphorylase (MTAP) codeletion occurs frequently in non-small cell lung cancer and other solid tumors, including glioblastoma and pancreatic ductal adenocarcinoma. Lung cancer remains the leading cause of cancer-related mortality, and fewer than 15% of patients with glioblastoma or pancreatic cancer survive 5 years, underscoring the need for more effective therapies. Protein arginine methyltransferase 5 (PRMT5) is a synthetic-lethal dependency in MTAP-null tumors and an attractive therapeutic target for CDKN2A/MTAP-deleted cancers. A new revolutionary class of inhibitors, referred to as methylthioadenosine (MTA)-cooperative PRMT5 inhibitors (PRMT5i), has shown promising results in ongoing early-phase clinical trials. Nonetheless, effective cancer treatment typically requires therapeutic combinations to improve response rates and defeat emergent resistant clones. Thus, we sought to determine whether perturbation of other pathways could improve the efficacy of MTA-cooperative PRMT5is (MTAC-PRMT5i). Using a paralog and single gene targeting CRISPR library, we screened MTAP-deleted cancers in the presence or absence of MTAC-PRMT5is. Loss of several genes sensitized cells to PRMT5 inhibition, including members of the MAPK pathway. Chemical inhibition of MAPK pathway members using KRAS, MEK, ERK, and RAF inhibitors synergized with PRMT5 inhibition to kill CDKN2A/MTAP-null, RAS-active tumors. Furthermore, MTAC-PRMT5is combined with either KRAS or RAF inhibitors led to complete responses in vivo, emphasizing the potential benefit for patients. Lastly, cell lines resistant to KRAS inhibition were not resistant to MTAC-PRMT5is and vice versa, suggesting noncross-reactive mechanisms of resistance. Overall, this study identifies therapeutic combinations with MTAC-PRMT5is that may offer significant benefits to patients.<br><br>SIGNIFICANCE: Combining PRMT5 and MAPK pathway inhibitors leads to complete, durable responses in lung cancer models, providing an effective therapeutic strategy for the 4-5% of cancer patients harboring CDKN2A/MTAP deletion and MAPK alterations. See related article by Drizyte-Miller et al., p. 3540.},
}
@article {pmid40684990,
year = {2025},
author = {Gustafsson, O and Krishna, S and Borate, S and Ghaeidamini, M and Liang, X and Saher, O and Cuellar, R and Birdsong, BK and Roudi, S and Estupiñán, HY and Alici, E and Smith, CIE and Esbjörner, EK and Spuler, S and de Jong, OG and Escobar, H and Nordin, JZ and Andaloussi, SEL},
title = {Advanced peptide nanoparticles enable robust and efficient delivery of gene editors across cell types.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {386},
number = {},
pages = {114038},
doi = {10.1016/j.jconrel.2025.114038},
pmid = {40684990},
issn = {1873-4995},
mesh = {Humans ; *Gene Editing/methods ; *Nanoparticles/chemistry/administration &amp; dosage ; HEK293 Cells ; CRISPR-Cas Systems ; *Cell-Penetrating Peptides/chemistry/administration &amp; dosage ; Silicon Dioxide/chemistry ; Induced Pluripotent Stem Cells/metabolism ; *Gene Transfer Techniques ; Integrases/genetics/administration &amp; dosage ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Efficient delivery of the CRISPR/Cas9 system and its larger derivatives, base editors, and prime editors remain a major challenge, particularly in tissue-specific stem cells and induced pluripotent stem cells (iPSCs). This study optimized a novel family of cell-penetrating peptides, hPep, to deliver gene-editing ribonucleoproteins. The hPep-based nanoparticles enable highly efficient and biocompatible delivery of Cre recombinase, Cas9, base-, and prime editors. Using base editors, robust and nearly complete genome editing was achieved in the human cells: HEK293T (96%), iPSCs (74%), and muscle stem cells (80%). This strategy opens promising avenues for ex vivo and, potentially, in vivo applications. Incorporating silica particles enhanced the system's versatility, facilitating cargo-agnostic delivery. Notably, the nanoparticles can be synthesized quickly on a benchtop and stored as lyophilized powder without compromising functionality. This represents an important advancement in the feasibility and scalability of gene-editing delivery technologies.},
}
@article {pmid40680607,
year = {2025},
author = {Eladl, O},
title = {CRISPR interference-mediated silencing of pro-inflammatory genes in autoimmune models: functional and biophysical evaluation with sustained effects in primary human PBMCs.},
journal = {International immunopharmacology},
volume = {163},
number = {},
pages = {115257},
doi = {10.1016/j.intimp.2025.115257},
pmid = {40680607},
issn = {1878-1705},
mesh = {Humans ; *Leukocytes, Mononuclear/immunology ; *Gene Silencing ; CRISPR-Cas Systems ; *Autoimmune Diseases/genetics/immunology/therapy ; CD40 Antigens/genetics ; Interleukin-6/genetics ; Interferon-gamma/genetics ; *Inflammation/genetics ; Cells, Cultured ; Cytokines/genetics ; },
abstract = {Autoimmune diseases are caused by unregulated immune stimulation and overproduction of inflammatory cytokines, leading to chronic tissue injury and disability. Current treatments are broad-acting and short-lived, highlighting the need for targeted molecular interventions with prolonged immunomodulatory effects. Here, we demonstrate CRISPR interference (CRISPRi), employing a catalytically inactive Cas9 fused to a KRAB repression domain, to potently and selectively silence major inflammatory genes-IL-6, CD40, and IFN-γ-in human immune cell lines. Utilizing biophysical methods such as surface plasmon resonance and fluorescence anisotropy, complemented by flow cytometric analysis, we validated functional interference with inflammatory signaling cascades and significant downregulation of immune activation markers. These findings indicate that CRISPRi not only represses gene expression but also significantly affects downstream cytokine-receptor interactions and immune cell activation. Compared to other gene silencing approaches, like siRNA and antisense oligonucleotides, CRISPRi showed greater specificity and longer duration, with sustained repression lasting up to 72 h post-treatment. Importantly, we confirmed these effects in primary human peripheral blood mononuclear cells (PBMCs), where CRISPRi significantly suppressed pro-inflammatory cytokine release and modulated immune cell phenotypes. These results highlight the potent, programmable, and sustained immunomodulatory potential of CRISPRi as a therapeutic for autoimmune diseases characterized by inflammation and immune dysregulation.},
}
@article {pmid40673842,
year = {2025},
author = {Johnson, MJ and Sumstad, D and Folsom, TD and Slipek, NJ and DeFeo, AP and Growe, M and Kadidlo, D and Thyagarajan, B and Starr, TK and Lou, E and Choudhry, M and Moriarity, BS and Webber, BR and McKenna, DH},
title = {Clinical manufacture of CRISPR/Cas9-based cytokine-induced SH2 protein knock-out tumor-infiltrating lymphocytes for gastrointestinal cancers.},
journal = {Cytotherapy},
volume = {27},
number = {10},
pages = {1229-1239},
doi = {10.1016/j.jcyt.2025.06.007},
pmid = {40673842},
issn = {1477-2566},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Lymphocytes, Tumor-Infiltrating/immunology/metabolism ; *Gastrointestinal Neoplasms/immunology/therapy/pathology/genetics ; *Cytokines ; Male ; Female ; Gene Knockout Techniques ; },
abstract = {INTRODUCTION: The prognosis of stage IV gastrointestinal (GI) carcinomas is poor with a 15% five-year survival rate for colorectal carcinomas. To improve efficacy of tumor infiltrating lymphocytes (TIL), we isolated mutation-reactive autologous TIL and employed CRISPR/Cas9 to knockout (KO) the intracellular checkpoint protein CISH, which has been shown to enhance T cell expansion, functional avidity, and cytokine polyfunctionality, with consequent durable regression of established tumors in an animal model.<br><br>MATERIALS &amp; METHODS: TIL cultures were initiated from resected tumor fragments and maintained for six weeks before harvest and cryopreservation. Candidate neoantigens were nominated by exome sequencing and peptides were used to identify mutation reactive (MR) TIL. Selected MR TIL were thawed and allowed to recover for 24-36 h in media with 10% AB serum, 6000 IU/mL IL-2, and 5 ng/mL IL-7 and IL-15 followed by stimulation with plate-bound anti-CD3/soluble anti-CD28 for 4 days. CISH KO was performed by electroporation of Cas9 mRNA and chemically modified single guide RNA. Between 5 -7.5 million viable cells were added to each 100 cm[2] G-Rex vessel containing 600 mL expansion media (with allogeneic feeder MNC:TIL = 100:1) and incubated for 6-8 days. Cultures were evaluated and split according to cell concentration criteria (and dose cohort) and incubated for an additional 6-8 days. On day 14, all of the cells were harvested, washed with buffer and cryopreserved (5% DMSO). Lot release testing included: viability, %CD3+, cytology review, Gram stain, sterility, endotoxin, mycoplasma, and interferon gamma (IFN-&#x3b3;) production. Additional testing included DNA sequencing to determine genomic CISH editing efficiency and a Western blot for determination of CISH protein loss.<br><br>RESULTS: Patients with GI cancers (colon [10], rectal [8], pancreatic [1], and esophageal [1]) underwent tumor collection. Nineteen of 22 tumor biopsies sampled from 20 patients total proceeded to KO/expansion. Final TIL product results (mean [SD], median [range]) were: viable count (x 10[10]) -3.25 (3.67), 1.95 (0.018-12.40); viable TIL fold expansion -327.1 (364.8), 153.1 (8-1454); % viability - 76 (13), 78 (43-92); % CD3 -94.4 (5.4), 95.8 (78.6-99.4); % CISH KO efficiency - 75 (29), 87 (0-96); % editing efficiency - 59.9 (24.8), 66.9 (0.4-86). Viability fell below 70% for five TIL products. All other lot release testing has met specification. Thirteen patients have received TIL; six patients were not treated due to disease progression prior to anticipated infusion.<br><br>CONCLUSION: The translation of CRISPR/Cas9-based CISH KO MR TIL from the basic research lab to current good manufacturing practices The (cGMP) facility was successful, allowing for optimized, large-scale expansion in support of a first-in-human clinical trial to treat patients with metastatic GI cancers (ClinicalTrials.gov Identifier: NCT04426669).},
}
@article {pmid40641457,
year = {2025},
author = {Su, P and Wu, L and Li, D and Song, W and Tao, D and Liu, L and Wang, Q and Gao, M and Xu, T and Liu, X and Xie, S and Zhang, X and Zhou, J and Miao, YL},
title = {CRISPR Screening Reveals a Novel Role for FOXH1 in Regulating Pluripotency of Porcine Embryonic Stem Cells.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {34},
pages = {e09495},
doi = {10.1002/advs.202509495},
pmid = {40641457},
issn = {2198-3844},
support = {2024YFA1802400//National Key R&amp;D Program of China/ ; 2022YFD1302200//National Key R&amp;D Program of China/ ; 32425051//National Natural Science Foundation of China/ ; 2662023DKPY001//Fundamental Research Funds for the Central Universities/ ; 2024040801020291//Knowledge Innovation Program of Wuhan-Basic Research/ ; },
mesh = {Animals ; Swine ; Cell Differentiation/genetics ; *CRISPR-Cas Systems/genetics ; *Embryonic Stem Cells/metabolism/cytology ; *Pluripotent Stem Cells/metabolism/cytology ; *Forkhead Transcription Factors/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Porcine extended potential stem cells (pEPSCs), which exhibit both self-renewal and pluripotency, are promising for application in both agricultural biotechnology and regenerative medicine. However, the molecular mechanisms governing these two interconnected properties remain elusive. Here, two types of CRISPR-Cas9 screenings are conducted in pEPSCs. This fitness screening identified several genes essential for cell viability, including PRMT1, MYBL2, and NASP. Concurrently, FACS-based screenings revealed genes crucial for pluripotency, such as SOX2, ZFP42, and FOXH1. Notably, it is demonstrated that FOXH1 is required for maintaining pluripotency in pEPSCs, which complements the understanding of its role in mesendoderm specification. pEPSCs lacking FOXH1 exhibited a flatter and more dispersed clonal morphology, accompanied by downregulation of pluripotency genes and upregulation of lineage-specific genes. Additionally, FOXH1 knockdown significantly impaired blastocyst formation during early pig embryogenesis. Functionally, the dual role of FOXH1 in pluripotency maintenance and cell differentiation is validated: FOXH1 preserves pluripotency by enhancing chromatin accessibility at pluripotency gene loci, while also influencing lineage specification through H3K4me3 modification at developmental related genes. Thus, these findings uncover a novel role of FOXH1 involved in the core regulatory network that orchestrates gene expression programs to maintain the pluripotency state of pEPSCs and provide valuable insights into categorizing gene function.},
}
@article {pmid40586425,
year = {2025},
author = {Han, Z and Wang, X and Yu, D and Wang, J and Sun, K and Wang, S and Zhang, Y and Feng, G and Li, W and Hai, T and Ren, J},
title = {Achieving scalable expansion of therapeutic porcine hepatocytes in vivo through serial transplantation.},
journal = {Animal models and experimental medicine},
volume = {8},
number = {7},
pages = {1337-1344},
pmid = {40586425},
issn = {2576-2095},
support = {2021YFA0805905//National Key Research and Development Program of China/ ; 2023YFC3404305//National Key Research and Development Program of China/ ; 2024YFA1107900//National Key Research and Development Program of China/ ; XDB1150000//the Strategic Priority Research Program of the Chinese Academy of Sciences/ ; YSBR-012//the CAS Project for Young Scientists in Basic Research/ ; NYHXGG2023AA01//Bingtuan Science and Technology Project/ ; },
mesh = {*Hepatocytes/transplantation/physiology ; Animals ; Swine ; Liver Regeneration ; CRISPR-Cas Systems ; Liver ; },
abstract = {The clinical application of hepatocyte transplantation has been significantly hindered by the scarcity of primary hepatocytes and the functional immaturity of in vitro-produced hepatocytes. By performing serial allogeneic hepatocyte transplantation in CRISPR/Cas9-mediated Fah-knockout pigs, we successfully achieved large-scale expansion of hepatocytes while maintaining their authentic biological characteristics. Particularly, the established model enables sustained in vivo liver reconstruction, concurrently ameliorating hepatic fibrosis and demonstrating functional microenvironmental remodeling. Moreover, through comprehensive single-cell transcriptomic profiling of 52 418 hepatocytes across transplant generations (F0-F2), we discovered that the cellular composition of these transplanted hepatocytes is similar to that of wild-type hepatocytes. The regenerated liver exhibits all six major hepatic cell types identical to the wild-type counterparts, with the characteristic lobular zonation patterns well preserved. Our research provides valuable insights into the large-scale expansion of physiologically functional hepatocytes in vivo without compromising their biological properties. This finding holds great promise for advancing the clinical application of human hepatocyte transplantation, potentially offering more effective treatment options for patients with liver diseases.},
}
@article {pmid40432605,
year = {2025},
author = {López-Manzaneda, S and Mencía, &#xc1; and Bonafont, J and Bassons-Bascuñana, A and García, M and Nyström, A and Duarte, B and Llames, S and Murillas, R and Modamio-Hoybjor, S and Morín, M and Soletto, L and Escamez, MJ and Moreno-Pelayo, MA and Rio, MD and Larcher, F},
title = {Safe and Efficacious Permanent Removal of Large COL7A1 Exons for Gene Reframing as a Reliable Therapeutic Strategy for Recessive Dystrophic Epidermolysis Bullosa.},
journal = {Human gene therapy},
volume = {36},
number = {17-18},
pages = {1211-1221},
doi = {10.1089/hum.2024.238},
pmid = {40432605},
issn = {1557-7422},
mesh = {*Collagen Type VII/genetics/metabolism ; *Epidermolysis Bullosa Dystrophica/genetics/therapy/pathology ; *Exons ; Humans ; *Gene Editing/methods ; Animals ; CRISPR-Cas Systems ; *Genetic Therapy/methods ; Mice ; DNA End-Joining Repair ; },
abstract = {Mutations leading to premature termination codons in COL7A1 are commonly associated with severe generalized recessive dystrophic epidermolysis bullosa (RDEB). Previous research, including our own, has indicated that removing mutated COL7A1 exons along with the consequent reframing of COL7A1 may not pose noticeable impact on protein function, offering a potential therapeutic strategy. However, investigations into the long-term in vivo effects of genome editing-mediated removal of mutant exons have only focused on the small exon 80 thus far. Hence, this study focuses on exons 73 and 105 of COL7A1 to explore whether targeted exon removal, through a CRISPR/Cas9-assisted, Non-homologous end joining (NHEJ)-mediated approach, could be extended to other larger exons. Introducing ribonucleoprotein complexes carrying Cas9 and optimized sgRNA guide pairs for each exon (73 and 105) through electroporation efficiently led to their removal, consequently restoring type VII collagen (C7) synthesis in RDEB primary patient cells carrying frameshift mutations in these exons. In vitro tests indicated the normal stability of the resulting C7 variants expressed at physiological levels, while in vivo analyses of regenerated skin grafted onto immunodeficient mice using E73 or E105 RDEB edited cells demonstrated the proper deposition of C7 at the basement membrane zone, thereby restoring normal dermo-epidermal adherence. This study enhances the broader potential of the exon deletion approach in the treatment of RDEB.},
}
@article {pmid40252393,
year = {2025},
author = {Almasoudi, HH},
title = {Therapeutic promise of CRISPR-Cas9 gene editing in sickle cell disease and β-thalassemia: A current review.},
journal = {Current research in translational medicine},
volume = {73},
number = {3},
pages = {103513},
doi = {10.1016/j.retram.2025.103513},
pmid = {40252393},
issn = {2452-3186},
mesh = {Humans ; *Anemia, Sickle Cell/therapy/genetics ; *beta-Thalassemia/therapy/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; Animals ; Mutation ; beta-Globins/genetics ; },
abstract = {Sickle cell disease (SCD and β-thalassemia (BT) affects millions of people worldwide. In addition, around 500,000 infants are born with SCD and 60,000 people are diagnosed with BT every year. Mutations in the hemoglobin subunit beta (HBB) gene are responsible for causing both BT and SCD. Indeed, the diversity of potential mutations in the HBB gene elucidates the diversity in clinical severity observed in individuals with BT and related morbidities. On the other hand, SCD takes place because of the alteration in a single amino acid at position 6 in the beta-globin chain, where a base substitution occurs from glutamic acid to valine, which eventually results in abnormal sickle hemoglobin. Conventional therapies for BT and SCD including pharmaceutical drugs and blood transfusion might temporarily improve the clinical severity of these diseases, however these therapies cannot cure the diseases. CRISPR-Cas9 (CC9) is revolutionizing genome engineering, offering promising therapeutic avenues for genetic diseases. Therefore, CC9-mediated gene therapy provides great hope in the treatment of both BT and SCD. CC9-mediated gene therapy has already demonstrated its effectiveness in correcting both SCD and BT-causing mutations. Moreover, CC9-mediated gene editing was found to be effective in reactivating the expression of hemoglobin F (HbF) and regulating LRF and BCL11A. A number of clinical trials with CC9 gene-edited therapies are being carried out to elucidate their potential in treating BT and SCD. Genetics and pathophysiological mechanisms of SCD and BT, the mechanism of CC9-mediated gene editing, and common delivery methods of the CC9 system have been discussed in this review. Moreover, an in-depth discussion on applications and the current status of CC9-mediated gene editing in SCD and BT along with current challenges and future perspectives have been provided.},
}
@article {pmid39500392,
year = {2025},
author = {Huang, M and Wang, K and Li, A and Zhu, X and Zhou, Z and Yang, C and Bi, C and Zhang, X},
title = {Mini and enhanced CRISPR activators for cancer therapies.},
journal = {Journal of advanced research},
volume = {75},
number = {},
pages = {151-161},
doi = {10.1016/j.jare.2024.10.027},
pmid = {39500392},
issn = {2090-1224},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/therapy/genetics ; Animals ; *Gene Editing/methods ; *Genetic Therapy/methods ; Cell Line, Tumor ; Caenorhabditis elegans/genetics ; Cell Proliferation/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcriptional Activation/genetics ; },
abstract = {INTRODUCTION: The RNA-guided nuclease Cas9 can be used as a programmable transcription activator, but there is still room for improvement in its effectiveness in eukaryotes, and its potential in cancer genetic therapy has been poorly investigated.<br><br>OBJECTIVES: We aim to construct optimized CRISPRa tools and detect their potential role in cancer therapy by screening 9aa-TAD.<br><br>METHODS: We selected a range of transcriptional coactivators for programmable activation and analyzed their effects on the expression of multiple endogenous genes using Flow cytometry and qRT-PCR. In order to improve the activation capacity of the CRISPRa tool, we fused the coactivators with the efficient dCas9-VPR system to construct a new activation system. Utilize RNA-seq to assess the activation specificity of genome-wide. To evaluate the value of the newly constructed activation system in cancer gene therapy, we activated the expression of the tumor suppressor genes PER2 and ZNF382, and performed changes in cancer cell proliferation qRT-PCR and clonal formation analysis.<br><br>RESULTS: In this study, we screened the NHR module from C. elegans, which demonstrated a high transcription activation capacity with a compact size compared to VP64. We successfully demonstrated its efficiency in activating endogenous genes in mammalian cells. Furthermore, we developed an enhanced fused variant called NHR-VP64-p65-Rta (NVPR), which showed even higher efficiency compared to the previously established VPR module, making it an effective CRISPRa tool. The dCas9-NVPR complex also exhibited high specificity on a genome-wide scale. Finally, we utilized the dCas9-NVPR tool to restore the expression of tumor suppressor genes PER2 and ZNF382, effectively inhibiting the malignant phenotype of cancer cells.<br><br>CONCLUSION: We have successfully developed and demonstrated a breakthrough CRISPRa tool with promising implications for cancer genetic therapy. This innovation expands the range of available gene editing tools and further validates the immense potential of CRISPR-based approaches in precision medicine.},
}
@article {pmid39487265,
year = {2025},
author = {Tycko, J and Van, MV and Aradhana, and DelRosso, N and Ye, H and Yao, D and Valbuena, R and Vaughan-Jackson, A and Xu, X and Ludwig, C and Spees, K and Liu, K and Gu, M and Khare, V and Mukund, AX and Suzuki, PH and Arana, S and Zhang, C and Du, PP and Ornstein, TS and Hess, GT and Kamber, RA and Qi, LS and Khalil, AS and Bintu, L and Bassik, MC},
title = {Development of compact transcriptional effectors using high-throughput measurements in diverse contexts.},
journal = {Nature biotechnology},
volume = {43},
number = {9},
pages = {1525-1538},
pmid = {39487265},
issn = {1546-1696},
support = {K00 DK126120/DK/NIDDK NIH HHS/United States ; R01 EB029483/EB/NIBIB NIH HHS/United States ; R01 HG011866/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *High-Throughput Screening Assays/methods ; HEK293 Cells ; Protein Domains ; *Transcription, Genetic ; Gene Expression Regulation ; },
abstract = {Transcriptional effectors are protein domains known to activate or repress gene expression; however, a systematic understanding of which effector domains regulate transcription across genomic, cell type and DNA-binding domain (DBD) contexts is lacking. Here we develop dCas9-mediated high-throughput recruitment (HT-recruit), a pooled screening method for quantifying effector function at endogenous target genes and test effector function for a library containing 5,092 nuclear protein Pfam domains across varied contexts. We also map context dependencies of effectors drawn from unannotated protein regions using a larger library tiling chromatin regulators and transcription factors. We find that many effectors depend on target and DBD contexts, such as HLH domains that can act as either activators or repressors. To enable efficient perturbations, we select context-robust domains, including ZNF705 KRAB, that improve CRISPRi tools to silence promoters and enhancers. We engineer a compact human activator called NFZ, by combining NCOA3, FOXO3 and ZNF473 domains, which enables efficient CRISPRa with better viral delivery and inducible control of chimeric antigen receptor T cells.},
}
@article {pmid39415058,
year = {2025},
author = {Chen, K and Han, H and Zhao, S and Xu, B and Yin, B and Lawanprasert, A and Trinidad, M and Burgstone, BW and Murthy, N and Doudna, JA},
title = {Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR-Cas9 ribonucleoprotein.},
journal = {Nature biotechnology},
volume = {43},
number = {9},
pages = {1445-1457},
pmid = {39415058},
issn = {1546-1696},
support = {RM1HG009490//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; UH3 NS115599/NS/NINDS NIH HHS/United States ; R01MH125979-01//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 MH125979/MH/NIMH NIH HHS/United States ; DISC2-14097//California Institute for Regenerative Medicine (CIRM)/ ; DISC2-14045//California Institute for Regenerative Medicine (CIRM)/ ; UG3 NS115599/NS/NINDS NIH HHS/United States ; UM1AI164559//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; },
mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; Mice ; *Nanoparticles/chemistry ; *Liver/metabolism ; *Lung/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; Humans ; Lipids/chemistry ; Liposomes ; },
abstract = {Lipid nanoparticle (LNP) delivery of clustered regularly interspaced short palindromic repeat (CRISPR) ribonucleoproteins (RNPs) could enable high-efficiency, low-toxicity and scalable in vivo genome editing if efficacious RNP-LNP complexes can be reliably produced. Here we engineer a thermostable Cas9 from Geobacillus stearothermophilus (GeoCas9) to generate iGeoCas9 variants capable of >100× more genome editing of cells and organs compared with the native GeoCas9 enzyme. Furthermore, iGeoCas9 RNP-LNP complexes edit a variety of cell types and induce homology-directed repair in cells receiving codelivered single-stranded DNA templates. Using tissue-selective LNP formulations, we observe genome-editing levels of 16‒37% in the liver and lungs of reporter mice that receive single intravenous injections of iGeoCas9 RNP-LNPs. In addition, iGeoCas9 RNPs complexed to biodegradable LNPs edit the disease-causing SFTPC gene in lung tissue with 19% average efficiency, representing a major improvement over genome-editing levels observed previously using viral or nonviral delivery strategies. These results show that thermostable Cas9 RNP-LNP complexes can expand the therapeutic potential of genome editing.},
}
@article {pmid39385008,
year = {2025},
author = {Wang, Y and Liu, KI and Liu, MM and Ooi, KH and Nguyen, TA and Chee, JE and Teo, SXD and He, S and Tay, JWD and Teo, SY and Liew, KS and Ge, XY and Ng, ZJ and Avagyan, H and Liu, H and Yi, Z and Chang, K and Kok, EPL and Chen, R and Yau, CE and Koh, JW and Wan, Y and Tan, MH},
title = {A circularly permuted CasRx platform for efficient, site-specific RNA editing.},
journal = {Nature biotechnology},
volume = {43},
number = {9},
pages = {1548-1561},
pmid = {39385008},
issn = {1546-1696},
support = {NRF2017-NRF-ISF002-2673//National Research Foundation Singapore (National Research Foundation-Prime Minister's office, Republic of Singapore)/ ; SSG 2021-03//National Research Foundation Singapore (National Research Foundation-Prime Minister's office, Republic of Singapore)/ ; T2EP30223-0028//Ministry of Education - Singapore (MOE)/ ; RG50/17//Ministry of Education - Singapore (MOE)/ ; NMRC/OFIRG/0017/2016//MOH | National Medical Research Council (NMRC)/ ; },
mesh = {*RNA Editing/genetics ; Humans ; Adenosine Deaminase/genetics/metabolism ; *Protein Engineering/methods ; *CRISPR-Cas Systems/genetics ; RNA-Binding Proteins/genetics ; *CRISPR-Associated Proteins/genetics ; HEK293 Cells ; },
abstract = {Inactive Cas13 orthologs have been fused to a mutant human ADAR2 deaminase domain at the C terminus to enable programmable adenosine-to-inosine (A-to-I) RNA editing in selected transcripts. Although promising, existing RNA-editing tools generally suffer from a trade-off between efficacy and specificity, and off-target editing remains an unsolved problem. Here we describe the development of an optimized RNA-editing platform by rational protein engineering, CasRx-based Programmable Editing of RNA Technology (xPERT). We demonstrate that the topological rearrangement of a CasRx K940L mutant by circular permutation results in a robust scaffold for the tethering of a deaminase domain. We benchmark our tool against the REPAIR system and show that xPERT exhibits strong on-target activity like REPAIRv1 but low off-target editing like REPAIRv2. Our xPERT platform can be used to alter RNA sequence information without risking genome damage, effect temporary cellular changes and customize protein function.},
}
@article {pmid39375445,
year = {2025},
author = {Song, J and Cho, MH and Cho, H and Song, Y and Lee, SW and Nam, HC and Yoon, TH and Shin, JC and Hong, JS and Kim, Y and Ekanayake, E and Jeon, J and You, DG and Im, SG and Choi, GS and Park, JS and Carter, BC and Balaj, L and Seo, AN and Miller, MA and Park, SY and Kang, T and Castro, CM and Lee, H},
title = {Amplifying mutational profiling of extracellular vesicle mRNA with SCOPE.},
journal = {Nature biotechnology},
volume = {43},
number = {9},
pages = {1485-1495},
pmid = {39375445},
issn = {1546-1696},
support = {HR22C1832//Korea Health Industry Development Institute (KHIDI)/ ; U01CA284982//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; DP2CA259675//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; 2021M3E5E3080379//National Research Foundation of Korea (NRF)/ ; U01 CA230697/CA/NCI NIH HHS/United States ; 2023R1A2C2005185//National Research Foundation of Korea (NRF)/ ; 1U01CA279858//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; 2021R1A2B5B03001416//National Research Foundation of Korea (NRF)/ ; 2021R1A2C1005342//National Research Foundation of Korea (NRF)/ ; R01 CA237500/CA/NCI NIH HHS/United States ; R01 CA239078/CA/NCI NIH HHS/United States ; R01CA264363//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; },
mesh = {*Extracellular Vesicles/genetics/metabolism ; Humans ; *RNA, Messenger/genetics ; *Mutation/genetics ; Animals ; Mice ; CRISPR-Cas Systems/genetics ; Colorectal Neoplasms/genetics ; Glioblastoma/genetics ; Lung Neoplasms/genetics ; Cell Line, Tumor ; Liquid Biopsy ; },
abstract = {Sequencing of messenger RNA (mRNA) found in extracellular vesicles (EVs) in liquid biopsies can provide clinical information such as somatic mutations, resistance profiles and tumor recurrence. Despite this, EV mRNA remains underused due to its low abundance in liquid biopsies, and large sample volumes or specialized techniques for analysis are required. Here we introduce Self-amplified and CRISPR-aided Operation to Profile EVs (SCOPE), a platform for EV mRNA detection. SCOPE leverages CRISPR-mediated recognition of target RNA using Cas13 to initiate replication and signal amplification, achieving a sub-attomolar detection limit while maintaining single-nucleotide resolution. As a proof of concept, we designed probes for key mutations in KRAS, BRAF, EGFR and IDH1 genes, optimized protocols for single-pot assays and implemented an automated device for multi-sample detection. We validated SCOPE's ability to detect early-stage lung cancer in animal models, monitored tumor mutational burden in patients with colorectal cancer and stratified patients with glioblastoma. SCOPE can expedite readouts, augmenting the clinical use of EVs in precision oncology.},
}
@article {pmid40950596,
year = {2025},
author = {Hadian, S and Smith, DL and Suproniene, S},
title = {Genomic and phenotypic profiling of an Artemisia endophyte: dual biostimulant and biocontrol activities in pea under Fusarium stress.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1643204},
pmid = {40950596},
issn = {1664-302X},
abstract = {OBJECTIVE: To evaluate the plant growth-promoting and disease-suppressing potential of Serratia sp. AR11, an endophytic bacterium isolated from Artemisia absinthium, through phenotypic assessment and whole-genome analysis in pea (Pisum sativum) under normal and Fusarium-stress conditions.<br><br>MATERIALS AND METHODS: Greenhouse experiments were conducted to assess the effects of AR11 inoculation on shoot and root growth, biomass, chlorophyll content, and Fusarium-induced stunting. Whole-genome sequencing was performed using the PacBio SMRT platform, followed by functional annotation to identify genes related to nutrient metabolism, secondary metabolite biosynthesis, and stress adaptation. Biosafety assessment included screening for virulence and antibiotic resistance genes.<br><br>RESULTS: AR11 inoculation significantly increased shoot and root length and biomass, while reducing Fusarium-induced stunting by over 70%. Under pathogen stress, treated plants showed a 67% increase in SPAD index compared to controls. Genome analysis revealed a complete 5.49 Mb circular genome with 5,175 protein-coding genes, including those for nitrogen metabolism, phosphate solubilization, siderophore production, and antifungal secondary metabolite biosynthesis. Additional features included stress-responsive regulatory systems, CRISPR-Cas loci, and DNA methylation patterns. No high-risk virulence or acquired antibiotic resistance genes were detected.<br><br>CONCLUSION: Serratia sp. AR11 is a safe, multifunctional endophytic bacterium with strong genomic and phenotypic evidence for enhancing pea growth and suppressing Fusarium-related diseases, making it a promising candidate for sustainable low-input agriculture.},
}
@article {pmid40946761,
year = {2025},
author = {Uddin, N and Ullah, MW and Li, K and Liu, F and Xin, X},
title = {Plant cell wall biosynthesis: Immune signaling, genome editing, and physiological implications for biomass valorization.},
journal = {Biotechnology advances},
volume = {},
number = {},
pages = {108714},
doi = {10.1016/j.biotechadv.2025.108714},
pmid = {40946761},
issn = {1873-1899},
abstract = {Plants continuously face biotic stress from pathogens, pests, and environmental challenges that threaten their survival and productivity. In response, plants have developed complex immune systems, with the cell wall playing a central role in defense. The plant cell wall not only provides mechanical strength but also acts as a dynamic barrier against pathogens, influencing both plant growth and immune responses. This review discusses the molecular mechanisms of cell wall biosynthesis, facilitated by multi-omics technologies, particularly the synthesis and regulation of lignin and other polysaccharides, which contribute to cell wall integrity and plant immunity. It explores the interplay between cell wall modifications and immune signaling pathways, highlighting the role of pattern recognition receptors in pathogen detection and defense activation. Additionally, the potential of genome editing, especially CRISPR-Cas, in enhancing cell wall characteristics to improve pathogen resistance and biomass utilization is discussed. With growing interest in lignocellulosic biomass as a renewable resource for biofuels and bioproducts, this review also addresses the challenges of biomass recalcitrance, focusing on biotechnological advancements to improve saccharification efficiency. Finally, the review proposes integrated strategies combining genetic modifications, biotechnological innovations, and sustainable practices to optimize lignocellulosic biomass for a bio-based economy, contributing to both agricultural resilience and sustainable energy production. As climate change accelerates, these technologies hold the promise of developing resilient crops and enhancing the capacity of the bioeconomy to mitigate environmental impacts.},
}
@article {pmid40946164,
year = {2025},
author = {Sun, J and Liu, K and Sheng, Y and Zhang, H and Wang, J and Wang, Y and Tian, R and Zhu, X and Tian, SS and Wang, P and Zhao, X and Liu, Y},
title = {High-Fidelity AaCas12b[Max] Enables the Development of an Engineered T Cell Therapy with Enhanced Safety and Functional Fitness.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.ymthe.2025.09.009},
pmid = {40946164},
issn = {1525-0024},
abstract = {CRISPR-Cas systems have transformed genome editing, yet the commonly used Streptococcus pyogenes Cas9 (SpCas9) is limited by off-target effects and chromosomal instability. Here, we characterize AaCas12b[MAX], an engineered Alicyclobacillus acidiphilus Cas12b variant, as a high-precision editing platform optimized for tumor infiltrating lymphocyte (TIL) therapy. Using an FDA-compliant safety assessment framework, we systemically compared AaCas12b[MAX]- and SpCas9-edited TIL products in terms of on-target efficiency, genome-wide off-target activity, and structural variants (SVs) formation. AaCas12b[MAX] achieved >80% on-target editing efficiency with undetectable off-target events and a 3.3-fold reduction in SVs relative to SpCas9. Mechanistic studies revealed different DNA repair kinetics in AaCas12b[MAX]-edited cells, reducing sustained DNA damage responses and chromosomal instability. Structural modeling suggested a more stable enzyme-sgRNA-DNA ternary complex, enabling stringent PAM specificity and minimal mismatch tolerance. Functionally, AaCas12b[MAX]-edited TILs exhibited superior therapeutic potential, including enhanced cellular fitness, a twofold increase in expansion capacity, and enrichment of stem-like tumor-reactive CD39[-]CD69[-]CD8[+] subsets. Together, these results establish AaCas12b[MAX] as a robust, clinically translatable platform that improves the safety and functional limitations of SpCas9, enabling the development of next-generation T cell therapies.},
}
@article {pmid40945731,
year = {2025},
author = {Jain, M and Pattnayak, AK and Aggarwal, S and Rai, P and Kavya, J and Chandrayan, S and Goel, M and Gaur, V},
title = {Branched DNA processing by a thermostable CAS-Cas4 from Thermococcus onnurineus: expanding biochemical landscape of nuclease activity.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {110701},
doi = {10.1016/j.jbc.2025.110701},
pmid = {40945731},
issn = {1083-351X},
abstract = {The adaptive immune function of CRISPR-Cas systems in bacteria and archaea is mediated through CRISPR-Associated Proteins (Cas). The adaptation module, typically involving Cas1, Cas2, and Cas4, helps integrate viral "spacer" sequences into the host genome. Cas4 proteins are classified into two types based on neighboring genes: CAS-Cas4, flanked by other cas genes, and Solo-Cas4, which exist independently. While CAS-Cas4 proteins are implicated in adaptation, they remain biochemically uncharacterized in archaea, unlike archaeal Solo-Cas4 proteins. This study biochemically characterizes TON_0321, a CAS-Cas4 protein from the Type IV-C CRISPR cassette of Thermococcus onnurineus. TON_0321 exhibits 5' to 3' exonuclease activity and unique structure-dependent endonuclease activity, shedding light on CAS-Cas4 functional diversity. A distinct spatial organization of the catalytic site, angled with the positively charged patch on the protein surface, enables TON_0321 to recognize branching points in DNA substrates. Furthermore, this spatial arrangement facilitates cleavage 2 to 3 nucleotides away from the branch point in the 5' direction, demonstrating structure-specific endonuclease activity.},
}
@article {pmid40943511,
year = {2025},
author = {Ma, Z and Ren, J and Liu, Q and Li, J and Zhao, H and Tibesigwa, DG and Matola, SH and Gulfam, T and Yang, J and Wang, F},
title = {Integrating Traditional Breeding and Modern Biotechnology for Advanced Forest Tree Improvement.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943511},
issn = {1422-0067},
abstract = {In the context of global climate change and efforts toward "carbon peak and carbon neutrality," forest resource protection and restoration have become fundamental to ecological civilization. The genetic improvement of trees, as the primary component of forest ecosystems, holds strategic importance for ecological security, resource supply, and carbon neutrality. Traditional tree breeding techniques, including selective and hybrid breeding, have established robust technical systems through extensive practice. However, these methods face limitations such as extended cycles, reduced efficiency, and constrained genetic gains in meeting contemporary requirements. Modern biotechnologies, including genomic selection (GS), gene editing (CRISPR/Cas9), and marker-assisted selection (MAS), substantially enhance the precision and efficiency of genetic improvement. Nevertheless, exclusive reliance on either traditional or modern methods proves insufficient for addressing complex environmental adaptation and rapid breeding requirements. Consequently, the integration of traditional breeding with modern biotechnology to develop intelligent, sustainable, and efficient breeding strategies has emerged as a central focus in tree genetics and breeding. An integrated "step-by-step" approach warrants promotion, supported by a multi-source data sharing platform, an optimized core germplasm repository, and a "climate-soil-genotype" matching model to facilitate the region-specific deployment of improved varieties.},
}
@article {pmid40943296,
year = {2025},
author = {Sheveleva, O and Butorina, N and Protasova, E and Medvedev, S and Grigor'eva, E and Melnikova, V and Kuziaeva, V and Minzhenkova, M and Tatarenko, Y and Lyadova, I},
title = {The Generation of iPSCs Expressing Interferon-Beta Under Doxycycline-Inducible Control.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943296},
issn = {1422-0067},
support = {&#x2116; 0088-2024-0013//government basic research program at the Koltzov Institute of Developmental Biology of the Russian Academy of Sciences in 2024/ ; },
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology/drug effects ; Humans ; *Doxycycline/pharmacology ; *Interferon-beta/genetics/metabolism ; Cell Differentiation/drug effects ; CRISPR-Cas Systems ; Cell Line ; },
abstract = {Type 1 interferons (IFN-Is) exhibit significant antiviral, antitumor, and immunoregulatory properties, demonstrating substantial therapeutic potential. However, IFN-Is are pleiotropic cytokines, and the available data on their effect under specific pathological conditions are inconclusive. Furthermore, the systemic administration of IFN-Is can result in side effects. Generating cells that can migrate to the pathological focus and provide regulated local production of IFN-Is could overcome this limitation and provide a model for an in-depth analysis of the biological and therapeutic effects of IFN-Is. Induced pluripotent stem cells (iPSCs) are a valuable source of various differentiated cell types, including human immune cells. In this study, we describe the generation of genetically modified human iPSCs with doxycycline-controlled overexpression of interferon β (IFNB1). Three IFNB1-overexpressing iPSC lines (IFNB-iPSCs) and one control line expressing the transactivator M2rtTA (TA-iPSCs) were generated using the CRISPR/Cas9 technology. The pluripotency of the generated cell lines has been confirmed by the following: (i) cell morphology; (ii) the expression of the pluripotency markers OCT4, SOX2, TRA 1-60, and NANOG; and (iii) the ability to spontaneously differentiate into the derivatives of the three germ layers. Upon the addition of doxycycline, all IFNB-iPSCs upregulated IFNB1 expression at RNA (depending on the iPSC line, 126-816-fold) and protein levels. The IFNB-iPSCs and TA-iPSCs generated here represent a valuable cellular model for studying the effects of IFN-β on the activity and differentiation trajectories of different cell types, as well as for generating different types of cells with controllable IFN-β expression.},
}
@article {pmid40938447,
year = {2025},
author = {Kang, X and Tian, F and Liu, X and Xiao, G and Cai, Y},
title = {CRISPR/Cas13 system-based entropy-driven DNAzyme switch powered DNA walking system for sensitive and direct rotavirus detection.},
journal = {Mikrochimica acta},
volume = {192},
number = {10},
pages = {663},
pmid = {40938447},
issn = {1436-5073},
support = {No. 2018QN035//Sichuan Provincial Administration of Traditional Chinese Medicine Medical Research Special 2018/ ; },
mesh = {*DNA, Catalytic/chemistry/metabolism/genetics ; *Rotavirus/isolation &amp; purification/genetics ; Entropy ; *CRISPR-Cas Systems ; Limit of Detection ; Metal Nanoparticles/chemistry ; *Biosensing Techniques/methods ; Gold/chemistry ; DNA Probes/chemistry/genetics ; Nucleic Acid Amplification Techniques/methods ; Humans ; },
abstract = {DNA walker-based strategies are confronted with significant challenges in harmonizing design complexity, sequence dependence, and amplification efficiency. This study describes the innovative design of a double-stranded DNA probe, named the "LW probe," which integrates a locked DNAzyme segment, enabling the coupling of the entropy-driven amplification (EDA) process with a DNAzyme-powered DNA walker. In the absence of the target, the "LW probe" remains in an inactive ("OFF") state. Upon encountering target rotavirus sequences, the LW probe receives the trans-cleavage activity of Cas13a/crRNA and undergoes a conformational change, transforming into an activated structure. This structural transition initiates the EDA process continuously, leading to the release of the DNAzyme segment. Subsequently, the released DNAzyme segment acts on the surface of gold nanoparticles (AuNPs), cleaving the "Substrate probe" and consequently liberating fluorescence signals. Distinct from traditional DNA walkers that rely exclusively on the EDA for product amplification, the proposed approach synergistically combines the high-precision target recognition capacity of the EDA process with the potent signal amplification efficiency of DNA walkers. This integration results in remarkable enhancements in both specificity, demonstrated by the ability to discriminate single-base mismatched sequences, and sensitivity, with a detection limit as low as 2.7 fM. By synergizing EDA with the DNAzyme-driven DNA walker, our method achieves high sensitivity, with a detection limit of 2.7 fM, outperforming or matching the performance of previous DNA walker-based systems. This system enables highly sensitive and specific detection of low-abundance rotavirus with robust stability, offering a promising platform for disease diagnosis and biomedical research.},
}
@article {pmid40938388,
year = {2025},
author = {Higa, L and Blank, M and Hampson, E and Matsuyama, J and Wilkes, K and Uehara, A and Bouwman, T and Lee, K and Wang, K and Muszynski, M and Du, ZY},
title = {Rapid assessment of CRISPR gRNAs with optimized protoplast transformation in Maize.},
journal = {Plant cell reports},
volume = {44},
number = {10},
pages = {212},
pmid = {40938388},
issn = {1432-203X},
support = {2121410//National Science Foundation/ ; },
mesh = {*Zea mays/genetics ; *Protoplasts/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Transformation, Genetic ; Plants, Genetically Modified ; Transfection ; },
abstract = {We developed an optimized CRISPR/Cas9 gene editing system using maize mesophyll protoplasts to enable rapid evaluation of guide RNA (gRNA) activity. Using the tropical inbred line Tzi8, we improved protoplast isolation and transfection protocols, achieving high yields of 17.88 × 10[6] viable protoplasts per gram fresh weight while extending post-transfection viability. Etiolated seedlings and vertical leaf cutting significantly enhanced protoplast recovery and viability. A transfection efficiency of ~ 50% was achieved using 10 µg of plasmid DNA; higher DNA inputs did not result in significant gains, resulting in a more resource-efficient approach. Protoplast viability was maintained for up to seven days post-transfection, allowing for downstream applications that require extended incubation. This optimized system was used to assess the editing efficiency of nine gRNAs targeting three key floral repressors (ZmCCT9, ZmCCT10, and ZmRap2.7) across four maize genotypes (Tzi8, CML277, B73, and B104). These floral repressor genes are involved in the photoperiod sensitivity of tropical maize, a major challenge in the effort to introduce tropical maize germplasm into temperate breeding programs. Editing efficiencies ranged from 0.4% to 23.7%, with some variation observed between gRNAs and genotypes. Although protoplast-based assays do not currently enable plant regeneration, this platform offers a rapid method for in vivo gRNA validation, reducing assay time from months to days. This work expands the gene editing toolkit for tropical maize, supporting efforts to overcome breeding barriers through gene editing.},
}
@article {pmid40934206,
year = {2025},
author = {Lima, DA and Costa-Silva, HM and Albergaria, KSS and Ribeiro, JM and Resende, DM and Santarossa, BA and Liarte, DB and Calderano, SG and Murta, SMF},
