@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.},
}

*White spot syndrome virus 1/pathogenicity/immunology/physiology/genetics
Animals
*Aquaculture/methods
*Penaeidae/virology/immunology
Immunity, Innate
Viral Vaccines/immunology

@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.},
}

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

@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.},
}

*Drug Discovery/methods
*Protein Engineering/methods
Humans
Animals
Recombinant Proteins/therapeutic use/chemistry/genetics

@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.},
}

Animals
*Muscle, Skeletal/metabolism
Muscle Development/genetics
*Poultry/genetics
Muscular Diseases/genetics
Transcriptome
CRISPR-Cas Systems
*RNA/genetics/metabolism

@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.},
}

*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

@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.},
}

*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

@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.},
}

*CRISPR-Cas Systems/genetics
*Artificial Intelligence
*Plant Viruses/genetics
*Gene Editing/methods
*Plant Diseases/virology/genetics
Crops, Agricultural/genetics/virology

@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.},
}

*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

@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‑2024‑0009//The Ministry of Science and Higher Education of the Russian Federation/ ; 075‑15‑2025‑51//The Kurchatov Genomic Center of ICG SB RAS/ ; },
mesh = {*Triticum/genetics/growth & 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.},
}

*Triticum/genetics/growth & 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

@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.},
}

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

@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.},
}

*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

@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.},
}

*Biosensing Techniques/methods
*Food Microbiology
Humans
*Foodborne Diseases/microbiology/diagnosis
Nucleic Acid Amplification Techniques
CRISPR-Cas Systems
Molecular Imprinting

@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.},
}

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

@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.},
}

*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

@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 & 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.},
}

Animals
Chickens/genetics
*CRISPR-Cas Systems
Cell Line
RNA, Messenger/genetics

@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 & 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.},
}

*Klebsiella pneumoniae/genetics/drug effects/isolation & 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

@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 & 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.},
}

*Electrochemical Techniques/methods
*CRISPR-Cas Systems/genetics
*Parvovirus B19, Human/genetics/isolation & purification
*DNA, Viral/analysis/genetics
*Biosensing Techniques/methods
Humans
Limit of Detection
CRISPR-Associated Proteins
*Endodeoxyribonucleases/genetics
Bacterial Proteins

@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.},
}

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

@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.},
}

*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

@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.},
}

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

@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 & dosage/chemistry ; Autophagy/drug effects ; Animals ; Autophagy-Related Protein 5/genetics ; *Neoplasms/immunology/drug therapy ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Naphthoquinones/administration & dosage/chemistry ; *Antineoplastic Agents/administration & dosage ; Hyaluronan Receptors/metabolism ; Polyethyleneimine/chemistry/administration & 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.},
}

Humans
*Copper/administration & dosage/chemistry
Autophagy/drug effects
Animals
Autophagy-Related Protein 5/genetics
*Neoplasms/immunology/drug therapy
CRISPR-Cas Systems
Cell Line, Tumor
*Naphthoquinones/administration & dosage/chemistry
*Antineoplastic Agents/administration & dosage
Hyaluronan Receptors/metabolism
Polyethyleneimine/chemistry/administration & dosage
Female
Mice

@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&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.},
}

*Saccharomyces cerevisiae/metabolism/genetics
*Zea mays/metabolism/chemistry
*Metabolic Engineering
Fermentation
CRISPR-Cas Systems
Saccharomyces cerevisiae Proteins/genetics/metabolism
*Monoterpenes/metabolism

@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.},
}

*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

@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.},
}

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

@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.},
}

*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

@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.},
}

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

@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 & Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 2336877//National Science Foundation (NSF)/ ; GM132087//U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; GM132087//U.S. Department of Health & 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 & 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.},
}

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 & control
Genome, Insect
*Genetic Fitness
Female

@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.

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.

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.},
}

*Neoplastic Cells, Circulating/pathology/metabolism
Humans
*Extracellular Vesicles/metabolism/chemistry
*CRISPR-Cas Systems
Biomarkers, Tumor/genetics

@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.},
}

Humans
HEK293 Cells
*Optogenetics/methods
Light
*CRISPR-Cas Systems
Flow Cytometry/methods

@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.},
}

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

@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.},
}

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

@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.},
}

*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

@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.

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.

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.

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.).},
}

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

@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.

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.

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.