title = {Glycosomal ABC transporter 3 (GAT3) deletion enhances the oxidative stress responses and reduces the infectivity of Trypanosoma cruzi.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {9},
pages = {e0013479},
pmid = {40934206},
issn = {1935-2735},
mesh = {*Trypanosoma cruzi/genetics/pathogenicity/drug effects/metabolism ; *Oxidative Stress ; *ATP-Binding Cassette Transporters/genetics/metabolism ; Animals ; *Protozoan Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mice ; Chagas Disease/parasitology ; Nitroimidazoles/pharmacology ; *Microbodies/metabolism/genetics ; Trypanocidal Agents/pharmacology ; Gene Knockout Techniques ; Gene Deletion ; },
abstract = {Glycosomes, peroxisome-like organelles in Trypanosoma cruzi, contain enzymes involved in various metabolic processes, including glycolysis. Glycosomal ABC transporters (GATs) play a vital role in maintaining metabolic homeostasis by facilitating metabolite exchange between glycosomes and the cytoplasm. GAT3 is a member of the GAT family, which also includes GAT1 and GAT2. GAT3 transcript levels are downregulated in benznidazole-resistant T. cruzi populations; however, its specific functions remain unknown. Therefore, in this study, we generated GAT3 single-knockout and null mutant lines of the T. cruzi Dm28c strain using the CRISPR/Cas9 system to investigate GAT3 roles in parasite biology. RT-qPCR revealed increased GAT2 transcript levels in the GAT3 null mutant line, without any changes in GAT1 levels. Our findings suggest that GAT3 is not essential for T. cruzi survival, as null mutant parasites showed no growth difference compared to the Cas9-expressing controls. Moreover, the GAT3 single-knockout line exhibited increased resistance to benznidazole, whereas the null mutant line exhibited benznidazole susceptibility similar to the control. Furthermore, both GAT3 single-knockout and null mutant lines showed increased tolerance to hydrogen peroxide-induced oxidative stress. In vitro infection assay of L929 murine fibroblasts revealed that the GAT3 null parasites exhibited a significantly lower infection rate and fewer intracellular amastigotes than the controls. Overall, GAT3 is crucial for T. cruzi infectivity and the regulation of oxidative stress responses, playing key roles in the metabolic regulation and pathogenicity of this parasite.},
}
@article {pmid40934013,
year = {2025},
author = {Zhang, Y and Liu, T and Zhang, P and Ni, B and Wang, X and Bai, L and Sun, W and Guan, Y and Xia, X and Cao, H and Gu, J},
title = {A rapid and accurate method for Helicobacter pylori detection via integrating LAMP assay with CRISPR/Cas12b detection by one-step in one-pot.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1611134},
pmid = {40934013},
issn = {2235-2988},
mesh = {*Helicobacter pylori/genetics/isolation &amp; purification ; Humans ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *Helicobacter Infections/diagnosis/microbiology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Limit of Detection ; },
abstract = {INTRODUCTION: Accurate and timely detection of Helicobacter pylori (HP) is crucial for the diagnosis and management of gastritis and other HP-associated gastrointestinal disorders. Conventional diagnostic methods, such as PCR and culture, require specialized equipment and expertise, limiting their applicability in resource-limited settings. There is a pressing need for a rapid, cost-effective, and user-friendly diagnostic platform for HP detection, particularly in point-of-care settings.<br><br>METHODS: We developed an integrated detection platform combining loop-mediated isothermal amplification (LAMP) with the CRISPR/Cas12b system in a single, one-step, one-pot reaction. The assay was optimized to function at a constant temperature of 58 &#xb0;C and provides results within 45 minutes. The clinical performance of the system was evaluated using 22 clinical samples, and its diagnostic accuracy was compared with conventional PCR.<br><br>RESULTS: The LAMP-CRISPR/Cas12b assay demonstrated a limit of detection (LOD) of 14.77 copies per test, with no cross-reactivity observed against potential interfering nucleic acids, ensuring 100% specificity for HP. Clinical validation revealed a concordance rate of 90.91% (20/22) between the LAMP-CRISPR/Cas12b platform and conventional PCR, supporting the diagnostic reliability of the system.<br><br>DISCUSSION: The integrated LAMP-CRISPR/Cas12b platform represents a promising alternative for the rapid and sensitive detection of HP. It combines the simplicity and rapidity of LAMP with the specificity of CRISPR/Cas12b, offering a robust, cost-effective, and high\-sensitivity diagnostic tool without the need for complex instrumentation. The method shows great potential for use in point-of-care testing (POCT) and could significantly enhance clinical practice by facilitating timely diagnosis and treatment of HP-related diseases.},
}
@article {pmid40825449,
year = {2025},
author = {Chowdhury, R and Roure, A and Darras, S},
title = {Towards functional genetics in the European amphioxus: Efficient CRISPR/Cas9 editing reveals Ascl1/2.1 requirement for peripheral nervous system development.},
journal = {Developmental biology},
volume = {527},
number = {},
pages = {218-225},
doi = {10.1016/j.ydbio.2025.08.012},
pmid = {40825449},
issn = {1095-564X},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Basic Helix-Loop-Helix Transcription Factors/genetics/metabolism ; *Lancelets/genetics/embryology ; *Peripheral Nervous System/embryology/metabolism ; Gene Expression Regulation, Developmental ; },
abstract = {Amphioxus, or cephalochordates, have a key phylogenetic position among chordates and serve as pivotal invertebrate models for investigating the evolutionary origins of vertebrate traits. Although functional genetic tools have recently been developed, their application has been limited to the Floridian and Asian species, Branchiostoma floridae and Branchiostoma belcheri, respectively. In this study, we established a CRISPR/Cas9-based genome editing protocol to generate F0 mosaic mutants (crispants) in the European amphioxus B. lanceolatum. As a proof of concept, we targeted the Bl-Ascl1/2.1 gene, a putative regulator of epidermal sensory neuron (ESN) development in the peripheral nervous system coding for a bHLH transcription factor. Using a novel microinjection method of the sgRNA/Cas9 complex in fertilized eggs and two-cell stage embryos, we demonstrated the disruption of Bl-Ascl1/2.1 that resulted in a partial to complete loss of ESNs. Importantly, this phenotype could be rescued by Bl-Ascl1/2.1 mRNA microinjection. These findings demonstrate the efficiency of CRISPR/Cas9-mediated gene editing in B. lanceolatum and establish a foundation for future functional studies in this emerging EvoDevo model.},
}
@article {pmid40770575,
year = {2025},
author = {Southard, KM and Ardy, RC and Tang, A and O'Sullivan, DD and Metzner, E and Guruvayurappan, K and Norman, TM},
title = {Comprehensive transcription factor perturbations recapitulate fibroblast transcriptional states.},
journal = {Nature genetics},
volume = {57},
number = {9},
pages = {2323-2334},
pmid = {40770575},
issn = {1546-1718},
support = {GM132083//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; DRG-2462-22//Damon Runyon Cancer Research Foundation (Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation)/ ; DP2 GM140925/GM/NIGMS NIH HHS/United States ; AWD-GC-259296//Damon Runyon Cancer Research Foundation (Cancer Research Fund of the Damon Runyon-Walter Winchell Foundation)/ ; GM140925//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; HG012103//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*Fibroblasts/metabolism ; Humans ; *Transcription Factors/genetics/metabolism ; Kruppel-Like Factor 4 ; *Transcription, Genetic ; Animals ; Mice ; Gene Expression Regulation ; Kruppel-Like Transcription Factors/genetics ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Chromatin/genetics ; },
abstract = {Cell atlas projects have revealed that common cell types often comprise distinct, recurrent transcriptional states, but the function and regulation of these states remain poorly understood. Here, we show that systematic activation of transcription factors can recreate such states in vitro, providing tractable models for mechanistic and functional studies. Using a scalable CRISPR activation (CRISPRa) Perturb-seq platform, we activated 1,836 transcription factors in two cell types. CRISPRa induced gene expression within physiological ranges, with chromatin features predicting responsiveness. Comparisons with atlas datasets showed that transcription factor perturbations recapitulated key fibroblast states and identified their regulators, including KLF2 and KLF4 for a universal state present in many tissues, and PLAGL1 for a disease-associated inflammatory state. Inducing the universal state suppressed the inflammatory state, suggesting therapeutic potential. These findings position CRISPRa as a nuanced tool for perturbing differentiated cells and establish a general strategy for studying clinically relevant transcriptional states ex vivo.},
}
@article {pmid40748247,
year = {2025},
author = {Zhang, Y and Yang, X and Wang, Y and Chen, F and Liu, Y and Jiang, H and Wang, Y and Hu, Y and Li, S},
title = {Establishment of a CRISPR/Cas12b-Based Multiple Cross Displacement Amplification Assay for the Rapid, Sensitive, and Specific Detection of Brucella ovis.},
journal = {ACS infectious diseases},
volume = {11},
number = {9},
pages = {2446-2456},
doi = {10.1021/acsinfecdis.5c00287},
pmid = {40748247},
issn = {2373-8227},
mesh = {*Brucella ovis/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Brucellosis/diagnosis/veterinary/microbiology ; Animals ; Sheep ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Sheep Diseases/diagnosis/microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Brucella ovis (B. ovis), a major pathogenic species within the Brucella genus, causes ovine epididymitis. Although the isolation and identification of B. ovis remain the gold standard for diagnosis, these methods are unsuitable for early detection. The traditional polymerase chain reaction (PCR) offers faster detection but requires specialized equipment such as PCR thermal cyclers and gel electrophoresis imagers, limiting its use in basic laboratories. Thus, developing rapid, sensitive, and specific diagnostic strategies is vital for preventing and controlling the spread of ovine brucellosis. In this study, we developed a diagnostic assay combining clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b with multiple cross displacement amplification (MCDA)─termed CRISPR/Cas12b-MCDA─for rapid, sensitive, and specific identification of B. ovis. In the CRISPR/Cas12b-MCDA system, MCDA amplicons containing protospacer adjacent motif (PAM) sites are recognized by the Cas12b/gRNA complex, which binds the target region and triggers trans-cleavage of a single-stranded DNA (ssDNA) reporter. The CRISPR/Cas12b-MCDA assay demonstrated a detection limit of 10 fg/μL for synthetic genomic DNA and exhibited 100% specificity for B. ovis, with no cross-reactivity against other Brucella or non-Brucella species. The preamplification for template extraction takes 20 min, then 5 min for uracil DNA glycosylase (UDG) digestion, and 45 min for MCDA amplification. The total detection time was 75 min using real-time fluorescence analysis and 90 min with a lateral flow biosensor (LFB). Additionally, the results were validated using UV visualization to confirm the CRISPR/Cas12b-MCDA results. Notably, both LFB and UV analyses are instrument-free, enhancing their accessibility. In conclusion, the CRISPR/Cas12b-MCDA assay is a simple, rapid, sensitive, specific, and reliable method for detecting B. ovis.},
}
@article {pmid40437754,
year = {2025},
author = {Xu, Y and Li, J and Wang, Z and Lu, R and Liu, Y and Wang, M and Li, H and Zhao, R and Feng, W},
title = {Ablation of dysmorphic neurons is a safe and effective treatment for focal cortical dysplasia II.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {9},
pages = {4414-4430},
pmid = {40437754},
issn = {1525-0024},
mesh = {*Epilepsy/genetics/pathology/therapy ; *Malformations of Cortical Development, Group I/genetics/pathology/therapy ; Proto-Oncogene Proteins c-akt/genetics ; Disease Models, Animal ; Animals ; Mice ; *Diphtheria Toxin/therapeutic use ; *Ablation Techniques ; *Heparin-binding EGF-like Growth Factor/genetics ; Humans ; HEK293 Cells ; TOR Serine-Threonine Kinases/genetics ; *Neurons/pathology ; CRISPR-Cas Systems ; *Gene Editing/methods ; },
abstract = {Focal cortical dysplasia type II (FCDII) is a leading cause of refractory epilepsy in children, yet treatment options remain limited. The most frequent genetic cause of FCDII is mosaic and somatic variants in genes of the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT)-mammalian target of rapamycin (mTOR) pathway, leading to hyperactivation of mTOR signaling. The presence of dysmorphic neurons (DNs) resulting from hyperactive mTOR signaling is critical for the development of epilepsy in FCDII. One critical therapeutic challenge and opportunity for FCDII is to selectively eliminate DNs. Here, we developed two strategies to specifically ablate DNs in FCDII mouse models, and the results demonstrate that DN ablation is sufficient to both prevent and eliminate epilepsy in mice. Moreover, the associated neurobehavioral abnormalities were also reversed following treatment. Therefore, our study provides proof-of-concept evidence that DN ablation is a highly promising approach for curing FCDII in the future.},
}
@article {pmid40943207,
year = {2025},
author = {Gilyazova, I and Korytina, G and Kochetova, O and Savelieva, O and Mikhaylova, E and Vershinina, Z and Chumakova, A and Markelov, V and Abdeeva, G and Karunas, A and Khusnutdinova, E and Gusev, O},
title = {Advances in Genomics and Postgenomics in Poultry Science: Current Achievements and Future Directions.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943207},
issn = {1422-0067},
support = {&#x2116; 075-15-2025-484//Government of Russian Federation/ ; },
abstract = {The poultry industry, a globally fast growing agricultural sector, provides affordable animal protein due to high efficiency. Gallus gallus domesticus are the most common domestic birds. Hybrid chicken breeds (crosses) are widely used to achieve high productivity. Maintaining industry competitiveness requires constant genetic selection of parent stock to improve performance traits. Genetic studies, which are essential in modern breeding programs, help identify genome variants linked to economically important traits and preserve population health. Next-generation sequencing (NGS) has identified millions of single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELs), enabling detection of genome-wide regions associated with selection traits. Recent studies have pinpointed such regions using broiler lines, laying hen lines, or pooled genomic data. This review discusses advances in chicken genomic and transcriptomic research focused on traits enhancing meat breed performance and reproductive abilities. Special attention is given to transcriptome studies revealing regulatory mechanisms and key signaling pathways involved in artificial molting, as well as metagenome studies investigating resistance to infectious diseases and climate adaptation. Finally, a dedicated section highlights CRISPR/Cas genomic editing techniques for targeted genome modification in chicken genomics.},
}
@article {pmid40943047,
year = {2025},
author = {Huang, C and Cheng, L},
title = {Unlocking Casein Bioactivity: Lactic Acid Bacteria and Molecular Strategies for Peptide Release.},
journal = {International journal of molecular sciences},
volume = {26},
number = {17},
pages = {},
pmid = {40943047},
issn = {1422-0067},
abstract = {Bioactive peptides encrypted in bovine β-casein display diverse physiological functions, including antihypertensive, antioxidative, antimicrobial, and immunomodulatory activities. These peptides are normally released during gastrointestinal digestion or microbial fermentation, especially by proteolytic systems of lactic acid bacteria (LAB). However, peptide yields vary widely among LAB strains, reflecting strain-specific protease repertoires. To overcome these limitations, the scientific goal of this study is to provide a comprehensive synthesis of how synthetic biology, molecular biotechnology, and systems-level approaches can be leveraged to enhance the targeted discovery and production of β-casein-derived bioactive peptides. Genome engineering tools such as clustered regularly interspaced short palindromic repeats associated system (CRISPR/Cas) systems have been applied to modulate gene expression and metabolic flux in LAB, while inducible expression platforms allow on-demand peptide production. Additionally, cell-free systems based on LAB lysates further provide rapid prototyping for high-throughput screening. Finally, multi-omics approaches, including genomics, transcriptomics, proteomics, and metabolomics, further help pinpoint regulatory bottlenecks and facilitate rational strain optimization. This review provides a comprehensive overview of bioactive peptides derived from bovine β-casein and highlights recent progress in LAB-based strategies-both natural and engineered-for their efficient release. These advances pave the way for developing next-generation functional fermented foods enriched with targeted bioactivities.},
}
@article {pmid40825952,
year = {2025},
author = {Vats, P and Kumar, R and Kumar, R and Kaushik, JK and Mohanty, AK and Kumar, S},
title = {Deciphering the role of MFGE8 in lactation using CRISPR-CAS9 based gene editing in Buffalo mammary epithelial cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {30194},
pmid = {40825952},
issn = {2045-2322},
support = {EMR/2017/000152//DST-SERB/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Female ; *Lactation/genetics ; *Epithelial Cells/metabolism ; *Buffaloes/genetics ; *Mammary Glands, Animal/cytology/metabolism ; *Milk Proteins/genetics/metabolism ; Proteomics ; Gene Knockout Techniques ; *Glycoproteins/genetics/metabolism ; Proteome ; },
abstract = {Milk fat globule EGF factor 8 (MFGE8) is a glycoprotein which plays a crucial role in mammary gland remodeling. Our group previously identified MFGE8 as a marker associated with high milk yielding cows. Here, we generated MFGE8 knock-out buffalo mammary epithelial cells (BuMEC) via CRISPR-cas9 technology to decipher its role in lactation. gRNA3 reduced MFGE8 expression with good efficiency which was confirmed at transcriptomic and proteomic level and the stable knock-out cells obtained were named mfge8-/-/gRNA3. The amplicon sequencing of the edited region using next generation sequencing (NGS) showed that 54% of total reads showed indels, 3-4 bp upstream to PAM site in 2nd exon. A total 4282 proteins were identified when proteome level changes were examined and 178 were found to be differentially expressed above and below a threshold of ≥ 1.5 and ≤ 0.6. Major DEPs were found to be associated with regulation of hydrolase activity, endopeptidase activity and cytoskeletal organization and some DEPs including FABP3, FABP4, FABP5, KNG1, MT2A, CD82, SLC7A1 and SERPINH1 belonged to genes associated with milk synthesis. To the best of our knowledge, this is the first study which provides a comprehensive proteome profile of MFGE8 knockout BuMEC and explores downstream effects of disruption of MFGE8 gene.},
}
@article {pmid40825942,
year = {2025},
author = {Countryman, AD and Doherty, CA and Herrera-Perez, RM and Kasza, KE},
title = {Endogenous OptoRhoGEFs reveal biophysical principles of epithelial tissue furrowing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7665},
pmid = {40825942},
issn = {2041-1723},
support = {F31 HD118793/HD/NICHD NIH HHS/United States ; F31HD118793//U.S. Department of Health &amp; Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; P40 OD018537/OD/NIH HHS/United States ; R35GM138380//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R35 GM138380/GM/NIGMS NIH HHS/United States ; Sloan Fellowship//Alfred P. Sloan Foundation/ ; Packard Fellowship//David and Lucile Packard Foundation (David &amp; Lucile Packard Foundation)/ ; CAREER Award//National Science Foundation (NSF)/ ; },
mesh = {Animals ; *Drosophila Proteins/metabolism/genetics ; Epithelium/embryology/metabolism ; *Drosophila melanogaster/embryology/genetics/metabolism ; Optogenetics/methods ; Morphogenesis ; Actomyosin/metabolism ; Animals, Genetically Modified ; *Rho Guanine Nucleotide Exchange Factors/metabolism/genetics ; Gastrulation ; Embryo, Nonmammalian/metabolism ; CRISPR-Cas Systems ; Signal Transduction ; Gene Expression Regulation, Developmental ; },
abstract = {During development, epithelia function as malleable sheets that undergo extensive remodeling to shape developing embryos. Optogenetic control of Rho signaling provides an avenue to investigate mechanisms of epithelial morphogenesis, but transgenic optogenetic tools can be limited by variability in expression levels and deleterious effects of transgenic overexpression on development. Here, we use CRISPR/Cas9 to tag Drosophila RhoGEF2 and Cysts/Dp114RhoGEF with components of the iLID/SspB optogenetic heterodimer, permitting light-dependent control over endogenous protein activities. Using quantitative optogenetic perturbations, we uncover a dose-dependence of tissue furrow depth and bending behavior on RhoGEF recruitment, revealing mechanisms by which developing embryos can shape tissues into particular morphologies. We show that at the onset of gastrulation, furrows formed by cell lateral contraction are oriented and size-constrained by basal actomyosin. Our findings demonstrate the use of quantitative, 3D-patterned perturbations of cell contractility to precisely shape tissue structures and interrogate developmental mechanics.},
}
@article {pmid40825812,
year = {2025},
author = {Netsawang, C and Tongbaen, M and Jearawiriyapaisarn, N and Leecharoenkiat, K},
title = {Precise correction of G6PD Viangchan mutation in iPSCs by prime editing strategy.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {30192},
pmid = {40825812},
issn = {2045-2322},
support = {B05F640126//The NSRF via the Program Management Unit for Human Resources &amp; Institutional Development, Research and Innovation/ ; 196807//Thailand Science research and Innovation Fund Chulalongkorn University/ ; },
mesh = {Humans ; *Glucosephosphate Dehydrogenase/genetics ; *Induced Pluripotent Stem Cells/metabolism ; *Gene Editing/methods ; HEK293 Cells ; *Glucosephosphate Dehydrogenase Deficiency/genetics/therapy ; *Mutation ; CRISPR-Cas Systems ; },
abstract = {Individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency hold a significant risk of severe hemolytic crises under oxidative stress. Currently, the definitive and curative treatment for the disorder has not been developed. Among over 200 G6PD variants, G6PD Viangchan (c.871 G > A) is the most prevalent and has been extensively studied in Southeast Asia. This study assessed the effectiveness of prime editing for correcting the G6PD Viangchan mutation in an established mutant HEK293T cell line and G6PD-deficient induced pluripotent stem cells (iPSCs). Using optimized modalities, prime editing achieved a high correction efficiency of over 25% in the HEK293T cells. In iPSCs, this gene editing tool yielded satisfactory correction outcomes, with approximately 5% corrected alleles. Our findings indicate that prime editing provides high precision, producing minimal by-products below baseline and showing undetectable off-target effects. Overall, prime editing has the potential to correct the G6PD Viangchan mutation, providing a valuable approach for future therapeutic strategies and the generation of isogenic cell lines to promote extensive studies in drug discovery and the pathogenesis of the G6PD variant.},
}
@article {pmid40825783,
year = {2025},
author = {Calvo-Villamañán, A and Sastre-Dominguez, J and Barrera-Martín, &#xc1; and Costas, C and San Millan, &#xc1;},
title = {Dissecting pOXA-48 fitness effects in clinical Enterobacterales using plasmid-wide CRISPRi screens.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7700},
pmid = {40825783},
issn = {2041-1723},
support = {ALTF 322-2022//European Molecular Biology Organization (EMBO)/ ; },
mesh = {*Plasmids/genetics ; *beta-Lactamases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Enterobacteriaceae/genetics/drug effects ; Humans ; Enterobacteriaceae Infections/microbiology/drug therapy ; *Genetic Fitness ; CRISPR-Cas Systems/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Carbapenems/pharmacology ; },
abstract = {Conjugative plasmids are the main vehicle for the spread of antimicrobial resistance (AMR) genes in clinical bacteria. AMR plasmids allow bacteria to survive antibiotic treatments, but they also produce physiological alterations in their hosts that commonly translate into fitness costs. Despite the key role of plasmid-associated fitness effects in AMR evolution, their origin and molecular bases remain poorly understood. In this study, we introduce plasmid-wide CRISPR interference (CRISPRi) screens as a tool to dissect plasmid-associated fitness effects. We design and perform CRISPRi screens targeting the globally distributed carbapenem resistance plasmid pOXA-48 in 13 different multidrug resistant clinical Enterobacterales. Our results reveal that pOXA-48 gene-level effects are conserved across clinical strains, and expose the key role of the carbapenemase-encoding gene, blaOXA-48, as the main culprit for pOXA-48 fitness costs. Moreover, our results highlight the relevance of postsegregational killing systems in pOXA-48 vertical transmission, and uncover new genes implicated in pOXA-48 stability (pri, korC, DNDJGHEP_13 and 14 and H-NS). This study sheds new light on the biology and evolution of carbapenem resistant Enterobacterales and endorses CRISPRi screens as a powerful method for studying plasmid-mediated AMR.},
}
@article {pmid40824957,
year = {2025},
author = {Naes, SM and Ab-Rahim, S and Mazlan, M and Syafruddin, SE and Mohtar, MA and Abuhamad, AY and Abdul Rahman, A},
title = {CRISPR/Cas9 mediated ENT2 gene knockout altered purine catabolic pathway and induced apoptosis in colorectal cell lines.},
journal = {PloS one},
volume = {20},
number = {8},
pages = {e0329501},
pmid = {40824957},
issn = {1932-6203},
mesh = {Humans ; *Apoptosis/genetics ; *CRISPR-Cas Systems ; *Purines/metabolism ; *Colorectal Neoplasms/genetics/metabolism/pathology ; Gene Knockout Techniques ; Cell Line, Tumor ; *Equilibrative-Nucleoside Transporter 2/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Xanthine Oxidase/metabolism ; HT29 Cells ; Hypoxanthine/metabolism ; Gene Expression Regulation, Neoplastic ; },
abstract = {Although purine metabolism is one of the most impacted pathways in colorectal cancer (CRC), little is known about the role of equilibrative nucleoside transporter 2 (ENT2) in CRC development and its association with the altered purine metabolism pathway. This study aimed to determine the role of ENT2 in altered purine metabolism in the early and late stages of CRC using CRISPR/Cas9 gene editing tools and a variety of functional experiments. The expression of ENT2 was significantly higher (P < 0.001) in all CRC cell lines as compared to the normal colon cells. The two CRC cell lines with the highest ENT2 expression, the early stage HT29 cells and the late stage DLD1 cells, were knocked out (KO) using the CRISPR/Cas9 tool. The hypoxanthine (HPX) level and the xanthine oxidase (XO) activity were significantly higher in both HT29/KO and DLD1/KO single cell-derived clones (P < 0.01). The increase in HPX level and XO activity were associated with an elevation in the reactive oxygen species (ROS) level. These data suggest that the ENT2 KO elevated the ROS levels induced apoptosis and impaired the cell proliferation of the early stage of CRC cell line, i.e., HT29/KO clonal cells. In this context, targeting ENT2 gene might be a potential strategy in CRC treatment by increasing the production of ROS and hence, inducing the apoptosis pathway.},
}
@article {pmid40823809,
year = {2025},
author = {Migliori, V and Bruntraeger, MB and Gyulev, IS and Lichou, F and Burgold, T and Gitterman, DP and Iwama, S and Trinh, AL and Washer, SJ and Jones, CP and Trynka, G and Bassett, AR},
title = {ONE-STEP tagging: a versatile method for rapid site-specific integration by simultaneous reagent delivery.},
journal = {Nucleic acids research},
volume = {53},
number = {15},
pages = {},
pmid = {40823809},
issn = {1362-4962},
support = {220540/Z/20/A/WT_/Wellcome Trust/United Kingdom ; //Sanger Translation Committee/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Induced Pluripotent Stem Cells/metabolism ; T-Lymphocytes/metabolism ; Integrases/genetics/metabolism ; },
abstract = {We present a novel, versatile genome editing method termed ONE-STEP tagging, which combines CRISPR-Cas9-mediated targeting with Bxb1 integrase-based site-specific integration for efficient, precise, and scalable protein tagging. Applied in human-induced pluripotent stem cells (hiPSCs), cancer cells and primary T cells, this system enables rapid generation of endogenously tagged proteins. By enhancing the nuclear localization signal of the catalytically superior eeBxb1 integrase and co-delivering a DNA-PK inhibitor, we achieved up to ∼90% integration efficiency at the ACTR10 locus in hiPSCs. ONE-STEP tagging is robust across loci and cell types and supports large DNA cargo integration, with efficiencies reaching 16.6% for a 14.4 kb construct. The method also enables multiplexed tagging of multiple proteins within the same cell and simultaneous CRISPR-based editing at secondary loci, such as gene knockouts or homology-directed repair. Importantly, we demonstrate successful application in primary T cells by targeting the T cell receptor locus while simultaneously knocking out B2M, a key step towards generating immune-evasive, off-the-shelf chimeric antigen receptor T cells. Additionally, we introduce a dual-cassette version of the method compatible with universal donor plasmids, allowing use of entirely off-the-shelf reagents. Together, these advances establish ONE-STEP tagging as a powerful tool for both basic and therapeutic genome engineering.},
}
@article {pmid40814254,
year = {2025},
author = {Nevot, G and Güell, M and Santos-Moreno, J},
title = {Critical Analysis of Preprints and Inquiry-Based Lessons Improve the Synthetic Biology Learning Experience.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {2878-2884},
pmid = {40814254},
issn = {2161-5063},
mesh = {*Synthetic Biology/education ; Humans ; *Problem-Based Learning/methods ; Students ; Universities ; CRISPR-Cas Systems/genetics ; },
abstract = {Synthetic biology is a transformative field crucial to address global challenges. It is highly interdisciplinary, integrating different subjects beyond biology. Therefore, traditional lecture-based teaching methods often fall short in effectively covering the diverse and rapidly evolving advancements in synthetic biology. We developed active learning workflows for complementing classic theoretical lectures in universities to improve the synthetic biology learning experience. We used preprints as an educational resource for the students to critically analyze differences comparing manuscripts and the final published work. In addition, we designed a practical laboratory session where students had to infer the logic behind CRISPRi-based gene circuits that they assembled, thus engaging with every step of the design-build-test-learn cycle. Following these activities, 90% of the students reported having improved critical analysis skills and 80% felt that they had learned a wide range of synthetic biology concepts. These approaches demonstrate the potential of innovative teaching for synthetic biology, which helps students with both technical and soft skills at the same time and has the potential to be adapted to other fields.},
}
@article {pmid40736249,
year = {2025},
author = {Zhao, Y and Guo, G and Sun, Y and Zhang, M and Yang, G and Liu, Z and Song, Y and Ghonaim, AH and Ma, N and Zhang, M and Jongkaewwattana, A and He, Q and Li, W},
title = {Membrane protein CRISPR screen identifies RPSA as an essential host factor for porcine epidemic diarrhea virus replication.},
journal = {Journal of virology},
volume = {99},
number = {8},
pages = {e0064925},
pmid = {40736249},
issn = {1098-5514},
mesh = {Animals ; *Porcine epidemic diarrhea virus/physiology ; *Virus Replication ; Swine ; *Coronavirus Infections/virology/veterinary/metabolism ; CRISPR-Cas Systems ; *Swine Diseases/virology/metabolism/genetics ; Host-Pathogen Interactions ; *Membrane Proteins/genetics/metabolism ; MAP Kinase Signaling System ; Gene Knockout Techniques ; Chlorocebus aethiops ; },
abstract = {UNLABELLED: Porcine epidemic diarrhea, caused by porcine epidemic diarrhea virus (PEDV), is one of the most devastating diseases in the global pig industry due to its high mortality rate in piglets. The host factors required for PEDV replication, including receptors, remain poorly understood. Here, we developed a porcine membrane-protein-scale CRISPR/Cas9 knockout (PigMpCKO) library and performed two rounds of PEDV infection. Ribosomal protein SA (RPSA), the known receptor of dengue virus, was found to be a potent host factor. Moreover, our research revealed that RPSA is involved in the replication stage of PEDV and not in the entry stage. Inhibitor and activator experiments demonstrated that knockout (KO) of RPSA downregulates the ERK1/2 signaling pathway to impair PEDV infection. Additionally, RNA sequencing data indicated that cellular lipid biosynthesis and lipid transport processes were significantly inhibited in the absence of RPSA during PEDV infection. Mechanistic studies revealed that the reduction in total cholesterol and triglyceride levels, resulting from RPSA KO, was partially mediated by the ERK1/2 pathway, leading to impaired lipid accumulation during PEDV replication. Interestingly, RPSA KO also significantly downregulated the expression of aminopeptidase N (APN) and inhibited infection by transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV), both of which belong to the swine enteric coronavirus group. In summary, our results establish RPSA as a novel host factor that is critical for coronavirus replication. This provides new insights into the mechanisms of virus-host interactions and paves the way for the development of broad-spectrum antiviral therapies.<br><br>IMPORTANCE: Swine enteric coronaviruses (SeCoVs) cause severe economic losses to the global swine industry and pose a potential threat to public health. Identification of receptors required for PEDV infection could develop novel targets for drug therapy and disease-resistant breeding. We conducted a CRISPR/Cas9 screen targeting membrane proteins in porcine kidney cells infected with PEDV to identify possible receptors and discovered numerous novel candidate host factors. Considering RPSA's known role as a receptor for multiple viruses, we focused on investigating its potential in coronavirus infection. Our results revealed that RPSA does not contribute to the entry stage but to the replication stage of coronavirus infection. We first reported the role that RPSA plays in the regulation of APN expression and lipid metabolism. RPSA is essential for PEDV and other SeCoVs replication, providing a novel insight into the search for the receptor of PEDV and identifying potential therapeutic targets for coronaviruses.},
}
@article {pmid40707359,
year = {2025},
author = {Campbell, RR and Green, M and Choi, EY and Wulff, AB and Siclair, AN and Khatri, S and Virata, G and Barrett, C and Key, S and Patel, S and Rowell, MB and Franco, D and Ganapathy-Kanniappan, S and Mathur, BN and Lobo, MK},
title = {Dopamine Receptor 1 Specific CRISPRa Mice Exhibit Disrupted Behaviors and Striatal Baseline Cellular Activity.},
journal = {eNeuro},
volume = {12},
number = {8},
pages = {},
pmid = {40707359},
issn = {2373-2822},
support = {R01 DA038613/DA/NIDA NIH HHS/United States ; R33 DA052101/DA/NIDA NIH HHS/United States ; },
mesh = {Animals ; *Receptors, Dopamine D1/genetics/metabolism ; Mice, Transgenic ; *Corpus Striatum/metabolism ; Mice ; Male ; Reward ; *Behavior, Animal/physiology ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; Motor Activity/physiology ; },
abstract = {The two main cell types in the striatum, dopamine receptor 1 and adenosine receptor 2a spiny projection neurons (D1-SPNs and A2A-SPNs), have distinct roles in regulating motor- and reward-related behaviors. Cre-selective CRISPR-dCas9 systems allow for cell-type specific, epigenomic-based manipulation of gene expression with gene-specific single guide RNAs (sgRNAs) and have potential to elucidate molecular mechanisms underlying striatal subtype mediated behaviors. Conditional transgenic Rosa26:LSL-dCas9-p300 mice were recently generated to allow for robust expression of dCas9-p300 expression with Cre-driven cell-type specificity. This system utilizes p300, a histone acetyltransferase which regulates gene expression by unwinding chromatin and making that region of the genome more accessible for transcription. Rosa26-LSL-dCas9-p300 mice were paired with Drd1-Cre and Ador2a-Cre mice to generate Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 mouse lines and underwent behavioral phenotyping when sgRNAs were not present. Both Drd1-Cre:dCas9-p300 and Ador2a-Cre:dCas9-p300 have cell-type-specific expression of spCas9 mRNA. Baseline behavioral assessments revealed that, under a sgRNA absent nontargeted state, Drd1-Cre:dCas9-p300 mice display repetitive spinning behavior, hyperlocomotion, and enhanced acquisition of reward learning in comparison with all genotypic littermates. In contrast, Ador2a-Cre:dCas9-p300 do not exhibit any changes in behavior in comparison with their littermates. Electrophysiological recordings of dorsal striatum D1-SPNs revealed that Drd1-Cre:dCas9-p300 mice have increased input resistance and increased spontaneous excitatory postsynaptic current amplitude, together suggesting greater excitatory drive of D1-SPNs. Overall, these data demonstrate the necessity to validate CRISPR-dCas9 lines for research investigations. Additionally, the Drd1-Cre:dCas9-p300 line has the potential to be used to study underlying mechanisms of stereotypy and reward learning.},
}
@article {pmid40941236,
year = {2025},
author = {Liu, Y and Xie, Y and Wang, Z and Gai, Z and Zhang, X and Chen, J and Lei, H and Xu, Z and Shen, X},
title = {A Simple, Rapid, and Contamination-Free Ultra-Sensitive Cronobacter sakazakii Visual Diagnostic Platform Based on RPA Combined with CRISPR/Cas12a.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {17},
pages = {},
doi = {10.3390/foods14173120},
pmid = {40941236},
issn = {2304-8158},
support = {2023YFF1105103//National Key Research and Development Program of China/ ; },
abstract = {CRISPR/Cas systems have made significant progress in the field of molecular diagnostics in recent years. To overcome the aerosol contamination problem brought on by amplicon transfer in the common two-step procedure, the "one-pot method" has become a major research hotspot in this field. However, these methods usually rely on specially designed devices or additional chemical modifications. In this study, a novel "one-pot" strategy was developed to detect the foodborne pathogen Cronobacter sakazakii (C. sakazakii). A specific sequence was screened out from the virulence gene ompA of C. sakazakii as the detection target. Combining with the recombinase polymerase amplification (RPA), a rapid detection platform for C. sakazakii based on the CRISPR/Cas12a system was established for the first time. The sensitivity of this method was determined from three different levels, which are 10[-4] ng/μL for genomic DNA (gDNA), 1.43 copies/μL for target DNA, and 6 CFU/mL for pure bacterial culture. Without any microbial enrichment, the detection limits for artificially contaminated cow and goat milk powder samples were 4.65 CFU/mL and 4.35 CFU/mL, respectively. To address the problem brought on by aerosol contamination in the common RPA-CRISPR/Cas12a two-step method, a novel pipette tip-in-tube (PTIT) method for simple and sensitive one-pot nucleic acid detection was further developed under the inspiration of the capillary principle. The RPA and CRISPR/Cas systems were isolated from each other by the force balance of the solution in a pipette tip before amplification. The detection limits of the PTIT method in pure bacterial culture and the spiked samples were exactly the same as that of the two-step method, but with no false positive cases caused by aerosol contamination at all. Compared with other existing one-pot methods, the PTIT method requires no additional or specially designed devices, or any chemical modifications on crRNA and nucleic acid probes. Therefore, the PTIT method developed in this study provides a novel strategy for realizing one-pot CRISPR/Cas detection easily and holds significant potential for the rapid point-on-care testing (POCT) application.},
}
@article {pmid40940734,
year = {2025},
author = {Cattin, E and Schena, E and Mattioli, E and Marcuzzo, S and Bonanno, S and Cavalcante, P and Corradi, F and Benati, D and Farinazzo, G and Cattaneo, M and De Sanctis, V and Bertorelli, R and Maggi, L and Giannotta, M and Pini, A and Vattemi, G and Cassandrini, D and Cavallo, M and Manferdini, C and Lisignoli, G and Fontana, B and Pace, I and Bruno, C and Roncarati, R and Fiorillo, C and Ferracin, M and Schirmer, EC and Recchia, A and Lattanzi, G},
title = {Profibrotic Molecules Are Reduced in CRISPR-Edited Emery-Dreifuss Muscular Dystrophy Fibroblasts.},
journal = {Cells},
volume = {14},
number = {17},
pages = {},
doi = {10.3390/cells14171321},
pmid = {40940734},
issn = {2073-4409},
support = {20223WFJJ3//Ministero dell'universit&#xe0; e della ricerca/ ; CUP B33C22001640007//Associazione Italiana DIstrofia Muscolare di Emery-Dreifuss - AIDMED/ ; CUP B33C22001640007//Associazione Alessandra Proietti OdV/ ; ECOSISTER Project. cod. ECS_00000033-CUP B89I22000650001//EU-funded PNRR/ ; ECOSISTER project ECS_00000033 - CUP E93C22001100001//EU-funded PNRR/ ; TREAT-LMNA 2019-004426-24//AIFA/ ; T3-AN-03 CUP: B53C22002520006 REGINA//Ministero della Salute/ ; RRC//The Italian Ministry of Health/ ; },
abstract = {Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in EMD, LMNA, SYNE1, SYNE2, and other related genes. The disease is characterized by joint contractures, muscle weakening and wasting, and heart conduction defects associated with dilated cardiomyopathy. Previous studies demonstrated the activation of fibrogenic molecules such as TGFbeta 2 and CTGF in preclinical models of EDMD2 and increased secretion of TGFbeta 2 in patient serum. A wide screening of patient cells suggested fibrosis, metabolism, and myogenic signaling as the most affected pathways in various EDMD forms. In this study, we show that alpha-smooth muscle actin-positive myofibroblasts are overrepresented in patient fibroblast cultures carrying EMD, LMNA, or SYNE2 mutations, and profibrotic miRNA-21 is upregulated. Upon CRISPR/Cas correction of the mutated EMD or LMNA sequence in EDMD1 or EDMD2 fibroblasts, respectively, we observe a reduced expression of fibrogenic molecules. However, in patient myoblasts, neither fibrogenic proteins nor miRNA-21 were upregulated; instead, miRNA-21-5p was downregulated along with muscle-specific miRNA-133b and miRNA-206, which have a crucial role in muscle cell homeostasis. These observations suggest that the conversion of laminopathic fibroblasts into a profibrotic phenotype is a determinant of EDMD-associated muscle fibrosis, while miRNA-206-dependent defects of laminopathic myoblasts, including altered regulation of VEGF levels, contribute to muscle cell deterioration. Notably, our study provides a proof-of-principle for the application of gene correction to EDMD1 and EDMD2 and presents EDMD1 isogenic cells that exhibit an almost complete rescue of a disease-specific miRNA signature. These cells can be used as experimental models for studying muscular laminopathies.},
}
@article {pmid40938972,
year = {2025},
author = {Abdirassilova, AA and Yessimseit, DT and Kassenova, AK and Abdeliyev, BZ and Zhumadilova, ZB and Tokmurziyeva, GZ and Kovaleva, GG and Abdel, ZZ and Meka-Mechenko, TV and Umarova, SK and Begimbayeva, EZ and Agzam, SD and Motin, VL and Reva, ON and Rysbekova, AK},
title = {Whole genome sequencing of Yersinia pestis isolates from Central Asian natural plague foci revealed the role of adaptation to different hosts and environmental conditions in shaping specific genotypes.},
journal = {PLoS neglected tropical diseases},
volume = {19},
number = {9},
pages = {e0013533},
doi = {10.1371/journal.pntd.0013533},
pmid = {40938972},
issn = {1935-2735},
abstract = {The genetic diversity and biovar classification of Yersinia isolates from Central Asia were investigated using whole-genome sequencing. In total, 98 isolates from natural plague foci were sequenced using the MiSeq platform. Computational pipelines were developed for accurate assembly of Y. pestis replicons, including small cryptic plasmids, and for identifying genetic polymorphisms. A panel of 99 diagnostic polymorphisms was established, enabling the distinction of dominant Medievalis isolates derived from desert and upland regions. Evidence of convergent evolution was observed in polymorphic allele distributions across genetically distinct Y. pestis biovars, Y. pseudotuberculosis, and other Y. pestis strains, likely driven by adaptation to similar environmental conditions. Genetic polymorphisms in the napA, araC, ssuA, and rhaS genes, along with transposon and CRISPR-Cas insertion patterns, were confirmed as suitable tools for identifying Y. pestis biovars, although their homoplasy suggests limited utility for phylogenetic inference. Notably, a novel cryptic plasmid, pCKF, previously associated with the strain of the population 2.MED0 from the Central-Caucasus high-altitude autonomous plague focus, was detected in a genetically distinct isolate of 2.MED1 population from the Ural-Embi region, indicating potential plasmid transfer across the 2.MED lineage. These findings emphasize the need for ongoing genomic surveillance to monitor the spread of virulence-associated genetic elements and to improve our understanding of Y. pestis evolution and ecology.},
}
@article {pmid40938680,
year = {2025},
author = {Yang, K and Wang, T and Zhu, Q and Shen, C},
title = {CRISPR/Cas-based detection strategies for tumor biomarker detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5ay01150g},
pmid = {40938680},
issn = {1759-9679},
abstract = {Tumor biomarkers, such as nucleic acids, proteins, extracellular vesicles (EVs) and circulating tumor cells (CTCs), can provide valuable information for tumor risk assessment, diagnosis, prognosis and recurrence monitoring. Currently, polymerase chain reaction (PCR)-based approaches and enzyme linked immunosorbent assay (ELISA) are typically used for detecting tumor biomarkers in clinics. However, PCR-based methods have limits in sensitivity and detection channels. Besides, ELISA suffers from cumbersome operation and limited sensitivity. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) systems are adopted for developing novel detection strategies due to the characteristics of high sensitivity, high specificity, simple operability and flexible programmability. Besides, CRISPR/Cas systems are amenable to combination with isothermal amplification techniques, primarily attributed to their compatibility and stability. Moreover, the combination of different CRISPR/Cas systems enables multiplex target detection. Therefore, CRISPR/Cas-based detection strategies have emerged as highly promising approaches for the sensitive, specific and multiplex detection of tumor biomarkers. In this review, we at first introduced the classification and working mechanisms of CRISPR/Cas systems. And then, we comprehensively summarized recently developed CRISPR/Cas-based detection strategies for tumor biomarkers. Besides, we reviewed detection strategies based on CRISPR/Cas systems for multiplex tumor biomarker detection. Furthermore, the challenges and prospects of existing CRISPR/Cas-based detection strategies were thoroughly discussed.},
}
@article {pmid40935887,
year = {2025},
author = {Alves, CRR and Das, S and Krishnan, V and Ha, LL and Fox, LR and Stutzman, HE and Shamber, CE and Kalailingam, P and McCarthy, S and Lino Cardenas, CL and Fong, CE and Imai, T and Mitra, S and Yun, S and Wood, RK and Benning, FMC and Roh, K and Lawton, J and Kim, N and Silverstein, RA and Ferreira da Silva, J and de la Cruz, D and Richa, R and Xie, J and Gray-Edwards, HL and Malhotra, R and Chung, DY and Chao, LH and Tsai, SQ and Maguire, CA and Lindsay, ME and Kleinstiver, BP and Musolino, PL},
title = {Treatment of a severe vascular disease using a bespoke CRISPR-Cas9 base editor in mice.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {40935887},
issn = {2157-846X},
support = {P01HL142494//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01NS125353//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; DP2CA281401//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; K01NS134784//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01NS125353//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01NS125353//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
abstract = {Pathogenic missense mutations in the alpha actin isotype 2 (ACTA2) gene cause multisystemic smooth muscle dysfunction syndrome (MSMDS), a genetic vasculopathy that is associated with stroke, aortic dissection and death in childhood. Here we perform mutation-specific protein engineering to develop a bespoke CRISPR-Cas9 enzyme with enhanced on-target activity against the most common MSMDS-causative mutation ACTA2 R179H. To directly correct the R179H mutation, we screened dozens of configurations of base editors to develop a highly precise corrective A-to-G edit with minimal deleterious bystander editing that is otherwise prevalent when using wild-type SpCas9 base editors. We create a murine model of MSMDS that shows phenotypes consistent with human patients, including vasculopathy and premature death, to explore the in vivo therapeutic potential of this strategy. Delivery of the customized base editor via an engineered smooth muscle-tropic adeno-associated virus (AAV-PR) vector substantially prolongs survival and rescues systemic phenotypes across the lifespan of MSMDS mice, including in the vasculature, aorta and brain. Our results highlight how bespoke mutant-specific CRISPR-Cas9 enzymes can improve mutation correction with base editors.},
}
@article {pmid40934884,
year = {2025},
author = {Gast, K and Barrangou, R},
title = {All systems go: CRISPR crosstalk for enhanced immunity.},
journal = {Cell host &amp; microbe},
volume = {33},
number = {9},
pages = {1470-1472},
doi = {10.1016/j.chom.2025.08.002},
pmid = {40934884},
issn = {1934-6069},
abstract = {In this issue of Cell Host &amp; Microbe, companion manuscripts from Margolis &amp; Meeske[1] and Smith &amp; Fineran[2] demonstrate that CRISPR-Cas systems have an unprecedented level of cooperative crosstalk between different subtypes, which enables primed spacer acquisition. These studies illustrate how CRISPR-Cas systems cooperate to enhance adaptive immunity in bacteria.},
}
@article {pmid40641152,
year = {2025},
author = {Robertson, NR and Lenert-Mondou, C and Leonard, AC and Tafrishi, A and Carrera, S and Lee, S and Aguilar, Y and Sanchez Zamora, L and Nguyen, T and Beltrán, J and Li, M and Cutler, SR and Whitehead, TA and Wheeldon, I},
title = {PYR1 Biosensor-Driven Genome-Wide CRISPR Screens for Improved Monoterpene Production in Kluyveromyces marxianus.},
journal = {ACS synthetic biology},
volume = {14},
number = {8},
pages = {2972-2978},
pmid = {40641152},
issn = {2161-5063},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Kluyveromyces/genetics/metabolism ; *Monoterpenes/metabolism ; Metabolic Engineering/methods ; *Fungal Proteins/genetics/metabolism ; Acyclic Monoterpenes/metabolism ; Genome, Fungal ; },
abstract = {Monoterpenes are valued for their roles as flavors, fragrances, insecticides, and energy-dense fuels. Microorganisms provide sustainable biosynthesis routes for these important molecules, but production levels remain limited. Here, we introduce a biosensor-driven microbial engineering strategy to enhance monoterpene production, specifically targeting geraniol. Using mutagenized libraries of the PYR1 receptor─a versatile biosensor from plant ABA signaling pathways with a malleable binding pocket─we screened 24 monoterpenes and identified PYR1 variants responsive to eight, including geraniol. A low background, highly selective geraniol-sensitive PYR1 variant was expressed in the thermotolerant yeast Kluyveromyces marxianus as a growth-based biosensor circuit, allowing for rapid strain engineering. By coupling the geraniol-sensitive PYR1 sensor with a genome-wide CRISPR-Cas9 mutagenesis approach, we identified six gene knockouts that enhance geraniol production, achieving up to a 2-fold increase in titer. This study demonstrates the power of the PYR1 biosensor platform to enable rapid strain engineering and the identification of mutants that improve the titer of a desired metabolite.},
}
@article {pmid40580745,
year = {2025},
author = {Asadi-Sarabi, P and Rismani, E and Shabanpouremam, M and Hendi, Z and Nikoubin, B and Rahimi, S and Taleb, M and Khosravi, A and Zarrabi, A and Hassan, M and Vosough, M},
title = {Hypoimmunogenic pluripotent stem cells: A game-changer in cell-based regenerative medicine.},
journal = {International immunopharmacology},
volume = {162},
number = {},
pages = {115134},
doi = {10.1016/j.intimp.2025.115134},
pmid = {40580745},
issn = {1878-1705},
mesh = {Humans ; *Regenerative Medicine/methods ; *Pluripotent Stem Cells/immunology/transplantation ; Animals ; Gene Editing ; *Cell- and Tissue-Based Therapy/methods ; CRISPR-Cas Systems ; },
abstract = {Hypoimmunogenic pluripotent stem cells (hPSCs) represent a transformative innovation in regenerative medicine, offering solutions to the longstanding challenge of immune rejection in cell-based therapies. Through advanced gene-editing techniques, particularly CRISPR/Cas9, hPSCs are engineered to downregulate or eliminate the expression of major histocompatibility complex (MHC) molecules while upregulating immunomodulatory proteins such as HLA-G, PD-L1, and CD47. These modifications enhance immune evasion and create the foundation for universal donor cells. Compared to conventional cell therapies that rely on lifelong immunosuppression, hPSC-based strategies offer safer, more sustainable, and patient-friendly solutions by minimizing the risks of infection, malignancy, and drug toxicity. Beyond immune compatibility, critical challenges persist, including the risk of tumorigenicity, off-target genetic alterations, and ethical considerations surrounding genome editing. Recent advances, such as the integration of suicide gene systems and sensitive monitoring assays, offer promising strategies to enhance the safety and functional stability of hPSC-derived therapies. This review comprehensively discusses the molecular engineering of hPSCs, their biomedical applications, safety strategies, ethical implications, and the evolving regulatory frameworks needed for clinical translation. By addressing both the scientific and societal dimensions, hPSCs have the potential to revolutionize personalized and off-the-shelf regenerative treatments, provided that rigorous safeguards are implemented.},
}
@article {pmid40931807,
year = {2025},
author = {Wang, Y and Phelps, A and Godbehere, A and Evans, B and Takizawa, C and Chinen, G and Singh, H and Fang, Z and Du, ZY},
title = {Revolutionizing Agriculture With CRISPR Technology: Applications, Challenges, and Future Perspectives.},
journal = {Biotechnology journal},
volume = {20},
number = {9},
pages = {e70113},
doi = {10.1002/biot.70113},
pmid = {40931807},
issn = {1860-7314},
support = {//MBBE Molecular Biotechnology Lab/ ; HAW05047-H//USDA National Institute of Food and Agriculture/ ; 6114549//USDA REEU-Technology/ ; 5605280//CTAHR Agricultural Research and Extension Stations (CARES)/ ; //Undergraduate Research Opportunities Program (UROP)/ ; 5605280//U.S. Department of Agriculture/ ; 6114549//U.S. Department of Agriculture/ ; HATCH project HAW05047-H//U.S. Department of Agriculture/ ; },
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Agriculture/methods/trends ; Crops, Agricultural/genetics ; Livestock/genetics ; Aquaculture ; },
abstract = {CRISPR technologies are rapidly transforming agriculture by enabling precise and programmable modifications across a wide range of organisms. This review provides an overview of CRISPR applications in crops, livestock, aquaculture, and microbial systems, highlighting key advances in sustainable agriculture. In crops, CRISPR has accelerated the improvement of traits such as drought tolerance, nutrient efficiency, and pathogen resistance. In livestock and aquaculture, CRISPR has enabled disease-resistant pigs and poultry, hornless cattle, and fast-growing, stress-tolerant fish. Engineered microbes are also being leveraged to enhance nitrogen fixation and reduce input reliance. We examine the evolution of CRISPR tools, such as base and prime editing, multiplex editing, and epigenome modulation, that expand precision and control beyond traditional gene knockouts. These innovations offer significant advantages over conventional breeding, yet challenges remain, including off-target effects, delivery efficiency, and regulatory variability across countries. The review also explores emerging directions such as novel Cas variants and AI-integrated breeding platforms for high-throughput trait discovery. Together, these developments demonstrate the transformative potential of CRISPR technology to reshape agriculture, not only by enhancing productivity and resilience but also by reducing environmental impacts. With responsible implementation, CRISPR-enabled innovations are well-positioned to support global food security and sustainability targets by 2050.},
}
@article {pmid40931602,
year = {2025},
author = {Pan, L and Wang, P},
title = {DNA nanotechnology-enabled bioanalysis of extracellular vesicles.},
journal = {Nanoscale horizons},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5nh00557d},
pmid = {40931602},
issn = {2055-6764},
abstract = {Extracellular vesicles (EVs) have emerged as valuable sources for liquid biopsy in disease diagnostics, given their protein and nucleic acid cargoes (e.g., miRNA, mRNA, glycoRNA) can serve as critical biomarkers. DNA nanotechnology, leveraging its inherent programmability, high specificity, and powerful signal amplification capability, offers a transformative approach for the bioanalysis of EVs. This review summarizes recent advances in DNA nanotechnology-based analytical methodologies for detecting EV-associated proteins and nucleic acids. We detail the underlying principles, applications, and performance of key strategies, including aptamer-based recognition, enzyme-free catalytic amplification circuits (e.g., HCR, CHA), enzyme catalytic amplification techniques (e.g., RCA, CRISPR-Cas systems), and DNA nanostructures-assisted amplification. The integration of these DNA tools into multiplexed detection platforms is also discussed. Finally, current challenges and future perspectives concerning clinical translation of EV detection are presented.},
}
@article {pmid40114032,
year = {2025},
author = {Tang, K and Zhou, L and Tian, X and Fang, SY and Vandenbulcke, E and Du, A and Shen, J and Cao, H and Zhou, J and Chen, K and Kim, HR and Luo, Z and Xin, S and Lin, SH and Park, D and Yang, L and Zhang, Y and Suzuki, K and Majety, M and Ling, X and Lam, SZ and Chow, RD and Ren, P and Tao, B and Li, K and Codina, A and Dai, X and Shang, X and Bai, S and Nottoli, T and Levchenko, A and Booth, CJ and Liu, C and Fan, R and Dong, MB and Zhou, X and Chen, S},
title = {Cas12a-knock-in mice for multiplexed genome editing, disease modelling and immune-cell engineering.},
journal = {Nature biomedical engineering},
volume = {9},
number = {8},
pages = {1290-1308},
pmid = {40114032},
issn = {2157-846X},
support = {RF1DA048811//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; K99CA282989//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; F30CA250249//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; RF1 DA048811/DA/NIDA NIH HHS/United States ; R01 CA231112/CA/NCI NIH HHS/United States ; DP2 CA238295/CA/NCI NIH HHS/United States ; T32 GM007205/GM/NIGMS NIH HHS/United States ; F30 CA250249/CA/NCI NIH HHS/United States ; U54CA209992//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; DP2CA238295//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; K99 CA282989/CA/NCI NIH HHS/United States ; U54 CA209992/CA/NCI NIH HHS/United States ; R33 CA281702/CA/NCI NIH HHS/United States ; T32GM007205//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R33CA281702//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R33 CA225498/CA/NCI NIH HHS/United States ; R01CA231112//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; Mice, Inbred C57BL ; *Gene Knock-In Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Disease Models, Animal ; CRISPR-Cas Systems/genetics ; *Cell Engineering/methods ; Dependovirus/genetics ; Humans ; Dendritic Cells ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The pleiotropic effects of human disease and the complex nature of gene-interaction networks require knock-in mice allowing for multiplexed gene perturbations. Here we describe a series of knock-in mice with a C57BL/6 background and with the conditional or constitutive expression of LbCas12a or of high-fidelity enhanced AsCas12a, which were inserted at the Rosa26 locus. The constitutive expression of Cas12a in the mice did not lead to discernible pathology and enabled efficient multiplexed genome engineering. We used the mice for the retrovirus-based immune-cell engineering of CD4[+] and CD8[+] T cells, B cells and bone-marrow-derived dendritic cells, for autochthonous cancer modelling through the delivery of multiple CRISPR RNAs as a single array using adeno-associated viruses, and for the targeted genome editing of liver tissue using lipid nanoparticles. We also describe a system for simultaneous dual-gene activation and knockout (DAKO). The Cas12a-knock-in mice and the viral and non-viral delivery vehicles provide a versatile toolkit for ex vivo and in vivo applications in genome editing, disease modelling and immune-cell engineering, and for the deconvolution of complex gene interactions.},
}
@article {pmid40930534,
year = {2025},
author = {Lejars, M and Maeda, T and Guillier, M},
title = {EASY-edit: a toolbox for high-throughput single-step custom genetic editing in bacteria.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
doi = {10.1093/nar/gkaf883},
pmid = {40930534},
issn = {1362-4962},
support = {/ERC_/European Research Council/International ; 818750//European Union's Horizon 2020 research and innovation/ ; //CNRS/ ; ANR-11-LABX-0011//Initiative d'Excellence/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; *CRISPR-Cas Systems ; Operon ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genes, Reporter ; },
abstract = {Targeted gene editing can be achieved using CRISPR-Cas9-assisted recombineering. However, high-efficiency editing requires careful optimization for each locus to be modified, which can be tedious and time-consuming. In this work, we developed a simple, fast and cheap method: Engineered Assembly of SYnthetic operons for targeted editing (EASY-edit) in Escherichia coli. Highly efficient editing of the different constitutive elements of the operons can be achieved by using a set of optimized guide RNAs and single- or double-stranded DNA repair templates carrying relatively short homology arms. This facilitates the construction of multiple genetic tools, including mutant libraries or reporter genes. EASY-edit is also highly modular, as we provide alternative and complementary versions of the operon inserted in three loci which can be edited iteratively and easily combined. As a proof of concept, we report the construction of several fusions with reporter genes confirming known post-transcriptional regulation mechanisms and the construction of saturated and unbiased mutant libraries. In summary, the EASY-edit system provides a flexible genomic expression platform that can be used both for the understanding of biological processes and as a tool for bioengineering applications.},
}
@article {pmid40930528,
year = {2025},
author = {Jiang, Q and Jin, S and Qin, Z and Zhang, J and He, R and Chen, Z and Qiao, B and Qiao, J and Liu, Y},
title = {CRISPR/Cas12a DTR system: a topology-guided Cas12a assay for specific dual detection of RNA and DNA targets.},
journal = {Nucleic acids research},
volume = {53},
number = {17},
pages = {},
doi = {10.1093/nar/gkaf893},
pmid = {40930528},
issn = {1362-4962},
support = {2022YFC2304304//National Key Research and Development Program of China/ ; 2023DJC136//Science and Technology Innovation Talent Plan of Hubei Province/ ; 2025AFB825//Natural Science Foundation of Hubei Province/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *MicroRNAs/genetics/analysis ; *DNA/genetics/analysis ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *RNA/genetics/analysis ; *Bacterial Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas12a technology has revolutionized molecular diagnostics. However, existing Cas12a systems depend on continuous target DNA activation, which limits them to single-target detection. In this study, we developed a novel topology-guided Cas12a system, the double-target responsive (DTR) system, capable of being activated by noncontiguous dual RNA/DNA targets. The DTR system employs two split CRISPR RNA (crRNA) fragments and two Cas12a proteins that cooperatively reconstitute upon recognizing two nucleic acid activators. We demonstrated the DTR system's ability to specifically detect dual nucleic acid substrates in a single readout, achieving a detection limit of 78 fM for RNA and exceptional specificity for single-nucleotide variations. Additionally, we successfully applied the DTR system to clinical samples, enabling simultaneous detection of two oral squamous cell carcinoma-related microRNAs (miR-155 and miR-let-7a), thereby distinguishing healthy individuals from patients. This work establishes an efficient Cas12a-based platform for sensitive, simultaneous, and discriminative detection of RNA and DNA targets, enhancing the versatility of Cas12a in analytical detection and clinical diagnosis.},
}
@article {pmid40929845,
year = {2025},
author = {Chang, Y and Ding, J},
title = {CircRNA knockout/knockdown tools in molecular biology research.},
journal = {Biochemical and biophysical research communications},
volume = {783},
number = {},
pages = {152607},
doi = {10.1016/j.bbrc.2025.152607},
pmid = {40929845},
issn = {1090-2104},
abstract = {Circular RNAs (circRNAs), characterized by their covalently closed circular architecture, represent a unique class of endogenous RNA molecules that serve as pivotal regulators in post-transcriptional gene regulation in organisms. Accumulating evidence has established their potential as promising diagnostic biomarkers across various human pathologies, including but not limited to malignant neoplasms, neurodegenerative disorders, and metabolic dysregulation.By inhibiting circRNA expression, we can better understand their functions and their impact on related biological processes. Over the past decade, remarkable advancements have emerged in circRNAs manipulation technologies, including siRNA, DNAzyme, and CRISPR-Cas systems emerging as powerful tools for precise circRNAs editing in both experimental models and preclinical studies. In this review, we summarize the advantages and identification of these editing methods and discuss future challenges and prospects.},
}
@article {pmid40927181,
year = {2025},
author = {Ziemann, M and Mitrofanov, A and Stöckl, R and Alkhnbashi, OS and Backofen, R and Hess, WR},
title = {Analysis of tracrRNAs reveals subgroup V2 of type V-K CAST systems.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf020},
pmid = {40927181},
issn = {2633-6693},
abstract = {Clustered regularly interspaced palindromic repeats (CRISPR)-associated transposons (CAST) consist of an integration between certain class 1 or class 2 CRISPR-Cas systems and Tn7-like transposons. Class 2 type V-K CAST systems are restricted to cyanobacteria. Here, we identified a unique subgroup of type V-K systems through phylogenetic analysis, classified as V-K_V2. Subgroup V-K_V2 CAST systems are characterized by an alternative tracrRNA, the exclusive use of Arc_2-type transcriptional regulators, and distinct differences in the length of protein domains in TnsB and TnsC. Although the occurrence of V-K_V2 CAST systems is restricted to Nostocales cyanobacteria, it shows signs of horizontal gene transfer, indicating its capability for genetic mobility. The predicted V-K_V2 tracrRNA secondary structure has been integrated into an updated version of the CRISPRtracrRNA program available on GitHub under https://github.com/BackofenLab/CRISPRtracrRNA/releases/tag/2.0.},
}
@article {pmid40678944,
year = {2025},
author = {Zhu, C and Huang, Q and Fu, R and Xun, Z and Ou, Q and Xianyu, Y and Liu, C},
title = {A Triple-Modal Biosensing Strategy for Hepatitis B Virus Based on Mg[2+]-Mediated Modulation of CRISPR/Cas12a and Au@Pt Nanoparticles.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {36},
pages = {e05341},
doi = {10.1002/smll.202505341},
pmid = {40678944},
issn = {1613-6829},
support = {82030063 82372317 82372316//National Natural Science Foundation of China/ ; 2023Y4004//University-Industry Research Cooperation Project of Science and Technology of Fujian Province/ ; 2025C02124//"Pioneer" and "Leading Goose" R&amp;D Program of Zhejiang/ ; 2023QH1102//Startup Fund for Scientific Research of Fujian Medical University/ ; 2024QH2043//Startup Fund for Scientific Research of Fujian Medical University/ ; },
mesh = {*Hepatitis B virus/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; *Metal Nanoparticles/chemistry/ultrastructure ; *Gold/chemistry ; *CRISPR-Cas Systems/genetics ; *Magnesium/chemistry ; DNA, Viral ; *Platinum/chemistry ; Nucleic Acid Amplification Techniques ; Humans ; },
abstract = {Hepatitis B virus (HBV) infection remains a significant global public health issue, and rapid detection of HBV DNA is crucial for disease prevention and control. However, traditional methods for HBV DNA detection are limited by their reliance on precise instruments and single readout, which can hardly meet the requirements of on-site detection. In this study, the Mg[2+]-enhanced trans-cleavage activity of clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a) is reported and coupled with loop-mediated isothermal amplification (LAMP) and Au@Pt nanoparticles as a signaling reporter for on-site detection of HBV DNA. This triple-modal biosensing strategy enables multiple signal readouts including UV-vis spectrum, RGB value, and temperature with high sensitivity and accuracy. The linear detection ranges using UV-vis spectroscopy, RGB color recognition, and photothermal modes are from 50 to 10,000 copies µL[-1], with the limitation of detection of 24.07, 39.65, and 23.33 copies µL[-1], respectively. This biosensing strategy is further employed for the qualitative detection of HBV DNA in 48 serum samples, achieving sensitivities of 100%, 100%, and 95.24% for triple modes. This work offers a promising tool of next-generation LAMP-CRISPR/Cas12a for the rapid and portable detection of nucleic acids.},
}
@article {pmid40925176,
year = {2025},
author = {He, Z and Zhang, J and Kuang, S and Li, S and Wang, Y and Ding, J and Ma, Z and Zhang, B},
title = {Colloidal gold technology in viral diagnostics: Recent innovations, clinical applications, and future perspectives.},
journal = {Virology},
volume = {612},
number = {},
pages = {110686},
doi = {10.1016/j.virol.2025.110686},
pmid = {40925176},
issn = {1096-0341},
abstract = {Colloidal gold technology has revolutionized viral diagnostics through its rapid, cost-effective, and user-friendly applications, particularly in point-of-care testing (POCT). This review synthesizes recent advancements, focusing on its role in detecting respiratory viruses, hepatitis viruses, and emerging pathogens. The technology leverages the unique optical and physicochemical properties of gold nanoparticles (AuNPs), including localized surface plasmon resonance (LSPR) and high surface-to-volume ratios, to achieve rapid antigen-antibody recognition with visual readouts within 15 min. Innovations such as CRISPR-Cas-integrated lateral flow immunoassays (LFIAs), dual-mode plasmonic biosensors, and nanomaterials like CeO2-colloidal gold composites have enhanced sensitivity and multiplex capability, enabling simultaneous identification of co-circulating pathogens. Case studies highlight its efficacy in dengue serotyping, SARS-CoV-2 neutralizing antibody quantification, and HBV/HCV co-detection, demonstrating high clinical specificity. However, challenges persist, including the need for improved sensitivity; interference of sample matrix with immunity; false positives caused by cross-reactions; and limitations of semi-quantitative analysis. Recent progress in hybrid nanomaterial synthesis, surface functionalization, and device-level multiplexing-coupled with AI-driven data interpretation- promises to address these gaps. Future trends emphasize integration with surface-enhanced Raman scattering (SERS), microfluidics, and portable sensors to achieve sub-zeptomolar sensitivity and scalable deployment. By bridging nanotechnology with precision diagnostics, colloidal gold platforms are poised to redefine global viral surveillance, particularly in resource-limited settings, underscoring their indispensable role in pandemic preparedness.},
}
@article {pmid40922689,
year = {2025},
author = {Zhang, Q and Ren, J and Wu, S and Tan, Y and Wang, W and Feng, C and Zhao, L and Zhu, Z},
title = {Plasmid-Free CRISPR/Cpf1 Genome Editing With In Vivo T7 RNA Polymerase-Transcribed CRISPR RNA From Short Double-Stranded DNA.},
journal = {Biotechnology and bioengineering},
volume = {},
number = {},
pages = {},
doi = {10.1002/bit.70062},
pmid = {40922689},
issn = {1097-0290},
support = {//This study was funded by the National Key Research and Development Program (Grant Number: 2022YFA0911802); the National Natural Science Foundation of China (Grant Number: 22177018); the Liaoning Revitalization Talents Program (Grant Number: XLYC2203057) and the Fundamental Research Funds for the Central Universities (Grant Number: DUT23LAB104)./ ; },
abstract = {Plasmids are commonly employed in the delivery of clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) components for genome editing. However, the absence of heritable plasmids in numerous organisms limits the development of CRISPR/Cas genome editing tools. Moreover, cumbersome procedures for plasmid construction and curing render genome editing time-consuming. In this study, we developed a plasmid-free CRISPR/Cpf1 genome editing system for Saccharomyces cerevisiae and Starmerella bombicola. This system leveraged integrative expression of the Cpf1 nuclease and T7 RNA polymerase (T7RNAP), as well as the delivery of linear fragments including (i) a marker cassette for integration and selection, (ii) short double-stranded DNA (crDNA) for in vivo transcription of crRNA by T7RNAP, and (iii) donor DNA for homology-directed repair. We demonstrated that this editing system enabled efficient multiplexed and iterative genome editing without the need for marker recycling and plasmid curing. The use of short crDNA (87 bp) and donor DNA (≤ 120 bp), both readily prepared from ordered oligonucleotides via annealing or overlap extension, dramatically simplified the editing process. Successful implementation in S. bombicola, which lacks heritable plasmids for genetic engineering, highlighted the potential of this approach especially for genome engineering of genetically intractable organisms in a plasmid-free way.},
}
@article {pmid40920944,
year = {2025},
author = {Mangeot, PE and Ohlmann, T},
title = {[Harnessing retroviral engineering for genome reprogramming].},
journal = {Medecine sciences : M/S},
volume = {41},
number = {8-9},
pages = {647-656},
doi = {10.1051/medsci/2025098},
pmid = {40920944},
issn = {1958-5381},
support = {ANRS0516//ANRS/ ; },
mesh = {Humans ; *Retroviridae/genetics/physiology ; *Genetic Engineering/methods ; Genetic Vectors/genetics ; Animals ; CRISPR-Cas Systems ; Gene Editing/methods ; Genome, Human ; *Cellular Reprogramming/genetics ; },
abstract = {The accumulated knowledge on the biology of the HIV-1 virus has led to the emergence of technologies that exploit the architecture of retroviruses and their integration or vectorization properties. This field of study constitutes retroviral vectorology, democratized in laboratories by the use of lentiviral vectors. By hijacking retroviral assembly, other systems are emerging and are increasingly mentioned in recent literature. In particular, defective retroviral particles are capable of transiently delivering effectors that act on the genome: they thus appear to be more suitable tools for delivering genetic scalpels, whose persistence in the target cell or organism is not required. Since the description of the CRISPR Cas9 system in 2012, genome engineering techniques have continued to evolve in terms of capacity and reliability. Several derivatives of the CRISPR system can now modify the human genome with nucleotide-level precision. Introducing these effectors into the cell or organism remains a major technical challenge that vector scientists are striving to overcome. This review describes the major retroviral systems used for genome manipulation. Following an overview of genetic engineering techniques, we will see how researchers have developed a wide range of genomic tools by manipulating different processes in the retroviral architecture.},
}
@article {pmid40920796,
year = {2025},
author = {Nakagata, N and Nakao, S and Mikoda, N and Yamaga, K and Nakagawa, Y and Sakuma, T and Yamamoto, T and Takeo, T},
title = {Improved protocol for the vitrification and warming of rat zygotes by optimizing the warming solution and oocyte donor age.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0328718},
pmid = {40920796},
issn = {1932-6203},
mesh = {Animals ; Female ; *Zygote/physiology/drug effects/cytology ; *Vitrification ; Rats ; *Oocytes/cytology ; *Cryopreservation/methods ; Rats, Sprague-Dawley ; Embryo Transfer ; Fertilization in Vitro/methods ; Rats, Inbred F344 ; Male ; CRISPR-Cas Systems ; Sucrose/pharmacology ; },
abstract = {Zygotes are used to create genetically modified animals by electroporation using the CRISPR-Cas9 system. Such zygotes in rats are obtained from superovulated female rats after mating. Recently, we reported that in vivo-fertilized zygotes had higher cryotolerance and developmental ability than in vitro-fertilized zygotes in Sprague Dawley (SD) and Fischer 344 rats. To apply the in vitro-fertilized zygotes in creating genetically modified rats, we need to address their low cryotolerance and developmental ability. Hence, we evaluated the effects of warming solutions containing different sucrose concentrations (0-0.3 M) and the oocyte donor's age (3-7-week-old SD rats) on the viability of vitrified-warmed zygotes after in vitro fertilization and on developmental ability by embryo transfer in SD rats. A warming solution containing 0.1 M sucrose enhanced the survival rate of vitrified-warmed zygotes and their rate of development to two-cell embryos. Additionally, zygotes derived from 6- and 7-week-old female rats had higher cryotolerance and developmental ability than those from 3-week-old ones. Next, vitrified-warmed rat zygotes produced using the optimized protocol underwent genome editing by electroporation with Cas9 ribonucleoprotein and gRNA introduced to disrupt the Tyr gene. We then found that 86.5% of the pups derived from zygotes demonstrated mutation of the targeted gene. Therefore, the improved protocol for vitrifying and warming rat zygotes is useful for preserving and producing genetically modified rats.},
}
@article {pmid40891143,
year = {2025},
author = {Zheng, X and Zhai, Y and Chathurika, HAW and Ni, X and Lv, R and Wu, C and Sun, Z and Shen, Y and Zhang, CY and Zheng, P and Sun, J},
title = {A Highly Efficient 5S rRNA-CRISPR/Cas9 Genome Editing Toolkit in Acremonium chrysogenum.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {36},
pages = {22607-22616},
doi = {10.1021/acs.jafc.5c06429},
pmid = {40891143},
issn = {1520-5118},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Acremonium/genetics/metabolism ; *RNA, Ribosomal, 5S/genetics/metabolism ; Cephalosporins/biosynthesis ; Fungal Proteins/genetics/metabolism ; },
abstract = {Acremonium chrysogenum is an important industrial producer of cephalosporin C (CPC), and efficient genome editing tools are critical for its exploitation and metabolic engineering. Currently, CRISPR/Cas9 systems for A. chrysogenum employ heterologous promoters, including Aspergillus nidulans PgpdA or Aspergillus fumigatus AfU6p, to drive sgRNA expression. These systems often required additional sgRNA processing elements such as ribozymes or tRNAs, which increased cloning complexity and experimental workload. Here, we developed a simplified and highly efficient CRISPR/Cas9 genome editing system using the endogenous 5S rRNA promoter for sgRNA transcription in A. chrysogenum. This system obviated the need for processing elements and achieved up to 100% gene disruption efficiency, as demonstrated by targeting the sorB gene. Furthermore, this platform enabled 100% single gene deletion and efficient large-scale chromosomal deletion, up to 66.17 kb within the sorbicillinoid biosynthetic gene cluster, without donor DNA. To our knowledge, this represents the largest chromosomal deletion reported in A. chrysogenum to date. Moreover, the system also facilitated precise and iterative gene editing through homologous recombination-mediated marker replacement at the kusA locus. Overall, this 5S rRNA-CRISPR/Cas9 system provides a versatile, powerful, and efficient genome editing toolkit for functional genomics and strain improvement in A. chrysogenum.},
}
@article {pmid40877754,
year = {2025},
author = {Singuru, MMR and Bhattacharyya, P and Sriramakrishnan, HP and You, M},
title = {Sensitive Detection of Intercellular Tensile Forces via Cas12a-Assisted Membrane Molecular Probes.},
journal = {Nano letters},
volume = {25},
number = {36},
pages = {13519-13525},
doi = {10.1021/acs.nanolett.5c02983},
pmid = {40877754},
issn = {1530-6992},
mesh = {Cadherins/metabolism ; Humans ; *Cell Membrane/chemistry/metabolism ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Tensile Strength ; Mechanotransduction, Cellular ; Epithelial-Mesenchymal Transition ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *Molecular Probes/chemistry ; *Bacterial Proteins/metabolism/chemistry ; },
abstract = {Intercellular forces are critical for shaping cells, driving migration, and guiding tissue development and morphogenesis. However, these transient and low-intensity forces are still challenging to detect. Here, we developed a Force-Responsive Cas12a-assisted Tension Sensor (FRCTS), which leverages the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a technology to enable more reliable detection of cumulative molecular force events generated at cell-cell junctions. FRCTS incorporates a lipid-modified DNA hairpin to spontaneously anchor onto live-cell membranes. The hairpin unfolds upon molecular tension exerted by neighboring cells through an integrin or cadherin receptor and reveals a hidden strand to activate Cas12a. Cas12a activation leads to an irreversible cleavage of a fluorogenic reporter on the cell surface, causing cumulative cell membrane fluorescence signals for recording intercellular force events. After systematic optimization, we applied FRCTS to quantify E-cadherin/N-cadherin mechanical correlations during the epithelial-mesenchymal transition. This modular and sensitive FRCTS platform can potentially be used for assessing various intercellular mechanotransduction processes.},
}
@article {pmid40816522,
year = {2025},
author = {Meng, XQ and Xu, XL and Gao, Y and Deng, SL},
title = {Establishment of CRISPR/Cas9 lineage tracking technology for pig embryos.},
journal = {Molecular and cellular probes},
volume = {83},
number = {},
pages = {102046},
doi = {10.1016/j.mcp.2025.102046},
pmid = {40816522},
issn = {1096-1194},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Swine/genetics/embryology ; *Cell Lineage/genetics ; *Embryo, Mammalian/cytology/metabolism ; Single-Cell Analysis/methods ; },
abstract = {Understanding tissue development in pigs is critical for biomedical research and genetic engineering, particularly for modeling human disease. However, tracing developmental origins and reconstructing lineage trees for pig cells remains a significant challenge. Here, we present a high-resolution lineage tracing system that combines molecular barcoding with single-cell transcriptomics in pigs. Our system combines two key components: DNA barcodes (three CRISPR/Cas9 target sites and an 8-base pair intBC) integrated into the genome via piggyBac transposition, and a constitutive Cas9-EGFP cassette stably integrated at the Rosa26 locus using CRISPR/Cas12a. By combining lineage barcodes with single-cell RNA sequencing (scRNA-seq), we constructed an evolutionary lineage recorder that captures distinct cell states across developmental or differentiation trajectories. This system provides an essential tool for the subsequent construction of complete porcine cell fate maps. Our work provides a tool for studying porcine developmental biology, but also helps to optimize regenerative medicine strategies and improve the design of genetically engineered animal models.},
}
@article {pmid40815167,
year = {2025},
author = {Sunshine, S and Puschnik, A and Retallack, H and Laurie, MT and Liu, J and Peng, D and Knopp, K and Zinter, MS and Ye, CJ and DeRisi, JL},
title = {Defining the host dependencies and the transcriptional landscape of RSV infection.},
journal = {mBio},
volume = {16},
number = {9},
pages = {e0101025},
doi = {10.1128/mbio.01010-25},
pmid = {40815167},
issn = {2150-7511},
support = {//Chan Zuckerberg Biohub San Francisco/ ; F31AI150007//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {Humans ; *Respiratory Syncytial Virus Infections/virology/genetics/immunology ; *Respiratory Syncytial Virus, Human/genetics/physiology ; *Host-Pathogen Interactions/genetics ; Single-Cell Analysis ; CRISPR-Cas Systems ; Gene Expression Profiling ; },
abstract = {Respiratory syncytial virus (RSV) is a globally prevalent pathogen, causes severe disease in older adults, and is the leading cause of bronchiolitis and pneumonia in the United States for children during their first year of life. Despite its prevalence worldwide, RSV-specific treatments remain unavailable for most infected patients. Here, we leveraged a combination of genome-wide CRISPR knockout screening and single-cell RNA sequencing to improve our understanding of the host determinants of RSV infection and the host response in both infected cells and uninfected bystanders. These data reveal temporal transcriptional patterns that are markedly different between RSV-infected and bystander-activated cells. Our data show that expression of interferon-stimulated genes is primarily observed in bystander activated cells, while genes implicated in the unfolded protein response and cellular stress are upregulated specifically in RSV-infected cells. Furthermore, genome-wide CRISPR screens identified multiple host factors important for viral infection, findings which we contextualize relative to 29 previously published screens across 17 additional viruses. These unique data complement and extend prior studies that investigate the proinflammatory response to RSV infection, and juxtaposed to other viral infections, provide a rich resource for further hypothesis testing.IMPORTANCERespiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection in infants and the elderly. Despite its substantial global health burden, RSV-targeted treatments remain unavailable for the majority of individuals. While vaccine development is underway, a detailed understanding of the host response to RSV infection and identification of required human host factors for RSV may provide insight into combatting this pathogen. Here, we utilized single-cell RNA sequencing and functional genomics to understand the host response in both RSV-infected and bystander cells, identify what host factors mediate infection, and contextualize these findings relative to dozens of previously reported screens across 17 additional viruses.},
}
@article {pmid40767522,
year = {2025},
author = {Guo, G and Zhang, M and Xu, Z and Xi, P and Zhu, H and Evers, A and Lebbink, RJ and Lang, Y and He, Q and Huang, Y-W and Li, T and Bosch, BJ and Li, W},
title = {Genome-wide CRISPR screen reveals key role of sialic acids in PEDV and porcine coronavirus infections.},
journal = {mBio},
volume = {16},
number = {9},
pages = {e0162825},
doi = {10.1128/mbio.01628-25},
pmid = {40767522},
issn = {2150-7511},
mesh = {*Porcine epidemic diarrhea virus/physiology/pathogenicity/genetics ; Animals ; *Coronavirus Infections/virology/veterinary/metabolism ; Swine ; Humans ; *CRISPR-Cas Systems ; *Sialic Acids/metabolism ; *Swine Diseases/virology/metabolism ; Virus Internalization ; Sialyltransferases/genetics/metabolism ; *N-Acetylneuraminic Acid/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Host-Pathogen Interactions ; Cell Line ; },
abstract = {Porcine epidemic diarrhea virus (PEDV) is a globally distributed alphacoronavirus with economic importance that can cause severe watery diarrhea and even death in piglets. To identify host factors essential for PEDV infection, we performed a genome-wide CRISPR/Cas9 screen in human hepatocellular carcinoma cells (Huh7) using the highly virulent PEDV GIIb strain GDU. Several genes involved in the sialic acid and heparan sulfate biosynthesis pathway and cholesterol metabolism were highly enriched following PEDV selection. We validated that the host factor ST3 beta-galactoside alpha-2,3-sialyltransferase 4 (ST3GAL4), which catalyzes the transfer of sialic acid to sugar chains via α2,3-linked linkages, is important for PEDV infection. To systematically investigate the role of sialic acid in PEDV infection, we knocked out genes related to sialic acid synthesis. This led to a reduced abundance of sialic acid on the cell surface, which in turn inhibited PEDV adsorption and internalization. Furthermore, we found that both α2,3-linked and α2,6-linked sialic acids can serve as cellular attachment factors for PEDV. We conducted a glycan microarray screen to determine which sialoglycans are preferred by the PEDV spike protein. The results revealed that PEDV favors binding to α2,3-sialoglycans. Additionally, we found that not only current circulating PEDV strains but also other porcine coronaviruses rely on sialic acid for efficient infection. Collectively, our findings provide insights into critical host factors involved in PEDV infection and demonstrate that disrupting genes involved in sialic acid biosynthesis negatively affects the infectivity of multiple porcine enteric coronaviruses.IMPORTANCEA wide range of viruses utilize sialic acid as receptors. Sialic acid binding may serve as a key determinant of viral host range. Different viruses exhibit distinct preferences for specific types of sialic acid linkages. However, it remains unclear which specific subtypes of sialic acid are utilized during PEDV infection. In this study, we performed CRISPR-based genome-wide knockout screening and identified ST3GAL4 as a key host factor for PEDV infection. Furthermore, we found that both α2,3-linked and α2,6-linked sialic acids can function as attachment factors for PEDV infection. A glycan microarray screen revealed that PEDV S1 shows the strongest binding preference for α2,3-linked and α2,8-linked sialosides. Sialic acids were also implicated in infections by other porcine enteric coronaviruses. Overall, our findings advance our understanding of viral entry mechanisms of PEDV and other swine coronaviruses and may provide avenues for designing antiviral strategies.},
}
@article {pmid40744238,
year = {2025},
author = {Wen, Z and Yang, D and Yang, Y and Hu, J and Parviainen, A and Chen, X and Li, Q and VanDeusen, E and Ma, J and Tay, F},
title = {The path to biotechnological singularity: Current breakthroughs and outlook.},
journal = {Biotechnology advances},
volume = {84},
number = {},
pages = {108667},
doi = {10.1016/j.biotechadv.2025.108667},
pmid = {40744238},
issn = {1873-1899},
mesh = {Humans ; *Biotechnology/trends ; Gene Editing ; Artificial Intelligence ; Synthetic Biology ; Regenerative Medicine ; CRISPR-Cas Systems ; Brain-Computer Interfaces ; },
abstract = {Fueled by rapid advances in gene editing, synthetic biology, artificial intelligence, regenerative medicine, and brain-computer interfaces, biotechnology is approaching a transformative era often referred to as biotechnological singularity. CRISPR-based gene editing has revolutionized genetic engineering, enabling precise modifications for treating hereditary diseases and cancer. Synthetic biology facilitates sustainable biomaterial production and innovative therapeutic applications. Artificial intelligence accelerates drug discovery, enhances diagnostic accuracy, and personalizes treatment through deep learning models. Driven by stem cell research, regenerative medicine offers promising avenues for reversing aging and treating degenerative diseases. Brain-computer interfaces merge human cognition with technology, enabling direct neural control of prosthetics and expanding human-machine interactions. These breakthroughs, however, raise ethical, regulatory, and societal concerns, including equitable access, biosecurity risks, and the implications of human enhancement. The convergence of biological and computational technologies challenges traditional boundaries, necessitating comprehensive governance frameworks. By embracing responsible innovation, society can harness these advancements for transformative health interventions, environmental sustainability, and extended longevity. The realization of biotechnological singularity depends on interdisciplinary collaboration among scientists, policymakers, and the public to ensure that progress aligns with the well-being of humanity and ethical considerations.},
}
@article {pmid40552689,
year = {2025},
author = {Yao, B and Yang, Q and Snijders Blok, CJB and Daniels, MA and Doevendans, PA and Schiffelers, R and Sluijter, JPG and Lei, Z},
title = {Insights into pegRNA design from editing of the cardiomyopathy-associated phospholamban R14del mutation.},
journal = {FEBS letters},
volume = {599},
number = {17},
pages = {2543-2554},
doi = {10.1002/1873-3468.70097},
pmid = {40552689},
issn = {1873-3468},
support = {10250022110004//The Netherlands Organisation for Health Research and Development (ZonMw) Psider-Heart/ ; 202006170055//China Scholarship Council/ ; #725229//European Research Council (ERC), EVICARE project under H2020/ ; 2021/TTW/01038252//Netherlands Organization for Scientific Research (NWO) - Applied and Engineering Sciences (TTW)/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Calcium-Binding Proteins/genetics ; *Cardiomyopathies/genetics/pathology ; CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Mutation ; HEK293 Cells ; },
abstract = {Prime editing (PE) represents a transformative genome-editing technology and enables precise insertions, deletions, and base substitutions without introducing double-strand breaks, thereby reducing undesired indels and off-target effects. Despite advancements in enhanced prime editors and optimized prime editing guide RNAs (pegRNAs), designing effective pegRNAs remains a major challenge. The phospholamban (PLN) R14del mutation is associated with cardiomyopathies, making it a crucial target for precise gene-editing strategies. In this study, we explored pegRNA features that contribute to high editing efficiency using the FluoPEER.PLN R14del reporter cell line. Through systematic screening, we identified three pegRNAs with significantly enhanced editing efficiency. Our findings underscore the importance of pegRNA secondary structure and stability in optimizing prime editing, providing valuable insights into precise gene correction strategies.},
}
@article {pmid40920775,
year = {2025},
author = {Tan, Y and Kumagai-Takei, N and Shimizu, Y and Yamasaki, A and Hara-Yamamoto, M and Mitani, S and Ito, T},
title = {Targeting the Exon2 splice cis-element in PD-1 and its effects on lymphocyte function.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0331468},
pmid = {40920775},
issn = {1932-6203},
mesh = {*Programmed Cell Death 1 Receptor/genetics/metabolism ; Humans ; *Exons/genetics ; CRISPR-Cas Systems ; *CD8-Positive T-Lymphocytes/metabolism/immunology ; *RNA Splicing ; Cytokines/metabolism ; RNA Precursors/genetics ; Lymphocyte Activation ; Cell Proliferation ; },
abstract = {T-cell therapies have proven to be a promising treatment option for cancer patients in recent years, especially in the case of chimeric antigen receptor (CAR)-T cell therapy. However, the therapy is associated with insufficient activation of T cells or poor persistence in the patient's body, which leads to incomplete elimination of cancer cells, recurrence, and genotoxicity. By extracting the splice element of PD-1 pre-mRNA using biology based on CRISPR/dCas13 in this study, our ultimate goal is to overcome the above-mentioned challenges in the future. PD-1 plays an important role in controlling T cell responses and is expressed at the cell surface of T cells following activation. The receptor PD-1 interferes with T cell receptor (TCR) signaling following interaction with PD-L1. The outcome of stimulation via PD-1 leads to decreases in cytokine secretion and cell proliferation. We extracted the RNA region of PD-1 pre-mRNA using CD8+T cell lines and examined the effect of targeting the Exon2 splice cis-element on the production of cytokines in the present study. In particular, the production of IFN-γ, TNF-α, GM-CSF was lower in RNA-targeted cells than in non-targeted cells, but the cytokine secretion capacity and cell proliferation were maintained in RNA-targeted cells. These results suggested that the use of the RNA editing technology, CRISPR/dCas13 strategy offers a novel approach to mitigate genotoxicity in lymphocytes with cytokine production and cell proliferation.},
}
@article {pmid40919784,
year = {2025},
author = {Leite, VLM and Faria, AR and Guerra, CF and Souza, SdSR and Freitas, AdAR and Morais, JM and Merquior, VLC and Planet, PJ and Teixeira, LM},
title = {Hidden diversity in Enterococcus faecalis revealed by CRISPR2 screening: eco-evolutionary insights into a novel subspecies.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0142825},
doi = {10.1128/spectrum.01428-25},