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.},
}

*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

@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 & 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.},
}

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

@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 & 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.},
}

*CRISPR-Cas Systems/genetics
*RNA, Viral/genetics/analysis
*SARS-CoV-2/genetics/isolation & 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

@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.},
}

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

@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.},
}

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

@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.},
}

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

@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.},
}

*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

@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 & 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.},
}

Animals
*Cadherins/metabolism/genetics
Mice
*Brain/metabolism/embryology
Gene Knock-In Techniques
Gene Expression Regulation, Developmental
Mice, Transgenic
CRISPR-Cas Systems

@article {pmid41276731,
year = {2025},
author = {Eren Eroglu, AE and Toklu, K and Yasa, İ},
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 & 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 & 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.},
}

Animals
*Trichinellosis/diagnosis/parasitology/veterinary
*Trichinella/genetics/isolation & purification
*Molecular Diagnostic Techniques/methods/trends
CRISPR-Cas Systems
Humans

@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.},
}

*Printing, Three-Dimensional
Humans
Human papillomavirus 16/genetics
*CRISPR-Cas Systems/genetics
*Lab-On-A-Chip Devices
DNA, Viral/analysis/genetics
Equipment Reuse

@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.},
}

*Lathyrus/genetics
*Agrobacterium/genetics
*Plant Roots/genetics
*Gene Editing/methods
*Transformation, Genetic
CRISPR-Cas Systems
Plants, Genetically Modified/genetics

@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.},
}

*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

@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.},
}

*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

@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.},
}

Humans
*CRISPR-Cas Systems/genetics
*Pandemics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Point-of-Care Systems
Sensitivity and Specificity
Temperature

@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 & 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.},
}

*Biofilms/growth & development
*CRISPR-Cas Systems
*Food Safety/methods
Gene Editing/methods
*Food Microbiology/methods

@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.},
}

*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

@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.},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Knock-In Techniques/methods
Rats
*Integrases/metabolism/genetics
Biotinylation
Cell Membrane/metabolism

@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.},
}

*Halomonas/genetics/metabolism
*Amino Acids, Diamino/metabolism/biosynthesis/genetics
Metabolomics/methods
Gene Deletion
*CRISPR-Cas Systems/genetics
Betaine/metabolism
Bacterial Proteins/genetics/metabolism

@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.},
}

*Biosensing Techniques/methods
*CRISPR-Cas Systems/genetics
Humans
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats
DNA/genetics/analysis
Gene Editing

@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.},
}

*Osteoarthritis/therapy/genetics
*CRISPR-Cas Systems/genetics
Humans
Animals
*Genetic Therapy/methods
*Gene Editing/methods
Disease Models, Animal

@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 & 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.},
}

Humans
*Leukemia, Myeloid, Acute/genetics/therapy/diagnosis
*CRISPR-Cas Systems
*Gene Editing/methods
Animals
*Genetic Therapy/methods

@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 & 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.},
}

Animals
Dogs
*CRISPR-Cas Systems/genetics
*Adenoviridae Infections/diagnosis/veterinary/virology
*Adenoviruses, Canine/isolation & purification/genetics
Sensitivity and Specificity
*Nucleic Acid Amplification Techniques/methods
*Dog Diseases/diagnosis/virology
*Molecular Diagnostic Techniques/methods

@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.},
}

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

@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.},
}

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

@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.},
}

*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

@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.},
}

*Carbamates/analysis
*Aptamers, Nucleotide/genetics/chemistry
*CRISPR-Cas Systems
*Benzimidazoles/analysis
Limit of Detection
*Biosensing Techniques/methods
*Pesticide Residues/analysis
Reproducibility of Results

@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 Förderung 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.},
}

*Pyrophosphatases/metabolism/genetics
Humans
*Nucleotides/biosynthesis
*Uridine/metabolism/pharmacology
Phosphoribosyl Pyrophosphate/metabolism
CRISPR-Cas Systems
Pyrimidines/biosynthesis

@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.},
}

Humans
*CRISPR-Cas Systems/genetics
*Drug Resistance/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@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 & 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.},
}

*Gardnerella vaginalis/genetics/isolation & 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

@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.},
}

*Arabidopsis/genetics
*Chromosomes, Plant/genetics
*CRISPR-Cas Systems
Gene Editing
*Genome, Plant
Karyotype
Meiosis
Recombination, Genetic
Gene Fusion

@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 & 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 & 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.},
}

Animals
*Transplantation, Heterologous/methods
*Galactosyltransferases/genetics/deficiency
*Animals, Genetically Modified
Swine
*Graft Rejection/immunology/genetics/prevention & control
Gene Knockout Techniques
Humans
Heterografts/immunology

@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&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.},
}

*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

@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 & Human Services | National Institutes of Health (NIH)/ ; U54CA268084//U.S. Department of Health & 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.},
}

*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

@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.},
}

*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

@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.},
}

*CRISPR-Cas Systems
*Pyrimidines/chemistry/metabolism
*RNA/chemistry/metabolism
Humans
*Purines/chemistry/metabolism
Protein Biosynthesis
DNA-Directed RNA Polymerases/metabolism
Viral Proteins

@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.},
}

*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

@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.},
}

*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

@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.},
}

Humans
*Gene Editing/methods
HEK293 Cells
Animals
Dependovirus/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*Genome, Human
CRISPR-Cas Systems
Mice