pmid = {40919784},
issn = {2165-0497},
abstract = {Enterococcus faecalis is a commensal bacterium that colonizes the gut of humans and animals and is a major opportunistic pathogen, known for causing multidrug-resistant healthcare-associated infections (HAIs). Its ability to thrive in diverse environments and disseminate antimicrobial resistance genes (ARGs) across ecological niches highlights the importance of understanding its ecological, evolutionary, and epidemiological dynamics. The CRISPR2 locus has been used as a valuable marker for assessing clonality and phylogenetic relationships in E. faecalis. In this study, we identified a group of E. faecalis strains lacking CRISPR2, forming a distinct, well-supported clade. We demonstrate that this clade meets the genomic criteria for classification as a novel subspecies, here referred to as "subspecies B." Through a comprehensive pangenome analysis and comparative genomics, we explored the adaptive ecological traits underlying this diversification process, identifying clade-specific features and their predicted functional roles. Our findings suggest that the frequent isolation of subspecies B from meat products and processing facilities may reflect dissemination routes involving environmental contamination (e.g., water, plants, soil) from avian species. The absence of key virulence traits required for pathogenicity in mammals, particularly humans, and the lack of clinically relevant resistance determinants indicate that subspecies B currently poses minimal threat to public health compared with the broadly disseminated "subspecies A." Nevertheless, the unclear potential for genetic exchange between these subspecies and the frequent association of subspecies B with food sources calls for continued genomic surveillance of E. faecalis from a One Health perspective to detect and mitigate the emergence of high-risk variants in advance.IMPORTANCEExploring intraspecific genetic variability in generalist bacteria with pathogenic potential, such as Enterococcus faecalis, is a key to uncovering stable evolutionary trends. By screening the CRISPR2 locus across a representative set of genomes from diverse sources, this study reveals a previously unrecognized lineage within the population structure of E. faecalis, associated with underexplored nonhuman and nonhospital reservoirs. These findings broaden our knowledge of the species' genetic landscape and shed light on its adaptive strategies and patterns of ecological dissemination. By bridging phylogenetic patterns with variation in genetic defense systems and accessory traits, the study generates testable hypotheses about the genomic determinants and corresponding selective pressures that shape the species' behavior and long-term dissemination. This work offers new perspectives on the eco-evolutionary dynamics of E. faecalis and highlights the value of genomic surveillance beyond clinical settings, in alignment with One Health principles.},
}
@article {pmid40845666,
year = {2026},
author = {Wang, M and Zhou, W and Wang, M and Zhang, K},
title = {A highly sensitive ECL biosensor for NF-κB p50 detection based on entropy-driven amplification and CRISPR/Cas12a signal enhancement.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {167},
number = {},
pages = {109081},
doi = {10.1016/j.bioelechem.2025.109081},
pmid = {40845666},
issn = {1878-562X},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *NF-kappa B p50 Subunit/analysis ; Humans ; *Entropy ; *Luminescent Measurements/methods ; Electrochemical Techniques/methods ; Limit of Detection ; Exodeoxyribonucleases/metabolism ; Nucleic Acid Amplification Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Transcription factors, particularly NF-κB p50, play crucial roles in regulating gene expression and are involved in several diseases such as cancer, autoimmune disorders, and chronic inflammation. The sensitive detection of NF-κB p50 is essential for clinical diagnostics and therapeutic monitoring. In this study, we present an electrochemiluminescence (ECL) biosensor designed for the highly sensitive and specific detection of NF-κB p50. The biosensor integrates entropy-driven amplification and CRISPR/Cas12a-based signal enhancement to detect trace amounts of NF-κB p50. Upon detection of NF-κB p50, a ternary complex forms with a double-stranded DNA (dsDNA) probe, which prevents subsequent cleavage by exonuclease III (Exo III) and inhibits the CRISPR/Cas12a system. In the absence of NF-κB p50, Exo III digestion triggers entropy-driven amplification, which activates CRISPR/Cas12a, leading to enhanced electrochemical signals. The ECL biosensor demonstrated a detection limit of 0.56 pM, high selectivity, and excellent reproducibility. Furthermore, the biosensor successfully detected NF-κB p50 in complex biological samples, such as HeLa cell lysates, showcasing its potential for clinical applications in disease diagnostics.},
}
@article {pmid40845664,
year = {2026},
author = {Zhou, Y and Lv, Z and Geng, Y and Liu, R},
title = {An electrochemical RNA aptasensor based on sheet-like α-Fe2O3/Fe3O4 magnetic nanocomposites and CRISPR/Cas13a system for supersensitive detection of osteopontin.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {167},
number = {},
pages = {109085},
doi = {10.1016/j.bioelechem.2025.109085},
pmid = {40845664},
issn = {1878-562X},
mesh = {*CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry/genetics ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Nanocomposites/chemistry ; *Osteopontin/analysis/blood ; Limit of Detection ; Humans ; *Ferric Compounds/chemistry ; },
abstract = {Osteopontin (OPN) exhibits markedly elevated expression in malignant tumor tissues, rendering it a crucial tumor marker for cancer prevention and monitoring-underscoring the significance of its detection. This work proposed an electrochemical RNA aptasensor based on a novel sheet-like α-Fe2O3/Fe3O4 magnetic nanocomposites (MNCs) and CRISPR/Cas13a system to effectively detect OPN. The proposed aptasensor used the sheet-like α-Fe2O3/Fe3O4 MNCs as the conduction matrix and applied their magnetic property to accomplish self-assembly of the sensing element onto the electrode. The current change of this aptasensor depended on the activation degree of the CRISPR/Cas13a system, which correlated with the amount of OPN expression. Double-stranded RNA (Apt/Activator) was compelled disassembly due to the OPN's strong affinity of Apt, while single-stranded RNA (Activator) could be guided by crRNA to combine with Cas13a/crRNA and activated the enzymatic activity of Cas13a. Cas13a enzyme affected the current and electrical resistance by shearing the nucleic acid strands (Reporter) on the electrode, ultimately enabling the quantitative detection of OPN. The aptasensor demonstrated excellent selectivity, reproducibility, and stability, with the detection limit (LOD) of 0.33 pg·mL[-1] and the wide linear detection range of 1 pg·mL[-1] - 10 ng·mL[-1]. These results offer a novel idea for advancing tumor marker electrochemical biosensors.},
}
@article {pmid40757822,
year = {2025},
author = {Echavarria Galindo, M and Lai, Y},
title = {CRISPR-based genetic tools for the study of host-microbe interactions.},
journal = {Infection and immunity},
volume = {93},
number = {9},
pages = {e0051024},
doi = {10.1128/iai.00510-24},
pmid = {40757822},
issn = {1098-5522},
support = {26100423//Research Grants Council, University Grants Committee/ ; PF22-69790//Hong Kong PHD Fellowship Scheme/ ; R9829//Hong Kong University of Science and Technology (HKUST)/ ; },
mesh = {Humans ; Animals ; *CRISPR-Cas Systems ; *Host Microbial Interactions/genetics ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Microbiota/genetics ; *Host-Pathogen Interactions/genetics ; },
abstract = {CRISPR-based genetic tools have revolutionized our ability to interrogate and manipulate genes. These tools can be applied to both host and microbial cells, and their use can enhance our understanding of the dynamic nature of host-microbe interactions by uncovering their genetic underpinnings. As reviewed here, CRISPR-based tools are being used to explore the microbiome in an efficient, accurate, and high-throughput manner. By employing CRISPR screens, targeted genome editing, and recording systems to the study of host cells and microorganisms, we can gain critical insights into host defense mechanisms, potential vulnerabilities, and microbial pathogenesis, as well as essential or condition-specific genes involved in host-microbe interactions. Additionally, CRISPR-based genetic tools are being used in animal models to study host-microbe interactions in vivo. Recent advancements in CRISPR-derived technology can be combined with emerging techniques, such as single-cell RNA sequencing, to examine the complex interactions between hosts and microbes, shedding light on the role of the microbiome in health and disease. This review aims to provide a comprehensive overview of how these cutting-edge genetic tools are being used to investigate host-microbial systems, as well as their current limitations. Current research is likely to yield even more advanced genetic toolkits than those presently available, and these can serve researchers in identifying and exploring new therapeutic targets for diseases related to host-microbe interactions.},
}
@article {pmid40749504,
year = {2026},
author = {Ji, Z and Cheng, S and Li, W and Xing, Y and Tang, Z and Zhu, X and Wang, D and Hao, C and Wang, B and Shi, M},
title = {Ultrasensitive detection of miR-31 using a signal-on electrochemiluminescence biosensor based on CRISPR/Cas12a and MXene nanocomposites.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {167},
number = {},
pages = {109059},
doi = {10.1016/j.bioelechem.2025.109059},
pmid = {40749504},
issn = {1878-562X},
mesh = {*MicroRNAs/analysis/blood/genetics ; *Biosensing Techniques/methods ; Humans ; *Nanocomposites/chemistry ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; Limit of Detection ; *Luminescent Measurements/methods ; *Endodeoxyribonucleases/metabolism ; *CRISPR-Associated Proteins/metabolism ; Gold/chemistry ; Electrodes ; Metallocenes ; Bacterial Proteins ; Nitrites ; Transition Elements ; },
abstract = {MicroRNAs (miRNAs) have emerged as critical biomarkers for early cancer diagnosis due to their high specificity and stability in biological fluids. In this study, we report a highly sensitive and specific electrochemiluminescence (ECL) biosensor for the detection of miR-31, a miRNA closely associated with non-small cell lung cancer. The sensing platform integrates a PEI-Ru@Ti3C2@AuNPs-modified electrode with a ferrocene-labeled DNA probe (DNA1-Fc) to construct a target-responsive signal-on system. Upon recognition of miR-31, an isothermal strand displacement amplification (ISDA) reaction is initiated, producing abundant double-stranded DNA (dsDNA) that activates the CRISPR/Cas12a complex. The trans-cleavage activity of Cas12a then cleaves the DNA1-Fc probes on the electrode surface, removing the quenching ferrocene moiety and restoring the Ru-based ECL signal. Under optimized conditions, the biosensor exhibited a wide dynamic range from 10 aM to 100 pM and a remarkably low detection limit of 1.67 aM. The system also showed excellent specificity against homologous miRNAs, and its applicability was successfully validated in spiked human serum samples, achieving high recovery and reproducibility. The synergistic combination of nanomaterial-enhanced ECL emission, isothermal nucleic acid amplification, and CRISPR-based enzymatic cleavage provides a powerful strategy for ultrasensitive nucleic acid detection. This work offers a promising approach for early cancer diagnosis and has great potential for clinical translation and point-of-care testing.},
}
@article {pmid40719649,
year = {2025},
author = {Chen, M and Choi, HK and Goldston, LL and Hou, Y and Jiang, C and Lee, KB},
title = {Advanced Cancer Liquid Biopsy Platform for miRNA Detection in Extracellular Vesicles Using CRISPR/Cas13a and Gold Nanoarrays.},
journal = {ACS nano},
volume = {19},
number = {35},
pages = {31438-31456},
doi = {10.1021/acsnano.5c06940},
pmid = {40719649},
issn = {1936-086X},
mesh = {*MicroRNAs/genetics/analysis/blood ; Humans ; *Extracellular Vesicles/chemistry/metabolism/genetics ; *Gold/chemistry ; *CRISPR-Cas Systems ; Liquid Biopsy/methods ; *Colorectal Neoplasms/diagnosis/genetics ; Biomarkers, Tumor/genetics/blood ; Epithelial Cell Adhesion Molecule/metabolism ; Cell Line, Tumor ; Metal Nanoparticles/chemistry ; },
abstract = {Liquid biopsy is a transformative, noninvasive tool for cancer diagnosis and monitoring, with the potential to revolutionize personalized medicine. In this study, we introduce an advanced liquid biopsy platform for highly sensitive and selective detection of extracellular vesicle (EV) microRNAs (miRNA-21 and miRNA-23a) as biomarkers for colorectal cancer. The platform combines two innovations: (1) gold nanoarrays with epithelial cell adhesion molecule (EpCAM)-specific aptamers to selectively isolate tumor-derived EVs from plasma and (2) CRISPR/Cas13a-encapsulated liposomes that fuse with EVs to form nanoscale reactors. Upon fusion, the CRISPR/Cas13a complex is activated by target miRNA, triggering trans-cleavage of RNA reporters and generating an amplified fluorescence signal for enhanced detection sensitivity. The assay achieves a linear detection range of 10 to 10[6] EVs/μL and a detection limit of 2.5 × 10[1] EVs/μL on the gold nanoarray. Its performance relies on three strategies: (i) EpCAM-mediated tumor EV enrichment, (ii) CRISPR/Cas13a-based collateral activity for ultrasensitive miRNA detection, and (iii) fluorescence signal enhancement via localized nanoreactors. Validation with a 2D SW480 cell model, a 3D vascularized tumor spheroid (VTS) model, and clinical plasma samples confirms diagnostic accuracy, with miRNA quantification comparable to RT-qPCR but without the need for labor-intensive RNA extraction and amplification. By integrating nanotechnology with CRISPR-based diagnostics, this platform bridges research and clinical translation, improving diagnostic precision and streamlining workflows. Future development will focus on multiplexed biomarker detection and single-EV analysis to reveal insights into EV heterogeneity and function in cancer. This technology supports the application in precision oncology, offering a tool for early detection, treatment monitoring, and therapeutic decision-making.},
}
@article {pmid40653285,
year = {2025},
author = {Monteiro, JP and Vacante, F and De Pace, AL and Bennett, M and Rodor, J and O'Carroll, D and Wu, JC and Quertermous, T and Baker, AH},
title = {Targeting Cis-regulatory elements for CRISPR-mediated transcriptional activation of the human MIR503HG locus.},
journal = {Vascular pharmacology},
volume = {160},
number = {},
pages = {107521},
doi = {10.1016/j.vph.2025.107521},
pmid = {40653285},
issn = {1879-3649},
mesh = {Humans ; *MicroRNAs/genetics/metabolism ; *Transcriptional Activation ; *CRISPR-Cas Systems ; *RNA, Long Noncoding/genetics/metabolism ; Lentivirus/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Vectors ; *Gene Editing/methods ; *Regulatory Elements, Transcriptional ; Human Umbilical Vein Endothelial Cells/metabolism ; },
abstract = {Advances in genome annotation have revealed a striking increase in the number and complexity of non-coding RNA (ncRNA) genes, particularly multi-transcript loci that harbor long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) within the same genomic region. These loci can often function as coordinated regulatory units, with their transcription modulated by shared cis-acting regulatory elements (CREs). Traditional gene manipulation approaches, which typically target individual transcripts, are insufficient to capture the full regulatory and therapeutic potential of these complex loci. Here, we present "cis-ON" a single-vector lentiviral delivery system based on CRISPR activation (CRISPRa), designed to simultaneously upregulate multiple noncoding RNA transcripts by targeting a single CRE. We focused on the evolutionarily conserved MIR503HG locus, which encodes seven lncRNA isoforms and hosts the miR-424/503 cluster, both implicated in various cellular processes, including proliferation, angiogenesis, and endothelial-to-mesenchymal transition. Using integrative analysis of histone marks (H3K27Ac, H3K4Me3), DNase hypersensitivity, and CAGE-seq data, we identified the primary promoter of the MIR503HG locus. A dual fluorescent reporter screen selected optimal single guide RNAs (sgRNAs) for targeting this region. We then engineered cis-ON, a novel lentiviral system combining dCas9-VPR and sgRNA to enable a streamlined single-vector delivery approach. Transduction of primary human endothelial cells with this system robustly activated the entire locus including the MIR503HG isoforms and co-embedded miRNAs miR-424 and miR-503, demonstrating coordinated transcriptional regulation. Additionally, the neighboring LINC00629 lncRNA locus remained unaffected, highlighting regulatory specificity. This approach demonstrates the feasibility of modulating complex ncRNA loci across a ∼ 10 kb genomic region by targeting a single CRE, bypassing limitations of transcriptspecific strategies. By enabling simultaneous upregulation of lncRNAs and miRNAs, the cis-ON platform provides a streamlined strategy for restoring regulatory networks disrupted in disease.},
}
@article {pmid39992014,
year = {2025},
author = {Chen, RH and Bai, Y and Shi, LD and Liu, XY and Li, DL and Di, M and Duan, JX and Zhang, ZH and Xu, JL and He, ZQ and Li, K},
title = {Functional exploration of the Sex combs reduced gene in Gryllus bimaculatus.},
journal = {Insect molecular biology},
volume = {34},
number = {5},
pages = {608-618},
doi = {10.1111/imb.12987},
pmid = {39992014},
issn = {1365-2583},
mesh = {Animals ; *Insect Proteins/genetics/metabolism ; Female ; CRISPR-Cas Systems ; Male ; Gene Knockout Techniques ; *Homeodomain Proteins/genetics/metabolism ; },
abstract = {The Hox gene Sex combs reduced (Scr) is recognized as a key factor in the development of the head and thorax in insects. However, its function in the growth, development and morphogenesis of Gryllus bimaculatus remains poorly understood. This study aimed to explore the function of the Scr gene in G. bimaculatus by using CRISPR/Cas9 technology to generate an Scr gene knock-out strain. Intercrossing the G0 generation knock-out individuals with wild-type individuals yielded the G1 generation to screen the mutant strain. It was found that the knock-out of the Scr gene had a severe impact on the growth and development of G. bimaculatus, resulting in high mortality and making it difficult to obtain Scr[-/-] mutants. Therefore, heterozygous individuals (Scr[+/-]) with 1 bp deleted were obtained for investigation. The results showed that the Scr deletion led to ectopic segment formation in the G0 generation. In the G2 generation, it was observed that stable Scr[-/-] strains displayed abnormal embryonic development, characterized by enlarged, blackened and lethal eggs during embryogenesis. During the post-embryonic stage, Scr[-/-] mutants exhibited abnormalities in body segmentation, particularly in the head-thorax region, resulting in a dorsal ridge structure. Furthermore, some Scr[+/-] individuals exhibited a dorsal ridge during the nymphal stage. Notably, this characteristic did not persist into the adult stage. Our findings highlight the distinct but crucial roles of the Scr gene in both embryonic and post-embryonic growth and development of G. bimaculatus.},
}
@article {pmid40918426,
year = {2025},
author = {Senbadejo, TY and Ntiamoah Osei, S and Isawumi, A},
title = {Snapshot of Defense Systems in Multidrug Resistant Klebsiella pneumoniae.},
journal = {microPublication biology},
volume = {2025},
number = {},
pages = {},
pmid = {40918426},
issn = {2578-9430},
abstract = {Bacterial defense mechanisms protect pathogens from host immunity, bacteriophages, and harsh environments. This study investigates defense systems in multidrug-resistant Klebsiella pneumoniae from Ghanaian hospital ICUs, focusing on CRISPR-Cas, restriction-modification (R-M), and toxin-antitoxin (TA) systems. Genomes of environmental (NS2) and clinical (PS4) strains were sequenced and analyzed using PADLOC, defensefinder, and TADB3.0. NS2 carries 12 defense systems, including CRISPR-Cas, while PS4 has five. Both possess diverse RM and TA systems. These strains, resistant to six antibiotic classes, encode clinically significant defense systems, suggesting microbial exchange between fomites and humans, potentially increasing infection risks in ICU environments requiring targeted surveillance.},
}
@article {pmid40918304,
year = {2025},
author = {Dash, B and Bhuyan, SS and Sahoo, RK and Swain, N and Jeughale, KP and Sarkar, S and Verma, RL and Parameswaran, C and Devanna, BN and Samantaray, S},
title = {CRISPR/Cas-mediated genome editing: playing a versatile role in mitigating the challenges of sustainable rice improvement.},
journal = {3 Biotech},
volume = {15},
number = {10},
pages = {327},
pmid = {40918304},
issn = {2190-572X},
abstract = {Just as Gregor Mendel's laws of inheritance laid the foundation for modern genetics, the emergence of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas systems has catalyzed a new era in precision genome engineering. CRISPR/Cas has revolutionized rice (Oryza sativa L.) breeding by enabling precise, transgene-free edits to improve yield, nutrition, and stress tolerance. Advanced tools like base and prime editing further refine these capabilities, offering powerful solutions for climate-resilient agriculture and global food security. The review synthesizes the CRISPR-mediated strategies for improving resistance against major biotic (bacterial blight, blast, sheath blight) and abiotic (drought, salinity, submergence, nutrient deficiency) stresses. Additionally, we explore the critical prerequisites for efficient genome editing in rice, ranging from target site design, PAM specificity, delivery systems (like Agrobacterium, RNPs, and nanoparticle-mediated delivery), to screening and validation of mutants. This review also highlights recent breakthroughs in multiplex genome editing for complex traits, including the development of haploid inducer lines and clonal seed technology. Haploid inducers accelerate breeding by producing homozygous lines without tissue culture, while engineered apomixis enables clonal propagation of elite hybrids. Beyond technical dimensions, this review underscores the broader socio-economic and regulatory implications of genome-edited rice, addressing the emerging ethical concerns, intellectual property issues, farmer access, and equitable technology dissemination in resource-limited agricultural regions. As the global policy landscape transitions to accommodate CRISPR-edited crops, transparent regulatory frameworks, stakeholder engagement, and public perception will play pivotal roles in ensuring sustainable, safe, and inclusive adoption of genome editing in agriculture.},
}
@article {pmid40918251,
year = {2025},
author = {Zu, H and Sun, R and Li, J and Guo, X and Wang, M and Guo, W and Wang, X},
title = {Integrated CRISPR-Cas12a and RAA one-pot visual strategy for the rapid identification of Streptococcus equi subspecies equi.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1526516},
pmid = {40918251},
issn = {2235-2988},
mesh = {*Streptococcus equi/genetics/isolation &amp; purification/classification ; Horses ; *CRISPR-Cas Systems ; Animals ; *Streptococcal Infections/veterinary/diagnosis/microbiology ; *Horse Diseases/diagnosis/microbiology ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism/genetics ; *Molecular Diagnostic Techniques/methods ; Iceland ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; Streptococcus ; CRISPR-Associated Proteins ; },
abstract = {Strangles, a highly contagious disease caused by Streptococcus equi subspecies equi (S.equi), significantly impacts horse populations worldwide, with Iceland as the only exception. This disease poses serious threats to equine health and results in considerable economic losses. Consequently, the accurate, sensitive, and rapid detection of S.equi from clinical samples is essential for early warning and effective disease management. This study introduces a novel detection method that integrates recombinase-aided amplification (RAA) with CRISPR/Cas12a technologies. We specifically designed RAA primers and CRISPR RNA to target the eqbE gene of S.equi, and we have carefully optimized the reaction systems for this purpose. The newly established visual diagnostic method has shown to be highly effective, demonstrating 97.14% specificity and 100% sensitivity, with the capability to detect as few as 5.6×10[0] copies of the target. This is the first study to propose the combined application of RAA and CRISPR/Cas12a for the on-site rapid detection of S.equi. This is the first study to propose the combined application of RAA and CRISPR/Cas12a for the on-site rapid detection of S.equi, which enables visual point-of-care diagnosis of Strangles.},
}
@article {pmid40918142,
year = {2025},
author = {Golinelli, G and Scholler, J and Roussel-Gervais, A and Šakić, A and Ilmjärv, S and Song, D and Gabunia, K and Ji, M and Fan, TJ and Gupta, A and Deshmukh, M and Berjis, A and Cuoghi Costantini, R and Apodaca, K and Sheppard, NC and Kili, S and Dominici, M and Alessandrini, M and June, CH and Levine, BL},
title = {Multiplex engineering using microRNA-mediated gene silencing in CAR T cells.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1647433},
pmid = {40918142},
issn = {1664-3224},
mesh = {*MicroRNAs/genetics ; Animals ; Humans ; *Receptors, Chimeric Antigen/genetics/immunology ; *Immunotherapy, Adoptive/methods ; Mesothelin ; *Gene Silencing ; Mice ; *T-Lymphocytes/immunology/metabolism ; Cell Line, Tumor ; Gene Editing ; CRISPR-Cas Systems ; Receptors, Antigen, T-Cell/genetics ; *Pancreatic Neoplasms/therapy/immunology/genetics ; Xenograft Model Antitumor Assays ; GPI-Linked Proteins/genetics/immunology ; },
abstract = {BACKGROUND: Multiplex gene-edited chimeric antigen receptor (CAR) T-cell therapies face significant challenges, including potential oncogenic risks associated with double-strand DNA breaks. Targeted microRNAs (miRNAs) may provide a safer, functional, and tunable alternative for gene silencing without the need for DNA editing.<br><br>METHODS: As a proof of concept for multiplex gene silencing, we employed an optimized miRNA backbone and gene architecture to silence T-cell receptor (TCR) and major histocompatibility complex class I (MHC-I) in mesothelin-directed CAR (M5CAR) T cells. The efficacy of this approach was compared to CD3&#x3b6; and &#x3b2;2-microglobulin (&#x3b2;2M) CRISPR/Cas9 knockout (KO) cells. miRNA-expressing cassettes were incorporated into M5CAR lentiviral vectors, enabling combined gene silencing and CAR expression. Antitumor activity was evaluated using in vitro assays and in vivo pancreatic ductal adenocarcinoma models.<br><br>RESULTS: Silenced (S) M5CAR T cells retained antitumor functionality comparable to, and in some cases exceeding, that of KO cells. In vivo, S M5CAR T cells achieved tumor control with higher persistence and superior metastasis prevention. In vitro assays demonstrated enhanced resistance to alloreactive natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs).<br><br>CONCLUSIONS: Titratable multiplex gene silencing via targeted miRNAs offers an alternative to gene editing for CAR T cells, with potential advantages in potency, persistence, metastasis prevention, and immune evasion for allogeneic products. This strategy may overcome tumor-induced immunosuppression while avoiding the risks associated with DNA double-strand breaks.},
}
@article {pmid40916896,
year = {2025},
author = {Courtier-Orgogozo, V},
title = {Risks associated with CRISPR homing gene drive.},
journal = {Comptes rendus biologies},
volume = {348},
number = {},
pages = {211-227},
doi = {10.5802/crbiol.182},
pmid = {40916896},
issn = {1768-3238},
mesh = {*Gene Drive Technology/adverse effects ; Humans ; Animals ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Ecosystem ; Gene Editing ; },
abstract = {CRISPR homing gene drive is a disruptive biotechnology developed over the past decade with potential applications in public health, agriculture, and conservation biology. This technology relies on an autonomous selfish genetic element able to spread in natural populations through the release of gene drive individuals. However, it has not yet been deployed in the wild. In this review, we examine the key risks associated with CRISPR homing gene drives. First, we explore technical limitations, where gene drives might not be as efficient as intended, and cases where mitigation strategies may not be able to block a gene drive. Then, we present four main categories of adverse effects: (a) ecological risks, corresponding to unintended consequences on ecosystems and non-target populations; (b) sociological risks, i.e. concerns over public perception, governance, and societal acceptance; (c) risks associated with research activities; and (d) risks associated with malevolent usage. Regulatory aspects are not addressed here. This article provides a foundation for evaluating gene drive risks to ensure responsible and informed decision-making.},
}
@article {pmid40916704,
year = {2025},
author = {Ficoseco, CMA and Chieffi, D and Montemurro, M and Bavaro, A and Rizzello, CG and Nader-Macias, MEF and Fadda, S and Fanelli, F and Fusco, V and Vignolo, GM},
title = {Genomic Characterisation of Limosilactobacillus fermentum CRL2085 Unveiling Probiotic Traits for Application in Cattle Feed.},
journal = {Environmental microbiology reports},
volume = {17},
number = {5},
pages = {e70176},
doi = {10.1111/1758-2229.70176},
pmid = {40916704},
issn = {1758-2229},
support = {//This work was financially supported by the Joint Bilateral Agreement CNR/CONICET (ITALY-Argentina) "Lactic Acid Bacteria as bioprotective agents against zoonotic pathogens in the meat chain" Biennial Programme 2023-2024/ ; },
mesh = {Animals ; *Probiotics ; Cattle ; *Limosilactobacillus fermentum/genetics/isolation &amp; purification/metabolism ; *Animal Feed/microbiology ; *Genome, Bacterial ; Genomics ; Genomic Islands ; Bacterial Adhesion ; },
abstract = {Limosilactobacillus fermentum CRL2085, isolated from feedlot cattle rations, displayed high efficiency as a probiotic when administered to animals. A comprehensive genomic analysis was performed to elucidate the genetic basis underlying its probiotic potential. Fifteen genomic islands and CRISPR-Cas elements were identified in its genome. Pan-genomic analysis highlighted the dynamic evolution of this species, and clustering based on the nucleotide genomic similarity only partially correlated with the source of isolation or the geographic origin of the strains. Several genes known to confer probiotic properties were identified, including those related to adhesion, resistance to acidic pH and bile salts, tolerance to oxidative stress, metabolism/transport of sugars and other compounds, and genes for exopolysaccharide biosynthesis. In silico analysis of antimicrobial resistance genes and virulence determinants confirmed the safety of this strain. Moreover, genes related to B-group vitamins biosynthesis and feruloyl esterase hydrolase were also found, showing the nutritional contribution of the strain, which also showed moderate adhesion capability, exopolysaccharide production when grown with sucrose, and the capacity to metabolise 42 out of 95 carbon substrates tested. This data provides the genetic basis for deciphering the mechanisms beyond the benefits demonstrated by its use during cattle intensive raising and confirms its promising role as a probiotic.},
}
@article {pmid40914328,
year = {2025},
author = {Rathored, J and Budhbaware, T},
title = {Integrative Strategies Against Multidrug-Resistant Bacteria: Synthesizing Novel Antimicrobial Frontiers for Global Health.},
journal = {Microbial pathogenesis},
volume = {},
number = {},
pages = {108018},
doi = {10.1016/j.micpath.2025.108018},
pmid = {40914328},
issn = {1096-1208},
abstract = {Concerningly, multidrug-resistant bacteria have emerged as a prime worldwide trouble, obstructing the treatment of infectious diseases and causing doubts about the therapeutic accidentalness of presently existing drugs. Novel antimicrobial interventions deserve development as conventional antibiotics are incapable of keeping pace with bacteria evolution. Various promising approaches to combat MDR infections are discussed in this review. Antimicrobial peptides are examined for their broad-spectrum efficacy and reduced ability to develop resistance, while phage therapy may be used under extreme situations when antibiotics fail. In addition, the possibility of CRISPR-Cas systems for specifically targeting and eradicating resistance genes from bacterial populations will be explored. Nanotechnology has opened up the route to improve the delivery system of the drug itself, increasing the efficacy and specificity of antimicrobial action while protecting its host. Discovering potential antimicrobial agents is an exciting prospect through developments in synthetic biology and the rediscovery of natural product-based medicines. Moreover, host-directed therapies are now becoming popular as an adjunct to the main strategies of therapeutics without specifically targeting pathogens. Although these developments appear impressive, questions about production scaling, regulatory approvals, safety, and efficacy for clinical employment still loom large. Thus, tackling the MDR burden requires a multi-pronged plan, integrating newer treatment modalities with existing antibiotic regimens, enforcing robust stewardship initiatives, and effecting policy changes at the global level. The international health community can gird itself against the growing menace of antibiotic resistance if collaboration between interdisciplinary bodies and sustained research endeavours is encouraged. In this study, we evaluate the synergistic potential of combining various medicines in addition to summarizing recent advancements. To rethink antimicrobial stewardship in the future, we provide a multi-tiered paradigm that combines pathogen-focused and host-directed strategies.},
}
@article {pmid40912804,
year = {2025},
author = {Qiu, Y and Li, X and Fan, M and Tang, H and Zhang, S and Huang, W and Han, Z and Wang, S and Peng, H and Xiao, Y and Ye, X and Wang, K},
title = {Modification of starch traits in commercial wheat through TaWaxy gene editing.},
journal = {Carbohydrate polymers},
volume = {368},
number = {Pt 1},
pages = {124105},
doi = {10.1016/j.carbpol.2025.124105},
pmid = {40912804},
issn = {1879-1344},
mesh = {*Triticum/genetics/metabolism/chemistry ; *Gene Editing/methods ; Amylose/genetics ; *Starch/genetics/metabolism/chemistry ; *Plant Proteins/genetics/metabolism ; Mutation ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {Amylose content (AC) is a key determinant of wheat quality, and the TaWaxy gene determined amylose synthesis with a dose-dependent effect on AC. In this study, the TaWOX5 gene, which significantly enhances wheat transformation efficiency, was combined with CRISPR/SpCas9 system to generate TaWaxy mutants in a commercial winter wheat Jimai 22. Seven transgene-free mutant types were produced, compared to only three transgene-free mutants in the spring wheat variety Ningchun 4. The TaWaxy mutants from the two varieties showed decreased ACs ranging from 0 to 19.05 %. Results demonstrated that the Waxy-B1 protein has the most significant effect on amylose synthesis. The mutants with TaWaxy-abd, TaWaxy-ab, and TaWaxy-bd alleles showed waxy wheat trait. Interestingly, the TaWaxy-b mutant from Jimai 22 exhibited a waxy trait, unlike the TaWaxy-b mutant from Ningchun 4. Transmission electron microscope and scanning electron microscopy showed increased B-type starch granules in mutant grains. The mutants displayed varying effects on bread, cake, cookie, and noodle quality. All mutants showed decreased quality in bread and cake production, while TaWaxy-ad-JM and TaWaxy-b-NC mutants showed improved noodle and cookie quality. The generated mutants provide optimized optimized amylose content, enhancing noodle and biscuit quality as a practical alternative to blending.},
}
@article {pmid40912763,
year = {2025},
author = {Joshi, R and Paliwal, T and Sharma, S and Kapoor, DU and Prajapati, BG},
title = {Chitosan polyplexes for targeted gene delivery: From mechanisms to clinical applications.},
journal = {Carbohydrate polymers},
volume = {368},
number = {Pt 1},
pages = {124080},
doi = {10.1016/j.carbpol.2025.124080},
pmid = {40912763},
issn = {1879-1344},
mesh = {*Chitosan/chemistry ; Humans ; *Gene Transfer Techniques ; Animals ; Genetic Therapy/methods ; DNA/chemistry ; },
abstract = {As a diverse natural polymer called Chitosan, it created ground-breaking advancements in nucleic acid therapeutic delivery techniques for handling essential DNA and RNA delivery hurdles. The article investigates how nucleic acids form stable polyplexes with chitosan through electrostatic bonds, as well as explores their chemical and biological properties. The review explores how molecular weight, combined with the degree of deacetylation, combined with advanced functionalization strategies, help enhance delivery results. Cellular uptake and specific targeting of polyplexes are improved through research into different targeting methods, which include ligand attachment, charge manipulation, and cell-penetrating peptides. Endosomal release of polyplexes is enabled by pH-sensitive and redox-sensitive polyplex architectures, while simultaneous control strategies are achieved through these delivery systems. The field of nucleic acid therapy now includes applications with CRISPR-Cas delivery and mRNA vaccines, and gene silencing therapy. The safety assessment encompasses cytotoxicity testing alongside immunogenicity evaluation and biodegradability assessment, which is conducted alongside direct comparison with alternative polymeric delivery vehicles. The potential for clinical use and targeted delivery, and combined smart transport systems establishes chitosan polyplexes as an innovative solution for nucleic acid drug delivery according to recent research findings.},
}
@article {pmid40911134,
year = {2025},
author = {Jiang, C and Li, Y and Yu, P and Fang, M and Huang, D and Fang, X and Xu, Z},
title = {A rapid and ultrasensitive CRISPR/Cas12a-based assay for the accurate identification of T-even type phages.},
journal = {Biotechnology letters},
volume = {47},
number = {5},
pages = {95},
pmid = {40911134},
issn = {1573-6776},
support = {2021YFC2103200//the National Key Research and Development Program of China/ ; 22404149//the National Natural Science Foundation of China/ ; 2023M742994//the China Postdoctoral Science Foundation/ ; GZC20232319//the Postdoctoral Fellowship Program of CPSF/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *T-Phages/genetics/isolation &amp; purification/classification ; Limit of Detection ; Wastewater/virology ; },
abstract = {Phage contamination poses a significant threat to industrial fermentation, leading to substantial economic losses. Virulent T-even type phages (T2/T4/T6) represent particularly concerning biological hazards in fermentation systems. This paper developed a novel CRISPR/Cas12a-based system integrated with recombinase polymerase amplification (RPA), enabling ultrasensitive identification of T-even type phages. This method targeted the TerL gene of T-even type phages as a detection marker. The optimized RPA-CRISPR assay demonstrated exceptional sensitivity with a limit of detection (LOD) reaching 1 aM for synthetic targets. Besides, this system achieved detection thresholds of 1 and 10 PFU/μL for T2 and T4 phages, respectively. Comparative validation with quantitative PCR (qPCR) confirmed the method's reliability through strong correlation in the detection for both spiked and wastewater samples. The detection platform exhibited remarkable potential for rapid, sensitive monitoring of T-even type phages contamination in fermentation processes, offering promising application prospects for quality control in biochemical industries.},
}
@article {pmid40904109,
year = {2025},
author = {Clabby, T and Tesson, F and Gaborieau, B and Bernheim, A},
title = {Why do bacteria accumulate antiphage defence systems?.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240082},
pmid = {40904109},
issn = {1471-2970},
support = {//MSDAVENIR/ ; //Pasteur Institute/ ; /ERC_/European Research Council/International ; },
mesh = {*Bacteria/classification/genetics/immunology/virology ; *Bacteriophages/genetics/physiology ; Ecology ; CRISPR-Cas Systems ; Gene Transfer, Horizontal ; Interspersed Repetitive Sequences ; *Phage Therapy ; },
abstract = {While it is well established that bacterial genomes encode multiple and diverse antiphage systems, the reasons for their co-occurrence and their heterogeneous distribution remain debated. This review examines why bacteria accumulate antiphage systems and how this influences phage-bacteria interactions, particularly in the context of phage therapy. Two main hypotheses may explain this phenomenon: (i) the pan-immunity hypothesis, which suggests that defence system accumulation provides protection against phage predation at the community level, and (ii) mobile genetic element (MGE) competition, where defence systems primarily protect intra-bacterial MGEs against other ones rather than the bacterial host itself. The ecological context also influences the distribution of antiphage systems, with defencee accumulation shaping phage-bacteria interactions in diverse communities but playing a lesser role at the species level, potentially explaining why multiple defences do not strongly limit phage host range in therapeutic settings. Finally, we address the challenges in understanding the drivers shaping the distribution of defence systems across bacterial genomes (expressions, costs, etc.) and their implications for elucidating the ecological role of defence systems and optimizing phage therapy strategies.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}
@article {pmid40889469,
year = {2025},
author = {Li, Y and Dou, Y and Lu, Z and Wang, Y and Zhou, H and Li, T},
title = {CRISPR/Cas12a-functionalized silicon nanowires field-effect transistor sensor for ultra-sensitive detection of pathogen nucleic acids.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117936},
doi = {10.1016/j.bios.2025.117936},
pmid = {40889469},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *Nanowires/chemistry/ultrastructure ; *Silicon/chemistry ; *CRISPR-Cas Systems/genetics ; Transistors, Electronic ; *Bacillus anthracis/genetics/isolation &amp; purification/pathogenicity ; Limit of Detection ; *DNA, Bacterial/genetics/isolation &amp; purification/analysis ; *Endodeoxyribonucleases/chemistry ; Bacterial Proteins/chemistry ; CRISPR-Associated Proteins ; },
abstract = {Rapid, sensitive, and accurate detection of pathogen nucleic acids is critical for ensuring public safety and health. Nevertheless, current methods still encounter significant challenges. Field-effect transistor (FET) biosensors are renowned for high sensitivity, rapid response, and label-free detection. However, when employed for the direct detection of long-chain DNA extracted from pathogens, these sensors exhibit low recognition efficiency, poor accuracy, and prolonged reaction times. To address these limitations, we propose a novel silicon nanowires FET sensing strategy functionalized with the CRISPR/Cas12a system. The Cas12a/crRNA complex rapidly scans and precisely cleaves target sequences within long double-stranded DNA (dsDNA). This mechanism effectively mitigates detection performance degradation caused by nucleic acid folding and entanglement, thereby significantly enhancing both sensitivity and accuracy. Additionally, Cas12a/crRNA cleaves long dsDNA into specific-length fragments, thereby ensuring their distribution within the Debye length and enhancing signal consistency. Using this approach, we successfully achieved quantitative detection of Bacillus anthracis dsDNA within 10 min, with a detection limit at the attomolar (aM) level. Furthermore, the correlation coefficient between detection results of real whole-genome samples and digital PCR reached 0.912, validating the reliability of this strategy. In summary, this strategy provides a highly valuable reference for the direct detection of pathogen nucleic acids.},
}
@article {pmid40886437,
year = {2025},
author = {Xu, Q and Wang, X and Gu, Z and Duan, N and Jiang, S and Wu, S and Yuan, W},
title = {An electrochemical/colorimetric sensor for 5-methyltetrahydrofolate based on a screened specific aptamer and DNA walker-driven CRISPR/Cas12a cascade amplification.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117931},
doi = {10.1016/j.bios.2025.117931},
pmid = {40886437},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Colorimetry/methods ; *Aptamers, Nucleotide/chemistry ; Electrochemical Techniques/methods ; Humans ; Limit of Detection ; CRISPR-Cas Systems/genetics ; *Tetrahydrofolates/analysis/isolation &amp; purification/chemistry ; Gold/chemistry ; DNA, Single-Stranded/chemistry ; Endodeoxyribonucleases/chemistry/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {5-Methyltetrahydrofolate (5-MTHF), the primary bioactive form of folate (vitamin B9), played a vital role in human metabolism. In this work, an electrochemical/colorimetric dual-mode aptasensor for 5-MTHF was constructed by combining a DNA Walker-driven CRISPR-Cas12a trans-cleavage system. A 5-MTHF aptamer D1a was obtained through Capture-SELEX with subsequent trimming of non-binding regions, which exhibiting high affinity and specificity. CuMOF@CuO@RuO2@IrO2, serving as a multifunctional indicator with high conductivity and peroxidase-like activity, was immobilized on a gold electrode (AuE) via ssDNA. The DNA Walker and nicking enzyme (Nt.BbvCI) continuously released Cas12a activators, triggering Cas12a-mediated cleavage of ssDNA on the AuE, leading to reduced electrochemical signals. The released materials were collected for colorimetric detection. The detection limits were determined to be 0.044 ng/mL (electrochemical) and 0.114 ng/mL (colorimetric). This dual-mode aptasensor demonstrated high selectivity, sensitivity, and stability for 5-MTHF detection, establishing a novel sensing platform for 5-MTHF analysis in food samples. The proposed aptasensor exhibited excellent sensitivity, selectivity, and reproducibility. This strategy offered a promising platform for 5-MTHF analysis in nutritional evaluation and dietary supplementation guidance.},
}
@article {pmid40886435,
year = {2025},
author = {He, X and Liang, J and Zhang, J and Fang, W and Liu, J and Zhang, M and Wang, L and Song, C},
title = {CRISPR/Cas13a triggered-DNA walker amplified SERS sensor for ultrasensitive detection of cancer-related exosomal miRNA.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117924},
doi = {10.1016/j.bios.2025.117924},
pmid = {40886435},
issn = {1873-4235},
mesh = {Humans ; *MicroRNAs/isolation &amp; purification/genetics/blood ; *Biosensing Techniques/methods ; *Exosomes/chemistry/genetics ; Limit of Detection ; *Spectrum Analysis, Raman/methods ; CRISPR-Cas Systems/genetics ; *Neoplasms/genetics/diagnosis/blood ; DNA/chemistry ; DNA, Catalytic/chemistry ; },
abstract = {Accurate quantification of cancer-related miRNA in exosomes offers a promising approach for early and effective cancer diagnosis. However, reliably detecting extremely low-abundance exosomal miRNAs in complex bodily fluids remains a significant challenge. Herein, a CRISPR/Cas13a triggered-DNA walker amplified SERS sensor has been proposed for detection of cancer cell-derived exosomal miRNA-106a. The sensor comprises three main components: SERS Tags, SERS capture substrates and the CRISPR/Cas13a system. In the presence of miRNA-106a, the CRISPR/Cas13a trans-cleavage is activated, and the cleavage product further enables the DNAzymes to 'walk' on the SERS Tags, leading to the linking of the SERS Tags to a SERS capture substrate, ultimately generating significantly amplified SERS signal. The proposed SERS sensor exhibits good detection capability for miRNA-106a, i.e., rapid detection time within 80 min, wide linear response ranging from 100 aM to 1 nM, limit of detection (LOD) as low as 53.16 aM, good specificity, excellent reproducibility, and satisfactory recovery rates in human serum. Furthermore, the exosomal miRNA-106a derived from gastric cancer cells were detected and a LOD as low as 6.1 × 10[3] particles/mL of exosome was achieved, and the sensing results of the clinical serum samples underscore the potential of accurately differentiate between cancer patients and healthy individuals via analyzing cancer-related exosomal miRNAs, which indicates that the proposed SERS sensor can be a powerful tool for exosomal miRNA detection and holds good potential for precise liquid biopsy of tumor-derived exosomes.},
}
@article {pmid40882515,
year = {2025},
author = {Li, QN and Huang, HR and Li, RY and Hou, XY and Yang, QF and Jiang, HX and Cai, QL and Kong, DM},
title = {Position-independent single-nucleotide polymorphism discrimination by CRISPR/Cas12a via rational activator strand engineering.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117929},
doi = {10.1016/j.bios.2025.117929},
pmid = {40882515},
issn = {1873-4235},
mesh = {*Polymorphism, Single Nucleotide/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Single-nucleotide polymorphisms (SNPs) are critical biomarkers for disease diagnosis and genetic research, yet their sensitive and specific detection remains challenging. Here, we report a rational activator strand design strategy that significantly enhances the SNP discrimination capability of CRISPR/Cas12a-based biosensing systems. By systematically optimizing the length of the crRNA-complementary region and the architecture of the 3'-terminal random extension sequence, we developed an engineered CRISPR/Cas12a platform capable of discrimination SNPs with single-nucleotide resolution, regardless of mutation position. Our optimized activator strand (ssAS13+3-X) leverages the "RESET" effect (random extending sequences enhance trans-cleavage activity) enables simple one-pot detection of low-abundance mutations (0.1 %) without target pre-amplification, offering significant advantages over conventional SNP detection methods in clinical settings. The single-stranded flexibility and length tolerance of the 3'-terminal extension further ensure broad applicability across diverse genomic contexts. This work not only deepens our fundamental understanding of CRISPR/Cas12a regulation, but also provides a versatile and streamlined platform for applications in molecular diagnostics, pathogen surveillance, and precision medicine.},
}
@article {pmid40865357,
year = {2025},
author = {Wang, H and Wang, Z and Wu, M and Chen, M and He, X and Deng, L and Huang, J and Lin, R and Mao, X and Li, Q and Sheng, Y},
title = {CrispHunter-enabled systematic crRNA design and structure-optimized hairpin probes for enhanced CRISPR-Cas12a detection of Burkholderia pseudomallei.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117912},
doi = {10.1016/j.bios.2025.117912},
pmid = {40865357},
issn = {1873-4235},
mesh = {*Burkholderia pseudomallei/genetics/isolation &amp; purification/pathogenicity ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Limit of Detection ; *Melioidosis/diagnosis/microbiology ; *RNA, Bacterial/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Effective control of genetically diverse pathogens necessitates rapid and accurate diagnostics, as their genomic variability undermines assay reliability and complicates public health interventions. Herein, we introduce a CRISPR-Cas12a-based molecular diagnostic platform integrating robust in silico crRNA design via our CrispHunter pipeline with a high-performance detection module featuring structure-optimized hairpin probes. We validated this approach using Burkholderia pseudomallei, whose high pathogenicity, genetic diversity, and diagnostic evasion exemplify the challenges of detecting heterogeneous pathogens. Applying CrispHunter to 3245 publicly available B. pseudomallei genomes, we systematically identified 12 highly conserved crRNA candidates and optimized a penta-crRNA strategy that reduced the limit of detection (LOD) from the previously reported 100 pM to 7.5 pM, thereby minimizing the risk of false negatives due to pathogen genomic diversity. Engineered hairpin-structured molecular beacons with iteratively optimized loop and stem lengths further enhanced assay performance, lowering the LOD to 2.1 pM without pre-amplification while improving signal-to-noise ratio by 42-fold, response sensitivity by 47-fold, and reducing time-to-positive from 56 to 3 min with excellent specificity. Employing recombinase polymerase amplification further enhanced sensitivity, lowering the LOD to 14.82 copies/μL, which is significantly below the concentrations typically found in clinical specimens. The modular architecture of CrispHunter, together with the optimized CRISPR-Cas12a detection platform, provides a versatile framework for rapid, sensitive, and specific molecular diagnostics. This strategy enhances detection of B. pseudomallei and can be readily adapted for the diagnosis of other genetically diverse pathogens, supporting broader applications in infectious disease management and public health.},
}
@article {pmid40848338,
year = {2025},
author = {Hiniduma, K and De Silva, PIT and Canete, R and Vora, P and Gunathillaka, H and Clement, O and Shawky, SM and Rouge, JL and Mosa, IM and Steffens, DC and Manning, K and Breno, D and Rusling, JF},
title = {ECL-CRISPR array for multiplexed detection of miRNAs.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117855},
doi = {10.1016/j.bios.2025.117855},
pmid = {40848338},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation &amp; purification/blood ; Humans ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Limit of Detection ; *Luminescent Measurements/methods/instrumentation ; *CRISPR-Cas Systems/genetics ; *Alzheimer Disease/genetics/blood/diagnosis ; Equipment Design ; },
abstract = {We describe here an electrochemiluminescent (ECL) array for individually detecting 3 miRNAs utilizing CRISPR/Cas13a. Detection involves binding a target miRNA to Cas 13a protein that includes the RNA complement to the target, This activated Cas13a then cleaves a poly-RNA rich in r-Guanosine to produce electrochemiluminescent (ECL) activators that increases ECL output proportional to target miRNA concentration. Specifically, poly-r-guanosine (poly-r-G) is cleaved by the collateral RNase activity of Cas13a to generate small poly-r-G fragments that are efficient in activating ECL of (bis-2,2'-bipyridyl) ruthenium polyvinylpyridine ([Ru(bpy)2PVP10] (ClO4)2) (RuPVP) films on sensor electrodes at +1.1 V vs. Ag/AgCl. The 3D-printed array was used to detect three Alzheimer's disease (ALZ) miRNA biomarkers (30e-5p, 34c-3p and 200c-5p). ECL is generated in the 3D-printed array designed with reference, counter and four separate RuPVP sensor electrodes. Detection limits for miRNAs were 7.4 fg/mL to 7 pg/mL with high sensitivities in linear dynamic ranges from 70 pg/mL to 70 μg/mL. Limits of detection (LOD) were 42 pg/mL, 0.074 fg/mL, and 0.15 fg/mL for miR30e-5p, miR34c-5p, and miR200c-3p, respectively. Spike recovery studies and patient plasma analyses after RNA extraction gave high accuracy and specificity, and excellent correlation with a referee CRISPR fluorescence method.},
}
@article {pmid40834995,
year = {2025},
author = {De Marco, F and Sebastian, IR and Napoleone, A and Molin, A and Riedl, M and Bydlinski, N and Motheramgari, K and Hussein, MK and Kramer, L and Kelly, T and Jostock, T and Borth, N},
title = {A genome-scale CRISPR deletion screen in Chinese hamster ovary cells reveals essential regions of the coding and non-coding genome.},
journal = {Metabolic engineering},
volume = {92},
number = {},
pages = {194-207},
doi = {10.1016/j.ymben.2025.08.007},
pmid = {40834995},
issn = {1096-7184},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Genome ; *CRISPR-Cas Systems ; Cricetinae ; *Gene Deletion ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Sequence Deletion ; },
abstract = {The biopharmaceutical sector relies on CHO cells to investigate biological processes and as the preferred host for production of biotherapeutics. Simultaneously, advancements in CHO cell genome assembly have provided insights for developing sophisticated genetic engineering strategies. While the majority of these efforts have focused on coding genes, with some interest in transcribed non-coding RNAs (e.g., microRNAs and lncRNAs), there remains a lack of genome-wide systematic studies that precisely examine the remaining 90 % of the genome and its impact on cellular phenotypes. This unannotated "dark matter" includes regulatory elements and other poorly characterized genomic features that may be potentially critical for cell behaviour. In this study, we deployed a genome-scale CRISPR screening platform with 112,272 paired guide RNAs targeting 14,034 genomic regions for complete deletion of 150 kb long sections. This platform enabled the execution of a negative screen that selectively identified dying cells to determine regions essential for cell survival. By using paired gRNAs, we overcame the intrinsic limitations of traditional frameshift strategies, which will likely have little or no effect on the non-coding genome. This study revealed 427 regions essential for CHO cell survival, many of which currently lack gene annotation or known functions. For these regions, we present their annotation status, transcriptional activity and annotated chromatin states. Selected regions, particularly those lacking all of the above, were individually deleted to confirm their essentiality. This work sheds a novel light on a substantial portion of the mammalian genome that has been traditionally difficult to investigate and therefore neglected. Notably, the fact that the deletion of some of these regions is lethal to cells suggests they encode critical regulatory functions. A better genome-wide understanding of these functions could open new avenues for engineering cells with improved bioprocess relevant properties.},
}
@article {pmid40803011,
year = {2025},
author = {Jiang, Y and Wu, J and Xiang, X and Wei, J and Cheng, K and Cui, L and Xu, H and Li, Z},
title = {A cleavage-gated terminal exposure-driven CRISPR-RCA self-amplifying system for ultra-fast DNA detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117857},
doi = {10.1016/j.bios.2025.117857},
pmid = {40803011},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *DNA, Viral/genetics/isolation &amp; purification/analysis ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Humans ; CRISPR-Associated Proteins/chemistry ; Endodeoxyribonucleases/chemistry ; Bacterial Proteins ; },
abstract = {The one-pot detection technology based on nucleic acid isothermal amplification combined with CRISPR has a significant advantage in on-site detection of infectious diseases. It is superior to quantitative polymerase chain reaction (qPCR) due to its lack of temperature variation and significantly faster reaction speed. Nevertheless, Cas proteins compete with amplification enzymes for reaction substrates, which results in the signal amplification effect being less than expected. To overcome this limitation, we have developed a single-tube self-amplifying system driven by cleavage-gated terminal exposure based on CRISPR and rolling circle amplification (AURORA), enabling ultra-fast and sensitive monkeypox virus (MPXV) detection. This method innovatively designs a dual-function probe (DF probe). In the presence of the target, the trans-cleavage activity of Cas12a is activated, only cleaving the single-stranded DNA to expose the 3' terminal of the DF probe. The cleaved DF probe hybridizes with the circular DNA template and is bound by phi29 DNA polymerase to initiate RCA. Here, we utilize the characteristics of Cas12a and phi29 DNA polymerase acting on substrates in different strand states to avoid substrate competition between the two enzymes in a single-tube reaction. This assay can achieve ultra-fast signal amplification of MPXV DNA within 8 min, with a limit of detection (LOD) of 88 aM (53 copies/μL). Combining the viral nucleic acid thermal lysis method, it is possible to achieve results from sample input to output in 10 min. The AURORA detection strategy was further used to detect MPXV in clinical samples (36 MPXV samples), and the results were completely consistent with qPCR. The AURORA system features ultra-fast and precise detection, providing a more efficient tool for the prevention and control of severe infectious diseases.},
}
@article {pmid40752104,
year = {2025},
author = {Pavlikova, L and Krepop, J and Sulova, Z and Breier, A and Seres, M},
title = {Differential UVC radiation sensitivity in multidrug-resistant l1210 cells: Insights into p53 and Bcl-XL expression/function.},
journal = {Journal of photochemistry and photobiology. B, Biology},
volume = {271},
number = {},
pages = {113229},
doi = {10.1016/j.jphotobiol.2025.113229},
pmid = {40752104},
issn = {1873-2682},
mesh = {*Ultraviolet Rays ; *Tumor Suppressor Protein p53/genetics/metabolism ; *bcl-X Protein/metabolism/genetics ; Animals ; Mice ; Cell Line, Tumor ; *Drug Resistance, Multiple/radiation effects ; Drug Resistance, Neoplasm/radiation effects ; Doxorubicin/pharmacology ; DNA Damage/radiation effects ; Vincristine/pharmacology ; Apoptosis/radiation effects/drug effects ; CRISPR-Cas Systems ; Radiation Tolerance ; },
abstract = {Multidrug-resistant (MDR) variants of L1210 cells, selected for resistance to vincristine (R) or doxorubicin (D), exhibit elevated ABCB1 (P-glycoprotein) expression but differ in UVC sensitivity. D cells resemble parental L1210 (S) cells, whereas R cells are significantly more UVC vulnerable. To investigate this, we analyzed the expression of genes involved in DNA damage response, Trp53 family members, cyclin-dependent kinase inhibitors (p15, p21), Bcl-2 family genes, and DNA repair genes in S, R, and D cells before and after UVC irradiation. The most striking difference was the absence of Trp53 expression in R cells at both mRNA and protein levels, while S and D cells expressed this gene. Instead, R cells uniquely expressed Trp63. CRISPR/Cas9-generated p53-null mutants of S and D cells showed increased UVC-induced cell death, but their sensitivity did not reach that of R cells. Bcl-XL, a protein linked to resistance against UVC-induced apoptosis, was also reduced at both transcript and protein levels in R cells. Pharmacological inhibition of Bcl-XL in S and D cells with A-1155463 and A-1331852 enhanced UVC-induced cell death but did not replicate the high sensitivity observed in R cells. Our findings suggest that the heightened UVC sensitivity of R cells results from a combined deficiency of p53 and Bcl-XL, impairing DNA damage response and apoptosis. These results reveal distinct molecular adaptations in MDR variants and provide insight into the mechanisms underlying differential UVC sensitivity.},
}
@article {pmid40835108,
year = {2025},
author = {Shan, X and Zhang, X and Tao, B and Song, Y and Zhu, Z and Hu, W and Chen, J},
title = {CRISPR/Cas9-mediated editing of ptprfb (protein tyrosine phosphatase receptor type fb) reveals its regulatory role in zebrafish spermatogenesis.},
journal = {Comparative biochemistry and physiology. Part B, Biochemistry &amp; molecular biology},
volume = {280},
number = {},
pages = {111146},
doi = {10.1016/j.cbpb.2025.111146},
pmid = {40835108},
issn = {1879-1107},
mesh = {Animals ; *Zebrafish/genetics/physiology ; Male ; *CRISPR-Cas Systems ; *Spermatogenesis/genetics ; *Gene Editing ; *Zebrafish Proteins/genetics/metabolism ; Spermatozoa/metabolism ; },
abstract = {Gonadal development and gamete maturation are essential for fish reproduction. The protein tyrosine phosphatase receptor type Fb (Ptprfb) is a member of the tyrosine phosphatase family. In the present study, we used CRISPR/Cas9 to mutate ptprfb in zebrafish. A significantly reduced natural fertilization rate of sperm from mutant fish was observed. The mutant fish produced fewer sperm with shorter flagella, and a smaller proportion of sperm could be activated. RNA-seq analysis revealed abnormal expression of some genes in testicular cells, such as hemoglobin subunit βA1 (hbba1), myosin heavy chain 11b (myh11b), and transgelin (tagln), as well as some genes involved in focal adhesion formation. These findings demonstrate that ptprfb contributes to spermatogenesis in zebrafish, and its dysfunction can adversely affect both the quantity and quality of sperm.},
}
@article {pmid40190036,
year = {2025},
author = {Wang, J and Xu, K and Liu, T and Zhao, H and Jamal, MA and Chen, G and Huo, X and Yang, C and Jiao, D and Wei, T and Huang, H and Zhao, H and Guo, J and Wang, F and Zhang, X and Liu, K and Qu, S and Wang, G and Guo, H and Chen, G and Zhao, HY and Zeng, Z and Dou, K and Wei, HJ},
title = {Production and Functional Verification of 8-Gene (GGTA1, CMAH, β4GalNT2, hCD46, hCD55, hCD59, hTBM, hCD39)-Edited Donor Pigs for Xenotransplantation.},
journal = {Cell proliferation},
volume = {58},
number = {9},
pages = {e70028},
doi = {10.1111/cpr.70028},
pmid = {40190036},
issn = {1365-2184},
support = {202102AA310047//Major Science and Technology Project of Yunnan Province/ ; 2019YFA0110700//National Key Research and Development Program of China/ ; 2023-JCJQ-ZD-118-00//173 Basic Strengthening Program/ ; },
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Gene Editing/methods ; Swine ; Humans ; CRISPR-Cas Systems/genetics ; Galactosyltransferases/genetics ; Animals, Genetically Modified ; CD59 Antigens/genetics ; N-Acetylgalactosaminyltransferases/genetics ; Membrane Cofactor Protein/genetics ; Mixed Function Oxygenases ; },
abstract = {Gene-edited (GE) pig-to-human xenotransplantation continues to make breakthroughs, but which kind of gene combination is suitable for organ-specific transplantation remains unclear. In this study, we utilised CRISPR/Cas9 gene editing technology, PiggyBac transposon system, and serial somatic cell cloning technology to develop GTKO/CMAHKO/β4GalNT2KO/hCD46/hCD55/hCD59/hCD39/hTBM 8 gene-edited cloned (GEC) donor pigs and performed pig-to-non-human primate (NHP) transplantation to evaluate the effectiveness of these GEC pigs. The 8-GEC pigs were obtained by recloning with a 33-day-old 8-GEC fetus with O blood type, which was generated after cell transfection, screening of cell colonies, and somatic cell cloning. Molecular identification at DNA, mRNA, and protein levels confirmed successful 8-gene editing. Three copies of transgenes were identified by droplet digital polymerase chain reaction and whole genome sequencing, which were inserted into the introns of pig RFTN1 and MYO10 genes, as well as the intergenic region between PRLR and LOC110257300 genes of these 8-GEC pigs. The 8-GEC pigs also exhibited the ability of germline transmission when mated with our previously generated 4-GEC male pigs. Moreover, antigen-antibody binding assay and complement-dependent cytotoxicity assay demonstrated that 8-gene editing effectively reduced the immune incompatibility and kidney xenograft from 8-GEC pigs survived for 15 and 17 days in two NHPs, respectively. Postoperatively, the recipient serum antibodies IgA, IgG and IgM, complements C3 and C4, coagulation indicators PT, APTT, TT and FIB, as well as most electrolytes and liver function indicators remained relatively stable. Serum creatinine was normal within 10 days post operation. However, the kidney xenograft developed active antibody-mediated rejection at necropsy, characterised by the deposition of antibodies IgG and IgM, as well as complements C4d, C3c and C5b-C9, infiltration of CD68[+] macrophages, and micro-thrombotic embolism of glomerular capillaries, etc. In conclusion, we successfully developed fertile 8-GEC pigs, which effectively alleviated immune rejection and exerted life-supporting kidney function in the recipients.},
}
@article {pmid40909725,
year = {2025},
author = {Hong, A and Liu, M and Truta, A and Talaie, A and Smith, GR and Bondy-Denomy, J},
title = {Gabija restricts phages that antagonize a conserved host DNA repair complex.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.08.30.673261},
pmid = {40909725},
issn = {2692-8205},
abstract = {Anti-bacteriophage systems like restriction-modification and CRISPR-Cas have DNA substrate specificity mechanisms that enable identification of invaders. How Gabija, a highly prevalent nuclease-helicase anti-phage system, executes self- vs. non-self-discrimination remains unknown. Here, we propose that phage-encoded DNA end-binding proteins that antagonize host RecBCD sensitize phages to Gabija. When targeting temperate phage D3 in Pseudomonas aeruginosa, Gabija functions early by preventing phage genome circularization in a non-abortive manner. Phage and plasmid DNA-end sensitivity to Gabija is licensed by a phage exonuclease and ssDNA-annealing protein. Unrelated F8 and JBD30 phages are sensitized to Gabija by Gam_Mu, a distinct DNA end-binding protein that antagonizes loading of the host repair complex RecBCD. Escape phages lacking these end-binding proteins become protected from Gabija by RecBCD activities, which also prevent Gabija from targeting self-DNA. Therefore, we propose that Gabija antagonizes circularization of linear DNA devoid of RecBCD as a mechanism to identify foreign invaders.},
}
@article {pmid40543483,
year = {2026},
author = {Wang, H and Yan, Y and Codjia, CR and Dan, Y and Li, L and He, S and Yang, X and Chen, L and Liu, H and Wang, X},
title = {CRISPR-Cas13a coupled with recombinase aided amplification: Development of an ultrasensitive point - of - care detection method for visual diagnosis of egg drop syndrome virus.},
journal = {Talanta},
volume = {296},
number = {},
pages = {128470},
doi = {10.1016/j.talanta.2025.128470},
pmid = {40543483},
issn = {1873-3573},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Recombinases/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *Atadenovirus/isolation &amp; purification/genetics ; *Poultry Diseases/diagnosis/virology ; Chickens/virology ; *Point-of-Care Systems ; Limit of Detection ; },
abstract = {Egg Drop Syndrome Virus (EDSV) infection in poultry causes a significant drop in egg - laying rate, harming poultry farms' economic benefits. A sensitive and specific EDSV detection method is urgently needed in clinical practice. The CRISPR-Cas13a system can detect various targets specifically, and recombinase-aided amplification (RAA) can rapidly amplify nucleic acids. Therefore, in this experiment, the RAA was integrated with the CRISPR-Cas13a system to develop a novel, visual, ultrasensitive and point - of - care detection method for EDSV. In this experiment, the reaction system was optimized, and its sensitivity, specificity, repeatability, and clinical sample validation were conducted and evaluated. The results showed that the optimal concentration of the obtained Cas13a protein was 2.4 mg/mL, and that of crRNA 1 was 100 μg/μL. This method not only showed rapid detection (30-50 min), very high sensitivity, with the detection limit reaching 1 copy/μL, but also showed good specificity with no cross - reaction to Marek's Disease Virus (MDV), Infectious Laryngotracheitis Virus (ILTV), Avian Leukosis Virus (ALV), Chicken Anemia Virus (CAV), Astrovirus (AstV), H9N2 subtype of Avian Influenza Virus (H9N2 AIV), Fowl Adenovirus serotype 4 (FAdV-4), Fowl Adenovirus serotype 8 (FAdV-8) and Fowl Adenovirus serotype 11 (FAdV-11). Furthermore, the method displayed excellent repeatability, with the coefficient of variation of both intra - group and inter - group no more than 4 %. Evaluation of this method through 210 clinical samples found that compared with the traditional polymerase chain reaction (PCR) which is the industry standard, its positive coincidence rate was 100 %, the negative coincidence rate was 98.35 %, the overall coincidence rate was 98.57 %, and the kappa value was 0.94. This assay provides a potential point - of - care testing approach for the clinical detection, virology, and epidemiological studies of EDSV.},
}
@article {pmid40499384,
year = {2026},
author = {Yang, Y and Ji, X and Zhou, F and He, Z},
title = {A one-pot assay based on PAM-free recombinase polymerase amplification and CRISPR/Cas12a for rapid detection of SARS-CoV-2 N gene.},
journal = {Talanta},
volume = {296},
number = {},
pages = {128448},
doi = {10.1016/j.talanta.2025.128448},
pmid = {40499384},
issn = {1873-3573},
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Recombinases/metabolism/genetics ; *COVID-19/diagnosis/virology ; CRISPR-Associated Proteins/metabolism ; *Phosphoproteins/genetics ; Endodeoxyribonucleases/metabolism ; Coronavirus Nucleocapsid Proteins ; Bacterial Proteins ; },
abstract = {SARS-CoV-2 is a pathogenic virus, which exhibits high contagiousness. Therefore, a rapid and sensitive SARS-CoV-2 detection strategy is imperative. Herein, a one-pot assay by the combination of protospacer adjacent motif (PAM)-free recombinase polymerase amplification with CRISPR/Cas12a for detecting SARS-CoV-2 N gene was reported. To avoid the constraint of the PAM site for double-stranded DNA (dsDNA) in CRISPR/Cas12a system, we designed two individual crRNAs to hybridize with two different regions of the target sequence. The presence of N gene DNA was able to initiate the amplification of RPA, exposing the recognition site of crRNA and activating the Cas12a. Whereafter, the Cas12a activation resulted in the digestion of nontarget DNA reporters to induce significant fluorescence signal. The assay completed the detection of N gene DNA within 30 min. And a high sensitivity of 100 aM was obtained because of RPA amplification and Cas12a trans cleavage activity. Meanwhile, the proposed assay showed excellent specificity due to the site-specific recognition ability of CRISPR/Cas12a. More importantly, analysis of spiked samples verified the excellent practical application of the proposed method. Thus, the assay earned promising potential in molecular diagnostics.},
}
@article {pmid40909132,
year = {2025},
author = {Biswas, I},
title = {Ethical dimensions and societal implications: ensuring the social responsibility of CRISPR technology.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1593172},
pmid = {40909132},
issn = {2673-3439},
abstract = {CRISPR-Cas9 is a breakthrough genome-editing platform that can cut chosen DNA sequences with unprecedented speed, accuracy, and affordability. By reprogramming a single guide RNA, researchers now alter gene function, correct pathogenic variants, or introduce novel traits. Earlier tools such as zinc-finger nucleases and TALENs performed similar tasks but were significantly more complex and costly. Yet CRISPR's very power raises urgent ethical concerns: Who controls its use, and how can society prevent germ-line enhancement, eugenic selection, or unequal access that favors wealthy nations and patients? A well-publicized case of embryo editing already showed how premature, unregulated experiments can erode public trust. This perspective therefore frames CRISPR's scientific promise alongside its social responsibilities, arguing that proactive, globally coordinated governance is essential to unlock benefits while preventing new forms of genetic inequality.},
}
@article {pmid40907709,
year = {2025},
author = {Chugh, P and Soni, S and Ghanghas, N and Kumar, S and Mohan, H},
title = {Comprehensive insights into Japanese encephalitis virus: From molecular characterization to advanced detection and vaccine strategies.},
journal = {Antiviral research},
volume = {},
number = {},
pages = {106268},
doi = {10.1016/j.antiviral.2025.106268},
pmid = {40907709},
issn = {1872-9096},
abstract = {The Japanese encephalitis virus (JEV) remains a major cause of viral encephalitis in Asia, with significant morbidity and mortality. This review offers a comprehensive overview of the current landscape of JEV research, focusing on its genomic structure, protein composition, and global epidemiology. We highlight the complexity of JEV transmission and pathogenesis, examining the interplay of demographic factors and geographic spread. In particular, we assess the evolution of diagnostic methodologies from traditional molecular and serological techniques to emerging biosensor-based approaches, emphasizing advancements in sensitivity and rapidity. The application of CRISPR/Cas systems for JEV detection marks a promising frontier in molecular diagnostics. Additionally, we review the current status of JEV vaccines, discussing recent innovations in vaccine development aimed at enhancing immunogenicity and accessibility. Beyond prevention, a spectrum of antiviral strategies-including direct-acting antivirals, entry inhibitors, host-directed modulators, neuroprotective agents, and steroidal/synthetic compounds-has demonstrated potent in vitro and in vivo efficacy, targeting viral enzymes, structural proteins, and host pathways. This review underscores the critical role of advanced detection strategies and vaccines in controlling JEV, offering insights into ongoing efforts to mitigate its impact in endemic regions.},
}
@article {pmid40906807,
year = {2025},
author = {Han, Z and Huang, C and Luo, T and Mirkin, CA},
title = {A general genome editing strategy using CRISPR lipid nanoparticle spherical nucleic acids.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {36},
pages = {e2426094122},
doi = {10.1073/pnas.2426094122},
pmid = {40906807},
issn = {1091-6490},
support = {FA9550-22-1-0300//DOD | AF | AMC | AFRL | Air Force Office of Scientific Research (AFOSR)/ ; DMR-2428112//National Science Foundation (NSF)/ ; DMR-2308691//National Science Foundation (NSF)/ ; NSF ECCS-2025633//National Science Foundation (NSF)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Nanoparticles/chemistry ; Humans ; *Lipids/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Nucleic Acids/chemistry/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transfection ; Liposomes ; },
abstract = {Genome editing with CRISPR-Cas systems hold promise for treating a wide range of genetic disorders and cancers. However, efficient delivery of genome editors remains challenging due to the requirement for the simultaneous delivery or intracellular generation of Cas proteins, guide RNAs, and, in some applications, donor DNAs. Furthermore, the immunogenicity and toxicity of delivery vehicles can limit the safety and efficacy of genetic medicines. Here, we combine two nucleic acid delivery approaches to create CRISPR lipid nanoparticle-spherical nucleic acids (LNP-SNAs) that are both efficient and biocompatible. Compared to lipid nanoparticles (LNPs) lacking a surface-bound DNA shell, CRISPR LNP-SNAs exhibit two- to three-fold higher cellular uptake, reduced cytotoxicity, and improved gene transfection efficiency. Across multiple cell lines and genomic loci, CRISPR LNP-SNAs induce insertion-deletion mutations at average frequencies two- to three-fold higher than those observed with LNPs. When codelivered with donor templates, CRISPR LNP-SNAs enable homology-directed repair at an average efficiency of 21 ± 7%, a 2.5-fold improvement over LNPs (8 ± 4%). The ease of synthesis and biocompatibility of CRISPR LNP-SNAs highlight their potential as a versatile delivery platform for CRISPR-Cas and other gene therapies.},
}
@article {pmid40906401,
year = {2025},
author = {Luo, J and Lu, W and Liu, R and Zhang, S and Cao, J and Ma, C},
title = {From Panels to Pathogen Networks: The Expanding Role of Targeted Sequencing in Veterinary Medicine.},
journal = {Biology},
volume = {14},
number = {8},
pages = {},
pmid = {40906401},
issn = {2079-7737},
abstract = {Targeted sequencing, a pivotal branch of next-generation sequencing (NGS), enables the selective enrichment of specific genomic regions and has demonstrated significant advantages in the detection of animal pathogens. This review systematically explores the underlying principles of targeted sequencing, various enrichment strategies-including PCR amplification, probe hybridization, and CRISPR-Cas systems-and their key applications in veterinary pathogen diagnostics. Due to its high throughput, sensitivity, and cost-effectiveness, targeted sequencing has been successfully applied in the multiplex detection of pathogens in economically significant livestock, such as cattle, as well as in the surveillance of antimicrobial resistance (AMR) genes, pathogen typing, and source tracing. It is particularly effective in identifying mixed infections and low-abundance pathogens. Nonetheless, wide application is restricted by some factors, like incomprehensive reference databases, cost-effectiveness, and limited application in primary-level laboratories. Further development directions are AI-based panel design, multimodal diagnostic platform integration, standard workflow construction, and introduction of a multi-omics method. Such progress focuses on enhancing the targeted sequencing scalability and precision consistent with the "One Health" initiative objective.},
}
@article {pmid40905590,
year = {2025},
author = {Cheng, L},
title = {Topology-Engineered Guide RNAs for Programmable Control of CRISPR/Cas Activity.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {},
number = {},
pages = {e202511756},
doi = {10.1002/anie.202511756},
pmid = {40905590},
issn = {1521-3773},
support = {2025YFA0920900//National Key R&amp;D Program of China/ ; XDB0960103//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; BNLMS-CXTD-202401//Beijing National Laboratory for Molecular Sciences/ ; 22537005//National Natural Science Foundation of China/ ; },
abstract = {CRISPR/Cas systems have transformed genome editing, yet achieving precise temporal and conditional control remains challenging. Traditional strategies involving linear guide RNAs (gRNAs) modified with multiple chemical groups throughout their strands often face limitations such as heterogeneous reaction outcomes, irreversibility, and variable editing efficiencies. To overcome these issues, topology-engineered guide RNAs (TE-gRNAs) have emerged, featuring defined structural architectures including polymeric, circular, and dendrimer-like topologies that enable precise spatial control, reversibility, and programmable activation of CRISPR activity. By selectively incorporating physical or chemically responsive linkers and stimuli-sensitive groups at specific sites, TE-gRNAs facilitate dynamic and conditional genome editing that can be activated or deactivated with external triggers such as light or chemical signals. These engineered RNA structures significantly improve synthesis feasibility, stability, reduce off-target effects, and provide unprecedented control over gene editing processes. Recent advancements in TE-gRNAs demonstrate their broad applicability in synthetic biology, functional genomics, and therapeutic interventions, highlighting their potential to achieve precise spatiotemporal modulation of CRISPR systems. This review summarizes the current strategies, benefits, and challenges associated with TE-gRNAs, and discusses future directions for enhancing their performance and utility in complex genome editing applications.},
}
@article {pmid40848498,
year = {2026},
author = {Chen, J and Shi, K},
title = {A fluorescent aptasensor for accurate and sensitive detection of glyphosate based on asymmetrically competitive CRISPR/Cas12a with phosphorothioate-modified G-quadruplex.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {345},
number = {},
pages = {126840},
doi = {10.1016/j.saa.2025.126840},
pmid = {40848498},
issn = {1873-3557},
mesh = {Glyphosate ; *Glycine/analogs &amp; derivatives/analysis ; *G-Quadruplexes ; *Aptamers, Nucleotide/chemistry/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; Spectrometry, Fluorescence/methods ; *Fluorescent Dyes/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Accurate detection of glyphosate (GLY) residues is essential for protecting public health, ensuring food safety, and preventing environmental pollution caused by excessive or improper herbicide application. Based on the asymmetric CRISPR/Cas12a system as a signal amplification technique, with phosphorothioate-modified hairpin G-quadruplex (psHG4) acting as a signal probe, a platform named ACC-GLY is developed for the sensitive and accurate detection of GLY. In the designed ACC-GLY platform, the target GLY specifically binds to the aptamer, and the cascading signal amplification strategy, driven by the DNase activity of a single Cas12a, is initiated by two competitive guide RNAs. Under the influence of Cas12a's DNase activity, the psHG4 probe is cleaved, releasing the psG4 sequence. The released psG4 sequence then binds to Thioflavin T (ThT), forming a complex that generates a fluorescence signal. Under optimal conditions, the detection platform can specifically detect GLY at a concentration as low as 0.3 pM. The detection platform demonstrates significant capability in detecting GLY in tap water and corn samples, highlighting its vast potential for applications in environmental monitoring and food safety. To the best of our knowledge, this is the first study to utilize the CRISPR/Cas12a system for GLY detection.},
}
@article {pmid40795705,
year = {2026},
author = {Li, H and Tang, X and Li, F and Yang, M and Jing, H and Li, L and Liu, J and Cao, Z and Yang, X and Wang, J},
title = {Neutrophil-inspired CRISPR/dCas9 nanomedicine to program self-destructing and bystander killing of tumor cell for selective cancer therapy.},
journal = {Biomaterials},
volume = {325},
number = {},
pages = {123619},
doi = {10.1016/j.biomaterials.2025.123619},
pmid = {40795705},
issn = {1878-5905},
mesh = {*Neutrophils/metabolism ; Humans ; Animals ; *CRISPR-Cas Systems/genetics ; *Bystander Effect ; *Nanomedicine/methods ; *Neoplasms/therapy/pathology ; Cell Line, Tumor ; Leukocyte Elastase/metabolism/genetics ; Mice ; Female ; },
abstract = {Achieving high efficiency, selectivity, and durability remains a major challenge in the development of innovative cancer treatments, as current clinical therapies often fall short. Inspired by neutrophil-mediated tumor clearance, we engineered a nanocarrier-mediated CRISPR/dCas9 system to activate endogenous expression of neutrophil elastase (ELANE) for precise cancer therapy. Although ELANE was upregulated in both normal and tumor cells, selective killing occurred only in tumor cells through a histone H1.0-dependent self-destructing mechanism. Additionally, secreted ELANE further efficiently eliminated neighboring tumor cells via bystander killing. ELANE-mediated tumor cell death also induced immunogenic responses, potently enhancing antitumor immunity and synergizing with anti-PD-L1 therapy to inhibit tumor metastasis. This study presents a novel CRISPRa-based therapeutic strategy that mimic neutrophil function to achieve potent, selective, and durable tumor eradication through combined mechanisms of self-destruction, bystander killing and immune activation.},
}
@article {pmid40904932,
year = {2025},
author = {Wang, HM and Xu, SJ and Cai, BY and Qiu, WY and Lu, H and Tang, YD},
title = {Highly efficient gene editing of Feline herpesvirus 1 using CRISPR/Cas9 combined with FACS.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1660446},
pmid = {40904932},
issn = {2235-2988},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *Varicellovirus/genetics ; Cats ; *Flow Cytometry/methods ; Thymidine Kinase/genetics ; Genes, Reporter ; Herpesviridae Infections/virology/veterinary ; Green Fluorescent Proteins/genetics ; },
abstract = {Feline herpesvirus 1 (FHV-1) is a major causative agent of feline viral rhinotracheitis and ocular lesions. Due to its large DNA genome, the construction of recombinant FHV-1 viruses presents considerable challenges for conventional methodologies. In this study, we implemented an integrated strategy combining CRISPR/Cas9-mediated gene editing with fluorescence-activated cell sorting (FACS) to enable the rapid and efficient generation of recombinant FHV-1 viruses. Specifically, the thymidine kinase (tk) gene was disrupted by inserting a monomeric Cherry (mCherry) reporter gene, and the glycoprotein E (gE) gene was similarly interrupted through the incorporation of a green fluorescent protein (GFP) reporter. The CRISPR/Cas9 system enables precise, site-specific genomic modifications, while FACS allows for effective enrichment and isolation of the desired recombinant viral populations. This combined approach significantly reduces the time required for recombinant virus generation from weeks to days, thereby offering substantial potential to expedite vaccine development and advance functional genomics research.},
}
@article {pmid40904117,
year = {2025},
author = {Koonce, KC and Mauritzen, JJ and Hitz, IF and Vangsgaard, EF and Putz, EHM and Wajn, AS and Leth, FH and Høyland-Kroghsbo, NM},
title = {The H-NS homologues MvaT and MvaU repress CRISPR-Cas in Pseudomonas aeruginosa.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240073},
pmid = {40904117},
issn = {1471-2970},
support = {//Danmarks Frie Forskningsfond/ ; //Lundbeck Foundation/ ; },
mesh = {*Pseudomonas aeruginosa/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics/metabolism ; *DNA-Binding Proteins/genetics/metabolism ; Trans-Activators ; },
abstract = {CRISPR-Cas is an adaptive immune system of bacteria and archaea that protects against foreign genetic elements. In Escherichia coli and Salmonella, CRISPR-Cas is inhibited by the conserved global repressor the histone-like nucleoid structuring protein (H-NS), which blocks the expression of AT-rich horizontally acquired genes. While the opportunistic pathogen Pseudomonas aeruginosa harbours two partially redundant H-NS homologues, MvaT and MvaU, their role in CRISPR-Cas regulation in this bacterium remains unexplored. Here, we demonstrate that in the absence of both MvaT and MvaU, CRISPR-Cas activity increases more than tenfold, as measured by a reduction in the transformation efficiency of a CRISPR-targeted plasmid. Importantly, we find that in the absence of MvaT and MvaU, Cas proteins are already produced at low cell density prior to the onset of quorum sensing-mediated activation of CRISPR-Cas, which occurs at high cell density. Moreover, the ∆mvaT ∆mvaU mutant has a significantly reduced growth rate, known to independently increase CRISPR-Cas activity. In addition to regulating CRISPR-Cas, the absence of MvaT and MvaU affects phage-host interactions, including enhancing the adsorption of the LPS-binding phage JBD44, highlighting their broader role in coordinating bacterial defence mechanisms.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}
@article {pmid40904116,
year = {2025},
author = {Hoikkala, V and Chi, H and Grüschow, S and Graham, S and White, MF},
title = {Diversity and abundance of ring nucleases in type III CRISPR-Cas loci.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240084},
pmid = {40904116},
issn = {1471-2970},
support = {/ERC_/European Research Council/International ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Bacteria/genetics/enzymology ; *Archaea/genetics/enzymology ; *Endonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {Most type III CRISPR-Cas systems facilitate immune responses against invading mobile genetic elements such as phages by generating cyclic oligoadenylates (cOAs). Downstream effectors activated by cOAs are typically non-specific proteins that induce damage to essential cellular components, thereby preventing phage epidemics. Owing to these toxic effects, it is crucial that the production and concentration of cOAs remain under tight regulatory control during infection-free periods or when deactivating the immune response after clearing an infection. Type III CRISPR loci often encode enzymes known as ring nucleases (RNs) that bind and degrade specific cOAs, while some effectors are auto-deactivating. Despite the discovery of several classes of RNs, a comprehensive bioinformatic analysis of type III CRISPR-Cas loci in this context is lacking. Here, we examined 38 742 prokaryotic genomes to provide a global overview of type III CRISPR loci, focusing on the known and predicted RNs. The candidate RNs Csx16 and Csx20 are confirmed as active enzymes, joining Crn1-3. Distributions and patterns of co-occurrence of RNs and associated effectors are explored, allowing the conclusion that a sizeable majority of type III CRISPR systems regulate cOA levels by degrading the signalling molecules, which has implications for cell fate following viral infection.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}
@article {pmid40904105,
year = {2025},
author = {Elliott, JFK and Cozens, K and Cai, Y and Waugh, G and Watson, BN and Westra, E and Taylor, TB},
title = {Phage susceptibility to a minimal, modular synthetic CRISPR-Cas system in Pseudomonas aeruginosa is nutrient dependent.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240473},
pmid = {40904105},
issn = {1471-2970},
support = {//UK Government's Horizon Europe funding guarantee/ ; //Royal Society/ ; //Philip Leverhulme Prize/ ; /BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Pseudomonas aeruginosa/virology/genetics ; *CRISPR-Cas Systems ; *Pseudomonas Phages/physiology/genetics ; *Nutrients/metabolism ; *Bacteriophages/physiology ; },
abstract = {CRISPR-Cas systems can provide adaptive, heritable immunity to their prokaryotic hosts against invading genetic material such as phages. It is clear that the importance of acquiring CRISPR-Cas immunity to anti-phage defence varies across environments, but it is less clear if and how this varies across different phages. To explore this, we created a synthetic, modular version of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. We used this synthetic system to test CRISPR-Cas interference against a panel of 13 diverse phages using engineered phage-targeting spacers. We observed complete protection against eight of these phages, both lytic and lysogenic and with a range of infectivity profiles. However, for two phages, CRISPR-Cas interference was only partially protective in high-nutrient conditions, yet completely protective in low-nutrient conditions. This work demonstrates that nutrient conditions modulate the strength of CRISPR-Cas immunity and highlights the importance of environmental conditions when screening defence systems for their efficacy against various phages.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}
@article {pmid40904104,
year = {2025},
author = {David, E and Plantady, C and Poissonnier, S and Elliott, JFK and Kenck, E and Le Boulch, J and Gutierrez, A and Chevallereau, A},
title = {Systematic functional assessment of anti-phage systems in their native host.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240067},
pmid = {40904104},
issn = {1471-2970},
support = {//ATIP-Avenir/ ; //Agence Nationale de la Recherche/ ; //Idex Paris Cit&#xe9;/ ; //Emergence ville de Paris/ ; },
mesh = {*Escherichia coli/virology/genetics ; *Coliphages/physiology ; *Bacteriophages/physiology ; DNA Restriction-Modification Enzymes/genetics ; },
abstract = {Bacterial resistance to bacteriophages (phages) relies on two primary strategies: preventing phage attachment and blocking post-attachment steps. These post-attachment mechanisms are mediated by diverse defence systems, including DNA-degrading systems such as restriction-modification and CRISPR-Cas, along with abortive infection systems that induce cell death or dormancy. Computational analyses suggest that bacterial genomes encode multiple defence systems, which may act synergistically to enhance phage resistance. However, the regulation, interactions and ecological roles of these systems in native hosts remain poorly understood. This study explored the role of eight predicted defence systems in the clinical isolate NILS69 of Escherichia coli by testing its susceptibility to 93 phages. Infectivity and adsorption assays using mutants defective in these systems revealed that only PD-T4-3 and restriction-modification systems restricted phages that were able to adsorb. The restriction-modification system acted via a predicted type IV endonuclease and was also able to limit plasmid conjugation if the plasmid was transferred from a donor strain lacking a methylase, which is the hallmark of type I, II or III restriction-modification systems. Other defence systems showed no detectable activity, likely owing to phage specificity, environmental regulation or cofactor requirements. These findings underscore the need for further studies to investigate the regulation and ecological roles of bacterial defence systems in their native host contexts.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}
@article {pmid40904102,
year = {2025},
author = {Pons, BJ and Łapińska, U and Lopes-Domingues, I and Chisnall, MAW and Westra, ER and Pagliara, S and van Houte, S},
title = {Phage provoke growth delays and SOS response induction despite CRISPR-Cas protection.},
journal = {Philosophical transactions of the Royal Society of London. Series B, Biological sciences},
volume = {380},
number = {1934},
pages = {20240474},
pmid = {40904102},
issn = {1471-2970},
support = {/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; /MRC_/Medical Research Council/United Kingdom ; //UK Government's Horizon Europe funding guarantee/ ; //Leverhulme Trust/ ; },
mesh = {*Pseudomonas aeruginosa/virology/growth &amp; development/genetics ; *CRISPR-Cas Systems ; *Pseudomonas Phages/physiology ; *SOS Response, Genetics ; *Bacteriophages/physiology ; },
abstract = {Bacteria evolve resistance against their phage foes with a wide range of resistance strategies whose costs and benefits depend on the level of protection they confer and on the costs for maintainance. Pseudomonas aeruginosa can evolve resistance against its phage DMS3vir either by surface mutations that prevent phage binding or through CRISPR-Cas immunity. CRISPR immunity carries an inducible cost whose exact origin is still unknown, and previous work suggested it stems from the inability of the CRISPR-Cas system to completely prevent phage DNA injection and subsequent gene expression before clearing the phage infection. However, the bacterial processes involved are still unknown, and we hypothesize that CRISPR-immunity-associated costs could come from increased mortality rate or reduced growth ability compared with surface-resistant bacteria. To tease apart these two mechanisms with divergent ecological consequences, we use a novel microfluidics-based single-cell approach combined with flow cytometry methods to monitor the effects of phage exposure on the survival and growth of its host. We observed that while CRISPR immunity protects from phage-induced lysis, it cannot prevent phage-induced division lag, filamentation and SOS response activation in a subpopulation of the host bacteria. These results suggest that the costs associated with CRISPR immunity at the population level are caused by heterogeneity in phage-induced growth defects.This article is part of the discussion meeting issue 'The ecology and evolution of bacterial immune systems'.},
}
@article {pmid40903118,
year = {2025},
author = {Zhou, J and Shi, X and He, C and Zheng, X and Yuan, R and Yang, X},
title = {SERS biosensor based on the Cas13a assisted entropy-driven system and lychee-like Fe-TiO2 with excellent exciton capture and separation.},
journal = {Analytica chimica acta},
volume = {1372},
number = {},
pages = {344442},
doi = {10.1016/j.aca.2025.344442},
pmid = {40903118},
issn = {1873-4324},
mesh = {*Titanium/chemistry ; *Spectrum Analysis, Raman/methods ; *Entropy ; *Biosensing Techniques/methods ; *Iron/chemistry ; CRISPR-Cas Systems ; *MicroRNAs/analysis ; Limit of Detection ; },
abstract = {BACKGROUND: Entropy-Driven Circuits (EDC), distinguished by their spontaneous operation and absence of enzymatic reactions, represent a superior strategy for integration with CRISPR/Cas systems, as they obviate the potential for interference among various enzymes during the process of DNA amplification and CRISPR/Cas system integration. Due to the wide band gap of TiO2, its response to visible light is limited, and owing to its high crystallinity and exceptionally stable crystal lattice, the charge transfer (CT) process in TiO2 is suboptimal.<br><br>RESULTS: In this study, lychee-like Fe-TiO2 was firstly prepared to serve as Raman enhanced substrate, facilitating exciton capture and separation to exhibit an excellent Surface-enhanced Raman spectroscopy (SERS) performance. It is proven that the incorporation of Fe results in a significantly narrower band gap for TiO2, facilitating exciton resonance. The amount of Fe in TiO2 was optimized to fabricate a SERS biosensor for detection of miRNA-21 based on the Cas13a assisted entropy-driven system. The detection limit of miRNA was 43.88&#xa0;fmol/L.<br><br>SIGNIFICANCE: This work proposes a sensing strategy that integrates the CRISPR/Cas system with EDC, leveraging a semiconductor substrate exhibiting superior SERS performance to provide a stable Raman signal, thereby enabling highly sensitive detection of miRNA-21, which has a potential application in disease early warning and treatment.},
}
@article {pmid40902823,
year = {2025},
author = {Zhou, X and Diao, R and Li, X and Ziegler, CA and Gramelspacher, MJ and Freddolino, L and Hou, Z and Zhang, Y},
title = {Cas9 senses CRISPR RNA abundance to regulate CRISPR spacer acquisition.},
journal = {Nature},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41586-025-09577-9},
pmid = {40902823},
issn = {1476-4687},
abstract = {Prokaryotes create adaptive immune memories by acquiring foreign DNA snippets, known as spacers, into the CRISPR array[1]. In type II CRISPR-Cas systems, the RNA-guided effector Cas9 also assists the acquisition machinery by selecting spacers from protospacer adjacent motif (PAM)-flanked DNA[2,3]. Here, we uncover the first biological role for Cas9 that is independent of its dual RNA partners. Following depletion of crRNA and/or tracrRNA, Neisseria apoCas9 stimulates spacer acquisition efficiency. Physiologically, Cas9 senses low levels of crRNA in cells with short CRISPR arrays - such as those undergoing array neogenesis or natural array contractions - and dynamically upregulates acquisition to quickly expand the small immune memory banks. As the CRISPR array expands, rising crRNA abundance in turn reduces apoCas9 availability, thereby dampening acquisition to mitigate autoimmunity risks associate with elevated acquisition. While apoCas9's nuclease lobe alone suffices for stimulating acquisition, only full-length Cas9 responses to crRNA levels to boost acquisition in cells with low immunity depth. Finally, we show that this activity is evolutionarily conserved across multiple type II-C Cas9 orthologs. Altogether, we establish an auto-replenishing feedback mechanism in which apoCas9 safeguards CRISPR immunity depth by acting as both a crRNA sensor and a regulator of spacer acquisition.},
}
@article {pmid40902040,
year = {2025},
author = {Li, L and Dai, H and Sun, R and Zhang, Z and Zhang, B},
title = {MicroRNAs as Biotechnological Targets for Future Food Security and Agricultural Sustainability.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c07100},
pmid = {40902040},
issn = {1520-5118},
abstract = {MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate gene expression, playing key roles in plant growth, development, and stress responses. Their regulatory functions make miRNAs ideal targets for enhancing crop yield, quality, and stress tolerance using biotechnologies, such as transgenic overexpression and CRISPR/Cas genome editing. By targeting multiple genes, miRNAs address complex agricultural challenges effectively. This review focuses on the diverse roles of miRNAs in enhancing crop productivity and resilience; miRNAs are an important biotechnological target for ensuring food security and agricultural sustainability. We also highlight transgenic and CRISPR/Cas genome editing approaches to demonstrate miRNA-driven trait improvements, such as drought/salinity tolerance, pest resistance, and nutrient use efficiency. Due to the quick development of advanced biotechnology tools, both upregulated and downregulated miRNAs can be manipulated for optimizing agronomic traits. Challenges including off-target effects, regulatory barriers, and environmental concerns are analyzed with strategies proposed to overcome them. By leveraging miRNA technologies, this perspective emphasizes their transformative potentials in achieving sustainable agriculture and global food security.},
}
@article {pmid40901825,
year = {2025},
author = {Thiam, R and Ceballos, MS and Beneke, T and Kuk, N and Pasquier, G and Crobu, L and Jeffares, DC and Vergnes, B and Barckmann, B and Sterkers, Y},
title = {A novel Leishmania infantum reference strain for gene editing and the study of visceral leishmaniasis.},
journal = {PloS one},
volume = {20},
number = {9},
pages = {e0327390},
pmid = {40901825},
issn = {1932-6203},
mesh = {*Leishmania infantum/genetics/growth &amp; development/pathogenicity ; *Leishmaniasis, Visceral/parasitology ; *Gene Editing/methods ; Animals ; Humans ; CRISPR-Cas Systems ; Mice ; Macrophages/parasitology ; Genome, Protozoan ; },
abstract = {Parasites of the Leishmania donovani complex are responsible for visceral leishmaniasis, a vector-borne disease transmitted through the bite of female phlebotomine sand flies. As well as the human hosts, these parasites infect many mammals which can serve as reservoirs. Dogs are particularly important reservoirs. Transmission is widespread across Asia, Africa, the Americas, and the Mediterranean basin, including South of France. Visceral leishmaniasis poses a fatal threat if left untreated. Research into the pathophysiology of this neglected disease is of prime importance, as is the development of new drugs. In this study, we evaluated the growth, differentiation, and macrophage infectivity of four L. donovani complex strains and identified L. infantum S9F1 (MHOM/MA/67/ITMAP263, clone S9F1) as a well-adapted strain for genetic engineering studies. We present here the genome sequence and annotation of L infantum S9F1 T7 Cas9, providing the scientific community with easy access to its genomic information. The data has been integrated into the LeishGEdit online resource to support primer design for CRISPR-Cas9 experiments. We now aim to make this strain widely available to foster studies of visceral leishmaniasis.},
}
@article {pmid40788276,
year = {2025},
author = {Gao, H and Zhang, Y and Wang, Y and Wu, Y and Zheng, Z and Quan, F and Han, Q and Li, Y and Zhang, K},
title = {Integration of CRISPR/Cas12a and Cas13a in one pot for ratiometric calibration of single-nucleotide variations.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {72},
pages = {13675-13678},
doi = {10.1039/d5cc03931b},
pmid = {40788276},
issn = {1364-548X},
mesh = {*Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; Calibration ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Humans ; },
abstract = {Accurate detection of single-nucleotide variations (SNVs) plays a pivotal role in medical diagnosis. Herein, by integrating Cas12a and Cas13a to simultaneously detect wild-type and mutated sites on a single RNA strand, we developed an innovative one-pot SNV analysis method, namely iCasdrop, which is capable of reducing non-specific signals induced by the wild type sequence and achieving ratiometric calibration of SNVs.},
}
@article {pmid40599075,
year = {2025},
author = {Zhang, T and Cai, L and Chu, Z and She, A and Yang, J and Su, X},
title = {Rapid and Sensitive Detection of miRNA by Single-Molecule Fluorescence Dequenching Assay with Target Recycled CRISPR/Cas12a Amplification System.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {35},
pages = {e2412228},
doi = {10.1002/smll.202412228},
pmid = {40599075},
issn = {1613-6829},
support = {32271521//National Natural Science Foundation of China/ ; 31971361//National Natural Science Foundation of China/ ; 2022YFC2603902//State Key Research Development Program of China/ ; PT2406//Fundamental Research Funds for the Central Universities/ ; 2023-NHLHCRF-YXHZ-ZRMS-05//National High Level Hospital Clinical Research Funding/ ; },
mesh = {*MicroRNAs/genetics/metabolism/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; Fluorescence ; Carcinoma, Non-Small-Cell Lung/genetics ; Lung Neoplasms/genetics ; Female ; Cell Line, Tumor ; },
abstract = {Dysregulated miRNAs play a critical role in the development of cancers. A rapid and sensitive single-molecule fluorescence dequenching assay combined with a CRISPR/Cas12a-based target recycling amplification system for miRNA detection is developed. This single-molecule assay detects miRNAs down to ≈10 fM within 10 min. An automated single-molecule fluorescent puncta analysis procedure is also created, improving the signal-to-noise ratio by 3.76-fold compared to traditional hidden Markov model (HMM)based methods. The clinical applicability of this technique is demonstrated. Two key miRNA targets associated with non-small cell lung cancer (NSCLC) and ovarian cancer (OC) from 2867 datasets of the TCGA database are screened. Validation is initially conducted at the cell line level, followed by testing with tissue and blood samples from 10 patients with NSCLC and OC. The assay demonstrated high diagnostic accuracy, with receiver operating characteristic curves (area under the curve (AUC) > 0.93) and significant statistical differentiation (p < 0.001) between cancer and healthy samples. This method's exceptional sensitivity and speed highlight its potential for early cancer diagnostics and personalized medicine.},
}
@article {pmid40536333,
year = {2025},
author = {Zhao, G and Li, Z and Zhao, MJ and Li, SY and Xia, Q and Xu, S and Zhang, Y and Wang, Y and Li, F and Liu, YL and Guo, YH and Xu, RX and Zhou, H and Zhou, H and Ding, WW and Wang, YC and Miao, Y and Wang, Z},
title = {A High-Fidelity RNA-Targeting Cas13X Downregulates Connexin43 in Macroglia: A Novel Neuroprotective Strategy for Glaucoma.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {33},
pages = {e15856},
doi = {10.1002/advs.202415856},
pmid = {40536333},
issn = {2198-3844},
support = {82301215//National Natural Science Foundation of China/ ; 32271043//National Natural Science Foundation of China/ ; 32471057//National Natural Science Foundation of China/ ; 82171047//National Natural Science Foundation of China/ ; 2018SHZDZX01//Shanghai Municipal Science and Technology Major Project/ ; },
mesh = {*Connexin 43/genetics/metabolism ; *Glaucoma/genetics/metabolism/therapy ; Animals ; Mice ; Retinal Ganglion Cells/metabolism ; Disease Models, Animal ; CRISPR-Cas Systems/genetics ; Down-Regulation ; Intraocular Pressure ; Mice, Inbred C57BL ; Humans ; Optic Nerve/metabolism ; },
abstract = {Glaucoma is a neurodegenerative disease characterized by the progressive degeneration of retinal ganglion cells (RGCs) and their axons, ultimately leading to irreversible vision loss. Elevated intraocular pressure (IOP) is one of the significant risk factors in glaucoma; however, neurodegeneration continues even after effective IOP management, underscoring the need for neuroprotective therapies. This study investigates the role of connexin43 (Cx43), which is extensively expressed in retinal macroglia, in regulating microglial activation and optic nerve degeneration in glaucoma. A high-fidelity CRISPR-Cas13 (hfCas13X) system is employed to selectively target and knock down Cx43 expression in macroglia. The findings reveal that Cx43-mediated ATP release through hemichannels exacerbates microglial activation and neuroinflammation, thereby contributing to RGC loss. Notably, in a mouse model of chronic ocular hypertension (COH) glaucoma, knocking down Cx43 in macroglia using the hfCas13X system significantly promoted the survival of RGCs and the integrity of the optic nerve, and improved visual function. The hfCas13X system, which offers high-fidelity RNA editing with minimal off-target effects, represents a novel and promising therapeutic strategy for glaucoma, highlighting the potential of gene editing technologies in the management of neurodegenerative diseases.},
}
@article {pmid40534129,
year = {2025},
author = {Nicosia, L and Harrison, PT},
title = {CRISPR for cystic fibrosis: Advances and insights from a systematic review.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {9},
pages = {4091-4112},
doi = {10.1016/j.ymthe.2025.06.021},
pmid = {40534129},
issn = {1525-0024},
mesh = {*Cystic Fibrosis/genetics/therapy ; Humans ; *Gene Editing/methods ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Mutation ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cystic fibrosis (CF) is a severe genetic disorder caused by loss-of-function mutations in the CFTR gene. Gene-editing approaches have the potential to correct such mutations. This systematic review outlines the mechanisms of the main CRISPR-based technologies, and, through cross-study comparisons, analyzes 27 research articles that applied them to target CF-causing variants. We report and discuss the strategy design, target cell selection, editing efficiency, prevalence of editing byproducts, and levels of CFTR functional restoration achieved in each work, with the aim of providing technical insights for further exploration of CRISPR-based gene-editing approaches. Our findings show that the F508del and W1282X mutations were the most extensively studied CF-causing variants, though over fifteen mutations were targeted overall. The majority of works under review explored the use of homology-directed repair or base editing, with a growing number of studies reporting efficient prime editing. Some studies tackled multiple individual mutations, compared different editors, or tested strategies across various models, while others focused on approaches that rescue CFTR function without directly correcting a mutation. Several works also proposed strategies that could address multiple variants with a single approach, while others highlighted technical difficulties in editing certain regions of the CFTR gene. This cross-study comparison also emphasizes the need for standardized reporting of editing efficiency and functional recovery, and stresses the importance of further single-cell RNA sequencing and in vivo studies to reach clinically relevant conclusions. As gene-editing techniques continue to evolve, and with over 60 ongoing CRISPR-based clinical trials, there is growing optimism for meaningful advancements in CF gene-editing therapeutics.},
}
@article {pmid40518666,
year = {2025},
author = {Yang, Y and Fu, Z and Deng, S and Wu, G and Wang, C and Luo, X and Kang, R and Chen, Y and Peng, C and Zhang, P and Cui, K and Wan, F and Wang, J and Zhou, Q and Chen, W and Xiong, Y and Ma, W and Songyang, Z and Liang, P},
title = {RNA-DNA hybrid binding domain broadens the editing window of base editors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {33},
number = {9},
pages = {4431-4446},
doi = {10.1016/j.ymthe.2025.06.024},
pmid = {40518666},
issn = {1525-0024},
mesh = {*Gene Editing/methods ; Humans ; Animals ; Mice ; *DNA/genetics/metabolism/chemistry ; CRISPR-Cas Systems ; Ribonuclease H/genetics/metabolism ; *RNA/genetics/metabolism/chemistry ; },
abstract = {Adenine base editors (ABEs) and cytosine base editors (CBEs) are prominent tools for precise genome editing but are hindered by limited editing activity at positions proximal to the protospacer adjacent motif (PAM). This study investigates the potential of enhancing base editors editing activity by fusing them with RNA-DNA hybrid binding domains (RHBDs). Specifically, fusing ABE8e with the RHBD of Homo sapiens RNaseH1 (RHBD1) significantly increased A-to-G editing efficiency in the PAM-proximal region (A9-A15) by up to 3.5-fold, while reducing off-target cytosine editing. Additionally, RHBD1 is compatible with ABEmax, BE4max, and dual base editor (eA&amp;C-BEmax), enhancing their editing activity at the PAM-proximal bases. Notably, RHBD1-fused BE4max led to a 3.1-fold improvement in C-to-T editing efficiency at PAM-proximal region (C9-C12). Furthermore, we demonstrated that RHBD1-fused ABE8e could effectively edit disease-related single nucleotide variations (SNVs) in human cells and validated its efficacy in adult mouse liver. These findings highlight the significance of the RHBD in expanding editing window and the applicability of base editors for gene therapy and disease modeling.},
}
@article {pmid40493884,
year = {2025},
author = {Loke, J and Kim, PG and Nguyen, TTP and Boileau, M and McConkey, M and Miller, A and Shin, W and Hergott, CB and Ericsson, M and Nordstrom, A and Llopis, PM and Armstrong, SA and Mancias, JD and Ebert, BL},
title = {An in vivo barcoded CRISPR-Cas9 screen identifies Ncoa4-mediated ferritinophagy as a dependence in Tet2-deficient hematopoiesis.},
journal = {Blood},
volume = {146},
number = {10},
pages = {1174-1186},
doi = {10.1182/blood.2024028033},
pmid = {40493884},
issn = {1528-0020},
mesh = {*CRISPR-Cas Systems ; *Hematopoiesis/genetics ; *Ferritins/metabolism/genetics ; *DNA-Binding Proteins/genetics/deficiency/metabolism ; Animals ; *Nuclear Receptor Coactivators/metabolism/genetics ; Dioxygenases ; Mice ; *Proto-Oncogene Proteins/genetics/deficiency ; Hematopoietic Stem Cells/metabolism ; Mice, Knockout ; *Autophagy ; Iron/metabolism ; Humans ; },
abstract = {TET2 is among the most commonly mutated genes in both clonal hematopoiesis and myeloid malignancies; thus, the ability to identify selective dependencies in TET2-deficient cells has broad translational significance. Here, we identify regulators of Tet2 knockout (KO) hematopoietic stem and progenitor cell (HSPC) expansion using an in vivo CRISPR-Cas9 KO screen, in which nucleotide barcoding enabled large-scale clonal tracing of Tet2-deficient HSPCs in a physiologic setting. Our screen identified candidate genes, including Ncoa4, that are selectively required for Tet2 KO clonal outgrowth compared with wild type. Ncoa4 targets ferritin for lysosomal degradation (ferritinophagy), maintaining intracellular iron homeostasis by releasing labile iron in response to cellular demands. In Tet2-deficient HSPCs, increased mitochondrial adenosine triphosphate production correlates with increased cellular iron requirements and, in turn, promotes Ncoa4-dependent ferritinophagy. Restricting iron availability reduces Tet2 KO stem cell numbers, revealing a dependency in TET2-mutated myeloid neoplasms.},
}
@article {pmid40492429,
year = {2025},
author = {Meng, R and Li, J and Wang, W and Liang, D and Li, Z and Mao, C and Li, Q and Zhang, Y and Chen, H and Tang, J and Hu, P and Niu, Q and Huang, X and Shen, B and Zhang, J},
title = {Engineered Cas12j-8 is a Versatile Platform for Multiplexed Genome Modulation in Mammalian Cells.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {33},
pages = {e02593},
doi = {10.1002/advs.202502593},
pmid = {40492429},
issn = {2198-3844},
support = {2022YFC2702705//National Key Research and Development Project of China/ ; 82221005//Creative Research Groups of China/ ; 32371546//National Natural Science Foundation of China/ ; 82071434//National Natural Science Foundation of China/ ; 81971398//National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; HEK293 Cells ; *CRISPR-Associated Proteins/genetics ; },
abstract = {Cas12j-8 is a compact Cas nuclease discovered from the metagenome of giant bacteriophages, consisting of only 717 amino acids and recognizing the '5-TTN-3' protospacer adjacent motif (PAM) sequence. However, its low gene editing efficiency in mammalian cells limits its application in therapeutic gene editing. To address this limitation, structure-guided mutagenesis is employed to replace key negatively charged residues with arginine, strengthening DNA binding. The resulting quintuple mutant, engineered Cas12j-8 (enCas12j-8), demonstrates robust on-target editing efficiency comparable to LbCas12a while maintaining low off-target effects. Cytosine base editors (CBEs) and adenine base editors (ABEs) are developed using enCas12j-8, achieving up to 29.54-fold C-to-T and 36.57-fold A-to-G conversion efficiency compared with the wild-type at the dominated sites, respectively. Notably, enCas12j-8 enables multiplexed editing of three genomic loci simultaneously via a single crRNA array, achieving efficiencies comparable to single-guide approaches. Additionally, enCas12j-8-ABE facilitates the disruption of splice acceptor sites, effectively inducing exon skipping in the SOD1 gene. This strategy holds potential significance for therapeutic genome modulation. These findings establish enCas12j-8 as a versatile, high-precision tool for genome engineering, combining efficient delivery, multiplexing capability, and compatibility with diverse editing modalities.},
}
@article {pmid40901634,
year = {2025},
author = {Jiang, X and Wang, X and Shen, S and Hou, S and Yu, C},
title = {3D Genome Engineering: Current Advances and Therapeutic Opportunities in Human Diseases.},
journal = {Research (Washington, D.C.)},
volume = {8},
number = {},
pages = {0865},
doi = {10.34133/research.0865},
pmid = {40901634},
issn = {2639-5274},
abstract = {Dynamic chromatin 3-dimensional (3D) conformation is a key mechanism regulating gene expression and cellular function during development and disease. Elucidating the structure, functional dynamics, and spatiotemporal organization of the 3D genome requires integrating multiple experimental approaches, including chromatin conformation capture techniques, precise genome manipulation tools, and advanced imaging technologies. Notably, CRISPR/Cas systems have emerged as a revolutionary genome-editing platform, offering unprecedented opportunities for manipulating 3D genome organization and investigating disease mechanisms. This review systematically examines recent advances in CRISPR-based mammalian 3D genome engineering and explores the therapeutic potential of 3D genome engineering strategies in disease intervention.},
}
@article {pmid40900077,
year = {2025},
author = {Yu, Y and Zhang, Z and Zhai, Z and Sun, B and Yang, D and Wang, Z and Lin, Q and Zhou, X and Zhao, J},
title = {A Novel Mouse Model of Granular Corneal Dystrophy Type II Reveals Impaired Autophagy and Recapitulates Human Pathogenesis.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {66},
number = {12},
pages = {7},
doi = {10.1167/iovs.66.12.7},
pmid = {40900077},
issn = {1552-5783},
mesh = {Animals ; *Corneal Dystrophies, Hereditary/genetics/pathology/metabolism ; *Autophagy/physiology ; *Disease Models, Animal ; Mice ; Microscopy, Electron, Transmission ; Tomography, Optical Coherence ; Transforming Growth Factor beta/genetics/metabolism ; Blotting, Western ; Extracellular Matrix Proteins/genetics/metabolism ; Humans ; *Mutation ; Mice, Inbred C57BL ; Slit Lamp Microscopy ; CRISPR-Cas Systems ; Phenotype ; *Cornea/pathology ; },
abstract = {PURPOSE: To develop and characterize a novel mouse model of granular corneal dystrophy type II (GCD2) using CRISPR/Cas9 technology and explore the underlying pathogenesis of transforming growth factor-beta-induced protein (TGFBIp) aggregation.<br><br>METHODS: CRISPR/Cas9 technology was employed to introduce the R124H mutation in the TGFBI gene of mice. Genomic sequencing and polymerase chain reaction confirmed the mutation. Phenotypic characteristics were evaluated through slit-lamp examination, optical coherence tomography, histological analysis, electron microscopy, and immunofluorescence, comparing wild-type (WT), heterozygous (HE), and homozygous (HO) mice. Transcriptome sequencing was conducted to identify the pathogenesis of GCD2. The findings were further validated through western blotting and transmission electron microscopy.<br><br>RESULTS: The R124H mutation in TGFBI was successfully introduced, with breadcrumb-like deposits observed in the corneas of mutant mice, with HO mice displaying more severe phenotypes than HE mice. TGFBIp levels were elevated in HE and HO mice (both P < 0.001). Histological and electron microscopy analyses revealed abnormal collagen arrangement and TGFBIp deposits in the corneal stroma of the HE and HO mice. Transcriptome analysis indicated that the TGFBI-R124H mutation was associated with impaired autophagy, endocytosis, and extracellular matrix signaling. Additional experiments confirmed autophagy-related markers LC3 and SQSTM1 were upregulated in the corneas of mutant mice, accompanied by increased autophagosome formation in corneal keratocytes, indicating impaired autophagy flux in HE and HO mice.<br><br>CONCLUSIONS: We established a GCD2 mouse model caused by the R124H mutation using CRISPR/Cas9, providing a reliable platform for understanding pathogenesis for GCD2.},
}
@article {pmid40897812,
year = {2025},
author = {Wolter, JM and James, LM and Boeshore, SL and Mao, H and McCoy, ES and Ryan, DF and Fragola, G and Taylor-Blake, B and Stein, JL and Zylka, MJ},
title = {AAV-dCas9 vector unsilences paternal Ube3a in neurons by impeding Ube3a-ATS transcription.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1332},
pmid = {40897812},
issn = {2399-3642},
support = {631904//Simons Foundation/ ; 1R01NS109304//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; T32HD040127//U.S. Department of Health &amp; Human Services | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; },
mesh = {Animals ; *Dependovirus/genetics ; Mice ; *Neurons/metabolism ; Humans ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Genetic Vectors/genetics ; *Angelman Syndrome/genetics/therapy ; *Transcription, Genetic ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Male ; *RNA, Long Noncoding/genetics ; },
abstract = {Angelman syndrome (AS) is a debilitating neurodevelopmental disorder caused by loss of maternally-inherited UBE3A. In neurons, paternally-inherited UBE3A is silenced in cis by a long non-coding RNA called Ube3a-ATS. Here, we found that Neisseria meningitidis Cas9 with two mutations (D15A and H587A) in the nuclease domains (dNmCas9) can unsilence the dormant paternal Ube3a allele in mouse and human neurons when targeted to Snord115 snoRNA genes located in introns of Ube3a-ATS. Importantly, dNmCas9 disrupted Ube3a-ATS with a non-template bias and in the absence of a chromatin modifying domain, supporting a transcriptional interference mechanism. When packaged into an adeno-associated virus (AAV) vector, dNmCas9 exhibited dose-dependent Ube3a-ATS knock-down and paternal Ube3a unsilencing in vitro and in vivo. This vector also partially rescued the hind limb clasp phenotype when delivered to neonatal AS model mice. Collectively, our study underscores the potential of dCas9-based therapeutics without chromatin repression domains to mediate transcriptional downregulation.},
}
@article {pmid40897312,
year = {2025},
author = {Bircheneder, M and Parniske, M},
title = {Engineering and comparison of cas12a-based genome editing systems in plants.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {5},
pages = {e70410},
pmid = {40897312},
issn = {1365-313X},
support = {401867691//Deutsche Forschungsgemeinschaft/ ; //Bayerisches Staatsministerium f&#xfc;r Unterricht und Kultus/ ; },
mesh = {*Gene Editing/methods ; *Arabidopsis/genetics ; Nicotiana/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; *Lotus/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Plants, Genetically Modified ; },
abstract = {While Cas9 and Cas12a are both RNA-guided endonucleases used for genome editing, only Cas12a is able to process pre-crRNA via its additional ribonuclease activity. This feature reduces the complexity of Cas12a versus Cas9-based genome editing systems thus providing an attractive alternative for generating site-specific mutations in plants. Here we aimed to improve the efficiency of the cas12a-based generation of two double-strand breaks flanking the open reading frame of a target gene, leading to its full deletion. To this end, we compared the relative impact of different components on cas12a-based gene deletion efficiency in three different eudicotyledons, Arabidopsis thaliana, Lotus japonicus, and Nicotiana benthamiana. We detected the highest cas12a-based editing efficiency with a combination of suitable promoters for crRNA and cas12a expression, a tandem terminator to control cas12a expression, a re-coded cas12a, adapted to the codon usage of Arabidopsis and engineered to carry introns, and encoding a Cas12a flanked by a nuclear localization signal at both ends. Our work revealed the high potential for improving cas12a-based genome editing systems for plant genetic research.},
}
@article {pmid40896718,
year = {2025},
author = {Madsen, CK and Hanak, T and Aronsson, H and Brinch-Pedersen, H},
title = {Rapid one-step CRISPR-cas vector assembly by isothermal spacer removal linearization and sequence-ligation independent cloning (ISRL-SLIC).},
journal = {MethodsX},
volume = {15},
number = {},
pages = {103567},
pmid = {40896718},
issn = {2215-0161},
abstract = {CRISPR-Cas genome editing is a powerful tool in various fields, but current cloning methods can be time-consuming due to the frequent use of intermediate entry vectors and multiple steps involving restriction enzymes and ligases. These multiple steps can create a bottleneck in CRISPR-Cas experiments. In response to this challenge, we propose a highly efficient streamlined approach, which enables simultaneous linearization of the acceptor plasmid and protospacer cloning in a single isothermal reaction. This eliminates the need for entry vectors, pre-linearization of vectors, and in vitro ligation, thus significantly simplifying the cloning process. The method can be applied to clone short synthetic oligos for single protospacer constructs or multiple amplicons for multiplex genome editing designs. Either way, researchers can proceed directly to Escherichia coli transformation after a one-hour isothermal reaction and recover the final construct within two days. By combining the advantages of sequence-ligation independent cloning (SLIC) cloning with a streamlined workflow, our approach facilitates rapid and efficient construction of CRISPR-Cas vectors and holds the promise of accelerating research and development in genome editing and related fields. To expedite the cloning of constructs, we propose a rapid one-step CRISPR-Cas vector assembly method that combines isothermal spacer removal with a sequence-ligation-independent cloning reaction. We could show that Isothermal Spacer Removal Linearization and Sequence-Ligation Independent Cloning (ISRL-SLIC) can create single, double and triple protospacer constructs in one reaction with scalability. The ISRL-SLIC reaction delivers clones under a broad range of oligo concentration making it a robust and time saving alternative to other methods for constructing CRISPR-Cas vectors.},
}
@article {pmid40895203,
year = {2025},
author = {Kesarwani, P and Sundar, D},
title = {Conformational changes induced by K949A mutation in the CRISPR-Cas12a complex drives an effective target-binding mechanism.},
journal = {Current research in structural biology},
volume = {10},
number = {},
pages = {100173},
pmid = {40895203},
issn = {2665-928X},
abstract = {The CRISPR/Cas system is a potential tool for genome editing, yet it faces challenges due to off-target activity caused by mismatches at specific positions. However, Off-target activity can be minimized by optimal design of guide RNA (gRNA) but there remains a possibility of unintended cleavage, highlighting the role of the Cas nuclease in off-target recognition and binding the target site. This study focuses on comparing the conformational dynamics and stability of Wildtype, RR, RVR, RRm and RVRm variants of AsCas12a with gRNA-DNA bound complexes. It was found that the cross-correlation coefficient between His1167 of the NUC domain and Thr384 of the REC II domain significantly increased after the K949A mutation compared to other variants. The extensive spread of principal components also revealed flexibility in both Cas nuclease and gRNA-DNA hybrid of RVR variant and wildtype AsCas12a whereas the confined clusters in PCA plot suggests increased stability in both the variants after mutation. This study shows the role of K949A mutation in improving stability of PAM variants and predicted critical residues such as His1167, Thr384 and Ser959, in inducing stability in mutants of PAM variants.},
}
@article {pmid40893972,
year = {2025},
author = {},
title = {Correction to "High-efficiency genome editing of an extreme thermophile Thermus thermophilus using endogenous type I and type III CRISPR-Cas systems".},
journal = {mLife},
volume = {4},
number = {4},
pages = {470},
doi = {10.1002/mlf2.70010},
pmid = {40893972},
issn = {2770-100X},
abstract = {[This corrects the article DOI: 10.1002/mlf2.12045.].},
}
@article {pmid40891977,
year = {2025},
author = {Li, X and Zhong, Y and Jin, C and Chen, X and Cui, X and Xu, Y and Fan, Y and Song, F and Cen, P and Dong, L and Yu, K and He, Q and Wang, J and Hu, S and Zhang, XY and Li, C and Tian, M and Zhang, H},
title = {CRISPR/Cas9-Engineered Triple-Fusion Reporter Gene Imaging System for Monitoring Transplanted Neural Progenitor Cells in Ischemic Stroke.},
journal = {Radiology},
volume = {316},
number = {3},
pages = {e250305},
doi = {10.1148/radiol.250305},
pmid = {40891977},
issn = {1527-1315},
mesh = {Animals ; *Neural Stem Cells/transplantation ; Rats ; Male ; *Ischemic Stroke/diagnostic imaging/therapy ; *CRISPR-Cas Systems/genetics ; *Genes, Reporter/genetics ; Magnetic Resonance Imaging/methods ; Humans ; Disease Models, Animal ; Rats, Sprague-Dawley ; Positron Emission Tomography Computed Tomography/methods ; },
abstract = {Background Neural progenitor cell therapy holds great potential for repairing brain damage induced by ischemic stroke, and molecular imaging plays a crucial role in evaluating the therapeutic efficacy of neural progenitor cell transplantation. However, the presence of the blood-brain barrier significantly limits the effectiveness of such imaging methods. Purpose To enable long-term monitoring of transplanted human neural progenitor cells (hNPCs) in a rat model of ischemic stroke by combining a clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9)-engineered triple-fusion (TF) reporter gene system with a noninvasive adenosine agonistic micelle (AM)-based probe delivery strategy. Materials and Methods Between January 2021 and May 2025, 60 male rats that were 2 months old were included. Thirty-seven rats with stroke were administered either TF human NPCs (hNPCs) or vehicle (culture media) and underwent MRI, bioluminescence imaging, PET/CT, and neurologic assessments at weeks 1, 2, 4, and 8 after transplantation. Comparisons between groups were determined by t tests, one-way analysis of variance, linear regression, and linear mixed-effects model. Results TF-hNPCs proliferated within the ischemic rat brain (week 8 vs week 1, bioluminescence imaging and PET: P < .001 and P = .02, respectively) and exhibited progressive migration and maturation by 8 weeks after transplantation (proportion of microtubule-associated protein 2-positive TF-hNPCs at week 8 vs week 4: 94.08% ± 3.02 vs 85.47% ± 6.54, respectively [P = .04]; proportion of doublecortin-positive TF-hNPCs at week 4 vs week 2: 83.90% ± 2.84 vs 59.74% ± 0.55, respectively [P = .02]). Moreover, TF-hNPC transplantation increased glucose (fluorine 18 fluorodeoxyglucose) uptake in the ischemic brain (TF-hNPCs vs vehicle at week 4, 0.58 ± 0.04 vs 0.37 ± 0.05, respectively [P = .008]; TF-hNPCs vs vehicle at week 8, 0.52 ± 0.06 vs 0.29 ± 0.02, respectively [P = .01]) and attenuated neurologic deficits compared with the vehicle group (neurologic score, TF-hNPCs vs vehicle at week 8: 9.6 ± 0.25 vs 7.6 ± 0.3, respectively; P = .003). Conclusion A CRISPR/Cas9-engineered TF reporter gene imaging system combined with a noninvasive AM-based approach enabled in vivo monitoring of transplanted human NPCs in a rat model of ischemic stroke. © RSNA, 2025 Supplemental material is available for this article. See also the editorial by Chapelin in this issue.},
}
@article {pmid40857331,
year = {2025},
author = {Farr, GH and Reid, W and Hasegawa, EH and Azzam, A and Young, I and Li, ML and Olson, AK and Beier, DR and Maves, L},
title = {A systems genetics approach identifies roles for proteasome factors in heart development and congenital heart defects.},
journal = {PLoS genetics},
volume = {21},
number = {8},
pages = {e1011579},
pmid = {40857331},
issn = {1553-7404},
mesh = {Animals ; Zebrafish/genetics/embryology ; *Proteasome Endopeptidase Complex/genetics/metabolism ; *Heart Defects, Congenital/genetics/pathology ; *Heart/embryology/growth &amp; development ; Humans ; *Zebrafish Proteins/genetics/metabolism ; Protein Interaction Maps/genetics ; Gene Expression Regulation, Developmental ; Transcriptome/genetics ; Mutation ; CRISPR-Cas Systems ; },
abstract = {Congenital heart defects (CHDs) occur in about 1% of live births and are the leading cause of infant death due to birth defects. While there have been remarkable efforts to pursue large-scale whole-exome and genome sequencing studies on CHD patient cohorts, it is estimated that these approaches have thus far accounted for only about 50% of the genetic contribution to CHDs. We sought to take a new approach to identify genetic causes of CHDs. By combining analyses of genes that are under strong selective constraint along with published embryonic heart transcriptomes, we identified over 200 new candidate genes for CHDs. We utilized protein-protein interaction (PPI) network analysis to identify a functionally-related subnetwork consisting of known CHD genes as well as genes encoding proteasome factors, in particular POMP, PSMA6, PSMA7, PSMD3, and PSMD6. We used CRISPR targeting in zebrafish embryos to preliminarily identify roles for the PPI subnetwork genes in heart development. We then used CRISPR to create new mutant zebrafish strains for two of the proteasome genes in the subnetwork: pomp and psmd6. We show that loss of proteasome gene functions leads to defects in zebrafish heart development, including dysmorphic hearts, myocardial cell blebbing, and reduced outflow tracts. We also identified deficits in cardiac function in pomp and psmd6 mutants. These heart defects resemble those seen in zebrafish mutants for known CHD genes and other critical heart development genes. Our study provides a novel systems genetics approach to further our understanding of the genetic causes of human CHDs.},
}
@article {pmid40779935,
year = {2025},
author = {Wu, J and Li, X and Yin, X and Hu, J and Zhou, P and Zhong, X and Wu, M},
title = {Rapid detection of plasma exosomal LncRNA CASC9 for HCC using RT-RPA-CRISPR/Cas12a assay.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {266},
number = {},
pages = {117085},
doi = {10.1016/j.jpba.2025.117085},
pmid = {40779935},
issn = {1873-264X},
mesh = {Humans ; *Carcinoma, Hepatocellular/blood/diagnosis/genetics ; *Liver Neoplasms/blood/diagnosis/genetics ; *Exosomes/genetics/metabolism ; *RNA, Long Noncoding/blood/genetics ; Biomarkers, Tumor/blood/genetics ; Male ; Female ; Middle Aged ; ROC Curve ; CRISPR-Cas Systems/genetics ; alpha-Fetoproteins ; Real-Time Polymerase Chain Reaction/methods ; Aged ; },
abstract = {Hepatocellular carcinoma (HCC) is a leading cause of cancer-related mortality worldwide. Early detection is essential for improving patient outcomes. Long non-coding RNAs (lncRNAs) in plasma exosomes have emerged as promising non-invasive biomarkers. However, sensitive detection methods remain limited. Plasma exosomes were isolated and validated using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot (WB). RNA sequencing identified CASC9 as the most significantly upregulated exosomal lncRNA in HCC patients. Its diagnostic value was evaluated using real-time quantitative PCR (RT-qPCR) and a novel RT-RPA-CRISPR/Cas12a fluorescence assay. Diagnostic performance was assessed through receiver operating characteristic (ROC) curve analysis and compared with alpha-fetoprotein (AFP). Exosomal CASC9 levels were significantly elevated in HCC patients and correlated with tumor size, stage, and number (P < 0.001). ROC analysis demonstrated that CASC9 had superior diagnostic accuracy (area under the curve [AUC] = 0.822) compared to AFP (AUC = 0.795), with further improvement when combined (AUC = 0.875). The RT-RPA-CRISPR/Cas12a assay achieved a detection limit of 0.1 copies/μL, outperforming RT-qPCR. When combined with RT-qPCR and AFP, the method achieved an AUC of 0.987 against normal controls and 0.975 against benign cases. Plasma exosomal CASC9 is a promising diagnostic biomarker for HCC. The RT-RPA-CRISPR/Cas12a assay offers a rapid, ultra-sensitive, and clinically feasible detection strategy.},
}
@article {pmid40764055,
year = {2025},
author = {Stewart-Ornstein, J and Irby, MJ and Lilieholm, MK and Laprise, D and Collier, MD and Aunins, T and Harjanto, D and Chang, AN and Reyon, D and Duffield, JS},
title = {3'-end ligation sequencing is a sensitive method to detect DNA nicks at potential sites of off-target activity induced by prime editors.},
journal = {Genome research},
volume = {35},
number = {9},
pages = {2064-2075},
pmid = {40764055},
issn = {1549-5469},
mesh = {Humans ; *Gene Editing/methods ; *Sequence Analysis, DNA/methods ; Genome, Human ; CRISPR-Cas Systems ; *DNA/genetics ; DNA Breaks, Double-Stranded ; },
abstract = {Gene editing makes precise changes in DNA to restore normal function or expression of genes; however, the advancement of gene editing to the clinic is limited by the potential genotoxicity of off-target editing. To comprehensively identify potential sites in the genome that may be recognized by gene editing agents, in vitro approaches, in which the editor is combined with human genomic DNA and sites where editing may occur are identified biochemically, are important tools. Existing biochemical approaches for off-target discovery recognize double-stranded breaks generated by nuclease-based gene editors such as SpCas9, but novel approaches are needed for new editing modalities, such as prime editing, that nick one strand of DNA. To fill this gap, we have developed 3'-end ligation sequencing (PEG-seq), which can identify prime editor-induced nicks throughout the genome on in vitro digested human genomic DNA to identify potential off-target sites. Here we show that PEG-seq is an important addition to the off-target detection toolkit, enabling off-target discovery for DNA nicking gene editors such as prime editors.},
}
@article {pmid40757665,
year = {2025},
author = {Carlsson, PO and Hu, X and Scholz, H and Ingvast, S and Lundgren, T and Scholz, T and Eriksson, O and Liss, P and Yu, D and Deuse, T and Korsgren, O and Schrepfer, S},
title = {Survival of Transplanted Allogeneic Beta Cells with No Immunosuppression.},
journal = {The New England journal of medicine},
volume = {393},
number = {9},
pages = {887-894},
doi = {10.1056/NEJMoa2503822},
pmid = {40757665},
issn = {1533-4406},
mesh = {Humans ; Male ; *Diabetes Mellitus, Type 1/therapy/surgery ; *Islets of Langerhans Transplantation/methods ; Transplantation, Homologous ; Gene Editing ; Adult ; *Graft Survival ; CRISPR-Cas Systems ; Insulin/metabolism ; *Insulin-Secreting Cells/transplantation ; },
abstract = {The need to suppress a patient's immune system after the transplantation of allogeneic cells is associated with wide-ranging side effects. We report the outcomes of transplantation of genetically modified allogeneic donor islet cells into a man with long-standing type 1 diabetes. We used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 12b (Cas12b) editing and lentiviral transduction to genetically edit the cells to avoid rejection; the cells were then transplanted into the participant's forearm muscle. He did not receive any immunosuppressive drugs and, at 12 weeks after transplantation, showed no immune response against the gene-edited cells. C-peptide measurements showed stable and glucose-responsive insulin secretion. A total of four adverse events occurred, none of which were serious or related to the study drug. (Funded by the Leona M. and Harry B. Helmsley Charitable Trust; EudraCT number, 2023-507988-19-00; ClinicalTrials.gov number, NCT06239636.).},
}
@article {pmid40744866,
year = {2025},
author = {Schmal, M and Kramer, LTS and Mach, RL and Mach-Aigner, AR and Zimmermann, C},
title = {Providing a toolbox for genomic engineering of Trichoderma aggressivum.},
journal = {Microbiology spectrum},
volume = {13},
number = {9},
pages = {e0096625},
pmid = {40744866},
issn = {2165-0497},
support = {P 34036//Austrian Science Fund/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; Genome, Fungal ; Transformation, Genetic ; *Trichoderma/genetics ; *Genetic Engineering/methods ; Hypocreales/genetics ; Agaricus ; Plasmids/genetics ; Fungal Proteins/genetics ; Plant Diseases/microbiology ; Genomics/methods ; },
abstract = {Fungi belonging to the genus of Trichoderma have a long history of infecting crops of edible fungi and reducing the yield. Trichoderma aggressivum is the main causal agent of green mold disease in Agaricus bisporus. Despite its infamous role as a persistent and aggressive contamination in A. bisporus crops, T. aggressivum can also be used as a biocontrol agent or as a promoter of plant growth. In order to work efficiently with T. aggressivum on a molecular biology level, a transformation protocol is required. This study provides a detailed protocol on how to perform a transformation in T. aggressivum using plasmid DNA for ectopic integration. In addition, a Cas9-RNP-based approach has been established for genome editing. We performed two transformations to confirm the usability of the hph gene as well as the pyr4 gene from Trichoderma reesei as selection markers. First, we integrated the hph gene ectopically and determined the overall transformation efficiency. In the second transformation experiment, we disrupted the ornithine-5' phosphate decarboxylase gene of T. aggressivum by using the CRISPR-Cas9 approach. In total, four candidates showed the expected uridine auxotrophy and resistance to 5-FOA. Additionally, the genomic locus around the CRISPR target sites was sequenced to determine the exact circumstances around the gene disruption. To complete the genetic toolbox for T. aggressivum, the pyr4 gene of T. reesei was tested as a suitable selection marker in one of the generated uridine auxotrophic strains.IMPORTANCEResearchers need an efficient tool for genomic manipulation to investigate the fundamental biology of mycoparasitism of T. aggressivum and its correlation to secondary metabolites. We provide a protocol for transformation of T. aggressivum and successfully demonstrated transformation of T. aggressivum using a plasmid and genome editing applying a Cas9-RNP-based strategy. Simultaneously, we established two selection markers, the hph gene and pyr4 gene from T. reesei. By applying these methods, we give researchers the tools needed to investigate T. aggressivum on a deeper level. Possible applications include activation of biosynthetic gene clusters of secondary metabolites to determine the biosynthetic pathway and biotechnological applications of these compounds.},
}
@article {pmid40646716,
year = {2025},
author = {Coirry, C and Manessier, J and Clot, C and Mortier, M and Fauvarque, MO and Taillebourg, E},
title = {The deubiquitinase USP36 funtions through catalytic-dependent and catalytic-independent mechanisms in Drosophila.},
journal = {Genetics},
volume = {231},
number = {1},
pages = {},
pmid = {40646716},
issn = {1943-2631},
support = {ANR-17-EURE-0003//French National Research Agency/ ; //IDEX Universit&#xe9; Grenoble Alpes/ ; //GRAL PhD Operating Costs/ ; },
mesh = {Animals ; *Drosophila Proteins/genetics/metabolism ; Male ; *Drosophila melanogaster/genetics/growth &amp; development ; *Ubiquitin Thiolesterase/genetics/metabolism ; Spermatogenesis/genetics ; CRISPR-Cas Systems ; Female ; Proto-Oncogene Proteins c-myc/metabolism/genetics ; Deubiquitinating Enzymes/metabolism/genetics ; },
abstract = {Deubiquitinases (DUBs) form a specific class of proteases removing ubiquitin from target proteins. They are involved in the regulation of many cellular processes including cell growth and proliferation. Among them, USP36 is a key regulator of the oncogenic transcription factor c-Myc, preventing its degradation by the proteasome. These 2 proteins form an evolutionary conserved complex providing the opportunity to investigate USP36 mechanisms of action in vivo in a genetically tractable model such as Drosophila melanogaster. Null mutants of dUsp36 die early during larval development and exhibit severe growth defects. Strikingly, we report here that flies expressing a catalytically inactive version of dUSP36 produced by CRISPR/Cas9 gene editing survive to adulthood with only minor growth defects, yet males are infertile. This finding indicates that dUSP36 deubiquitinating activity is dispensable for cell growth but essential for spermatogenesis. Our results thus reveal that dUSP36 functions through both catalytic-dependent and catalytic-independent mechanisms, highlighting a dual mode of action with implications for the understanding of DUBs mechanism of action.},
}
@article {pmid40555854,
year = {2025},
author = {Basit, A and Liu, A and Zheng, W and Zhu, J},
title = {A review on the mechanism and potential diagnostic application of CRISPR/Cas13a system.},
journal = {Mammalian genome : official journal of the International Mammalian Genome Society},
volume = {36},
number = {3},
pages = {709-726},
pmid = {40555854},
issn = {1432-1777},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacteria/genetics ; CRISPR-Associated Proteins/genetics ; },
abstract = {Clustered regularly Interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form a natural immune defense system in prokaryotic species, with approximately 90% of archaea and 40% of bacteria possessing these systems, highlighting their widespread role in microbial immunity. Among these, the CRISPR/Cas13a system, guided by a single-stranded RNA (crRNA), selectively targets RNA sequences and has shown immense potential in developing sensitive diagnostic tools. Recent advancements have combined Cas13a with amplification methods and lateral flow detection (CRISPR/Cas13a-LFD), improving its application for rapid and accurate RNA detection. In this review, we explore the history, structure, and functional mechanism of the CRISPR/Cas13a system, focusing on its diagnostic capabilities. We compare CRISPR/Cas13a to conventional diagnostic approaches, highlighting their advantages in sensitivity, specificity, speed, and flexibility for point-of-care application. Given the rapid development of CRISPR-based diagnostics in recent years, the Cas13a system shows great potential as a next-generation platform for accurate, portable, and cost-effective detection of viral and bacterial diseases. Furthermore, we address the existing challenges, including reliance upon amplification and off-target effects, and highlight the need for ongoing research to develop amplification-free systems suitable for clinical application.},
}
@article {pmid40541099,
year = {2025},
author = {Tian, J and Wang, Y and Zhou, Z and Li, Y and Chang, C and Meng, F and Shi, W and Li, H and Wang, W and Wei, Q and Liu, H and Yang, Z and Xiao, S},
title = {Genome-wide CRISPR/Cas9 screen identifies host factors for Newcastle disease virus replication.},
journal = {Poultry science},
volume = {104},
number = {9},
pages = {105421},
pmid = {40541099},
issn = {1525-3171},
mesh = {Animals ; *Newcastle disease virus/physiology ; *Virus Replication/genetics ; *CRISPR-Cas Systems ; Mice ; *Newcastle Disease/virology/genetics ; Cell Line ; },
abstract = {Viruses rely strictly on host factors to determine their susceptibility. Newcastle disease virus (NDV), an avian pathogen, can infect a wide range of avian, mammalian and human cell lines. However, NDV can only infect certain mammals and humans, and it does not cause disease in mammals or mice. A comprehensive identification of NDV-susceptible host genes that promote NDV infection is lacking. Here, we performed a mouse genome-wide CRISPR knockout (GeCKO) screening in murine fibroblast L929 cells infected with NDV. Fifty host genes were highly enriched in the screening. Using a viral minigenome assay and gene overexpression as well as single guide RNA (sgRNA) knockout, one of the genes was shown to predominantly promote viral replication. This gene is the solute carrier family 35 member A1 (SLC35A1), which is a cytidine monophosphate (CMP)-Sia transporter involved in the synthesis of sialic acid (SA). Knockout of SLC35A1 in L929 cells decreased the expression of the NDV receptors α2,3-SA and α2,6-SA on the cell surface, resulting in significant reductions in viral adsorption, internalization and replication. Furthermore, the knockout of a six-residue stretch, [82]LGSPKE[87], of SLC35A1 in cells specifically reduced the expression of the α2,6-SA receptor but not the α2,3-SA receptor, which decreased viral adsorption and replication. The reconstitution of SLC35A1 in the knockout cells completely recovered the α2,3-SA receptor, partially recovered the α2,6-SA receptor and almost completely recovered viral replication. In chicken fibroblast DF-1 cells, siRNA-mediated knockdown of chicken SLC35A1 reduced the expression levels of both the α2,6-SA and α2,3-SA receptors, decreasing viral replication. Our research indicated that SLC35A1 is a key host factor that promotes NDV replication. The CRISPR/Cas9 system can be used to identify essential host factors for the replication of intracellular pathogens. This study provides valuable insights into host susceptibility to NDV.},
}
@article {pmid40527130,
year = {2025},
author = {Jin, S and Wang, X and Liu, X and Xu, Y and Wang, W and Chen, W and Chang, H and Li, Z and Geng, Z},
title = {Effective IHH gene knockout by CRISPR/Cas9 system in chicken DF-1 cells.},
journal = {Poultry science},
volume = {104},
number = {9},
pages = {105433},
pmid = {40527130},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics ; *CRISPR-Cas Systems ; *Hedgehog Proteins/genetics/metabolism ; *Gene Knockout Techniques/veterinary ; *Avian Proteins/genetics/metabolism ; Cell Line ; },
abstract = {Indian hedgehog (IHH) gene codes an important signal molecule mediating chondrogenesis and bone development in chickens, which are key factors that affect body weight and several other significant economic traits. The aim of this study was to construct an IHH knockout cell model using CRISPR-associated protein 9 (CRISPR/Cas9) technology to further analyze the function of IHH. TA cloning was used to screen the single-guide RNA (sgRNA1) [45 %] and sgRNA3 (30.8 %) with the highest targeting efficiency. Monoclonal cells were selected by flow cytometry for TA cloning sequencing to construct the IHH knockout cell model. Quantitative PCR (qPCR) was used to detect the changes in downstream gene expression levels after IHH knockout. TA cloning sequencing results showed that the IHH knockout cell model was successfully constructed, and two mutation types were generated with a 100 % mutation rate. In addition, qPCR results revealed that the expression of patched 1 (PTCH1), smoothened, frizzled class receptor (Smo), glioma-associated oncogene homolog 1 (Gli1), glioma-associated oncogene homolog 2 (Gli2), and osteopontin (OPN) was significantly lower in the IHH knockout group, while that of type II collagen (Col Ⅱ) was significantly higher. These results lay a theoretical foundation for the successful application of knockout technology in poultry functional genomics research and provide a stable knockout cell line model for further study of chicken IHH gene function.},
}
@article {pmid40891974,
year = {2025},
author = {Chapelin, F},
title = {CRISPR-based Triple-Modality Imaging Ushers a New Era for Stem Cell Tracking in Stroke.},
journal = {Radiology},
volume = {316},
number = {3},
pages = {e252546},
doi = {10.1148/radiol.252546},
pmid = {40891974},
issn = {1527-1315},
mesh = {Humans ; *Cell Tracking/methods ; *Stroke/diagnostic imaging/therapy ; *Multimodal Imaging/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Stem Cells ; *CRISPR-Cas Systems ; },
abstract = {"Just Accepted" papers have undergone full peer review and have been accepted for publication in Radiology. This article will undergo copyediting, layout, and proof review before it is published in its final version. Please note that during production of the final copyedited article, errors may be discovered which could affect the content.},
}
@article {pmid40891659,
year = {2025},
author = {Takizawa, A and Foeckler, J and Knapp, E and Grzybowski, M and Geurts, AM and Carroll, J and Merriman, DK},
title = {Successful Generation of Germline Tyrosinase Gene Edited Thirteen-Lined Ground Squirrels (Ictidomys tridecemlineatus Mitchill 1821).},
journal = {Molecular reproduction and development},
volume = {92},
number = {9},
pages = {e70055},
doi = {10.1002/mrd.70055},
pmid = {40891659},
issn = {1098-2795},
support = {//This study was supported by the National Eye Institute of the National Institutes of Health (NIH) under award number T32EY014537, U24EY029891 and by the National Center for Advancing Translational Sciences, National Institutes of Health, Award Number UL1TR001436./ ; },
mesh = {Animals ; *Monophenol Monooxygenase/genetics/metabolism ; *Sciuridae/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Female ; Male ; *Germ-Line Mutation ; *Germ Cells/metabolism ; },
abstract = {Thirteen-lined ground squirrels (Ictidomys tridecemlineatus Mitchill 1821; 13-LGS) are useful diurnal rodent models of human cone-mediated vision due to their cone photoreceptor-dominant retinas. To develop the 13-LGS as a better model of inherited human visual disorders, we report a gene-editing protocol targeting the 13-LGS tyrosinase (Tyr) gene. CRISPR/Cas9 microinjection into donor embryos, followed by transfer to pseudo-pregnant recipients, yielded two Tyr-mutated founders. Mating these two to wild-type 13-LGS resulted in 22 offspring, of which five were genotyped with either a 17-bp deletion, 1-bp insertion, or 7-bp deletion Tyr mutation. These results demonstrated that this valuable mammalian model is amenable to germline gene editing by conventional methods.},
}
@article {pmid40891290,
year = {2025},
author = {Deng, Z and Sha, R and Qin, H and Shang, Y and Yuan, A and Xie, W and Peng, H},
title = {A CRISPR Cas protein coronated AuNP nanostructure for enhanced uptake efficiency into cells.},
journal = {Chemical communications (Cambridge, England)},
volume = {},
number = {},
pages = {},
doi = {10.1039/d5cc03658e},
pmid = {40891290},
issn = {1364-548X},
abstract = {The effectiveness of nanotechnologies is often limited by their non-specific aggregation in biological environments. We developed a protein coronated nanostructure by functionalizing AuNPs with nucleic acid scaffolds and CRISPR Cas proteins, significantly enhancing nanoparticle stability and cellular uptake efficiency, making it a promising tool for imaging and biomedical applications.},
}
@article {pmid40890415,
year = {2025},
author = {Laidoudi, Y and Davoust, B and Lepidi, H and Levasseur, A},
title = {Emergence of the zoonotic bacterium Necropsobacter rosorum in nutria Myocastor coypus with implications for wildlife and human health.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {32252},
pmid = {40890415},
issn = {2045-2322},
mesh = {Animals ; Humans ; Phylogeny ; Genome, Bacterial ; France/epidemiology ; *Pasteurellaceae/genetics/isolation &amp; purification/classification/pathogenicity ; Animals, Wild/microbiology ; *Zoonoses/microbiology ; Whole Genome Sequencing ; *Rodentia/microbiology ; *Bacterial Zoonoses/microbiology/epidemiology ; },
abstract = {The nutria (Myocastor coypus), a semi-aquatic rodent native to South America, poses significant ecological and agricultural threats as an invasive species in France, where it continues to proliferate despite sustained control efforts. A fatal case of pneumonia in a nutria from Marseille (France) prompted a microbiological investigation that led to the isolation, taxonomic classification, genomic characterization, and phylogenetic analysis of Necropsobacter rosorum. Whole-genome sequencing of the N. rosorum strain RG01 revealed a genome size of 2,505,657 base pairs and 2303 predicted open reading frames, showing high similarity to other publicly available N. rosorum genomes. Comparative pan-genomic analysis indicated a high level of genomic conservation among N. rosorum strains. The presence of putative virulence factors and a CRISPR-Cas system suggests both pathogenic potential and adaptive defense mechanisms against bacteriophage predation. This study also explored the genetic epidemiology of members of the Pasteurellaceae family, highlighting a considerable overlap between species infecting animals and humans. Among the 408,387 sequence records retrieved from GenBank, 62.1% were deemed suitable for genomic epidemiological analysis. Notably, N. rosorum was underrepresented, with only 13 entries spanning nine countries and three host types, revealing critical gaps in current surveillance and research. Collectively, these findings contribute to a better understanding of the microbiology and epidemiology of N. rosorum and Pasteurellaceae-associated infections, and underscore the importance of integrated, genomics-informed approaches for the monitoring, control, and prevention of zoonotic diseases.},
}
@article {pmid40890128,
year = {2025},
author = {Álvarez-Pérez, JC and Sanjuán-Hidalgo, J and Arenas, AM and Hernández-Navas, I and Benitez-Cantos, MS and Andrades, A and Calabuig-Fariñas, S and Jantus-Lewintre, E and Paz-Ares, L and Ferrer, I and Medina, PP},
title = {High-fidelity Cas9-mediated targeting of KRAS driver mutations restrains lung cancer in preclinical models.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {7080},
pmid = {40890128},
issn = {2041-1723},
support = {LAB-AECC-2018//Fundaci&#xf3;n Cient&#xed;fica Asociaci&#xf3;n Espa&#xf1;ola Contra el C&#xe1;ncer (Scientific Foundation, Spanish Association Against Cancer)/ ; B-CTS-480-UGR20, C-EXP-051-UGR23, C-CTS-149-UGR23//Universidad de Granada (University of Granada)/ ; },
mesh = {*Lung Neoplasms/genetics/therapy/pathology ; Animals ; Humans ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Carcinoma, Non-Small-Cell Lung/genetics/therapy/pathology ; Mice ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Gene Editing/methods ; *Genetic Therapy/methods ; Mutation ; Female ; Adenoviridae/genetics ; },
abstract = {Missense mutations in the 12[th] codon of KRAS are key drivers of lung cancer, with glycine-to-cysteine (G12C) and glycine-to-aspartic acid (G12D) substitutions being among the most prevalent. These mutations are strongly associated with poor survival outcomes. Given the critical role of KRAS in lung cancer and other cancers, it remains as a major target for the development of new and complementary treatments. We have developed a CRISPR-High Fidelity (HiFi)-Cas9-based therapy strategy that can effectively and specifically target KRAS[G12C] and KRAS[G12D] mutants, avoiding KRAS[WT] off-targeting and affecting KRAS downstream pathways, thereby significantly reducing tumorgenicity. The delivery of HiFiCas9 components via ribonucleoprotein particles (RNPs) and adenovirus (AdV) effectively abrogates cell viability in KRAS-mutant Non-Small Cell Lung Cancer (NSCLC) preclinical models, including 2D and 3D cell cultures, cell-derived xenografts (CDX), and patient-derived xenograft organoids (PDXO). Our in vitro studies demonstrate that HiFiCas9-based therapy achieves superior KRAS inhibition compared to Sotorasib and effectively circumvents certain resistance mechanisms associated with Sotorasib treatment. Moreover, in vivo delivery using adenoviral particles significantly suppresses tumor growth in preclinical NSCLC models. Collectively, our findings establish HiFiCas9 as an effective therapeutic strategy with promising clinical applications, especially if in vivo delivery methods are further optimized.},
}
@article {pmid40825745,
year = {2025},
author = {Zhuang, S and Huang, X and Diao, M and Dong, R and Dong, Y and Liu, J and Liu, Y},
title = {CONVERT: Dynamic crRNA Reconfiguration for Universal One-Pot CRISPR/Cas12a-Based Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {34},
pages = {18776-18785},
doi = {10.1021/acs.analchem.5c03577},
pmid = {40825745},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *RNA/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Current one-pot CRISPR diagnostics necessitate meticulous control of nucleic acid hybridization parameters or extensive modification of CRISPR components to achieve complete enzymatic silencing, a fundamental bottleneck limiting their robustness and generalizability. Here, we resolve this challenge through dynamic crRNA reconfiguration, a paradigm-shifting strategy that exploits the intrinsic structural duality of CRISPR RNA. We present CONVERT (CRISPR One-Pot Nucleic acid detection Via Engineered crRNA Reconfiguration Technology), a universal platform where nontarget intact crRNA acts as a universal suppressor, achieving complete Cas12a inhibition during RPA (recombinase polymerase amplification) by irreversible enzyme sequestration. Target detection is initiated through programmable conversion to split crRNA activators, bypassing sequence-specific design constraints entirely. This crRNA conformational switching mechanism is implemented via photocleavable linker cleavage and subsequent assembly with presupplied truncated crRNA (tcrRNA), activating trans-cleavage signal amplification. Three transformative advances emerge: First, the endogenous crRNA engineering eliminates dependency on exogenous nucleic acid blockers or chemical modifications, reducing optimization costs and time. Second, near-total enzymatic suppression ensures zero cross-talk between amplification and detection phases. Third, the unified workflow enables contamination-resistant operation through spatiotemporal control, requiring no physical partitioning. Validated for Enterocytozoon bieneusi detection, the system demonstrates attomolar sensitivity of 1 aM, 100% diagnostic concordance with qPCR, and absolute specificity against related pathogens. By effectively decoupling CRISPR regulation from sequence-context constraints through rational crRNA structural plasticity, this work establishes a generalized framework for next-generation molecular diagnostics.},
}
@article {pmid40742143,
year = {2025},
author = {Liu, G and Wu, J and Yang, B and Kong, J and Li, Y and Chen, C and Hu, J and Sun, Z and Hou, Y and Sun, R and Li, D},
title = {Ultrasensitive eccDNA Detection for Tumor Diagnostics by Using CRISPR/Cas12a-Coupled Nested PCR.},
journal = {Analytical chemistry},
volume = {97},
number = {34},
pages = {18527-18536},
doi = {10.1021/acs.analchem.5c02316},
pmid = {40742143},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Polymerase Chain Reaction/methods ; *DNA, Circular/genetics/blood/analysis ; Limit of Detection ; *Biomarkers, Tumor/genetics/blood ; *Neoplasms/diagnosis/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Extrachromosomal circular DNA (eccDNA) has emerged as a novel biomarker for cancer detection due to its tumor-specific amplification and stable structure in circulation. However, its clinical application is hindered by extremely low abundance in biofluids and the lack of robust detection techniques. To address this, we screened for tumor-associated eccDNA biomarkers and developed NPCC (Nested PCR-CRISPR/Cas12a), a novel method combining nested PCR for ultrasensitive amplification with CRISPR/Cas12a for sequence-specific detection. The assay employs two rounds of junction-specific PCR to enrich eccDNA, followed by CRISPR/Cas12a-mediated cleavage guided by target-specific crRNA. Validation using synthetic circular DNA standards demonstrated a limit of detection (LoD) of 10[-6] fM, representing a >100-fold improvement over conventional PCR, with no cross-reactivity to linear or genomic DNA fragments. In plasma samples from 88 cancer patients, NPCC successfully detected multiple tumor-specific eccDNAs, including the hepatocellular carcinoma marker eccDNA-HCC-1 (AUC = 0.8977). NPCC overcomes key technical barriers in liquid biopsy, offering a cost-effective, highly sensitive, and specific platform for noninvasive cancer diagnostics.},
}
@article {pmid40614014,
year = {2025},
author = {Shan, L and He, Q and Zhang, W and Zheng, X and Zhao, J and Yang, P and Mao, Q and Xia, H},
title = {CRISPR/Cas9-mediated promoterless gene targeting reduces lysosome storage in MPS VII mice.},
journal = {Science China. Life sciences},
volume = {68},
number = {9},
pages = {2697-2706},
pmid = {40614014},
issn = {1869-1889},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Glucuronidase/genetics/metabolism/blood ; *Mucopolysaccharidosis VII/therapy/genetics/metabolism ; *Gene Targeting/methods ; *Lysosomes/metabolism ; Disease Models, Animal ; Genetic Vectors/genetics ; Promoter Regions, Genetic ; Genetic Therapy/methods ; Actins/genetics ; Adenoviridae/genetics ; Liver/metabolism ; },
abstract = {Targeted gene integration mediated by CRISPR/Cas9 is a promising therapeutic strategy for monogenic autosomal recessive diseases. In this study, we established a novel all-in-one high-capacity adenovirus (HCAd) that can pack both CRISPR/Cas9 and donor DNA into the same vector and tested it on a mouse model of mucopolysaccharidosis type VII (MPS VII) caused by mutations in the β-glucuronidase (GUSB) gene. This system allowed targeted integration of promoterless GUSB in the mouse beta-actin gene (mActb) locus and the co-expression of GUSB with the self-cleaving peptide T2A (T2A) controlled by a strong endogenous mActb promoter. The in vivo results indicated that the serum GUSB level of MPS VII mice treated with a single intraperitoneal injection of the HCAd vector achieved 14% of that of wild-type mice, resulting in significant amelioration of lysosomal storage in the liver and spleen. Furthermore, the HCAd was injected intraventricularly in the brain of newborn MPS VII mice, leading to strongly positive GUSB enzyme staining in the choroid plexus and perivascular spaces of the periventricular regions and reduced lysosome storage. In summary, by using an all-in-one vector, the study provides a universal, one-for-all therapeutic for MPSVII, a disease caused by different mutations of the GUSB gene.},
}
@article {pmid40377870,
year = {2025},
author = {Amen, RA and Hassan, YM and Essmat, RA and Ahmed, RH and Azab, MM and Shehata, NR and Elgazzar, MM and El-Sayed, WM},
title = {Harnessing the Microbiome: CRISPR-Based Gene Editing and Antimicrobial Peptides in Combating Antibiotic Resistance and Cancer.},
journal = {Probiotics and antimicrobial proteins},
volume = {17},
number = {4},
pages = {1938-1968},
pmid = {40377870},
issn = {1867-1314},
mesh = {Humans ; *Gene Editing/methods ; *Neoplasms/microbiology/therapy/drug therapy ; *Microbiota ; *Antimicrobial Peptides/pharmacology ; CRISPR-Cas Systems ; Animals ; *Drug Resistance, Microbial ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The growing crisis of antibiotic resistance and the increasing incidence of cancer have prompted the exploration of innovative approaches, such as gene editing and antimicrobial peptides (AMPs). The human microbiome is integral to various aspects of health, disease, and therapeutic development, influencing metabolic pathways, immune function, and pathogen resistance. Recent advances in gene editing technologies, particularly CRISPR (clustered regularly interspaced short palindromic repeats), have opened new avenues for leveraging the microbiome to address complex medical challenges, including combating multidrug-resistant pathogens and cancer. The microbiome plays a crucial role in combating antibiotic resistance by modulating microbial communities, influencing pathogen survival and susceptibility to treatments. This review explores the microbiome's dynamic role in metabolic regulation, its contribution to cancer management, and how AMPs help maintain homeostasis and exhibit emerging anticancer properties, supported by both preclinical findings and clinical evidence. Additionally, CRISPR-based microbiome engineering offers potential to enhance host-microbiome interactions, optimizing therapeutic outcomes. The integration of microbiome metagenomics and proteomics has led to the discovery of novel AMPs with targeted anticancer effects. Innovative strategies, such as engineered probiotics and CRISPR-based microbiome engineering, present exciting prospects for next-generation therapies. Despite these advances, the translation of microbiome-based therapies into clinical settings remains challenging due to ethical, regulatory, and ecological hurdles. This review underscores the transformative potential of microbiome-based interventions, emphasizing the role of personalized medicine in maximizing therapeutic efficacy. Furthermore, we also address critical research gaps, limitations, and future directions, including optimizing AMP stability, delivery, and bioavailability, as well as overcoming the regulatory and ethical challenges in clinical translation.},
}
@article {pmid40326293,
year = {2025},
author = {Lazarides, K and Engel, JL and Meseonznik, M and Feng, T and Choi, AH and Yu, Y and Liu, S and Meier, SR and Zhang, H and Shen, B and Tjin Tham Sjin, R and Whittington, DA and McMillan, BJ and Doyon, B and Pan, X and Wilker, E and Huang, A and Andersen, JN and Mallender, WD and Bandi, M},
title = {CRISPR Screens Identify POLB as a Synthetic Lethal Enhancer of PARP Inhibition Exclusively in BRCA-Mutated Tumors.},
journal = {Molecular cancer therapeutics},
volume = {24},
number = {9},
pages = {1466-1479},
doi = {10.1158/1535-7163.MCT-24-0822},
pmid = {40326293},
issn = {1538-8514},
mesh = {Humans ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; Animals ; Mice ; *BRCA2 Protein/genetics ; *Synthetic Lethal Mutations ; Female ; Cell Line, Tumor ; *BRCA1 Protein/genetics ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Mutation ; Apoptosis/drug effects ; },
abstract = {PARP inhibitors (PARPi) are an approved class of anticancer therapeutics that inhibit the activities of PARP1/2 and produce synthetic lethality in BRCA1/2-mutated cancers because of the absence of a functional homologous recombination-dependent DNA repair pathway. Although PARPis have led to successful clinical outcomes, two thirds of patients develop acquired resistance, limiting long-term utility as maintenance therapy. Motivated by this clinical need, we utilized a CRISPR target discovery screening platform to identify DNA polymerase beta (POLB) as a gene that acts selectively and synergistically with PARPis in BRCA1/2-mutated cancers and found that POLB knockout (KO) along with PARPi treatment enhanced loss of viability in BRCA1/2-mutant and BRCA2-null cells but not in isogenic BRCA1/2 wild-type cells. Overexpression of either POLB wild-type or catalytically inactive mutants confirmed that perturbation of both the polymerase and lyase catalytic activities of POLB are required for synergistic PARP-BRCA synthetic lethality. Mechanistically, POLB KO was associated with an increase in single- and double-strand DNA breaks, cell-cycle arrest, and apoptosis when in combination with PARP inhibition. The translational nature of this interaction was further examined using murine xenograft models of BRCA1-mutant and BRCA2-null cell lines, wherein the combination of POLB KO and niraparib led to profound tumor regression and prevented tumor regrowth even after cessation of treatment. Together, these results suggest that POLB is a synergistic enhancer of the synthetic lethal interaction between PARP and BRCA and support POLB as a promising therapeutic target for improving antitumor responses to PARPis in homologous recombination-deficient cancers.},
}
@article {pmid40889353,
year = {2025},
author = {Yu, Y and Yuan, Q and Liu, Z and Tong, B and Shi, S},
title = {Enhancement of Free Fatty Acids Production in Rhodotorula toruloides Using the CRISPR/Cas9-Based Base Editor.},
journal = {ACS synthetic biology},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00359},
pmid = {40889353},
issn = {2161-5063},
abstract = {Rhodotorula toruloides is a promising cell factory to produce various value-added chemicals, including fatty acid derivatives. However, their metabolic engineering development has been hindered by the limited availability of genetic tools. In this study, an accurate and specific gene-editing tool, CRISPR/Cas-based cytidine base editor (CBE) system, was developed for the first time in R. toruloides to broaden its genetic toolbox. The target gene was disrupted by introducing a premature stop codon via C to T mutation. This system achieved single-gene disruption efficiencies of up to 90% and successfully disrupted four genes in parallel with 5% efficiency, marking a breakthrough in multiplexed editing for this yeast. To enable iterative engineering, an inducible Cre-loxP system was integrated, achieving an over 70% selection marker recycling efficiency. Application of this system enabled the construction of uracil-auxotrophic strains. Furthermore, the CBE system was employed to disrupt four genes involved in lipid metabolism, resulting in an engineered strain capable of producing 512.3 mg/L of free fatty acids, thereby demonstrating the utility of the CBE system as an efficient genome editing tool in R. toruloides. The study provides valuable tools to expand the genetic toolbox of R. toruloides and paves the way for fully exploiting its metabolic engineering potential.},
}
@article {pmid40888633,
year = {2025},
author = {Kelleher, AM and Kim, HI and Bayammagari, GS and Davis, DJ and Spencer, TE},
title = {A Cxcl15 Cre Recombinase Mouse Model Useful to Study Gland Development in the Uterus.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {63},
number = {5},
pages = {e70026},
doi = {10.1002/dvg.70026},
pmid = {40888633},
issn = {1526-968X},
support = {HD042311/NH/NIH HHS/United States ; HD112315/NH/NIH HHS/United States ; },
mesh = {Animals ; Female ; *Integrases/genetics/metabolism ; Mice ; Hepatocyte Nuclear Factor 3-beta/genetics/metabolism ; *Uterus/metabolism/growth &amp; development ; *Endometrium/metabolism/growth &amp; development ; Pregnancy ; CRISPR-Cas Systems ; },
abstract = {The mammalian uterus contains glands in the endometrium that develop only or primarily after birth. In the mouse, endometrial glands govern post implantation pregnancy establishment via regulation of blastocyst implantation, stromal cell decidualization, and placental development. Here, we describe a new uterine glandular epithelium (GE) specific Cre recombinase mouse line that is useful to study endometrial gland development and function. Utilizing CRISPR-Cas9 genome editing, improved Cre recombinase (iCre) was inserted into the endogenous C-X-C motif chemokine ligand 15 (Cxcl15) gene. Cxcl15 mRNA, Cxcl15 protein, and Cxcl15-iCre recombinase activity were specific to the developing GE of the uterus. Cxcl15-iCre mice were crossed with floxed Foxa2 mice to conditionally delete Foxa2 specifically in the glands of the neonatal mouse uterus. This conditional deletion of Foxa2 in the developing neonatal uterus resulted in adult mice that lacked Foxa2 in the GE of the uterus, and the adult mice were infertile. The studies described here establish that Cxcl15-iCre mice are a valuable resource to elucidate and explore mechanisms regulating the development and function of glands in the uterus.},
}
@article {pmid40887498,
year = {2025},
author = {Gwon, LW and Badon, IW and Lee, Y and Kim, HJ and Lee, SH},
title = {Advances in large-scale DNA engineering with the CRISPR system.},
journal = {Experimental &amp; molecular medicine},
volume = {},
number = {},
pages = {},
pmid = {40887498},
issn = {2092-6413},
support = {the Chung-Ang University Research Grants in 2024//Chung-Ang University (CAU)/ ; },
abstract = {In recent years, DNA engineering technology has undergone significant advancements, with clustered regularly interspaced short palindromic repeats (CRISPR)-based target-specific DNA insertion emerging as one of the most rapidly expanding and widely studied approaches. Traditional DNA insertion technologies employing recombinases typically involve introducing foreign DNA into genes in vivo by either pre-engineering recognition sequences specific to the recombinase or through genetic crossing to incorporate the requisite recognition sequence into the target gene. However, CRISPR-based gene insertion technologies have advanced to streamline this engineering process by combining the CRISPR-Cas module with recombinase enzymes. This process enables accurate and efficient one-step insertion of foreign DNA into the target gene in vivo. Here we provide an overview of the latest developments in CRISPR-based gene insertion technologies and discusses their potential future applications.},
}
@article {pmid40885801,
year = {2025},
author = {Zirman, A and Abed El-Nabi, M and Samuel, E and Anavy, L and Yakhini, Z and Nachman, I and Rak, R},
title = {Pooled CRISPR screens identifies key regulators of bovine stem cell expansion for cultured meat.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1313},
pmid = {40885801},
issn = {2399-3642},
support = {20-04-0001//Ministry of Agriculture and Rural Development (Israeli Ministry of Agriculture and Rural Development)/ ; },
mesh = {Animals ; Cattle ; Cell Proliferation/genetics ; *Mesenchymal Stem Cells/cytology/metabolism/physiology ; *CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cells, Cultured ; *Meat ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Knockout Techniques ; PTEN Phosphohydrolase/genetics ; Tumor Suppressor Protein p53/genetics ; In Vitro Meat ; },
abstract = {Cultured meat presents a sustainable alternative to traditional meat production but faces significant challenges in scalability and cost efficiency. A key limitation is the restricted proliferation capacity of bovine mesenchymal stem cells (bMSCs), a widely used cell source in the field. Using a pooled, lentiviral CRISPR knockout screen, we interrogated 3000 CRISPR guides targeting 600 genes involved in stem cell regulation or proliferation. Notably, knockouts of TP53 and PTEN significantly increased proliferation rates and delayed senescence. Validation with individual gene knockouts confirms these effects, showing enhanced growth but reduced differentiation potential. We also identified chondrogenic differentiation as a promising target whose repression may further promote MSC expansion. These findings demonstrate the utility of CRISPR screening for optimizing bovine stem cell traits and offer a path toward more scalable cultured meat production in the future.},
}
@article {pmid40669350,
year = {2025},
author = {Suvá, M and Bastón, JI and Wiedenmann, EA and Pose Ortiz de Rozas, MB and Jordán, R and Ghetti, A and Viale, DL and Moro, LN and Vichera, GD},
title = {Use of an exogenous DNA-free system to generate MSTN-KO calves by CRISPR/Cas9 and SCNT.},
journal = {Reproductive biology},
volume = {25},
number = {3},
pages = {101050},
doi = {10.1016/j.repbio.2025.101050},
pmid = {40669350},
issn = {2300-732X},
mesh = {Animals ; Cattle/genetics ; *Nuclear Transfer Techniques/veterinary ; *Myostatin/genetics ; *CRISPR-Cas Systems ; *Gene Editing/veterinary/methods ; *Gene Knockout Techniques/veterinary ; Animals, Genetically Modified ; Female ; Fibroblasts ; Male ; },
abstract = {This study aimed to obtain myostatin (MSTN)-knockout calves, while avoiding the risk of exogenous DNA integration during gene editing. To achieve this, we combined CRISPR/Cas9 ribonucleoprotein gene editing with somatic cell nuclear transfer (SCNT) technology. In the first experiment (E1), we compared the gene editing efficiency of four gRNAs targeting different coding regions of the MSTN gene using plasmid-based CRISPR/Cas9 in bovine fetal fibroblasts (BFF-E1 cells). The highest bioinformatically-predicted editing rate (BPE) was obtained with gRNA2 (96 %), which was subsequently used for further experiments. Next, embryos were produced by SCNT using BFF-E1-edited cells as nuclear donors. Sanger sequencing of the embryos showed biallelic MSTN editing. In the second experiment (E2), plasmid-based editing was replaced with CAS9 protein and trac:crRNA oligoribonucleotides. Editing efficiency was assessed on one edited bovine fetal fibroblast line (BFF-E2-male[ed]) and two edited bovine mesenchymal stem lines (MSC-E2-male[ed] and MSC-E2-fem[ed]) derived from price-winning animals. BPEs were 58.8 %, 31 % and 59 % in fibroblast and MSC cells, and 64 %, 73.3 %, and 66.6 % in SCNT embryos derived from BFF-E2-male[ed], MSC-E2-male[ed] and MSC-E2-fem[ed], respectively. Transfer of 35 MSC-E2-fem[ed] embryos to recipient cows, resulted in the birth of one MSTN-edited calf with a heterozygous genotype. A second-generation clone was subsequently produced, using a fibroblast sample as nuclear donor. In conclusion, we established an efficient protocol for generating high rates of edited blastocysts with a desirable genetic background, resulting in the birth of two MSTN-knockout calves. This study provides a foundation for gene editing to improve productive or biomedical traits.},
}
@article {pmid40639438,
year = {2025},
author = {Bhatia, P and Mohd, A and Agrawal, I and Katiyar, H and Goel, A and Aich, M and Chakraborty, D and Veerapu, NS},
title = {Rapid and efficient generation of viral genome knock-in cell lines using the CRISPR-Cas9 system to produce infectious virus.},
journal = {Journal of virological methods},
volume = {338},
number = {},
pages = {115219},
doi = {10.1016/j.jviromet.2025.115219},
pmid = {40639438},
issn = {1879-0984},
mesh = {*CRISPR-Cas Systems ; Humans ; *Genome, Viral ; Cell Line ; *Gene Knock-In Techniques/methods ; *Hepatitis B virus/genetics ; Genotype ; Virus Replication ; Hepacivirus/genetics ; Antiviral Agents/pharmacology ; Gene Editing ; },
abstract = {Several medically significant viruses are difficult to propagate with conventional laboratory host systems, limiting their availability for detailed characterization, antiviral screening, and functional studies. A range of methods can be used to generate viruses, such as creating sophisticated cell lines, organoid cultures, and the utilization of animal models. Here, we report the generation and characterization of CRISPR-Cas9 edited Huh7 stable cell lines engineered to carry and express overlength HBV genotypes A, B, C and D and full HEV genomes in the AAVS1 site. Viral polymerase inhibitors and IFN-α significantly reduced the production of viral genomes and proteins from the edited cells. The virus released by the edited cells was infectious in permissive cell lines and could be blocked by neutralizing antibodies. This approach can extend to other viruses, like HCV genotype 3, that are hard to culture or to culturable viruses, like Dengue, for vaccine production.},
}
@article {pmid40629854,
year = {2025},
author = {Lin, ZC and Yang, SC and Tran, TTP and Fang, JY},
title = {Animal models of psoriasis for novel drug discovery: a literature update.},
journal = {Expert opinion on drug discovery},
volume = {20},
number = {9},
pages = {1193-1208},
doi = {10.1080/17460441.2025.2528959},
pmid = {40629854},
issn = {1746-045X},
mesh = {*Psoriasis/drug therapy/physiopathology/genetics/pathology ; Animals ; Humans ; *Disease Models, Animal ; *Drug Discovery/methods ; Imiquimod/administration &amp; dosage ; Mice ; Gene Editing/methods ; CRISPR-Cas Systems ; Cytokines/metabolism ; Mice, Knockout ; },
abstract = {INTRODUCTION: Psoriasis is a chronic, immune-mediated inflammatory skin disorder with a multifactorial pathogenesis involving keratinocyte proliferation, dysregulated immune responses, and vascular remodeling. The development of effective therapeutics mainly relies on preclinical models that can reproduce disease-relevant mechanisms.<br><br>AREAS COVERED: This review outlines current in vivo psoriasis models, including spontaneous mutation models, transgenic and knockout mice, xenotransplantation systems, and cytokine-induced and imiquimod-induced models. Each model is evaluated for its ability to replicate key histological and immunological features of human psoriasis, such as acanthosis, immune cell infiltration, and cytokine network activation. The utility of CRISPR/Cas9 gene editing in generating targeted models is also discussed, thus highlighting its potential use for mechanistic studies. Finally, this review also emphasizes the limitations in translational applicability and the need for multimodel validation strategies regarding psoriasis. This article was based on a comprehensive literature search using PubMed, Scopus, and Google Scholar databases, covering publications from January 2015 to March 2025.<br><br>EXPERT OPINION: Despite extensive model development, no single system fully mimics human psoriatic disease. The imiquimod-induced model remains widely used due to its practicality, although it better reflects acute inflammation compared with chronic pathology. The combination of complementary models and the incorporation of human-derived tissues or immune components may improve translational relevance. Advances in genome editing and humanized systems are likely to shape the future of psoriasis research and therapeutic discovery.},
}
@article {pmid40885742,
year = {2025},
author = {Valdez, I and O'Connor, I and Patel, D and Gierer, K and Harrington, J and Ellis, E and Caponetti, SA and Sebra, RP and Valley, HC and Coote, K and Mense, M and Marro, SG and Jiang, T},
title = {A streamlined base editor engineering strategy to reduce bystander editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {8115},
pmid = {40885742},
issn = {2041-1723},
support = {R00HL153940//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; 005363I223//Cystic Fibrosis Foundation (CF Foundation)/ ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; Adenosine Deaminase/genetics/metabolism ; },
abstract = {Base editing (BE) can permanently correct over half of known human pathogenic genetic variants without requiring a repair template, thus serving as a promising therapeutic tool to treat a broad spectrum of genetic diseases. However, the broad activity windows of current base editors pose a major challenge to their therapeutic application. Here, we show that integrating a naturally occurring oligonucleotide binding module into the deaminase active center of TadA-8e, a highly active deoxyadenosine deaminase, enhances its editing specificity. When conjugated with a Cas9 nickase or alternative PAM Cas9 variants, the engineered TadA variant-TadA-NW1-consistently achieves robust A-to-G editing efficiencies within an editing window consisting of four nucleotides, substantially narrower than the 10-bp editing window of the TadA-8e-derived ABEs. Moreover, compared to ABE8e, ABE-NW1 shows significantly decreased Cas9-dependent and -independent off-target activity while maintaining similar on-target editing efficiency. Further, TadA-NW1 can be reprogrammed to perform desired cytidine deamination and adenine transversion within a restricted editing window. Finally, in a cystic fibrosis (CF) cell model, ABE-NW1 outperforms existing ABEs in accurately and efficiently correcting the CFTR W1282X variant, one of the most common CF-causing mutations. In all, we engineered a suite of base editors with refined activity windows, enabling more precise base editing. Importantly, this study presents a streamlined genome editor re-engineering strategy to accelerate the development of therapeutic base editing.},
}
@article {pmid40885054,
year = {2025},
author = {Tang, Z and Gong, F and Feng, Y and Shan, X and Yi, K and Xu, H and Zhou, F and Ji, X and He, Z},
title = {Enhancing CRISPR/Cas-mediated detection of nucleic acids using PNIPAM-based reporters.},
journal = {Biosensors &amp; bioelectronics},
volume = {289},
number = {},
pages = {117917},
doi = {10.1016/j.bios.2025.117917},
pmid = {40885054},
issn = {1873-4235},
abstract = {Currently most conventional reporters in CRISPR/Cas system, including fluorophore-quencher (FQ) and magnetic bead (MB)-based reporters, encounter limitations in terms of sensitivity and compatibility. To overcome these challenges, we developed novel reporters for CRISPR/Cas systems based on thermo-responsive poly(N-isopropylacrylamide) (PNIPAM). Below the lower critical solution temperature (LCST), PNIPAM-based reporters exhibited a liquid state and can be cleaved by Cas proteins in a homogeneous reaction, preserving function and structure of Cas proteins while effectively accelerating the reaction kinetics. Based on this, we designed three dual-enzyme amplification strategies for ultra-sensitive RNA detection, where RNA-activated LbuCas13a cleaved PNIPAM-based reporters to release enzymes (HRP or ALP) or Cas12a activators as the first amplification, followed by thermal separation to initiate secondary enzymatic amplification. Using SARS-CoV-2 RNA as a model target, these strategies achieved a limit of detection (LOD) as low as ∼1 fM, representing a 100-fold improvement over the traditional CRISPR/Cas13a system, while its excellent practical applicability was validated by spiked recovery assay and specific analysis. Overall, this work proposed novel PNIPAM-based reporters that not only could be applied to various individual CRISPR/Cas systems, but also enable integration with downstream amplification steps through their thermal separation properties, advancing the development of next-generation CRISPR/Cas-based molecular diagnostic tools.},
}
@article {pmid40884843,
year = {2025},
author = {Hou, X and Wang, T and Li, Y and Cui, A and Kong, Y and Zhu, Y and Fang, H and Wang, C and Liao, W},
title = {NaCl promotes tomato fruit coloring by relieving SlSR3-induced transcriptional inhibition of lycopene synthesis-related genes.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {5},
pages = {e70450},
doi = {10.1111/tpj.70450},
pmid = {40884843},
issn = {1365-313X},
support = {31860568//the National Natural Science Foundation of China/ ; 32072559//the National Natural Science Foundation of China/ ; 32260353//the National Natural Science Foundation of China/ ; 32360743//the National Natural Science Foundation of China/ ; YB2022004//the Fostering Foundation for the Excellent PH.D. Dissertation of Gansu Agricultural University/ ; GSCS-2022-Z03//the Research Program Sponsored by the State Key Laboratory of Aridland Crop Science of China/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism/drug effects/physiology ; *Lycopene/metabolism ; *Fruit/metabolism/genetics/drug effects/physiology ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant/drug effects ; *Sodium Chloride/pharmacology/metabolism ; Chlorophyll/metabolism ; Carotenoids/metabolism ; Transcription Factors/metabolism/genetics ; Pigmentation/drug effects ; Salt Stress ; },
abstract = {Although salt stress has an adverse effect on plant growth and development, mild salt stress acts as an elicitor of biosynthesis and thus improves fruit quality. To date, the role and mechanism of NaCl in accelerating tomato (Solanum lycopersicum) fruit coloring remain unclear. This study found that 50 mM NaCl treatment (moderate salt stress) reduced the chlorophyll content, increased the carotenoid and lycopene content, and accelerated tomato color transition without decreasing yield. Moreover, NaCl treatment downregulated calmodulin-binding transcription activator (CAMTA5)/signal responsive (SR3). Knockout of SlSR3 by CRISPR/Cas 9 (sr3 mutant) accelerated chlorophyll degradation and carotenoid and lycopene accumulation and upregulated chlorophyll degradation (PPH) and lycopene synthesis (PSY2, PDS, and ZDS) genes in tomato fruit, thereby accelerating tomato coloring. However, SlSR3 overexpression had the opposite effect. Although NaCl treatment decreased chlorophyll, increased carotenoids, and upregulated PPH, PSY2, PDS, and ZDS in wild type and OE-sr3 fruit, these changes were not observed in sr3 mutant fruit. Therefore, PPH, PSY2, PDS, and ZDS might be involved in SR3-regulated coloring under moderate salt stress. Further results showed that SlSR3 could directly bind to the promoter of PSY2 and ZDS via the CG-1 domain, thereby downregulating PSY2 and ZDS. However, NaCl treatment reversed the transcriptional inhibition of SlSR3 on PSY2 and ZDS expression, thereby upregulating PSY2 and ZDS. Collectively, our results suggest that the promoting effects of NaCl on fruit coloring may be dependent on SlSR3-induced transcriptional regulation of lycopene synthesis-related genes PSY2 and ZDS. Therefore, our study provides a better understanding of the function of SlSR3 in tomato coloring and offers insights on the molecular mechanism underlying the effects of moderate salt stress on tomato color transformation.},
}
@article {pmid40884799,
year = {2025},
author = {Yu, Y and Jin, M and Yuan, W and Gong, Y and Li, S and Qin, X and Hou, J and Liu, J and Liu, S and Li, H and Chu, Y and Wang, Y and Zhang, Y and Fang, F and Hao, W and Gu, Y and Fan, Q and Lin, J and Pang, D and Zhang, X},
title = {Engineered crRNA Drives RPA-T7-CRISPR/Cas14a Cascade for Ultrasensitive Detection of ctDNA PIK3CA H1047R.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e07126},
doi = {10.1002/advs.202507126},
pmid = {40884799},
issn = {2198-3844},
support = {82073410 82272623//National Natural Science Foundation of China/ ; 2023ZX06C10//Key Special Projects of Heilongjiang Province Key Research and Development Program/ ; 2017-02//Nn10 Program of Harbin Medical University Cancer Hospital/ ; CYQN24010//Spring Goose Support Program of Heilongjiang Province/ ; },
abstract = {The early detection of circulating tumor DNA (ctDNA) at mutant allele frequencies below 0.1% remains a critical challenge, significantly impeding therapeutic decision-making. To address this limitation, TIDE-Cas14a-an innovative CRISPR/Cas14a-based duplex detection system is developed that integrates recombinase polymerase amplification (RPA) with T7 exonuclease-mediated strand displacement. By strategically engineering crRNAs with synthetic mismatches, the platform achieves single-nucleotide resolution, enabling specific discrimination of the PIK3CA H1047R (c.3140A>G) variant from other mutant subtypes and wild-type sequences at a detection limit of 0.01% with attomolar sensitivity. The system leverages T7 exonuclease's 5'→3' digestion to convert RPA amplicons into single-stranded targets, thereby activating Cas14a without requiring thermal cycling. Furthermore, clinical validation using 32 breast cancer patient samples demonstrated that TIDE-Cas14a achieves 100% sensitivity and specificity, comparable to droplet digital PCR. When deployed on a low-cost digital microfluidic chip, the assay completes ctDNA profiling within 60 min at 37 °C, effectively bridging the gap between complex laboratory testing and point-of-care diagnostics. The work repurposes the CRISPR/Cas system's inherent specificity constraints as a precision oncology tool, establishing a scalable platform for early cancer detection and therapeutic monitoring.},
}
@article {pmid40829362,
year = {2025},
author = {Anwar, S and Khan, S and Azmi, I and Islam, KU and Ahmad, T and Iqbal, J},
title = {CRISPR-based molecular detection of SARS-CoV-2, its emerging variants, and diverse pathogens.},
journal = {Diagnostic microbiology and infectious disease},
volume = {113},
number = {4},
pages = {117062},
doi = {10.1016/j.diagmicrobio.2025.117062},
pmid = {40829362},
issn = {1879-0070},
mesh = {Humans ; *SARS-CoV-2/genetics/isolation &amp; purification ; *COVID-19/diagnosis/virology ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *COVID-19 Nucleic Acid Testing/methods ; Point-of-Care Testing ; },
abstract = {Pathogenic viruses such as SARS-CoV-2 (SCoV-2), continue to pose a significant threat to human civilization. The lessons learnt from SCoV-2 infections have highlighted the requirement for robust and readily available diagnostic tools in order to limit the virus transmission and prevent future pandemics such as COVID-19. RT-qPCR-based detection is routinely used for sensitive and accurate diagnosis, which requires a sophisticated instrument, laboratory setup, and technical expertise. Though RT-qPCR is highly reliable and considered the gold standard for pathogen detection, it is costly, time-consuming, and unaffordable for the masses. Therefore, other reliable methods for nucleic acid-based detection with sensitivity, specificity, and accuracy on-par with RT-qPCR are required. Recent advancement in CRISPR technology promises its development as a POC testing device, providing a high-end, instrument-free, portable, and cost-effective workflow. Further, COVID-19 pandemic has encouraged the development of next-generation CRISPR-based diagnostics with a provision for home-testing which has resulted in the development of portable and smart-phone integrated hand-held devices which can detect various pathogenic infections in a shorter time frame than RT-qPCR. For diagnosing the presence of SCoV-2, CRISPR-based diagnostics (SHERLOCK/DETECTR) are quicker (30-60 min), less expensive ($5-15/test), and portable than RT-qPCR (90-180 min; $10-50/test) demonstrating equivalent specificity (100%) and near-equivalent sensitivity (93-100% for CRISPR-based diagnostics vs 95-100% for RT-qPCR). For high-sensitivity centralized testing, RT-qPCR is still the gold standard, but CRISPR works well in point-of-care settings because it requires little equipment (like lateral flow strips or heating blocks) and allows multiplexing. CRISPR-based diagnostics breakthrough platform like CARMEN leverages microfluidic technology to test 5,000 plus samples in a single run, unlike RT-qPCR, which requires separate reactions for each target.In this review, the advancement in CRISPR technology such as SHERLOCK, DETECTR, and other Cas-9-based diagnostics are highlighted which exclusively focuses on the CRISPR-based diagnostics to detect SCoV-2 and its emerging VOCs, highlighting their advantages and limitations compared to the gold-standard RT-qPCR.},
}
@article {pmid40784045,
year = {2025},
author = {Abdel-Malek, K and von Eisenhart-Rothe, F and Stiegmann, S and Gottmann, I and Doppler, SA and Schneider, S and Preisler, H and Lahm, H and Dreßen, M},
title = {Generation of two B2M knockout induced pluripotent stem cell lines (DHMi005-A-8 and DHMi005-A-9) using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103785},
doi = {10.1016/j.scr.2025.103785},
pmid = {40784045},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Humans ; *beta 2-Microglobulin/genetics/metabolism/deficiency ; *Gene Knockout Techniques ; Cell Line ; },
abstract = {The transplantation of cells into a recipient organism has many hurdles to overcome, including the problem of T-cell-triggered cellular immune defense. Cellular rejection is based on antigen presentation by the MHC-I-complex, which is recognized by cytotoxic T lymphocytes. Elimination of the MHC-I-complex by knocking out the B2M (Beta-2-microglobulin) subunit may be one way of reducing or even completely preventing the initial cellular immune response during transplantation. Using CRISPR/Cas9 we established one heterozygous and one homozygous B2M knockout induced pluripotent stem cell (iPSC) line as a first step towards more effective cell transplantation.},
}
@article {pmid40752255,
year = {2025},
author = {Ramachandran, H and Thomas, AC and Binder, S and Hildebrandt, B and Spitali, P and Rossi, A},
title = {Generation of two iPSC lines with pathogenic DMD nonsense mutations c.4729C&gt;T and c.5713G&gt;T.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103789},
doi = {10.1016/j.scr.2025.103789},
pmid = {40752255},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Codon, Nonsense/genetics ; *Muscular Dystrophy, Duchenne/genetics/pathology ; Cell Line ; *Dystrophin/genetics ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder characterized by progressive degeneration of skeletal and cardiac muscles, typically beginning in early childhood. Here, we describe the generation of two isogenic induced pluripotent stem cell (iPSC) lines engineered using CRISPR-Cas12 to introduce specific nonsense mutations in the DMD gene: c.4729C>T (p.Arg1577Ter) and c.5713G>T (p.Arg1905Ter). The edited iPSC lines retain normal karyotypes, express key pluripotency markers, and exhibit the capacity to differentiate into derivatives of all three germ layers. These models provide powerful tools for investigating DMD pathogenesis, uncovering mechanisms of genetic compensation, and evaluating potential therapeutic strategies.},
}
@article {pmid40752254,
year = {2025},
author = {Ludwik, KA and Valone, VF and Jahn, R and Jyrch, S and Lechner, L and Kühnen, P and Stachelscheid, H},
title = {Generation of an isogenic iPSC line via CRISPR correction of the POMC:W84X mutation for monogenic obesity modeling.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103786},
doi = {10.1016/j.scr.2025.103786},
pmid = {40752254},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Pro-Opiomelanocortin/genetics/metabolism ; *Obesity/genetics/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Mutation/genetics ; Cell Line ; Gene Editing ; Cell Differentiation ; },
abstract = {We report the generation of a genetically corrected induced pluripotent stem cell (iPSC) line, BIHi261-A-1, derived from the patient-specific iPSC line BIHi261-A carrying a homozygous truncating mutation in the POMC gene (POMC:W84X). This mutation causes monogenic obesity by disrupting proopiomelanocortin function. The pathogenic variant was corrected using CRISPR-Cas9 editing. The resulting iPSC line maintained a normal karyotype, expressed pluripotency markers, and retained the ability to differentiate into all three germ layers. BIHi261-A-1 provides valuable isogenic control for disease modeling and therapeutic research targeting POMC-related obesity and hypothalamic regulation of energy homeostasis.},
}
@article {pmid40752251,
year = {2025},
author = {Sikora, T and Patraskaki, M and Howden, S and Graham, A and Christodoulou, J and Linster, CL and Van Bergen, NJ},
title = {Generation and characterisation of four human NAD(P)HX epimerase (NAXE) knockout iPSC lines.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103782},
doi = {10.1016/j.scr.2025.103782},
pmid = {40752251},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Gene Knockout Techniques ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {Pathogenic variants in NAD(P)HX epimerase (NAXE) cause early-onset progressive encephalopathy with brain edema and/or leukoencephalopathy-1 (PEBEL1), an ultra-rare severe neurometabolic disorder resulting in death in infancy. The absence of functional NAD(P)HX epimerase leads to accumulation of S- and R-forms of NAD(P)HX, inhibiting key metabolic pathways. We have generated four NAXE-deficient cell lines via simultaneous CRISPR/Cas9-mediated gene knockout (KO) of NAXE and episomal reprogramming of control human fibroblasts into induced pluripotent stem cells (iPSCs). We have demonstrated loss of NAXE gene expression, characterized iPSC pluripotency and differentiation potential into three germ layers. This provides a suitable model for investigating disease mechanisms and therapies.},
}
@article {pmid40749620,
year = {2025},
author = {Bhat, PP and Inamdar, MS},
title = {Generation of beta actin reporter line (BJNhem20 ACTB-eGFP) in human embryonic stem cells BJNhem20 using CRISPR-Cas9 gene targeting.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103783},
doi = {10.1016/j.scr.2025.103783},
pmid = {40749620},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Actins/genetics/metabolism ; Cell Line ; *Green Fluorescent Proteins/metabolism/genetics ; *Genes, Reporter ; *Gene Targeting ; Gene Editing ; },
abstract = {Beta actin is a cytoskeletal protein that contributes to a wide range of cellular processes. Here we generated beta actin reporter knock-in in BJNhem20 human embryonic stem cell line by CRISPR Cas9 gene editing. The reporter mEGFP is integrated at the beta actin locus, tagging the N-terminal of the protein via a linker. The reporter line is a valuable tool to study beta actin dynamics during cellular process in human embryonic stem cells and to track cells by live imaging.},
}
@article {pmid40706185,
year = {2025},
author = {Park, SH and Suh, D and Kim, H and Lee, RR and Coscarella, IL and Oh, J and Kim, S and Kim, HP and Kwon, C and Lee, CJ and Park, SW and Lee, S},
title = {Establishment of a homozygous LMNA knock-out human induced pluripotent stem cell line using CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103779},
doi = {10.1016/j.scr.2025.103779},
pmid = {40706185},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Lamin Type A/genetics/metabolism/deficiency ; *CRISPR-Cas Systems/genetics ; Homozygote ; Cell Line ; Gene Editing ; Gene Knockout Techniques ; Frameshift Mutation ; },
abstract = {The LMNA gene encodes lamin A/C, essential components of the nuclear envelope that play crucial roles in maintaining nuclear architecture, mechanotransduction, and gene regulation. LMNA mutations are linked to laminopathies, affecting multiple organ systems, including muscle, adipose tissue, and the cardiovascular system. To investigate LMNA-related disorders, we generated a human-induced pluripotent stem cell (hiPSC) line with a homozygous LMNA frameshift mutation (c.351_352insA) using CRISPR/Cas9 genome editing. The edited hiPSCs retained normal colony morphology and expressed key pluripotency markers. This LMNA knockout hiPSC line provides a valuable model for studying lamin A/C functions in nuclear integrity, cellular homeostasis, and disease pathogenesis.},
}
@article {pmid40701116,
year = {2025},
author = {Figueiro-Silva, J and Eschment, M and Mennel, M and Abidi, A and Oneda, B and Rauch, A and Bachmann-Gagescu, R},
title = {CRISPR/Cas9-mediated generation of two isogenic CEP290-mutated iPSC lines.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103781},
doi = {10.1016/j.scr.2025.103781},
pmid = {40701116},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Mutation/genetics ; Cell Cycle Proteins/genetics ; *Cytoskeletal Proteins/genetics ; Cell Line ; *Antigens, Neoplasm/genetics/metabolism ; Cell Differentiation ; },
abstract = {CEP290 is an important human disease gene, as mutations are implicated in a broad spectrum of autosomal recessive ciliopathies, including Leber congenital amaurosis and Joubert, Meckel, Senior-LØken or Bardet Biedl syndromes. To create isogenic mutant human induced pluripotent stem cell (hiPSC) lines for disease modeling, we employed CRISPR/Cas9 to introduce disease-relevant mutations into the control hiPSC line HMGU1 (ISFi001-A). Thorough characterization of the lines, including the effect of the mutation at the mRNA and protein level, shows that these CEP290-mutant lines provide a useful resource for studying ciliopathy disease mechanisms and cilia biology through differentiation into diverse cell types and organoids.},
}
@article {pmid40701115,
year = {2025},
author = {Clua Provost, C and Greetham, L and Monzo, C and Monteil, A and Rovelet-Lecrux, A and Lehmann, S and Wallon, D and Garcia, V and Hirbec, H and Nivet, E and Crozet, C},
title = {Generation of two isogenic-corrected control cell lines (IRMBi001-A-1; IRMBi001-A-2) from Autosomal dominant Alzheimer's disease patient-derived iPSCs carrying a G217D mutation in presenilin 1 gene.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103780},
doi = {10.1016/j.scr.2025.103780},
pmid = {40701115},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology/pathology ; *Alzheimer Disease/genetics/pathology/metabolism ; *Presenilin-1/genetics/metabolism ; Cell Line ; *Mutation/genetics ; CRISPR-Cas Systems ; },
abstract = {Mutations in the preselinin1 (PSEN1) gene are responsible for rare autosomic dominant Alzheimer's disease (ADAD). We generated isogenic control cell lines from iPS cell line derived from ADAD patient carrying a G217D mutation in PSEN1 gene, with CRISPR Cas9 technology. The edited cell lines present the correction of the c.650G > A mutation, no chromosomal abnormalities and no evidence of off-target event. The IRMBi001-A-1 and IRMBi001-A-2 cell lines exhibit pluripotency markers expression and the ability to differentiate into the three germ layers. These two isogenic controls will be used as control to study the pathomechanistic of ADAD through various in vitro assays.},
}
@article {pmid40676141,
year = {2025},
author = {Adnani, M and Hong, SH and Galli, S and Mahajan, A and Lu, C and Abualsaud, N and Biermann, T and Li, Y and Rivera, A and Sebsebie, BS and Caprio, L and Kuwahara, L and Krawczyk, E and Tilan, JU and Lee, Y and Rodriguez, O and Wang, H and Jin, L and Regan, M and de Assis, S and Albanese, C and Pack, SD and Cavalli, LR and Kitlinska, J},
title = {Targeted CRISPR approach reveals an essential role for neuropeptide Y receptor Y5 in Ewing sarcoma extrapulmonary metastasis.},
journal = {Oncogene},
volume = {44},
number = {36},
pages = {3350-3363},
pmid = {40676141},
issn = {1476-5594},
support = {1RO1CA123211//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1R03CA178809//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1R01CA197964//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1R21CA198698//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P30-CA051008//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; S10 OD025153-01//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Sarcoma, Ewing/genetics/pathology/metabolism ; *Receptors, Neuropeptide Y/genetics/metabolism ; Animals ; Humans ; Mice ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Neuropeptide Y/metabolism/genetics ; Neoplasm Metastasis ; *Bone Neoplasms/genetics/pathology ; Cell Movement/genetics ; },
abstract = {Ewing sarcoma (ES) is a pediatric malignancy that lacks adequate therapies for its metastatic form. These tumors constitutively express neuropeptide Y (NPY) and its Y5 receptor (Y5R), which leads to elevated levels of the peptide in patients' serum. In animal models, xenografts secreting NPY metastasize to extrapulmonary niches, including bone; the phenotype associated with adverse prognosis in ES patients. To determine the role of the NPY/Y5R axis in ES extrapulmonary dissemination, we used a doxycycline-inducible CRISPR/Cas9 system to knockout Y5R in SK-ES-1 xenografts that metastasize to these niches. We have shown that metastases developing from heterogenous SK-ES-1/Y5R-sgRNA primary tumors in doxycycline-treated mice were initiated exclusively by SK-ES-1 clones with a functional NPY5R gene. Similarly, metastasis from wild type SK-ES-1 xenografts was associated with a selection of clones with NPY5R gene gain. In vitro assays identified Y5R-dependent ES cell motility driven by RhoA activation as the mechanism underlying the metastatic effects of NPY. In ES cell lines that secrete NPY, the autocrine NPY/Y5R loop was responsible for maintaining basal cell motility, while ES cells that do not release the peptide responded to the exogenous NPY. These data provide evidence for the crucial role of the NPY/Y5R axis in ES metastasis.},
}
@article {pmid40561734,
year = {2025},
author = {Silva, NSD and D'Antonio-Chronowska, A and Hernandez-Benitez, R and McCarron, AR and Karaca, E and Fang, K and Izpisua Belmonte, JC and Panopoulos, AD and Suzuki, K and Frazer, KA},
title = {Generation of a set of genetically modified long QT syndrome induced pluripotent stem cell lines carrying knock-in variants rs120074178 (KCNQ1 c.569G &gt; A; p.Arg190Gln) and rs137854600 (SCN5A c.4865G &gt; A; p.Arg1622Gln) and isogenic control lines.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103755},
doi = {10.1016/j.scr.2025.103755},
pmid = {40561734},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Long QT Syndrome/genetics/pathology/metabolism ; *NAV1.5 Voltage-Gated Sodium Channel/genetics/metabolism ; *KCNQ1 Potassium Channel/genetics ; Cell Line ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; },
abstract = {Long QT syndrome (LQTS) is an inherited channelopathy characterized by life-threatening arrhythmias. LQTS has many subtypes defined by the gene that contains the mutation, including LQT1 (KCNQ1), LQT2 (KCNH2), and LQT3 (SCN5A). Here, we used CRISPR/Cas9 technology to generate five isogenic human induced pluripotent stem cell (iPSC) lines, one line harboring an LQT1 variant rs120074178 (KCNQ1 c.569G > A), two lines harboring an LQT3 variant rs137854600 (SCN5A c.4865G > A), and two derived control lines.},
}
@article {pmid40513401,
year = {2025},
author = {Yu, X and Su, Z},
title = {Generation of a LEUTX-2A-mCherry knock-in H1 human embryonic stem cell line using CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103754},
doi = {10.1016/j.scr.2025.103754},
pmid = {40513401},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Knock-In Techniques ; Cell Line ; *Luminescent Proteins/genetics/metabolism ; Red Fluorescent Protein ; },
abstract = {LEUTX is exclusively expressed at the 8-cell stage and serves as a key regulator of human embryonic genome activation. Induced 8-cell-like cells (8CLCs) derived from pluripotent stem cells offer a tractable model to dissect the molecular transition from pluripotency to totipotency. However, current 8CLC induction protocols are hampered by low efficiency and cellular heterogeneity. To overcome these limitations, we developed a fluorescent reporter system that dynamically monitors endogenous LEUTX expression, facilitating the purification and functional characterization of bona fide 8CLCs. This tool enables systematic interrogation of the regulatory networks underlying totipotency acquisition.},
}
@article {pmid40483902,
year = {2025},
author = {Bai, R and Zhang, S and Gu, X and You, Y and Liu, X},
title = {Establishment of a TRPV2 knockout human embryonic stem cell line (WAe009-A-1Y) using episomal vector-based CRISPR/Cas9.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103744},
doi = {10.1016/j.scr.2025.103744},
pmid = {40483902},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *TRPV Cation Channels/genetics/metabolism/deficiency ; Cell Line ; Cell Differentiation ; Myocytes, Cardiac/metabolism/cytology ; *Plasmids/genetics/metabolism ; Gene Knockout Techniques ; Genetic Vectors/genetics/metabolism ; Female ; },
abstract = {We established a TRPV2-knockout human embryonic stem cell line (WAe009-A-1Y) using a non-integrating episomal CRISPR/Cas9 system. This cell line exhibits an 8-nucleotide frameshift deletion in TRPV2 exon 2, confirmed pluripotency (97.6 % SSEA4+ cells, trilineage differentiation), and a normal female karyotype (46, XX) at passage 30. TRPV2 ablation was validated in differentiated cardiomyocytes, showing >90 % mRNA reduction and absent protein expression. No off-target edits or mycoplasma contamination were detected. This cell resource (STR-authenticated, off-target-free) provides a robust in vitro model to study the biological function of TRPV2 in cardiac mechanotransduction and disease.},
}
@article {pmid40479877,
year = {2025},
author = {Grüner, TF and Ramachandran, H and Thomas, AC and Hildebrandt, B and Gläsker, S and Dobner, J and Rossi, A},
title = {CRISPR/Cas9-mediated editing of VHL in induced pluripotent stem cells: A model for early cell fate in von Hippel-Lindau syndrome.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103748},
doi = {10.1016/j.scr.2025.103748},
pmid = {40479877},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism ; *von Hippel-Lindau Disease/genetics/pathology/metabolism ; *Gene Editing/methods ; Cell Differentiation ; },
abstract = {The von Hippel-Lindau (VHL) tumor suppressor gene is crucial for cellular homeostasis, and its loss leads to VHL syndrome. To model early effects of VHL deficiency, we used CRISPR/Cas9 to generate human iPSC lines with heterozygous or homozygous out-of-frame deletions in exon 1. Both clones showed normal morphology, genomic stability, expression of undifferentiated markers, and tri-lineage differentiation potential. These models offer a valuable system to study early lineage specification and tumor initiation linked to VHL loss.},
}
@article {pmid40466381,
year = {2025},
author = {Hyraht, A and Zhan, F and Guo, R},
title = {Generation of WT1-tdTomato knock-in cynomolgus monkey embryonic stem cell line, WT1-205 using CRISPR/CAS9-based gene targeting.},
journal = {Stem cell research},
volume = {87},
number = {},
pages = {103742},
doi = {10.1016/j.scr.2025.103742},
pmid = {40466381},
issn = {1876-7753},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Macaca fascicularis ; *Embryonic Stem Cells/metabolism/cytology ; *Gene Knock-In Techniques ; Cell Line ; *WT1 Proteins/genetics/metabolism ; Cell Differentiation ; *Gene Targeting ; Red Fluorescent Protein ; },
abstract = {The WT1 gene is crucial for developing intermediate subtypes, including the kidney, gonad, and adrenal cortex. In this study, we generated a tdTomato knock-in cynomolgus embryonic stem cell line (cyESC) by inserting the tdTomato gene at the WT1 stop codon using CRISPR/Cas9 technology. The construct included a PGK-Neo selection cassette (LSL), which was excised by Cre recombinase. Differentiation into kidney- and gonadal-like cells showed tdTomato fluorescence co-localized with GATA4 and PAX2, markers for gonadal and kidney cells. WT1-positive cells also expressed related intermediate genes. This knock-in line provides a valuable tool for studying monkey intermediate mesodermal development.},
}
@article {pmid40884668,
year = {2025},
author = {Iwata, S and Iwamoto, T},
title = {Recql5-Deficient Mice as a Model for Studying Chromoanagenesis Phenomena.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2968},
number = {},
pages = {559-578},
pmid = {40884668},
issn = {1940-6029},
mesh = {Animals ; Mice ; *RecQ Helicases/genetics/deficiency ; *Chromosome Aberrations ; Disease Models, Animal ; CRISPR-Cas Systems ; DNA Repair ; Mice, Knockout ; },
abstract = {Complex chromosomal rearrangements (CCRs) present significant challenges and opportunities in cancer and congenital disease research. Reproducing these rearrangements experimentally in animal models has been challenging, limiting our insights into their mechanisms and impacts. Recql5 is a critical DNA helicase that participates in replication, transcription, and repair processes. We recently succeeded in facilitating CRISPR/Cas9-mediated induction of CCRs in mice, harboring Recql5 deletion. Some CCRs were accomplished by DNA repair mechanisms, including fork stalling and template switching (FoSTeS) and microhomology-mediated break-induced replication (MMBIR), characteristics reminiscent of chromoanasynthesis. This phenomenon is a part of chromoanagenesis, which includes other catastrophic chromosomal rearrangements. This chapter discusses the creation of CCR animal models, offering a new perspective for exploring the pathogenesis of chromosomal rearrangements. Recql5-mutant mice will prove to be a valuable tool for further genetic studies, potentially advancing our understanding of disease mechanisms and suggesting directions for future research.},
}
@article {pmid40884263,
year = {2025},
author = {Liu, H and Yin, H and Xiu, L and Wu, W and Hu, Q and Xia, Y and Garcia, B and Shifa, S and Chen, H and Li, M and Yin, K},
title = {One-Pot Isothermal Nucleic Acid Amplification Assisted CRISPR/Cas Detection Technology: Challenges, Strategies, and Perspectives.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e06716},
doi = {10.1002/advs.202506716},
pmid = {40884263},
issn = {2198-3844},
support = {ZDYF2022SHFZ321//Hainan Province Science and Technology Special Fund/ ; YG2024ZD02//Interdisciplinary Program of Shanghai Jiao Tong University/ ; 24J22800900//Science and Technology Innovation Action Plan of Shanghai/ ; 24142201300//Science and Technology Innovation Action Plan of Shanghai/ ; MDPDMT-2023-02//Key Laboratory of Milk and Dairy Products Detection and Monitoring Technology, State Administration for Market Regulation/ ; 2024QN083//Shanghai Municipal Health Commission of China/ ; BJ1-3000-24-0067//Shanghai Municipal Education Commission's Industry-Academia Practice Program for University Teachers in Shanghai/ ; },
abstract = {The cutting-edge CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/Cas (CRISPR-associated proteins) system, as an emerging molecular diagnostic technique, is driving revolutionary developments in the detection field due to its high specificity and efficiency. However, the CRISPR-based assays typically require the combination with an additional pre-amplification step based on isothermal nucleic acid amplification to meet the requirements of clinical diagnosis, which brings issues including complicated operation and the risk of aerosol contamination. To address these challenges, one-pot CRISPR platforms are emerging as an attractive solution to streamline workflows, enabling rapid, cost-effective, and high-sensitivity diagnostics. This review outlines the current status, challenges, and three key strategies to realize highly efficient one-pot CRISPR-based detection. In addition, further perspectives are outlined that will inspire new exploration and promote one-pot CRISPR/Cas detection as the next generation of diagnostic tools.},
}
@article {pmid40883783,
year = {2025},
author = {Lei, H and Du, S and Tong, X and Chan, WL and Leung, MHY and Bøifot, KO and Bezdan, D and Butler, DJ and Danko, DC and Green, DC and Hernandez, MT and Kelly, FJ and Lucaci, AG and Meydan, C and Nieto-Caballero, M and Ryon, K and Tierney, B and Udekwu, KI and Young, BG and Mason, CE and Dybwad, M and Lee, PKH},
title = {Global biogeography of airborne viruses in public transit systems and their host interactions.},
journal = {Microbiome},
volume = {13},
number = {1},
pages = {193},
pmid = {40883783},
issn = {2049-2618},
support = {SLL 20160933//Stockholm Health Authority/ ; R01AI151059 and U01DA053941//Igor Tulchinsky and the WorldQuant Foundation, US National Institutes of Health/ ; OPP1151054//Bill and Melinda Gates Foundation/ ; R1016-20F//Research Impact Fund, Hong Kong Research Grants Council/ ; 11214721 and 11206224//General Research Fund, Hong Kong Research Grants Council/ ; },
mesh = {*Viruses/genetics/classification/isolation &amp; purification ; *Air Microbiology ; Metagenomics/methods ; Genome, Viral ; Metagenome ; CRISPR-Cas Systems ; *Host Microbial Interactions/genetics ; Humans ; Phylogeography ; Cities ; },
abstract = {BACKGROUND: There is a diverse assemblage of microbes in air in built environments (BEs), but our understanding of viruses and their interactions with hosts in BEs remains incomplete. To address this knowledge gap, this study analyzed 503 metagenomes isolated from air samples from public transit systems in six global cities, namely Denver, Hong Kong, London, New York City, Oslo, and Stockholm. Viral genomes were recovered from samples via metagenomic binning, and viruses' taxonomy, functional potential, and microbial hosts were determined. The study also investigated correlations between virus and host abundances, the coevolution of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems and anti-CRISPR (Acr) proteins, and the potential impacts of auxiliary metabolic genes (AMGs) on hosts.<br><br>RESULTS: Airborne viruses in global BEs exhibited biogeographical variations in diversity, composition, function, and virus-host interactions. Nearly half of the vOTUs analyzed were from the Caulimoviridae family, while 31.8% of them could not be taxonomically classified. Diverse functions were identified within the vOTUs, together with antimicrobial resistance genes with the potential to confer resistance to various antibiotics and antimicrobial agents. Strong correlations were observed between vOTU and host abundances, with clear distinctions between virulent and temperate viruses. However, there was limited co-evolution of CRISPR-Cas systems and Acr proteins, which was likely due to the oligotrophic and physical conditions in the BEs and the dominance of vOTUs with a virulent lifestyle. Phage-encoded AMGs appeared to have the potential to enhance host fitness. These findings highlight biogeographical variations in airborne viruses in BEs and that physical and oligotrophic conditions in BEs drive virus survival strategies and virus-host coevolution.<br><br>CONCLUSION: There are biogeographical variations in airborne viruses in BEs in global cities, as physical and oligotrophic conditions in BEs drive virus survival strategies and virus-host coevolution. Moreover, the characteristics of airborne viruses in BEs are distinct from those of viruses found in other, more nutrient-rich ecosystems. Video Abstract.},
}
@article {pmid40883747,
year = {2025},
author = {Brettmann, E and Chen, F and Beishir, S and Garvey, G},
title = {Cytosine base editor-DNA binding domain fusions for editing window modulation in the RNP format.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {92},
pmid = {40883747},
issn = {1472-6750},
mesh = {*Cytosine/metabolism/chemistry ; *Gene Editing/methods ; Humans ; *DNA-Binding Proteins/genetics/metabolism/chemistry ; Thymine ; Recombinant Fusion Proteins/genetics/metabolism ; DNA/genetics ; CRISPR-Cas Systems ; },
abstract = {Base editing technologies allow for the precise and efficient installation of defined nucleotide substitutions into a target genome without the introduction of double strand breaks or DNA templates. Here we describe two recombinant, protein format cytosine base editors (CBEs) that efficiently catalyze the installation of cytosine-to-thymine edits, termed "Flexible" and "Precision." Flexible exhibits a wide editing window, while Precision uses a fused single-stranded DNA binding protein to narrow the editing window, lowering the risk of editing multiple cytosine residues at the target site. We show that co-transfection with uracil glycosylase inhibitor protein increases the proportion of substitutions that are C-to-T and the ratio of C-to-T editing to indel formation, thus reducing undesired editing outcomes. We use in vitro editing assays to characterize our editors and show a preference for cytosine residues preceded by thymine (TpC dinucleotides) and unmethylated cytosine residues.},
}
@article {pmid40883169,
year = {2025},
author = {Wachsmann, TLA and Qi, LS},
title = {CRISPR tools for T cells: targeting the genome, epigenome, and transcriptome.},
journal = {Trends in cancer},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.trecan.2025.08.001},
pmid = {40883169},
issn = {2405-8025},
abstract = {T cell therapy has curative potential for many cancers. Despite impressive clinical efficacy in hematological malignancies, current T cell therapy still faces challenges related to sustaining responses, antigen escape, cytotoxicity, limited accessibility, and difficulties in treating solid tumors. The advent of CRISPR (clustered regularly interspaced short palindromic repeats) technologies provides a promising solution to these challenges. CRISPR technologies have grown from merely tools for gene knockout to sophisticated tools that can engineer cells at various levels of the genome, epigenome, and transcriptome. In this review we discuss recent technological advancements and how their application to T cells has the potential to steer the next generation of cellular therapy. We highlight emerging applications and current technological limitations that future tool development aims to overcome.},
}
@article {pmid40882619,
year = {2025},
author = {Liu, J and Song, Y and Mei, M and Zhao, X and Wan, S and Xun, Q and Meng, Y and An, J and Li, G and Ding, Y and Ding, C},
title = {Gene editing unlocks superior mutants from once detrimental RFL for enhanced rice yield traits.},
journal = {The Plant journal : for cell and molecular biology},
volume = {123},
number = {5},
pages = {e70454},
doi = {10.1111/tpj.70454},
pmid = {40882619},
issn = {1365-313X},
support = {2024YFD2301500//National Key Research and Development Program of China/ ; },
mesh = {*Oryza/genetics/growth &amp; development ; *Gene Editing/methods ; *Plant Proteins/genetics/metabolism ; Mutation ; Gene Expression Regulation, Plant ; Phenotype ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {RICE FLORICULA LEAFY/ABERRANT PANICLE ORGANIZATION 2 (RFL/APO2) is a master regulator of panicle morphogenesis and development in rice. Traditionally, mutations in RFL have led to severe growth phenotypes and decreased rice yield, labeling it as detrimental. However, the present study challenged this perception by utilizing CRISPR/Cpf1 and single-base gene-editing technologies to generate a series of site-directed rfl mutants. Our findings revealed that the evolutionarily conserved sterile alpha motif (SAM) domain and DNA-binding domain (DBD), as well as the intron region of RFL, all play roles in regulating rice morphological development and yield traits. Specifically, introns and the SAM domain are primarily involved in panicle development, whereas the DBD and its key functional sites are closely associated with morphological development and yield. Notably, the amino acid at position 266 was found to be a critical site for RFL regulation of grain shape, significantly affecting grain weight, with changes in the expression levels of genes involved in grain length and panicle weight regulation, such as GRF1 and SPL16. This study not only expands our understanding of the role of RFL in monocot plants but also provides a novel perspective on how gene editing can transform a gene once considered detrimental to improve yield traits in cereal crops. These findings suggest that the number of genes available for optimizing rice phenotypes through gene editing can be significantly increased.},
}
@article {pmid40880534,
year = {2025},
author = {Du, SW and Palczewska, G and Dong, Z and Lauterborn, JC and Kaipa, BR and Yan, AL and Hołubowicz, R and Ha, S and Chen, PZ and Gall, CM and Zode, G and Liu, DR and Palczewski, K},
title = {TIGER: A tdTomato in vivo genome-editing reporter mouse for investigating precision-editor delivery approaches.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {35},
pages = {e2506257122},
doi = {10.1073/pnas.2506257122},
pmid = {40880534},
issn = {1091-6490},
support = {R01EY036994//HHS | NIH (NIH)/ ; R01EY034501//HHS | NIH (NIH)/ ; T32GM008620//HHS | NIH (NIH)/ ; F30EY033642//HHS | NIH (NIH)/ ; R01HD101642//HHS | NIH (NIH)/ ; UG3AI150551//HHS | NIH (NIH)/ ; U01AI142756//HHS | NIH (NIH)/ ; R35GM118062//HHS | NIH (NIH)/ ; RM1HG009490//HHS | NIH (NIH)/ ; Career-Starter Research Grants//Knights Templar Eye Foundation (KTEF)/ ; P30EY034070//HHS | NIH (NIH)/ ; HHMI//HHMI (HHMI)/ ; Unrestricted//Research to Prevent Blindness (RPB)/ ; N/A//Canadian Government | Natural Sciences and Engineering Research Council of Canada (NSERC)/ ; R01EY026177//HHS | NIH (NIH)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Mice ; CRISPR-Cas Systems ; *Genes, Reporter ; *Luminescent Proteins/genetics/metabolism ; Red Fluorescent Protein ; Mice, Transgenic ; Indoles ; Gene Transfer Techniques ; Genetic Vectors ; },
abstract = {In vivo genome editing has the potential to address many inherited and environmental disorders. However, a major hurdle for the clinical translation of genome editing is safe, efficient delivery to disease-relevant tissues. A modality-agnostic reporter animal model that facilitates rapid, precise, and quantifiable assessment of functional delivery and editing could greatly enhance the evaluation and translation of delivery technologies. Here, we present the development of the tdTomato in vivo genome-editing reporter (TIGER) mouse, a reporter strain that harbors an integrated and constitutively expressed mutated tdTomato gene in the Polr2a locus. The mutations (Q115X, Q357X) abolish fluorescence, but successful adenine base editing (ABE) or prime editing (PE) restores tdTomato fluorescence. This mouse model facilitates the tissue- and cell type-specific assessment of genome editing agent delivery. We describe several editing strategies validated in vitro and demonstrate efficient ABE and PE in vivo using viral and nonviral delivery vectors targeting four cell types within the mouse eye: the retinal pigment epithelium, photoreceptors, Müller glia, and the trabecular meshwork. We show direct editing characterization in the ocular tissues via in vivo and ex vivo two-photon confocal microscopy and verify the spectral and fluorescence lifetime properties of tdTomato reporter in other mouse tissues. Additionally, we demonstrate successful adeno-associated virus (AAV)-mediated PE of extraocular tissues, including hepatocytes, skeletal muscle, and brain neurons by intravenous injection. Thus, the TIGER mouse facilitates the direct development, comparison, and optimization of delivery platforms for efficient and productive ABE or PE broadly applicable in vivo across multiple tissues tested in this study.},
}
@article {pmid40879839,
year = {2025},
author = {Zhang, Y and Hao, F and Gao, Y and Song, W and Su, C and Guo, X and Liu, D},
title = {Validation of caprine H11 and the Rosa26 platform for transgene integration via CRISPR-based system: investigations on stable transgene expression and genetic biosafety.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {180},
pmid = {40879839},
issn = {1438-7948},
support = {32360811//National Natural Science Foundation of China/ ; U23A20226//National Natural Science Foundation of China/ ; 2021ZD0048//the Science and Technology Major Project of Inner Mongolia Autonomous Region of China/ ; 2023KYPT0014//the Science and Technology Program of Inner Mongolia Autonomous Region/ ; },
mesh = {Animals ; *Goats/genetics ; *CRISPR-Cas Systems ; *Transgenes ; *Gene Editing/methods ; Animals, Genetically Modified/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Female ; },
abstract = {CRISPR/Cas9 technology is an efficient tool for site-specific livestock gene editing. However, to minimize potential disruption of host genome function, exogenous genes should be integrated into well-characterized genomic loci, such as H11 or Rosa26, which have been empirically validated for stable transgene expression. This study established a multi-dimensional assessment system to evaluate the biological applicability of the H11 locus and the widely used Rosa26 targeting platform as sites for targeted integration of exogenous genes in goats. Donor cells carrying the enhanced green fluorescent protein (EGFP) reporter gene at the H11 and Rosa26 loci were generated via CRISPR/Cas9-mediated homology-directed repair; this was followed by somatic cell nuclear transfer to produce transgenic cloned embryos and healthy offspring. Multi-dimensional analyses revealed the following. At the cellular level, there was stable and efficient EGFP expression at integration sites, with donor cells maintaining normal cell cycle progression, proliferation capacity, and apoptosis levels, and with no alterations in the transcriptional integrity of adjacent genes. At the embryonic level, there was sustained EGFP expression across pre-implantation embryonic stages, with developmental metrics statistically indistinguishable from wild-type embryos. Finally, at the individual level, cloned offspring exhibited growth phenotypes consistent with wild-type counterparts, and EGFP showed broad-spectrum expression in eight tissues. This study establishes the first CRISPR/Cas9-based crossscale (cellular-embryonic-individual) validation in goats, demonstrating that the H11 and Rosa26 loci support efficient and stable transgene integration in goats. These results provide a precise and predictable technical framework for livestock genetic improvement.},
}
@article {pmid40879164,
year = {2025},
author = {Takano, S and Takenawa, S and Divya, N and Yan, K and Wen, X and Maehara, T and Nomura, N and Obana, N and Toyofuku, M and Usui, M and Ariyoshi, W and Okamoto, A},
title = {Enrichment of Horizontally Transferred Gene Clusters in Bacterial Extracellular Vesicles via Non-Lytic Mechanisms.},
journal = {The ISME journal},
volume = {},
number = {},
pages = {},
doi = {10.1093/ismejo/wraf193},
pmid = {40879164},
issn = {1751-7370},
abstract = {Bacterial extracellular vesicles are emerging as key mediators of horizontal gene transfer, enhancing microbial adaptability. A critical factor determining the effectiveness of horizontal gene transfer is the fraction of vesicles containing specific functional genes. However, the proportion of containing specific DNA fragments has not been adequately determined, which hinders the understanding of the conditions and mechanisms that facilitate the incorporation of specific genes into the vesicles and possible evolutionary roles of vesicle-derived DNA. Here, we demonstrate that enrichment of horizontally transferred genes into bacterial extracellular vesicles is driven by cellular processes by profiling the DNA content of hundreds of individual vesicles using a microdroplet-based sequencing technique. This approach revealed unique DNA profiles in vesicles from the oral pathogen Porphyromonas gingivalis, pinpointing genomic regions related to DNA reorganization such as CRISPR-Cas clusters. Comparative genomic and phylogenetic analyses of Porphyromonas genomes revealed traces of horizontal gene transfer in vesicle-enriched genes. Modulating vesicle-biogenesis routes, quantitative real-time PCR revealed that this selective enrichment was driven by blebbing-driven DNA packaging mechanisms rather than stress-induced lysis. Applied to dental plaque-derived bacterial extracellular vesicles, the droplet-based approach reveled O-antigen biosynthetic genes, key for host-bacterial interactions, were prevalent in the vesicles from Alcaligenes faecalis, suggesting the vesicles from this bacterium can modulate pathogenicity in oral biofilms through targeted DNA packaging. These findings suggest the prevalence of functionally relevant gene clusters in bacterial extracellular vesicles in oral microbiota and their evolutionary roles as DNA cargoes for modulating phage-bacterial and host-bacterial interactions via horizontal gene transfer.},
}
@article {pmid40879132,
year = {2025},
author = {Perlee, S and Ma, Y and Hunter, MV and Swanson, JB and Cruz, NM and Ming, Z and Xia, J and Lionnet, T and McGrail, M and White, RM},
title = {Identifying in vivo genetic dependencies of melanocyte and melanoma development.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {40879132},
issn = {2050-084X},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; R01CA229215/NH/NIH HHS/United States ; R01CA238317/NH/NIH HHS/United States ; DP2CA186572/NH/NIH HHS/United States ; ORIP R24OD020166/NH/NIH HHS/United States ; T32GM007739/NH/NIH HHS/United States ; F30CA265124/CA/NCI NIH HHS/United States ; 1K99CA266931/CA/NCI NIH HHS/United States ; T32CA254875/CA/NCI NIH HHS/United States ; F31CA271518/CA/NCI NIH HHS/United States ; Core Grant//Ludwig Institute for Cancer Research/ ; },
mesh = {Animals ; Zebrafish/genetics ; *Melanocytes/physiology/metabolism ; *Melanoma/genetics/pathology ; Animals, Genetically Modified ; Microphthalmia-Associated Transcription Factor/genetics/metabolism ; Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {The advent of large-scale sequencing in both development and disease has identified large numbers of candidate genes that may be linked to important phenotypes. We have developed a rapid, scalable system for assessing the role of candidate genes using zebrafish. We generated transgenic zebrafish in which Cas9 was knocked in to the endogenous mitfa locus, a master transcription factor of the melanocyte lineage. The main advantage of this system compared to existing techniques is maintenance of endogenous regulatory elements. We used this system to identify both cell-autonomous and non-cell-autonomous regulators of normal melanocyte development. We then applied this to the melanoma setting to demonstrate that loss of genes required for melanocyte survival can paradoxically promote more aggressive phenotypes, highlighting that in vitro screens can mask in vivo phenotypes. Our genetic approach offers a versatile tool for exploring developmental processes and disease mechanisms that can readily be applied to other cell lineages.},
}
@article {pmid40878556,
year = {2025},
author = {Hyeon, H and Hwang, S and Luo, Y and Shin, E and Yeom, JH and Kim, HM and Ryu, M and Lee, K},
title = {CRISPR-Cas technologies: Emerging tools from research to clinical application.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {8},
pages = {e2504012},
doi = {10.71150/jm.2504012},
pmid = {40878556},
issn = {1976-3794},
support = {//Chung-Ang University/ ; //National Research Foundation of Korea/ ; RS-2024-00461596//Ministry of Education/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; Animals ; RNA Editing ; },
abstract = {CRISPR-Cas technologies have emerged as powerful and versatile tools in gene therapy. In addition to the widely used SpCas9 system, alternative platforms including modified amino acid sequences, size-optimized variants, and other Cas enzymes from diverse bacterial species have been developed to apply this technology in various genetic contexts. In addition, base editors and prime editors for precise gene editing, the Cas13 system targeting RNA, and CRISPRa/i systems have enabled diverse and adaptable approaches for genome and RNA editing, as well as for regulating gene expression. Typically, CRISPR-Cas components are transported to the target in the form of DNA, RNA, or ribonucleoprotein complexes using various delivery methods, such as electroporation, adeno-associated viruses, and lipid nanoparticles. To amplify therapeutic efficiency, continued developments in targeted delivery technologies are required, with increased safety and stability of therapeutic biomolecules. CRISPR-based therapeutics hold an inexhaustible potential for the treatment of many diseases, including rare congenital diseases, by making permanent corrections at the genomic DNA level. In this review, we present various CRISPR-based tools, their delivery systems, and clinical progress in the CRISPR-Cas technology, highlighting its innovative prospects for gene therapy.},
}
@article {pmid40877941,
year = {2025},
author = {Hu, C and Zeng, Z and Bao, X and Li, D and Tai, H and Zeng, H and Luo, C and Tang, L and Chen, T and Zuo, S},
title = {Whole-gene CRISPR/cas9 library screen revealed targeting STAT6 increased the sensitivity of liver cancer to celecoxib via inhibiting arachidonic acid shunting.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {384},
pmid = {40877941},
issn = {1478-811X},
support = {82473969, 82160665, 82260535//the National Natural Science Foundation of China/ ; 82473969, 82160665, 82260535//the National Natural Science Foundation of China/ ; 82473969, 82160665, 82260535//the National Natural Science Foundation of China/ ; 2022M720929 and 2024M750674//China Postdoctoral Science Foundation/ ; 2022M720929 and 2024M750674//China Postdoctoral Science Foundation/ ; Xiaobohe J 2022 [061]//Startup Fund for PhD Scholars of Guizhou Medical University/ ; GCC[2023]002//Guizhou High-level Innovative Talents Supporting Program/ ; QJJ [2022] 020//the Continuous Support Fund for Excellent Scientific Research Platform of Colleges and Universities in Guizhou Province/ ; gyfynsfc-2021-4//the National Natural Science Foundation Cultivation Project of the Affiliated Hospital of Guizhou Medical University/ ; gyfykyc-2023-01//the Affiliated Hospital of Guizhou Medical University Leading Scholar Project/ ; ZK[2024] major 039//Guizhou Provincial Science and Technology Projects General/ ; },
mesh = {*Celecoxib/pharmacology/therapeutic use ; Humans ; *Liver Neoplasms/genetics/drug therapy/pathology/metabolism ; *STAT6 Transcription Factor/metabolism/genetics/antagonists &amp; inhibitors ; *CRISPR-Cas Systems/genetics ; Animals ; *Arachidonic Acid/metabolism ; Cell Line, Tumor ; Mice ; Drug Resistance, Neoplasm/drug effects/genetics ; Mice, Nude ; },
abstract = {Celecoxib, a selective COX-2 inhibitor, has demonstrated anti-liver cancer effects in various preclinical models and clinical traits. However, prolonged use of celecoxib can lead to drug resistance, necessitating higher doses to maintain efficacy